The synapse as a biochemical self-organizing micro-cybernetic unit

117

BRAIN RESEARCH

Review Arfic|e

THE SYNAPSE AS A BIOCHEMICAL SELF-ORGANIZING MICRO-CYBERNETIC UNIT

EUGENE ROBERTS

Department of Biochemistry, City of IIope Medical Center, Duarte, Calif. (U.S.A.) (Received October 4th, 1965)

I. INTRODUCTION

One of the chief motivations for engaging in a chemical study of the nervous system is the hope that the knowledge gained eventually might contribute to an understanding of the working of the human mind. The force of this motivation is very great, indeed, as seen from the excitement engendered by each new report of an 'abnormal' molecule in tissue fluids of schizophrenics, the report of a new mental 'wonder drug', or the enunciation of a new theory of memory. There is no doubt that the structure of society and the course of human events would be greatly altered by real knowledge at the molecular level of our mental machinery. However, the intense effort devoted to each new finding and the attendant narrowing of the perspective often has led to the delusion that the one and only solution has been achieved. Frequently this has resulted in a waste of effort on the part of the initial investigators and of those who subsequently have undertaken to test the validity of the results, because the observations have dealt with isolated chemical findings which were not related to a meaningful model or framework. The general solutions to the various problems of the functions of the nervous system of various species will come from an integrated understanding of the wiring diagrams (neuroanatomy), the electrical and chemical characteristics of the conducting units and transmitting mechanisms in the circuits (neurophysiology and neurochemistry), and the behavior of the organisms whose activities are regulated by them (psychology). In the case of human beings, the influence of a complex social en~:ironment cannot be neglected. The logical role of the neurochemist is to supply information which will describe the structure and organization of the conducting and transducing units in molecular terms and their function at a chemo-cybernetic level. II. THE SYNAPSE AS A COMMON GROUND FOR CHEMIST AND BIOLOGIST

It is obvious that the chemist must choose a meaningful functional unit of the Brain Research, 2 (1966) 1!7-166

118

E. ROBERTS

neural m~,chinery with which to work. One of the major obstacles to progress in the past has been the absence of a single model to which both the neurobiologist and neurochemist could relate. It would appear to me that the detailed examination of the organization of chemically transmitting synapses would give the chemist the best chance of obtaining data which would allow maximal opportunity for the development of correlations with observations from other disciplines. In view of the dearth of detailed biochemical information about functional synapses and of the paucity of techniques with which to obtain such information at present, it seemed best to attempt to construct a frankly speculative framework, consistent with most available data, which might serve as a basis for the design of new experimental approaches. A rather detailed formulation will be made of the "general" synapse as a self-organizing micro-cybernetic unit using known observations and biologically reasonable assumptions. The minimal functional unit which can be considered is the entire synaptic apparatus of which the essential elements are the pre- and postsynaptic endings, an extraneuronal compartment consisting of glial end-feet and extracellular space, and the blood vessels in the synaptic area. Obviously, if understanding is to be gained of what happens in interneuronal circuits in which the nodal points and sites of information transmittal are synapses, a thorough knowledge must be gained about what happens at individual synapses. It is hard to envision how chemical measurements performed on homogenates, slices, or even on individually dissected nerve cell bodies covered with axo-somatic synapses and containing dendritic stumps could give the required information. However, recent studies of the central nervous system of the leech have indicated the feasibility of combined morphological, physiological, and chemical approaches to the study of neuron-glial-extracellular space--capillary relationships ~9,61,84,85. The units and supporting elements of neural circuits are made up entirely of chemical substances of varying degrees of complexity. The syntheses of the macromolecular constituents which catalyze the multitude of ongoing intracellular chemical reactions and which form the structural components of the cells are under genetic control. The genetic expressivity of a cell is subject to complex environmental influences so that at any one time the state of the system is a resultant of the interaction of multiple genetic and epigenetic factors. In the circuits of the nervous system, in contrast to ordinary electrical circuits, the greatest proportion of the messages between the units is carried through the intervention of a variety of chemical transducers which must be formed, stored, liberated, bound to membranes, and eventually destroyed. III. A HIGHLY SIMPLIFIED SYNAPTIC MODEL DEALING ONLY WITH PRE- AND POSTSYNAPTIC RELATIONSHIPS

A. Overall physiological relationships The relationships to be discussed in this section for illustrative purposes, which are only a few of the many possible, include only those at the presynaptic and postBrain Research, 2 (1966) 117-166

I i9

THE SYNAPSE AS A MICRO-CYBERNETIC UNIT

synaptic neuronal sites. Upon excitation of a neuron there is presumed to be a release of transmitter (or transmitters) so that there is a vectorial flow from the depolarized axonal presynaptic endings onto the postsynaptic membranes of dendrites or soma or onto presynaptic endings of other axons. The nature of the sp~ific interaction which takes place between transmitter and membrane must be a function both of the chemical nature of the transmitter and of the structure and state of the reacting membrane. Excitation (nerve activity) occurs when the permeability of a membrane is changed in such a fashion that depolarization results. Inhibition results when the liberated substance blocks the depolarizing action of excitatory influences acting upon the same membrane. In general, it is believed that in the vertebrate central nervous system excitatory transmitter is liberated at axo-dendritic endings and that inhibitory transmitter may be liberated chiefly at axo-axonic and axo-somatic connections 31. It also has been suggested recently 91, 93 that immediately following depolarization a substance (or substances) which could act as a direct synaptic feedback inhibitor might be liberated from a bound or stored form from postsynaptic sites into the extraneuronal synaptic environment. The latter type of inhibition will play an important role in subsequent discussions because it is an inhibitory mechanism which could exist at all synapses,

A.

INACTIVE

~

T r ......

i,

INHIBITORY INFLUENCE

LEVEL

* PRE

-!

:

[

: .--...2...---

-

.....

~

EXCITATION I N H I Bi'TORY INFLUENCE .....

LEVEL

l

POST -

iJ,,, 't !

, I. . . . . .

T

PRESYNAPTIC

~ ......

.o.=,.o

D.

B.

't I

FACILITATORY i N F LUEN,.~= . . . . . LEVEL

: .;

L,_ . . . .

NEGATIVE FEEDBACK r" . . . . . . !'

INHIBITORY .I N F L U E N C E LEVEL

< ......

1 '

POSTSYNAPTIC r" . . . . . |'

FAC I L I T A T O R Y INFLUENCE ..... LEVEL

EXGITATION INHIBITORY I NFLUENCE LEVEL

.....

I t

J

"l !

i !

i

DE-

,,,

= L .....

FACILITATORY INFLUENCE ..... LEVEL

=

T J L .....

~g FAC ILI TATORY INFLUEI~;E ..... LEVEL

I

Fig. 1. The synapse operating as a hypothetical cybernetic physiologic urlh during one cycle of activity. This representation places emphasis on the role of the pre- and postsynapticaiiy liberated transmitters, which are designated on the diagram by the lines between the pre-and postsynaptic endings. !nhi',~itory and facilitatory influences at a particular synapse which do not originate within that synapse are indicated separately. Brain Research, 2 (1966)' 117-166

120

E. ROBERTS

regardless of the details of the neuronal circuits in which the synapses are found. The possib!e relationships are illustrated schematically in Fig. 1. The excitation is shown to occur first presynaptically and then postsynaptically. The substance (or substances) released from the presynaptic endings could act synergistically with other facilitatory (depolarizing) influences and antagonistically to the inhibitory (hyperpolarizing) influences. When an excitatory transmitter affects a postsynaptic membrane and depolarization results, it is suggested that an instantaneous postsynaptic liberation of an inhibitory substance may take place. Likewise, somewhat later, as a consequence of nerve activity taking place in the postsynaptic cell in some instances there may be a subsequent activation of interneurons which might have their presynaptic endings on the presynaptic (axonal) or postsynaptic (somatic) sides of the synapse being considered and which could exert a negative feedback effect by liberating an inhibitory substance (or substances). From physiological studies it has been deduced that the naturally occurring inhibitory substance (or substances) should produce an increase in the K ÷ and/or CI- ion conductances of the membranes upon which it impinges. In this manner free inhibitory transmitter would accelerate the rate of return to the resting potential of all depolarized membrane segments which it would contact and would stabilize (decrease sensitivity to stimulation) of undepolarized membrane segments. The concentrations of free excitatory and inhibitory transmitter would decrease at the synapse as a result of rapid removal and degradation, the ionic balances characteristic of the resting state would be restored, and the pre- and postsynaptic membranes would attain their pre-stimulus state. The negative feedback effects of the release of inhibitory transmitter onto a synapse following its activation, directly from the stimulated postsynaptic membrane, or from terminals of interneurons, or from both would prevent excessive presynaptic release of excitatory transmitter per stimulus and a too extensive and prolonged depolarization of the postsynaptic membrane. This would tend to ensure the accumulation of sufficient amounts of the various chemical messengers between presynaptic volleys to maintain normal capacity for activity at the synaptic junction. Such a system would serve to maintain a minimally fluctuating activity at a synapse under any given condition of afferent stimulation and metabolic state of the participating cells. For the maximally effective operation of a synaptic servo-mechanism there would be required a coordination of the sequential changes in properties of pre- and postsynaptic membranes with the formation, storage, release, and metabolic degradation of the various membrane-active substances involved. At the present time the two known substances which are believed possibly to serve as excitat~ry transmitters in the vertebrate central nervous system are acetylcholine and glutamic acid; the only likely candidate for inhibitory transmitter is ),-aminobutyric acid 60. Some other naturally occurring substances which are synaptically active but which do not seem to have the physiological properties expected of true transmitters in the central nervous system are substance P, serotonin, tryptamine, tyramine, histamine, dopamine, norepinephrine, and epinephrine. In addition to having direct effects on neuronal membranes, some of the above substances may have effects on the microcircu!ation in the regions of activated synapses; and several of them are known to produce increases in content of adenosine-3',5'-phosphate, a Brain Research, 2 (1966) 117-166

THE SYNAPSE AS A MICRO-CYBERNETIC UNIT

121

substance which may have a regulatory role in many cellular functions (see Appendix liD. Efforts are being made in many laboratories to identify more substances which_ may be excitatory and inhibitory transmitters. B. Ionic movements

The ionic mechanisms of excitation and inhibition have been discussed extensivelyal,ag,99. It is assumed in what follows that when the presynaptic endings are depolarized as a result of stimulation there is at first net inflow of Na + ions and possibly many other substances from the extraneuronal compartment (see Fig. 2). There is then an accelerated outward flow from the presynaptic endings of K + ions, excitatory transmitter, and possibly a variety of other substances into the surrounding extraneuronal environment and onto the surfaces of the postsynaptic sites as well as entry of C1- ions from the extraneuronal compartment. The excitatory transmitter and K + ions would act synergistically in depolarizing the postsynaptic membrane and, together with the other substances liberated, would act through the extraneuronal B. PRESYNAPTIC DEPOLARIZATION 1.

,./T";-;:-)-..,

/ A,

INACTIVE

0:

o.. . ~I /................',:i

SYNAPSE

*.

•

.,." /

•- ~i*~'.~I ~Gha... "',.. .: .:

:. .

__~" .'"" ".... "'"~i ~ _

~,............ ..

i<% ",,,

,

= ext,~cellu&lr

°

\

KEY OTHER SUBSTANCES

G. POSTSYNAPTIG DEPOLARIZATION AND PRESYNAPTIG REPOLARIZATION

.

..--;~:~; --...

,,

".

Inleoneuronol

• Presynopl|c

EI1foneuronol

0

A

,,," ,/

)

polori-

Posfsynopt ic

....

zotion

D. POSTSYNAPTIC REPOLARIZATION

/

: f o .. ; ............. .' t=
"

II

?

Rezotion

,orion

..'. . . . . . . . . . . .

~J~o~,.A..&,'°~I~

Oe ,orion

Fig. 2. A proposal for the time-sequence of the movement of ions and other substances between the components of a synapse during one cycle of activity. One c ~ deduce from part C-2 of this diagram that the ideal role for a postsynaptically liberated ,"¢¢dback inhibitor would be that of increasing the K + and/or Cl- ion conductance of the presynaptic membrane, since it would be released at a time that the presynaptic ending is being repe,larized and the intraneuronal/extraneuronal ratios of these ions are being reestablished.

Brain Research, 2 (1966) 117-166

122

E. ROBERTS

compartment upon the c!,~,~elylying capillaries in such a manner as to facilitate entry of O~ and various nutrients and exit of COs and waste metabolites. Immediately upon the passage of Na + ions to the inside of the presynaptic membrane there would occur a stimulation of the ion pump which moves Na + ions outwards and K + ions inward, and a rapid reestablishment of the resting intraneuronal/extraneuronal ion concentration would begin to take place. This latter series of events would be taking place at a time when the depolarized postsynaptic membrane would have begun to recapitulate the series of events described above for the presynaptic ending. Following depolarization and entry of Na + ions and possibly other substances, there would be release of K + ions, negative-feedback transmitter, and other substances from the postsynaptic site and the entry of CI- ions. The negative-feedback transmitter would have the property of interacting with membranes in such a manner as to increase specifically their conductance to K + and/or C1- ions whose equilibrium potential is close to the resting membrane potential, in this manner stabilizing all the presynaptic and postsynaptic endings that it would contact by 'attenuating the spread of depolarization' and by tending to 'clamp the membrane potential close to the resting level'3°,33. The transmitters would be metabolized rapidly after their release so that the duration of their effects would be coordinated with the changes which occur in membrane permeabilities, ionic distributions, etc. C. Expansion o f the synaptic model in terms o f transmitter relationships 1. A single synapse at which one excitatory and one inhibitory transmitter are active In order to employ the above model in such a manner that it would serve as a basis for the design of experimental approaches, more detailed proposals are required. In Figs. 3-6 are shown partial expansions of the scheme in Fig. 1 which can lead to studies of specific biochemical and pharmacological relationships. The scheme relates to a highly oversimplified consideration of a single synapse at which a single excitatory transmitter (E.T.) and a single inhibitory transmitter (I.T.) are active. Eventually it may be found that several E.T.'s and I.T.'s are active at individual synapses. In this section consideration will be given only to gross membrane effects and some general aspects of transmitter storage, release, and metabolism. Occasionally it has been helpful to think of the E.T. system in relation to acetylcholine and the I.T. system in relation to ,-uABA. However, ;t ,,,,,~t be ,,,,;,,,,.a out that in no instance have both of these substances yet been proven to operate naturally at a particular synapse and it is certain that these substances are not operative at all synapses. The synthesis of acety!cho!ine~ a .qnhqtanee e~rerting eYeitatnry action on some neural membranes, is catalyzed by choline acetylase and its hydrolysis b) cholinesterase, yABA, an inhibitory substance which acts by increasing conductances of membranes to K + and CI- ions, is made from L-glutamic acid by glutamic decarboxylase and degraded by ),ABA-a-ketoglutarate transaminase (see Ref. 92). Both ),ABA and acetylchoiine are tbund in the vertebrate CNS in free and bound forms and experiments have shown that when these substances are applied to neural tissue they can undergo physical binding to membranes. Brain Research, 2 (1966) 117-166

THE SYNAPSE AS A MICRO-CYBERNETIC UNIT

123

2. Regulation within the excitatory and inhibitory transmitter systems a. Presynaptic intraterminal events. Current evidence suggests that in some neurons, even in the resting state, there may be a liberation of E.T. from presynaptic terminals in a random fashion. Statistical analyses of data from many experiments indicate that the E.T. is liberated in packets or quanta of remarkably uniform size. Where E.T. is known to be acetylcholine, the quanta have been estimated to consist of several thousand molecules (see Ref. 31 for extensive discussion of relevant data). Depolarization of the presynaptic terminals by an impulse is associated with a great increase in the frequency of discharge of transmitter packets. Calcium is essential for the release of transmitter into the synaptic cleft; magnesium antagonizes the action of calcium and prevents the release. The frequency of discharge is decreased by hyperpolarizing currents to the presynaptic terminals, but it is not greatly altered by polarizing the postsynaptic membrane. Thus, the frequency of presynaptic release of packets of transmitter is a function of influences acting at the presynaptic membrane. Hypotheses about the mechanism of release of transmitter packets have been generally based on the electron microscopic observation of vesicular components in presynaptic endings in sections of fixed nervous tissue. It has been suggested that individual vesicles may contain within them the packets of transmitter and that those vesicles in immediate contact with the presynaptic membrane facing the synaptic cleft would be discharged during the passage of an impulse 31. However, recent electron microscopic studies in our laboratory with negatively stained preparations of centrifugally isolated nerve endings from mouse brain are more compatible with the existence in the nerve endings of a densely packed membrane system rather than of individually bounded spheroidal vesicles eg. Mild osmotic rupture of the isolated nerve endings leads to the budding out and breaking off of numerous membranous expansions which can then be isolated centrifugally as vesicles. Electron microscopic examination of the latter showed that in many instances two or more vesicles appeared to be connected by membranous strands, although some of the vesicles appeared as separate entities. In view of the still uncertain knowledge of the morphological basis of transmitter release, the following discussion will be made without the assumption of the existence and discharge of discrete structural vesicles. Rather, we may tentatively refer to the functional units, more generally, as presynaptic micro-volumes (PMV). One may view the whole individual presynaptic nerve ending as a rather large bounded volume of cytoplasm densely packed with membrane-like components to ~_1_._1 W[IlUII

. . . . . . . " . ,~ . . . . . L,., tEU,[1311Ill, l,[;;l 3 U U 3 1 , C ~ I t ~ 3

,,~al[

II,,:,,A ffl,tI.L.

1[,~ LJ--

tlh,~.JJ~,

r,,~ac, ,,,~o,w

r~f" ~ v~,

,.,,

fr~ncmitfor L... . . . . . . . . . . . . .

whlr..tl.

1~

rnad~

enzymatically in such a system, for the synthesis of which are required at least some precursors which must come from the extracellular environment, there would be expected to be gradients of both free and bound transmitter and of transmitter precursors~within the volume of the nerve ending~ the largest amounts being found at or near the surfaces facing the extracellular environment, the quantities decreasing progressively toward the geographical center of the bounded volume. It is consistent with available data to assume that E.T. may exist in presynaptic storage sites in free .

Brain Research, 2 (1966) 117-166

124

E. ROBERTS

and bound forms, which are in equilibrium with each other (Fig. 3), and that only those PMVs are in a suitable po,~;ition or physical state to be discharged which have attained their maximal level of E.T. At its maximal level the free E.T. could inhibit its own formation directly by inhibition of the enzyme which forms it or by some other mechanism (see Fig. 4 for l:,ossible control steps in E.T. formation). The net a. "MATURE" PMV

e. FILLING OF PRECURSOR COMPARTMENT

ME. ,

,,-----~ | :

m

¢.-.-

.=,

)

'

L--~-J •

/

..,

.

"

.

•

:..

, "

\ d. FILLING OF ,

'i'

r,

•

TRANSM ITTER COMPARTMENTS

"--------I'

:

"1

b. STI'"" ,,,~,_~, " " ~,v,,, ' : OF

-

"

|

/

"~

"MATURE" PMV

C. RELEASE OF TRANSM ITTER ;

,q.

4~ FREE

E.T. or

I.T.

Fig. 3. Hypothetical sequence of events in a presynapdc micro-volume (PMV). The events postulated take place after presynaptic stimulation and discharge of transmitter may be envisioned to occur either during the recharging of a previously discharged unit or during "maturation" of a new PMV which replays one previously discharged. to

Brain Research, 2 (1966) 117-166

THE SYNAPSE AS A MICRO-CYBERNETIC UNIT

125

inward movement by carrier-mediated transport mechanisms of substrates for the enzymes which form E.T. or its precursors also would be stopped, because each substance, which under a given set of conditions enters a membrane-bounded volume by such a mechanism, tends to attain a given maximal intracellular/extracella!ar concentration ratio. When this ratio is reached no further net movement would take place, although exchange between the compartments still could occur. The passage of the stimulus would cause all of the free E.T. and possibly some of the bound E.T. in a responding PMV to be liberated into the contiguous synaptic cleft. Providing the volumes of the individual PMVs in the presynaptie nerve endings of a given species are approximately the same, the above mechanism would be consistent with the observation that E.T. seems to be liberated in relatively discrete multimolecular units or quanta. The scheme in Fig. 3 shows some of the hypothetical intermediate stages in the recharging of an individual PMV unit with E.T. after stimulation; or alternatively, it can represent the stages in the 'maturation' of PMVs which will replace those that have been discharged. The greater the stimulus, the greater will be the number of fully charged PMVs that will liberate their load of transmitter into the synaptic cleft. It is obvious that a given presynaptic terminal could be stimulated in such a fashion, that it could maintain a constant rate of output of transmitter and also that the stimulation could be of such an intensity that the capacity to form transmitter would be outstripped and that progressively fewer quanta would be liberated per impulse. Although it has not yet been possible to study transmitter metabolism in individual presynaptic terminals, elegant studies have been made of acetylcholine metabolism of cat superior cervical ganglia12,13, which contain large numbers of presynaptic terminals. The finding that Na + ions play an important role both in the synthesis and release of acetylcholine is particularly significantlL With regard to acetylcholine synthesis: 'If it is the extracellular sodium that is important, then the most likely way in which sodium would promote ACh synthesis would be by maintaining the transport of metabolic substrates, such as glucose and choline, into the nerve endings. If it is the intracellular sodium concentration that is important . . . . . . the action of sodium might still best be thought of as on membrane transport. In this case the membrane involved might be at the surface, or within the nerve ending, or both; since choline acetylase is probably located in some membrane-bounded intracellular compartment. If the action of sodium were intracellular, then a simple means by which ACh synthesis could be geared to activity suggests itself. For as the intraterminal sodium rose as a result of activity, it would promote synthesis of ACh by allowing access of metabolic substrates to the enzymes involved in ACh formation. Since it may be presumed that the level of intracellular sodium rises with increasing neuronal activity, ACh synthesis would be kept in step with ACh release.' About acetylcholine release: ' . . . . . . it is attractive to suppose that sodium entry during the invasion of the nerve endings by an action potential facilitates ACh release by producing a local transient increase in sodium concentration at the inner surface of the terminal membrane, and that the facilitation ceases w~-,_,enthe sodium becomes diluted in the relatively large volume of terminal axoplasm. Furthermore, as the general level of intraterminal sodium rises during prolonged activity, there will also be a higher transient local conBrain Research, 2 (1966) 117-166

126

E. ROBERTS

ii:;!i!!:/:: ;~i:!i!~:

Presynopt i c Micro-volume ( PMV )

Synoptic cleft

Fig. 4. Possible points of feedback inhibition of transmitter formation. It is suggested that each substance in the biosynthetic sequence of the transmitter could potentially inhibit any or all of the steps preceding its own formation and that each intraterminally contained substance might inhibit the active transport of the extracellular precursors. This is presented only to indicate the type of experiment that could be performed once a transmitter has been identified and the reactions in its biosynthesis elucidated. ,1:.. T. : Excitator 3

iransmit~er I. T " arnhibitorx t r a n s m i t t e r

iI

L

INHIB

I I I I

I I I I I I I I I i I

I I id .

s,p~., ~ "

~1

I

,I

.

1t

.~m

/

~"~,_

~

~"~'~,

I

/

/

/

/

/t

/

/

/

/

/

¢

i I

Fig. 5. Expansion of Fig. I with emphasis on some details of regulation within the excitatory transmitter (E. T.) and inhibitory transmitter (I. T.) systems. Brain Research, 2 (1966) 117-166

THE SYNAPSE AS A MICRO-CYBERNETIC UNIT

127

centration at the inner surface of the membrane as sodium enters during the action potential, and thus more ACh will be released.' The above hypothesis may help explain the well-known potentiation of nerve activity (increased presynaptic release of E.T.) produced either by a single conditioning impulse or by repetitive stimulation (see Refs. 48 and 49 for discussion of this phenomenon). In the following discussion it will be assumed that the considerations mentioned above concerning the possible coordinations involved in storage, release, and synthesis of E.T. also would apply to I.T. b. Sequence o f excitatory and inhibitory transmitter events at a synapse. When a particular neuron is effectively stimulated there is a liberation into the cleft in a free form of some of the E.T. which is contained in the presynaptic terminals (Fig. 5). The free form of E.T. is bound to the apposing postsynaptic membrane, producing depolarization, and the largest proportion of the liberated transmitter is most likely destroyed rapidly by enzymes associated with the pie- or postsynaptic membranes or both. Under some circumstances (excessive stimulation, overproduction, blockade of destructive enzymes) the rate of release of free E.T. might exceed the rate of removal and some of the undegraded transmitter might be transported back into the presynaptic site and be available for use again. If the condition of excessive accumulation of E.T. should persist for long enough periods, it might act directly or indirectly at a genetic level (Fig. 6) as a repressor for the formation of messenger RNAs involved in the formation of the enzymes which form E.T. and/or as derepressor for the formation of the RNA messengers for enzymes involved in its destructive metabolism or conversion into other substances (i.e. endogenous inhibitor). I.T. would be released into the synapse only after postsynaptic depolarization (Fig. 5). It would decrease the responsivity of the presynaptic and postsynaptic membranes to the depolarizing influence of a given afferent stimulus, since presumably in the presence of I.T. fewer quanta of E.T. would be liberated per impulse and the sensitivity to depolarization of the postsynaptic membrane per quantum of E.T. would be ;ess than in the absence of I.T. Under ordinary circumstances the rate of removal of free I.T. either would be great enough to remove all of it from the synaptic region between impulses, or the rates of liberation and removal would be so coordinated that a relatively constant amount of free I.T. would be present at all times between stimulations, supplying a modulatory 'tone'*. An additional defense against the escalation of free I. T. content would derive from its inhibitory effect on depolarization o f ' ,,emt,~ne~, 1-. . . . increased amounts of free I.T. at a synapse would decrease its own release from postsynaptic storage sites or from presynaptic terminals of interneurons.

* It would be more likely that there would be a constant background level of free 7ABA at a particular synapse than of free acetylcholine, since the affinity of substrate for enzyme and turnover number of the 7ABA-transaminase, the 7ABA-destroying enzyme system, are much lower than those of cholinesterase. Brain Research, 2 (1966) 117-166

128

E. ROBERTS

3. Cross-regulation between the excitatory and inhibitory systems Some cross-regulations may exist between the E.T. and I.T. systems which may become particularly important when rapid, large increases either in I.T. or E.T. are produced by extensive blockade of the respective degradative enzymes with exogenously administered or endogenously formed (dashed lines, Fig. 5) inhibitors. Products may then be formed from free I.T. and E.T. which ordinarily may not be formed at all or which may exist only in low concentrations because of the relatively low affinity of E.T. and I.T. for the enzymes involved. If, when the breakdown of either E.T. or I.T. is inhibited, products are formed which can inhibit the breakdown of I.T. or E.T., respectively, the enhanced excitability or depression attributable to the gross increases in one or the other of these substances at a synapse would be balanced off by an increase in a physiologically equivalent quantity of the other. In contrast to the small rapidly-occurring adjustments (millisecond time scale) taking place during

I I I I I

I I !

) J

p#SS is S

/

p

/

/// //

/ ~"

I 111

/

/

'-J*--....

/

Fig. 6. Expansion of Fig. 5 with emphasis on pessible genetic regulatory factors in the excitatory (E. T.) and inhibitory (i. T.) systems.

Brain Research, 2 (1966) 117-166

THE SYNAPSE AS A MICRO-CYBERNETIC UNIT

129

normal synaptic activity, changes such as disctLssed above would be expected to be relatively great and to persist for long periods of time (minute-hour time scale). In the case of increased E.T., an initial period of increased excitability, as measured by some physiological parameter, would be followed by a gradual increase in I.T. content which would be paralleled by a return of excitability to normal levels. Similarly, when I.T. would be increased by blockade of its destruction, a period of decreased excitability would be followed by an increase in E.T. content and return of excitability to normal. In both of the cases discussed above the eventual result would be the maintenance of essentially normal synaptic activity in the presence of increased levels of both E.T. and I.T. If the latter suggestions are correct, good correlations between estimates of E.T. or I.T., alone, and synaptic excitability would be expected only at the beginning of the induction of changes in contents of E.T. or I.T. However, at all times changes from the control levels in the relative amounts of E.T. and l.T. should be well correlated with changes in excitability.

4. Changes in reactivity of membranes Another factor which must be important in regulation of synaptic activity is the state of the pre- and postsynaptic membranes. It can easily be imagined that the plasma membranes, which contain structural and enzymatic proteins, lipids, glycolipids, and polysaccharidesS6,115,116, would be subject to many local influences which might affect their physical state [pH, concentration of small charged molecu~os (organic and inorganic), hormones, availability of water, exogenously administezed drugs, etc.]. Let us suppose that in the environment of a particular synapse a change occurs which suddenly decreases the sensitivity of the presynaptic membranes to stimulation (see Fig. 5). The same stimulus which previously had been causing the liberation of a given amount of E.T. would liberate less than before, the effect on the postsynaptic membrane would be less, and less I.T. would be released following postsynaptic depolarization. The decreased liberation of I.T. would result in an increased sensitivity of both pre- and postsynaptic membranes to stimulation and a tendency to maintain normal responsivity of the synapse. Likewise, an environmental change producing a decreased sensitivity of the postsynaptic membrane would lead to less liberation of I.T. Conversely, an increased sensitivity of the membranes responding to stimulation would res,,~t in an increased release of !.T. and a heigbt,.n,.a inhlhlmry 'tone' which would tend to counterbalance the hypersensitivity and keep the resultant synaptic activity within the normal range. D. Types of studies which eventually may lead to experimental tests of the simplified synaptic model Eventually it may be possible to test some of the above hypotheses at intact individual synapses by ultramicro analyses of pertinent chemical variables performed simultaneously with measurements of electrical changes at pre- and postsynaptic endings. It has not been possible to do this to date in vertebrate synapses. The only experimental work which can be discussed with reference to the model presented

Brain Research,2 (1966) 117-166

130

E. ROBERTS

thus far in this paper is concerned with correlations obtained between gross chemical measurements in brains of animals and some aspect of behavior. Although far from ideal, it is not entirely unreasonable to make inferences about synaptic activity from some measurable parameters of behavior, as will be brought out in subsequent sections of this paper. The three examples cited in Appendix I of results in mice and dogs may be examples of experimental situations which will eventually afford opportunities for the performance of critical physiological and ultramicrochemical analyses. IV. SYNAPTIC CONNECTIVITY

In the preceding section there was no discussion of the effect of use or disuse on the efficacy of a synapse as an information transmitting unit. Increases or decreases in the probability of information exchange at a synapse may be equated with increases or decreases, respectively, in connectivity between the presynaptic and postsynaptic elements:. A key problem of function in the nervous system that may be approachable from the biochemical point of view is that of connectivity: how can the use of a particular synaptic connection between particular neurons produce an enhancing effect so that successive excitatory afferent signals arriving at the same ending become quantitatively more important relative to those from synaptic connections in the same microanatomical field which have received less use ?* The increase in relative importance with use also must be progressive, so that the dominance of activity by a particular input which begins at a small outlying dendritic branch eventually might extend to that of a dendritic trunk and, perhaps, even to that of the activity of the entire postsynaptic neuron. Disuse also might lead to a regressive loss of this dominance. A somewhat more complex problem is the understanding of what happens during establishment of facilitated interneuronal circuits which consist of series of facilitated individual connections resulting from the repeated action of patterns of extra- or intraorganismal changes. It is feasible to examine some of the factors which might be involved in the establishment, maintenance, and loss of connectivities between neural elements. In order to be able to think about the possible events at the synapse it was convenient to consider them from several points of view. The problems of temporary and permanent connectivity at a synapse will be examined at three levels: transmitter action and ion movement (physiological), total amount of surface apposition and transduction at a synapse (growth), and the genetic expressivity which determines the degrees of similarities of surface properties of cellular elements at the synapse and their affinities for each other (genetic).

* Hebb's assumption4S: 'When an axon of cell A is near enoug.h to e×c,'_)e a cell B and repeatedly or persistently takes part in firing it, some growth process or metabolic change takes place in one or both cells such that A's efficiency, as one of the cells firing B, is increased.' See also other pertinent quotations in Ref. 10.

Brain Research, 2 (1966) 117-166

THE SYNAPSE AS A MICRO-CYBERNETIC UNIT

131

A. General physiological considerations

A number of the pertinent physiological considerations about synaptic action have been discussed in previous sections. The idea which is most pertinent at this point is that when an excitatory transmitter affects a postsynaptic membrane and depolarization results, it is presumed that at all synapses there is an instantaneous postsynaptic liberation into the extraneuronal synaptic environment from a bound or stored form of a substance (or substances) which would act as synaptic feedback inhibitor (see Figs. 1 and 2)*. This would take place whether or not other feedback inhibition through interneurons, collaterals, etc., would take place at a particular synapse. The amount of free feedback transmitter liberated from an intraneuronal form into the synaptic region at any point of the depolarized neuronal unit would have a quantitative relationship to the degree of displacement from the resting potential at that point. Such a substance could be bound to both pre- and postsynaptic membranes on the sides facing the synaptic cleft in the high-Na + extraneuronal environment and would produce an increase in the K + and/or CI- conductances of membranes to which it is bound. In this manner it would accelerate the rate of return to the resting potential of all depolarized membrane sectors which it would contact. It would not only increase the rate of repolarization of depolarized presynaptic endings but also would stabilize on the same depolarized postsyn~:ptic segment those presynaptic endings which had not become depolarized. Once bound to membranes it could be destroyed or inactivated by enzymes at membrane surfaces to which it is bound, or the substance, itself, or a metabolite thereof might be transported into intraneuronal (and possibly intraglial) environments where it could be metabolized to yield some of the ATP necessary to replenish that employed by the mechanisms by which are reestablished the extra- and intraneuronal ionic concentrations characteristic of the resting state. As the concentrations of pre- and postsynaptically liberated transmitter substances would decrease at the synapse, the lability of the excitatory presynaptic membranes and stability of the postsynaptic sites would increase progressively with time toward normal until the next excitatory synaptic event would occur. In such a synaptic regulatory system the postsynaptic membrane would be an important participant in the limitation of excitation and reestablishment of both pre- and postsynaptic membrane potentials. Such a system would serve to maintain a minimally fluctuating activity at a synapse under any given condition of afferent stimulation and metabolic state of the responding cell. Thus, for a particular stimulus in a given neuroanatomical setting, in the presence of such a system there would be less pre- and postsynaptic depolarization and quicker repolarization of the membranes and recovery of presynaptic stores of transmitter than in its absence. This would be reflected in a smaller amplitude and greater frequency of response to any given sus* This concept of local synaptic feedback was first introduced by the author at a symposium of the Society of General Physiologists held at Oregon State University in 1962 (Ref. 93). It was presented in more detail at an AIBS symposium held at Princeton in 1963 and stated independently from a mathematical point of view by Uttley at the same meeting 57: similar consequences were envisioned, whether a strictly biological or mathematical point of view was employed as a starting point.

Brain Research, 2 (1966) 117-166

132

E. ROBERTS

tained stimulation, which in cybernetic terms would mean that with such postsynaptic chemical negative feedback more bits of information could be transmitted per unit time and that the potential discriminatory capacity of a given synaptic connection would be greater. The above postulate would have important consequences not only in the consideration of the relatively simple case of synapses and circuits of synapses in which an impulse is transmitted through the axonal endings of one cell upon the dendrites of another, but also in the situation of neurons which integrate the signals which arrive from multiple afferent sources and whose axonal endings impinge upon extensive dendritic arborizations of other neurons, making variable fractional contributions to the total of the depolarizing influences acting thereon. At this point of the discussion, how could it be envisioned that the use of a particular excitatory synaptic site would increase connectivity and disuse decrease it? As suggested above, liberation of the postsynaptic feedback inhibitory substance (or substances) at a synaptic site when postsynaptic activity follows or accompanies presynaptic activity at that site could maximize the potential for information exchange at that particular connection. However, if postsynaptic inhibitor were to be released all along a particular depolarized neuronal segment and liberated onto all of the presynaptic connections on it, whether or not they had been previously depolarized, there would be a differentiation between the active and inactive synapses. The probability of the undepolarized presynaptic sites serving as meaningful sources of information in the immediate micro- or millisecond future in the region of a depolarized postsynaptic site would be decreased, since the inhibitor would act in such a manner that a greater amount of stimulation would be required than before to elicit the release of an effective amount of excitatory transmitter. Further, the inhibitory substance might tend to persist for a longer time in the region of the undepolarized presynaptic endings than in the region of the depolarized endings because the rate of itg metabolic removal might be slower if its rate of penetration into potential metabolic sites would be, at least in part, dependent on membrane changes which occur when the presynaptic endings are depolarized. The net effect would be that those synaptic connections at which postsynaptic activity would take place without presynaptic depolarization would suffer a loss in connectivity. Thus, coordinated use of a particular synaptic connection might lead to an increase in its relative importance as a source of information in a particular neuronal milieu. The kind of increase in connectivity suggested above, if occurring in the a- oL__ ~ n t ;_e of other changes, would be only ephemeral. It soon would disappear a~ a result of changes in the nature of the incoming signals and the operation of the synaptic servo-mechanisms (see Figs. 1-6). However, as we shall see below, the above events would be expected to be occurring simultaneously with a multitude of other chemical changes which might serve to preserve the effects of synaptic experience for longer periods. B. Metabolic events The above view of synaptic events must be integrated with metabolic processes, in general, and particularly with those aspects which might be correlated with growth Brain Research, 2 (1966) 117-166

THE SYNAPSE AS A MICRO-CYBERNETIC UNIT

133

and regression, if some case is to be made for hypertrophy of use and atrophy of disuse at the synaptic level (see Refs. 10, 31, 45 and literature cited therein)*. In contrast to the soma and the large axonal and dendritic branches, in which there may be a rapid exchange of cytoplasmic material between various regions and a potential for rapid exchange with nuclear constituents, the fine outgrowths of the outermost tips of the axonal and dendritic extensions may lead an adventurous and precarious existence. Not only may they be in constant movement because of internally generated asymmetric forces but also they may be buffeted about by pressure changes reflecting vascular phenomena and by the pulsations of the surrounding glial cells. Far away from the central source of genetic information and not directly in contact with the blood supply they always may be delicately poised between growth and regression. If one were to design the most efficient scheme by which to achieve growth of nerve endings (pre- and postsynaptic) and glial cells as a result of impulse transmission at the synapse, one requirement would be that as a consequence of the events triggered by the impulse there would be the release of a rate-limiting reaction (or reactions) which would not only furnish the energy for the repolarization of the membranes and resynthesis of transmitters but which also would make available rate-limiting materials for the various biosynthetic reactions required in growth. Another requirement would be that all of these processes would be brought to a halt when, as a result of their operation, the same key reaction (or reactions) again would become inhibited. With each stimulation the process would be repeated. A third requirement would be that an extensive and relatively free exchange of material can take place among the vascular, extracellular, glial, and pre- and postsynaptic components of the synapse only when both the presynaptic and postsynaptic sites are activated, i.e. only when depolarization of the presynaptic site is followed or accompanied (but not preceded) by that of the postsynaptic site. Although not directly applicable to chemical changes at synapses, the inverse changes observed in enzyme activity, total protein, and RNA in neuron cell bodies and adhering glial cells during increased function are compatible with the suggestion that an exchange of material can take place between glial and neuronal elements (Ref. 52 and references therein; see also Refs. 40 and 50 for other interesting pertinent data about neuron-glial relationships). The total quantity of protoplasmic mass at particular pre- and postsynaptic sites and in the adjacent glial cells must be a function of the balance t~etween degradative and synthetic reactions. If the degradative reactions were to go on at a more or less constant slow rate and the synthetic reactions could occur for only a brief period after * Hebb 45: 'The most obvious and I believe much the most probable suggestion concerning the way in which one cell could become more capable of firing another is that synaptic knobs develop and increase the area of contact between the afferent axon and efferent soma ('soma' refers to dendrites and body, or all of the cell except its axon)'. Recent morphological studies of cortical cells during development (see Refs. 88 and 8% for example) and quantitative studies of the maturational aspects of dendritic patterns in these cells 9~ suggest that in the future it should be possible to assess the influence of enhanced or decreased stimulation (experience) on the size and numbers of synaptic connections in those cells, which are believed to play an important role in learning processes. Advances in tissue culture technology 15,21.a7 eventually may make it feasible to study with time-lapse cinematography the morphological consequences of stimulation of individual synaptic connections.

Brain Research, 2 (1966) 117-166

134

E. ROBERTS

each stimulation, then it could be that in the absence of stimulation there would be a slow loss of cell substance; at a certain level of synaptic activity there might be a maintenance at a constant level; and at greater levels increases in sizes of nerve endings and in sizes and numbers of glial cells at a synapse actually might take place*. Unlimited growth would not take place; eventually some environmental limitation (physical, circulatory, etc.) again would lead to a new state in which there would be an equilibrium between growth and regression. When there is growth of all neuronal elements and growth and, possibly, division of glial units at a synapse, it would be expected that connectivity would increase. The actual amount of physical contiguity between the neurons could increase either by extension of previous areas of appo-gition or by the sending out of protoplasmic extensions from both pre- and postsynaptic sides which would establish new contacts. The new areas of contacts might differentiate so that more presynaptic sites containing transmitter would be located at the presynaptic side and, therefore, a stimulus from the afferent neuron could cause the liberation of more transmitter (more transduction) per impulse and would produce a greater effect upon the postsynaptic membrane, etc. The increase in glial support would furnish the greater metabolic machinery necessary to maintain the larger units. However, if the presynaptic element at a synaptic junction were to become depolarized and would undergo the changes consequent to the depolarization, and the postsynaptic unit were not to undergo depolarization, for whatever reason, an actual loss in protoplasmic mass of the presynaptic ending might take place. This could easily be understood if it is assumed that the direct participation, or indirect effect, of a postsynapticaUy liberated substance (or substances) is essential for the acceleration of some rate-limiting process in the presynaptic site. Similarly, regressive changes could occur in a particular postsynaptic site which is depolarized because of activity at a distance from the synapse and which does not receive the benefits of local presynaptic and glial contributions necessary for some essential synthetic reactions. When a stimulus crosses a synapse the wheels of metabolism are set in motion in a coordinated way so that in a brief period all the havoc wrought by the passage of the impulse is repaired, and the machinery even possibly slightly improved, before the ~ext impulse arrives.

* In this connection an interesting concept has been suggested to the author by Dr. Joseph Altman. Let us suppose that in the developing cortex there are closely lying axodendritic appositions that because of the proximity of pre- and postsynaptic elements have a potentiality for becoming active synapses, and that some of them become active and undergo an increase in connectivity with which is associated an increase in size of the axodendritic knobs and an increase in number (and total volume) of glial support units. The physical changes in the vicinity of the "used" axodendritic appositions could cause sufficient separation of the "unused" ones in the vicinity so that the latter no longer could become effective synaptic units. In this manner growth at synapses as a result of use could be an important differentiating factor in the formation of neural pathways, many diffuse potentialities becoming restricted to a much smaller number of actual functional connections. Recently it has been shown by autoradiographic techniques utilizing tritiated thymidine that a larger number of newly formed glial cells is added per unit time to the cortex of rats reared from weaning in an enriched environment than in rats living in isolation in an environment with minimal stimulation z. The increase in glial elem~.nts probably largely accounts for the significantly greater thickness of the cerebral cortex over the controls observed in rats kept in an enriched environment2,~0,2e, 27.

Brain Research~ ? (!966) ! I7-i66

THE SYNAPSE AS A MICRO-CYBERNETIC UNIT

135

At the present time hope for achieving some integrated understanding of the biochemistry of the synapse would lie in identifying rate-limiting steps in reaction sequences or metabolic systems of functional significance. Some pertinent possibilities are discussed in Appendix II.

C. Neuronal specificity The final biochemical level at which the problem of neuronal connectivity must be examined is that of neuronal specificity. It is assumed for purposes of the present discussion that the chief basis for the affinity or Jack of atfmity of the surface of one cell for that of another must be sought in the chemical and physical characteristics of the surfaces involved. It also is assumed that all the neurons and glial cells of a particular organism have the same genetic potentialities for i:-aakh~g o~emical compounds which are important in plasma membrane structure (lipids, structural and enzymatic proteins, glycolipids, and polysaccharides; see Ref. 36 for pertinent analysis of liver cell plasma menbrane and Ref. 116 for possible molecular relationships in membrane structures). The details of assembly of the supramolecular units at membrane surfaces also may be, at least partially, regulated by enzymes whose specificity is under genetic control. However, it would be expected that the assembly process would be subject to many epigenetic local influences [pH, concentrations of small charged molecules (organic and inorganic), availability of water, etc.]. In addition, it would be expected that the living cell may be able to exercise a number of options in terms of the exact numbers and types of molecules employed in construction of surface structures and still be able to meet it~ needs in the particular environment in which it finds itself. This is clearly evident from the point of view of lipids, since the closest-packing arrangements of myelin lipids do not require there to be rigidly repeating patte~ ns of the individual constituents 115,ne. Therefore, some variability in the nature of the proteins in membranes overlying the lipids also must be allowable. This brings up the possibility of the existence of a mosaic of different structures even on the surface of one cell. Indeed, such nonuniformity or specialization of cell-surface regions in individual cells has been repeatedly seen morphologically in light and electron microscopic studies and has been detected by sensitive biological tests in cells of protazoon and metazoon species 22,a4. There is evidence, at least in some types of cells, that the surface properties can change in response to changes in environmental conditions. Since the environment is highly inhomogeneous at all phases of development from the fertilized egg, it is possible to imagine that local environmental conditions may influence genetic control of surface properties of cells, as well as many other characteristics (see Appendix II! for pertinent discussion). Large cells, like neurons, parts of which may be found in quite different environments in a tissue of a fully developed organism, might show local genetically determined differences in surface properties. Thus, one dendrite or even a portion of a dendrite of a particular neuron might have surface properties different from that of another. Since multiple environmental gradients exist from the time of earliest development, it would appear likely that no two neural cells in the finally formed organism are Brain Research, 2 (1966) 117-166

.~O

E. ROBERTS

identical in every respect. Differences probably could be detected with sufficiently subtle methods even in cells which have arisen from the same embryonic area and which eventually occupy adjacent sites and subserve similar functions in the adult organism. In post-embryonic neural tissue there are cells between which the attractive forces are greater than the repulsive forces, and these will establish contact with each other if sufficient proximity is achieved; while no lasting adhesion takes place between cells in which the repulsive forces supervene. The remarkable degree of specification in some neural relationships that must take place during development has been demonstrated in experiments with fishes and amphibians in studies on regeneration of the optic nerve and recovery of vision4,106,107. After section, the regenerating nerve fibers find their original pathways, which they follow until the axons connect with the exactly correct loci in the tectum so that there is not only a recovery of the topographic projection of the retina upon the tectum but also a recovery of color vision. The above specificity is retained in spite of a great degree of disarray of the fibers in the nerve produced by the original injury. These observations imply the existence of a high degree of differentiation at all levels of the optic system from the receptor elements to their final central connections and also suggest that at least one way in which this differentiation is expressed is in the determination of specific properties of the cell or surface membranes. During early development, the properties of a nerve cell may be altered through contact with end-organs, other nerve cells, or by factors in its environment x07. How does an exchange of information take place between a particular cell with other cells and with its extracellular environment so that in the course of its developmental history it takes on the very specific properties which make it different from its neighbors? One possibility is that in the highly plastic developmental period during establishment of contact of a nerve cell with another nerve cell or with a peripheral structure there may be an exchange of material as a result of which, among other constituents, the contacting cells may now synthesize constituents which initially are characteristic of the membrane of one, but which after the exchange become common to both. There is evidence in a variety of biological systems that not only relatively simple substances can pass between cells but also that entry, exit, and exchange of RNA, DNA, protein, both types of nucleoproteins, and even macromolecular organelles may take place. The probability of the exchanging cells being similar to each other with regard to various properties would be expected to increase in proportion to the exchange of chemical information. The opportunity for generally extensive exchange probably is greatest during the plasticity of early embryonic development, with the dominant effect at each contact being exerted by the most highly differentiated cell. This might serve as a partial basis for the development of selective affinity of a nerve for a particular peripheral structure during morphogenesis and for the affinity for each other of nerves in the central nervous system subserving similar structures. The above sort of exchange might lead to the selective activation (by repression, derepression, or a combination of both) of particular genes specifying the structures of variants of classes of substances which might be important in determining cell surface properties which are relevant to the adhesiveness of cells. In the limiting case, Brain Research, 2 (1966) 117-166

THE SYNAPSE AS A MICRO-CYBERNETIC UNIT

137

an embryonic cell with the genetic potential for making a number of surface proteins, gangliosidic and cerebrosidic structures, etc., depending on environmental circumstances, could be progressively restricted until it made only one of each type, no matter what environment it would encounter. From a large body of experimental evidence it may be concluded that the greater the similarity of surface structures ofcells the greater will be the degree of adhesiveness. It is beyond the scope of this presentation to deal in detail with this problem :(see Ref. 22 for review). However, it is clear that no simple analogy with antigen-antibody reactions is adequate to explain the various aspects of cell adhesiveness. The degrees of specification in the fully developed organism would vary. The very high degree probably would exist in primary sensory and motor pathways, as in the optic system of fishes and amphibians. Less specificity probably exists in nerve cells in the central nervous system which can make connections with more than one of the more highly specified primary systems and which correlate information from them. The properties of the less highly differentiated cells would be potentially more responsive to environmental changes, and such cells possibly could exhibit mosaicism with regard to surface properties in different regions. They also would have the capacity for further differentiation. An increase of specification could take place at synaptic contacts (pre- or post) in such cells if there were a flow from the more highly differentiated cells into the less differentiated ones of substances that could carry the information which would restrict metabolic patterns in such a way as to cause them to be more like the highly differentiated cells. In view of the specificity required, it is less likely that such information-carrying material would be found among the postulated synaptic transmitters, common metabolites, or inorganic ions than that it would belong to a class of substance which could change the recipient environment in such a way as to promote its own production while guiding the synthesis of a product which either, itself, would be part of the cell-surface complex or which could be active enzymatically in the production of such a substance. RNA of the messenger type would be a good candidate for this kind of substance, although, as mentioned in Appendix III, nonchromosomal DNA and phospholipoprotein also might be active in a similar manner. There are many pertinent examples from the field of virology (see papers in Ref. 41) in which the demonstration has been made of the ability of specific RNA or DNA to enter cells and to induce biosyntheses of specific macromolecular cellular constituents. Evidence even has been adduced recently for the entry of active bacterial RNA into chick embryo fibroblasts in primary culture 3. It may be assumed that at any synaptic ending the attractive forces are greater than the repulsive forces between the pre- and postsynaptic endings and among the neuronal and glial structures and the glial elements, themselves. A continuum of degrees of relatedness depending on the expression of discrete properties at the surfaces of the cells involved may exist between that which just barely allows contact to take place and that strong affinity which must exist when identical surfaces are apposed to each other. Therefore, ber,veen two cell surfaces at any point of successful adhesion the differences, in molecular terms, may range from none at all to the maximal total number of differences or combinations of differences which still allow adhesion to Brain Research, 2 (1966) 117-166

138

E. ROBERTS-

take place. Within this range, those units which have fewer differences between them would have greater affinities for each other than those which have a larger number. It now is possible to envision how the use of a synapse might increase the connectivity between the elements of the synapse by reducing differences in cell surfaces of the participating units. Let us recall that one of the requirements of the synaptic model which is being discussed is that an extensive exchange of protoplasmic substance may take place among the glial and neuronal elements only when a sequential or simultaneous depolarization and permeabilization of both the pre- and postsynaptic sites takes place. Following the presynaptic depolarization there is an outflow of various substances (see Figs. 1-6 for postulated sequence of synaptic events). It is possible to suggest that information-carrying molecules, such as messenger RNA, may be among the substances liberated and that these would pass into glial cells, and into postsynaptic sites during their depolarization. RNA actually has been shown to be present in axons a2. Likewise, similar types of molecules could pass into presynaptic sites and into the glial cells during postsynaptic activation. Glial information units might enter both the pre- and postsynaptic endings during the respective periods of increased permeability of the latterS0, 52. Without attempting to outline a detailed mechanism, it would appear likely that the probability of the participating cells being more similar to each other would be greater after such exchanges as postulated above than before. This increased likeness would be expressed, at least to some extent, in increased similarities of surface structures and greater adhesiveness of the components of the synaptic unit. The increased similarity in structure, occurring simultaneously with growth, would tend to increase the number of surface connections and their strength. The increase in likeness would be proportional to the amount of material exchanged, which in turn, within limits, would be a function of strength and frequency of stimulation. It is also possible to suggest that as a result of activity at the synapse there is an increased relatedness between the glial cells and the connective tissue and endothelial cells in their vicinity. In the case of exchange between less differentiated units with highly specified units, such as are found in primary sensory or motor tracts, the differentiating influence ordinarily would operate largely in the direction of the less differentiated unit, whether pre- or postsynaptic, the less differentiated neuronal elements becoming ~,ore like the more l'ighly differentiated ones. V. HYPOTHETICAL TIME-RELATIONS OF PHYSIOLOGICAL AND GROWTH CHANGES AND CHANGES IN GENETIC EXPRESSIVITY DURING USE AND DISUSE OF A SYNAPTIC JUNCTION

The physiological events at a synapse, which must precede all other changes, take place in a fraction of a second after the initiation of stimulation. It is a requirement of the present model that growth and increasing genetic relatedness of structures at the synapse could not take place without the previous occurrence of the physiological changes which accompany activation of the entire synapse. The processes possioty .t.1 . involved in maximizing the relative physiological effectiveness of a synaptic junction which is in use by comparison with the neighboring synapses which are not in use Brain Research, 2 (1966) 117-166

THE SYNAPSE AS A MICRO-CYBERNETIC UNIT

139

(increase in connectivity) have been alluded to in a previous section. They are all fast in that they are dependent on permeability changes and recovery processes which take place on a millisecond time-scale. The enzymatic reactions related to these changes also probably are extremely rapid. Under given conditions of continued stimulation and with other synaptic activity on the same neuronal segment remaining constant, the relative increase in physiological connectivity of a particular synapse must reach the maximal value attainable by it very quickly after the beginning of stimulation. This is illustrated schematically in the diagram in Fig. 7. For the sake of simplicity the stimulation must be assumed to be of uniform intensity and to be applied at some constant dnspecified frequency. Relatively little total stimulation would be required to bring the physiological connectivity to its maximal potential value. This kind of connectivity, dependent on rapid events, also would be lost rapidly after cessation of use of a particular synaptic connection. This is indicated by the steep slope of the lines drawn downward from a number of points on the curve.

Protein synthetic reactions (and other synthetic reactions) necessary for growth to begin probably can take place in the time-range of seconds after the removal of rate-limiting restrictions, which is postulated to occur during activity at a synapse. However, net growth (increase in protoplasmic mass) would occur only when the amount of cellular material made per unit time would begin to exceed that removed by degradative reactions. It is, of course, possible that anabolism and catabolism are mutually exclusive at a synapse so that degradative phenomena are stopped when synthesis begins. Nonetheless, the amount of biochemical organization required for orderly growth of cells to take place would make it very likely that sufficient stimulation of a synapse to allow it to achieve its maximal connectivity through growth would be required to take place over a much longer period than is needed to attain the comparable physiological level. Growth at a particular synapse might be opposed by the physical resistance of a tightly-packed environment and possibly limited by the vascular capacity of the immediate region. Eventually the negative, or growthrestricting influences, would counterbalance the growth potential at a particular synapse and growth would cease even with continued stimulation. Upon cessation of all stimulation at a synapse the amount of cell substance and the physical connectivity would decrease at a rate commensurate with the intensity of degradative processes tp.king place. It is presumed that such processes would take place at an extremely slow rate, if neither pre- or postsynaptic depolarization were to take place. However, if either presynaptic or postsynaptic activity were to occur alone at a synapse, Gne without the other, the total metabolic capacity of the synapse would not be mobilized and the rate of loss at the stimulated ending would be accelerated. In the latter instance the recovery processes would have to draw largely on energy derived from metabolic breakdown products of endogenously contained macromolecules. Proteins, polysaccharides, nucleic acids, and to a lesser extent lipids can be degraded by hydro. ivtic enzymes wmcn ' - ' - ' - are present, possibly ,.u.,,,h,~,~"""*-'"-am" 1,j.,,o,,,,.,._......,.,._:.,~-,~packets. By comparison with the physiological and growth changes discussed above it would be expected that qualitative changes in membrane properties resulting from Brain Research, 2 (1966) 117-166

! 40

E. ROBERTS

transfer of biochemical information during stimulation would be very slow in appearing at neural and glial membranes. Although the potential for such changes would be tran,,mitted at the same time as that for the physiological and growth changes in the brief period during which the synapse is activated (Fig. 7), much greater amounts of effective stimulation would be required before this potential could be expressed. This is because a change at this level would require not only the reorganization of some aspects of the intracellular environment and redirection toward the synthesis of a particular substance (or substances) to be included in the structure of newly produced membranes, but also would include the replacement of some closely related subCONSOLIDATION PROCESSES

Maximum

s,ooo,c

1 \

MIN

H

/

.owT. \

Bosol DAY

W E E K MONTH

YEAR

TIME AFTER FIRST EFFECTIVE " STIMULATION ( Log scale) Fig. 7. Hypothetical time-relations of physiological growth, and genetic changes resulting from the use and disuse of a synapse. The dark curves indicate the increases in connectivities presumed to occur when a particular synapse is activated by a stimulus of uniform intensity occurring at a constant frequency. The time for beginning of the growth curve was chosen as less than one minute because the time for synthesis of a hemoglobin molecule is estimated to be about 1 ming8; the time for the beginning of the genetic curve was less than an hour because the action of ecdysome on chromosomes is visible within approximately 30 min 56. The lighter lines extending downward from points on the curves are intended to show the decreases in connectivities from points attained on the curves as a function of time after cessation of all stimulation.

stances present in already existing membranes. It is likely that material active at a genetic level can enter and begin affecting the immediate environment (i.e. in a dendritic branch) within a matter of minutes, so that as a result of its entry there may be an acceleration in the rate of genetically guided production of a particular codecarrying RNA (or RNAs) and a complete exclusion of others which participate in synthesis of closely related or alternative products. The unfavored RNAs could persist for a period within a cell, even though they were no longer being synthesized. and could be making their proteins at a rate decelerating in proportion to the decrease Brain Research, 2 (1966) 117-166

141

THE SYNAPSE AS A MICRO-CYBERNETIC UNIT

in numbers of the RNA units. Even after the complete disappearance of the templates, the proteins which were synthesized thereon might persist in cell structures for long periods of time; and an even longer intracellular life-span would be expected for the macromolecular assemblies, such as membranes, containing substances made by template-synthesized enzymatically active proteins. Thus, for a long time when any new membrane would be made the materials would be 'phenotypically" mixed although the genetic expressivity of the cell already would be changed. Eventually all new membrane being synthesized would contain material like that of the contiguous dominant element, but portions of the old membrane struciarc would remain until the whole membrane would have been replaced. The greater the amount of activity at a synapse and the greater the growth, the more rapid would be the rate of attainment of similarity in the membrane surfaces apposed to each other. However, even with extensive use it is unlikely that a new connection between adhesive, but different, nerve endings would ever attain the connectivity at this level that is possible between two elements in a primary neuronal circuit that had established their contact during the plastic period of the early developmental stages. The genetic type of connectivity discussed above, once attained, might persist for the lifetime of synapse even if used only occasic, nally. Even with complete disuse it would disappear only when the regressive changes combined with local physical forces would lead to a physical separation or when some other close and genetically different connection were to become activated sufficiently so as to change the quality of one of the surfaces in such a manner as to make it incompatible with the maintenance of contact at the disused synapse. It is suggested from the above that the physiological, growth, and genetic aspects of connectivity are expressions of the same system at levels of progressively increasing resistance to change. VI. SOME TENTATIVE APPLICATIONS OF THE SYNAPTIC MODEL

,4. Consolidation processes

Insofar as there are changes at the synapse which persist beyond the period of the energy fl0ctuations external to the synapse which initiate these changes, there may be said to be memory at all of the levels (physiological, growth, and genetic) discussed above (see Fig. 7). However, it is apparent that the increase in synaptic connectivity which occurs as a result of the growth and genetic changes would be the most significant, particularly when both are occurring simultaneously, among those changes which last long enough for an outside observer to be able to detect their persistence in some behavioral variable. It was estimated from data obtained in nonneural systems (Fig. 7) that the growth process, itself, could begin in less than 1 min and that, therefore, the minimal period after the first stimulation which would be required for any kind of 'consolidation' to take place would be, perhaps, 15-30 sec. However, it might take on the order of 15-45 min for both growth and changes in genetic expressivity to achieve significant levels. Since synaptic events take place in milliseconds, it seems logical to consider that a single effective external stimulus that Brain Research, 2 (1966) 117-166

142

E. ROBERTS

is 'remembered' by a particular synaptic unit must have some continuing internal effect. It is believed by some that under some circumstances a single external event can produce the activation of a closed circuit (or closed circuits) of neurons causing the repetitive firing of the synapses between members of the chain (see references in 25, 39, 43, 78). In any such potential circuit of synapses there would be expected to be a variation in thresholds with regard to the amount of stimulation required to activate the individual synapses. Until the synapse with highest threshold is activated, a circuit, as such, will not fire, and only those synapses in the chain up to the inactive member will be able to 'remember' the impingement of a single externally applied stimulus. An entire circuit might be fired once by a single stimulus but might not fire again without further stimulation if events at the individual synaptic units are not coordinated in such a way that the time constants of all the synaptic processes are closely similar. Thus, if a second signal arrives at a synapse before it has recovered its excitability after the preceding activating event, the chain will be broken and the operation of the circuit will cease. On the other hand, if a chain can be formed in which the events occur with similar time-constants at the various synapses, a persistently operating circuit could be set up. The time of persistence of activity in such a circuit would depend on the rate of change of the degree of coordination between the units. The numereus complex changes which take place at each synapse as a result of unique relationships with its environment, which are not shared equally by all of the synapses in the circuit, would tend to produce incoordination and discontinuation of the operation of a circuit. The increase in connectivities with use would favor the continuation of the operation of a circuit. Consideration of the actual details of the circuits in the nervous systems of various species and their interrelationships must be left to the electrical engineers of the nervous system: the neuroanatomists, electrophysiologists, and cyberneticists. When a whole behaving organism is presented with a new effective stimulus, which consists of a perceived pattern of changes in the environment, it would be expected that many perseverating circuits would be set into activity. It may be assumed that the greater the number of pertinent circuits which would be operative under a given pattern of stimulation, the greater would be the probability of a rapid and efficient processing and analysis of the data for use by the perceiving organism and the more effective would be a subsequent response to a similar pattern (learning). If the assumption is correct that what happens in neuronal circuits is largely a reflection of the events in the synapses of these circuits, it should be possible to make reasonable predictions about the effects of various types of treatments on such complex neuronal functions, if their effects on synaptic function are known. Two general types of effects of the treatments would be expected- inhibitory and excitatory, or pathological. Inhibitory and facilitatory substances or procedures would exert their detectable influences by acting in such a manner that more or less stimulation, respectively, would be required to initiate firing of the synapse with the highest threshold, and, therefore, more or less stimulation would be needed to activate the entire circuit. The potential sites of these effects at the synapse and possible mechanisms of action are legion, as can be seen readily from the preceding discussion. Substances or procedures exerting Brain Research, 2 (1966) 117-166

THE SYNAPSE AS A MICRO-CYBERNETIC UNIT

143

pathological effects at the synapse, which may include high levels of those which in smaller amounts cause excitation or inhibition, would act by producing sufficient disorganization in synaptic function so that at least some synapses in key circuits would cease to function as effective cybernetic units, and, therefore, the circuits of which these synapses are a part also would not function. Complex effects could be obtained in such studies if levels of treatment were such that either inhibitory or facilitatory effects would be produced at some synapses at the same time that pathological effects are produced at others. Procedures producing convulsions and spreading depression would be included in the pathological category. It would be predicted from the formulation in Fig. 7 that the effects of electroconvulsive shock, which are destructive to the operation of coordinated neuronal circuits, would be exerted by preventin? the v.ttainment of each type of connectivity after the first application of stimulation. A maximal inhibiting effect on the attainment of the physiological connectivity (Fig. 7) could only be exerted if the convulsions were produced, at most, within a few seconds ~fter ~timulation. If interference with development of this kind of connectivity occurred, nothing could be learned from the experience because the growth and genetic type of connectivities at key synapses would fail to take place at all. If the convulsions were produced up to 20-30 sec after the stimulus the amnesic effect still would be very great because the growth processes might only have just begun, and up to approximately one-half hour such treatment would still decrease the retention of the experience effectively because the genetic type of connectivity would not have begun to be developed. At later times the effects of convulsions would be greatly reduced because all three major types of connectivities would have been increased in synapses of the responding circuits. In the synaptic model dealt with here, once some connectivity has been developed at the growth and genetic levels, it would not be expected that it would be lost during a period of gross depolarization such as must follow electroconvulsive shock or spreading depression. Particularly interesting in this regard are some findings which showed that one electroconvulsive shock given to rats 5 sec after a stimulus produced a remarkable amnesic effect on retention of effects of a simple punishing stimulus eg, and another similar study in which it was shown that some amnesic effect can be demonstrated for a period up to 26 rain 4e. When electroconvulsive shock was given 10 sec after the performance of a well-learned discrimination task there was no evidence of forgetting (discussion by McConnel157). Thus, the convulsions may not be interfering with synaptic circuits that are well established. Rather, the interfering effects of the treatment must be exerted upon the consolidation processes. The above results could be interpreted to mean that the behaviorally observed consolidation phenomena and the increases in the connectivities of synapses in the activated neuronal circuits, as discussed in this paper, are the same processes looked at from different points of view. The results obtained with the effects of chemicals and drugs also support the above ideas. Facilitatory influences would be expected to act in such a way that they would increase the rate of attainment of synaptic connectivities. It is most likely that this wouId be accomplished by overcoming the limitation imposed upon establishment of connectivity by some rate-limiting process within the synaptic unit. The maximal Brain ~,esearch, 2 (1966) 117-166

144

E. ROBERTS

rate of attainment of connectivity would be set by the maximal innate rate of the operation of the entire coordinated sequence of reactions which are schematized in Figs. 2-6 and discussed in general terms in the text. It would be expected that the maximal effects on development of connectivity produced by a variety of facilitatory treatments would be the same under a given set of experimental conditions with a given species (same sex, age, experiential history, etc.). If the rate-limitation on the establishment of synaptic connectivity were a synaptic property that shows a normal distribution among the members of the species to be tested, then the animals with the optimal level of this property would be least affected by the facilitatory treatments and those in which this property showed the greatest deviation from the optimal level would be most affected. The most effective facilitatory treatment would tend to ehminate the d,fferences between the latter two groups in such a manner that the animals of both groups would tend to show the maximal potential rate of development of synaptic connectivity with use. The inhibitory treatments would tend to reduce the rate of development of synaptic connectivity in both groups to the minimal level which is still compatible with normal function. The facilitatory treatments would accelerate the rate of attainment of connectivity only within those intensity or dose ranges which would allow the synapse to continue its functions as an effective cybernetic unit. A small increase above these ranges would begin to show destructive effects on the coordination of synaptic events, at least at some synapses in some of the circuits involved, so that the overall facilitatory effects would begin to be balanced off by the inhibitory effects. Finally, upon further progressive increases a level would be reached at which the pathological effects would predominate and only inhibition would occur. Several studies of learning are consistent with the above hypothesis about synaptic facilitation and inhibition. McGaugh found under a given set of experimental conditions in maze-learning that a strain of maze-bright ($1) rats made an average of 13 errors, maze-dull rats (Sa) made 33 errors, and a first generation cross of these strains (F1) made 24 errors. After treatment with 5,7-diphenyl-l,3-diazadamantan6-ol (1757 I.S.), a substance similar in its excitatory action on the central nervous system to strychnine, the following mean numbers of errors were observed" $1, 16; Sa, 17; F1, 17 (Ref. 76) The decreases in the means (facilitatory effects) for the Sa and F~ groups were statistically significant while the changes in the $1 group were not. Similarly, intraventricularly administered potassium, which facilitates learning, only brought the average of the learning rate of the potassium-treated animals up to that of the fastest learners of the control group; while calcium, which inhibits learning, only reduced the average to that of the slowest members of the control group (see Ref. 57, E. R. John). ACTH brought the learning capacity of an entire group of rats up to that shown ordinarily by only 40 % of the animals, and the steroid levels of the adrenals of the normally faster learners were found to be higher than those of the slower ones (see Ref. 57, S. Levine). Also maze learning can be facilitated by low doses of strychnine and disrupted by high doses 74. The above findings, and results of other experiments in which the stimulants were given at various times after a learning experience 75, are in accord with the hypothesis that the enhancement occurs because there is an increased persistence of activity in neural circuits as a result of a stimulus pattern •

IF

Brain Research, 2 (1966) 117-166

THE SYNAPSE AS A MICRO-CYBERNETiC UNIT

145

presented to the organism during a learning trial, and also are consistent with the idea that this has to do with a facilitatory influence at some synapses which could become limiting to the persistence of the circuits which contain them. The following substances are known to increase neural excitability and also to facilitate learning when given in moderate doses: strychnine, picrotoxin, amphetamine, 1757 I.S., thyroxine, nicotine, caffeine, potassium, and physostigrnine. Substances known to decrease neural excitability which also have been shown to inhibit learning are calcium, various barbiturates, ether, and carbon dioxide. Hypoxia, spreading depression, and electroconvulsive shock also inhibit both excitability and learning (see Ref. 43 for references). In line with the inhibitory effects of the latter treatments is the finding that irritative lesions produced by aluminum hydroxide cream placed on various parts of the cortex of monkeys slowed the learning of new problems remarkably, but did not disturb the memory of solutions to problems learned before production of the lesion (Refs. 108-110). These effects might be a result of interference with memory consolidation. One way in which such a focus could operate would be by producing conditions which would result in abnormal electrical activity in neural cells lying within the focus. The connectivities of the synapses between the irritated cells, possibly firing spontaneously, and their neighboring cells could be increased greatly by the continuing activity. These connectivities would be irrelevant to the operation of functionally meaningful circuits, such as normally may be activated during presentation of a specific external stimulus~ but might include a large enough number of neurons required in such circuits so that the probabili'y would be decreased greatly of producing persistently firing circuits necessary for learning solutions to specific new problems. Pertinent here also is the work in which production of a primary irritative lesion on the cerebral surface of one hemisphere after a period of time results in an independently active epileptiform region in the homotopic region of the opposite hemisphere, the independent 'mirror focus '7a,79. Experiments suggested that the spontaneous discharges in such a region were the results of impulses circulating in closed neural circuits. However, even when surgical isolation prevented the activity of the circuits, marked increases in neuronal excitability over normal could be demonstrated by various methods. These experiments are entirely consistent with the previously expressed view that increased connectivity would result from increased use of synapses. Continuous bombardment of neural pathways by the original epiieptogenic focus would increase progressively the connectivities of all successive units. In this way, each of the neurons would play a progressively greater fractional role in the firing of the next neuron in the circuit. It could be envisioned that eventually the firing of any neuron in the circuit could lead to the firing of the rest of the circuit. If any of the neurons were to possess a spontaneous rhythm they could become pacesetters for firing the entire circuit. When the circuits would be interrupted surgically and would cease to fire as units there would still be present synapses with greatly enhanced L~" _'aconnectivities between ,,,~,,-,-,,-~,,,.,,,.,,,owmcn arc fragments of the former circuits. It can be seen from Fig. 7 how these connectivities enhanced by long use could be preserved for prolonged periods even with complete disuse. Brain Research, 2 (1966) 117-166

146

E. ROBERTS

B. Conditioned response learning The neural basis for the time, relations between conditioned and unconditioned stimuli required for the establishment of conditioned responses has not been adequately established. The type of thinking with regard to connectivity in the model being considered can be applied to some preliminary considerations dealing with synaptic

a. CIRCUITS AT REST

.

e.

ESTABLISHED REFLEX

SYNAPSE

,,~

o,.o,,,, ,/~'27---.

6

/ SENSOIY ELEMENT

N

AXON

1t

I

I

'"'°'°'

•

•

t ""

. , - ~ ~ . . ~ . . . . . . --.

j

"..,.,...,j._, ..,,"

d~

/ d. REPEAT OF (C) WITH INCREASE IN CONNECTIVITY

b. CIRCUIT No.1 FIRED ALONE

\

./ •% .

N...

r"

.. "'~,.. ~....,,...~

\

C. BOTH CIRCUITS FIRED SIMULTANEOUSLY ; OR No. 1 JUST BEFORE No. 2

~-

(-(~)

\

<.)

j

_

'~,~ ~. ....,Fig. 8. Postulated sequence of simplest possible circuit activations and connectivity changes during the establishment of a conditioned response (see text for explanation).

events during the establishment of conditioned reflexes. Let us suppose that two wellestablished, innately determined neuronal cir:uits subserving different sensory modalities could be fired by the stimulation of particular receptors and that responses could be observed in different effectors. Terminal axonal branches of one or more of the neurons in the first circuit in some region of the brain could establish contact with the. dendritic branches of one or more neurons which are an integral part of the second Brain Research, 2 (1966) 117-166

147

T H E SYNAPSE AS A M I C R O - C Y B E R N E T I C U N I T

circuit. A highly simplified scheme is shown in Fig. 8a. There are known to be numerous individual cells in the mesencephalic reticular formation which respond to 'natural stimuli of different types, locations, and modalities (convergence)'. It is particularly interesting that weak natural stimuli are effective and that frequently several of them can influence the same reticular unit 8. Afferent signals coming from the firing of the first circuit might never, by themselves, achieve su~cient depolarization in the dendrites of the second circuit to activate the second circuit beyond the points of contact (Fig. 8b). However, if both circuits repeatedly were fired at the same time, there would be expected to be a frequent coincidence of effective depolarizations taking place at both the originally ineffective presynaptic endings and the apposed postsynaptic sites (Fig. 8c). If the first circuit would be fired only shortly before the second such a coincidence also would be expected to occur, since some perseveration in the first circuit might take place after its original activation. This could lead to a progressive buildup of connectivities (Fig. 8d) so that eventually the activation of the sensory receptor of the first circuit could lead not only to the elicitation of its own typical response but also of that ordinarily evoked by the stimulation of the receptor of the second circuit (Fig. 8e). On the other hand if the second circuit were fired first, according to the hypothesis of the present paper (see Figs. 1, 2, and 7 and related discussion) we would never expect the connectivities between the two circuits to be built up in the manner mentioned above. This would be because the inhibitory or feedback transmitter postulated to be liberated at the postsynaptic site would decrease the probability of the depolarization of the unfired presynaptic site, and whatever connectivity existed at axodendritic connections between the first and second circuits actually would be decreased. The above ideas are entirely in keeping with what we know of the conditioned reflex. In the o]iginal Pavlovian conditioning experiments it was found that the food or acid employed for elicitation of the salivary response could L)e substituted for by a variety of other stimuli, provided the other stimuli were applied simultaneously with the food or acid, or before. If food or acid were given first and the other stimuli even only a few seconds later, conditioning did not take place. When the new stimuli preceded the food or acid by a brief period, after a number of combined applications the new stimuli alone would cause as much flow of saliva as did the original meat or acid. The existence of polysensory systems in the brain within which .......

,~. . . . .

.~ ~i,~,~t. ~ , ~ r a i

~tlm,ll~ r,~n t~lr~

nl2r,:, ,,nrm

~inal~

~11~ ~nd

the_ w i d e

ranges of responsivities of the individual units within the various cell groups a undoubtedly make possible the establishment of myriads of conditioned relationships which are far too subtle to be detected by most present methods of behavioral analysis. Convincing evidence recently has been presented for the learning and retention of conditioned avoidance responses by the insect ventral nerve cord (headless locusts and cockroaches) a7 and by the isolated prothoracic ganglion of the cockroachaL The latter preparations are much simpler than those portions of the vertebrate CNS in which the integrative processes involved in learning take place. Further comminution of such neuronal systems to attain the simplest ones which will still support behavior that can be called learning eventually may enable the neurobiologist to Brain Research, 2

(1966) 1 1 7 - 1 6 6

148

E. ROBERTS

study the morphological, neurophysiological, and biochemical correlates of learning at individual synapses. VII. COMMENT

The type of synapse discussed in this paper in largely conjectural biochemical and biological terms is one which would be a suitable nodal element for circuits with plastic properties in which 'plasticity is introduced by allowing connectivity of the net, as well as operational modality of nodal elements to be use and time dependent'. In such networks there could be 'memory without record' (see Ref. 57, H. von Foerster).' Suffice it to say that the actual demonstration that synapses have the general properties suggested in this paper would take us a long way toward understanding many of the integrative functions of the nervous system which are observed in behaving organisms, but for the performance of which the machinery somehow previously has seemed inadequate without the postulation of a mechanism for the storage of memory in some sort of stimulus-directed coding at the molecular level (see Appendix iV). The study of the chemistry of thc synapse is under way in many laboratories. The techniques most frequently employed by biochemists involve a disintegration of neural tissue which is just extensive enough to allow the centrifugal separation of subcellular constituents into categories which are identifiable electron-microscopically with structures which are found to occur in sections of whole tissue. Analyses are then performed on the fractions for chemical variables of interest to the investigator. Although potentially there are many possible sources of artifacts at all phases of such studies, much useful information has been derived. Particularly exciting has been the separation of presynaptic elements and the study of the content of neuroactive substances contained therein. However, none of the procedures employed to date has given well-defined preparations of postsynaptic structures. Rather complex organized structures have been observed in postsynaptic regions of neurons 4a,117 but these have not appeared to survive as separable, identifiable structures with the preparative methods employed to date. The postulate that the postsynaptic components may play an important modulatory role in synaptic events has led us to institute efforts to be#n to investigate procedures by which isolation of postsynaptic neuronal structures might be achieved. Difficult problems must be faced in any attempts to determine whether or not there is a causal relationship between the alteration of a particular biochemical variable and a measurable synaptic or behavioral change in the organism in which tlle change is induced. The model presented requires that the balance between the excitatory and inhibitory transmitter systems, rather than the absolute level of either one, would be important in the regulation of activity at synapses. It is now necessary to turn from simpler and more comfortable courses of action and to undertake the multivariant analytical approaches which are worthy of the cybernetic control mechanisms that exist at synapses.

Brain Research, 2 (1966) 117-166

THE SYNAPSE AS A MICRO-CYBERNETIC UNIT

149

APPENDICES i"

1. Studies dealing with changes in 7ABA and acetylcholine contents and sensitivity of neuronal membranes 1. Elevation of 7ABA In a recent series of expe.rimentse3 the time-course of changes in brain content of 7ABA and sensitivity to electroconvulsive shock were studied in mice after administration of aminooxyacetic acid (AOAA), an inhibitor of FABA transaminase. The increase in FABA content as a function of time after AOAA administration (25 mg/kg) was biphasic. A plateau in FABA level was attained between 3 and 4 h, and a subsequent secondary rise took place between 4 and 6 h. Elevated values were still observed at the 24-h period. There was a remarkable decrease in electroshock sdzure incidence (75 mA stimulus) during the first 1½ h after AOAA. Subsequently, the susceptibility to seizures began to return to normal, attaining the control values at 6 h, at which time the },ABA content was maximal. In Fig. 9 is shown a 3-dimensional plot in which seizure incidence and whole brain },ABA contents (as % of control

SEIZURES (%) 100

.........:.. ::i~:~i:ii~!!il ~:i~:!i-/~:.,/.:.. '. ,

.............i \

_-...: i-

~-i • -!...;ix

i:.:/. !i".ii:~:.~.;..:_i( '::~.. i!.- " 2s

.~!:~!i:~:~i:~:;~i:f~

~.... /

I ~ ~

/

/

: ."$i: . .:-:..

.~;'~

o!' ~ - " ~ - ? ! " ; / ~ " " 0

1

2

.i:! .:.:../

/

/

/

-

L ,,oo

"//

/

"/ ,/

3

4

5

,.,B, CONTROL I

6

H AFTER AOAA F,_'~. 9. Tbzee-dimensio~! Pl~e~-ef ~o;. . . . . s,,,.~c:ptibility and y-aminobutyric acid (7ABA) content of brains of mice as a function of time after administration of 25 mg/kg of aminooxyacetic acid. Arrows point away from the inflection points of the curves from which they are drawn.

Brain Research, 2 (1966) 117-166

! 50

E. ROBERTS

values) are plotted as a function of time for 6 h after the administration of AOAA. Only during the first 1½-h period was there a correlation of decrease in seizure susceptibility with increase in ~,ABA content. Thereafter, the seizure susceptibility increased while the yABA content continued to rise. One of the possibilities suggested by the above data is that the increases in yABA levels and changes in seizure susceptibility after administration of AOAA are completely unrelated. Another possibility is that a relationship does exist, but that it is localized to a particular, small region in the brain or can be found only by analysis of those neuronal regions from which neuroactive substances are liberated during function in the CNS. These latter points are now being further approached in our laboratory by study of smaller brain regions and by analysis of isolated nerve endings from normal and AOAA treated mice with regard to 7ABA content and the activity of the enzymes involved in its metabolism. Another way to view the problem is consistent with the formulation in Fig. 5. 2,ABA may, indeed, be importantly involved in decreasing neuronal excitability soon after yABA transaminase blockade with AOAA, and total brain 7ABA may be directly related to the physiologically effective amounts of the substance; but compensatory increases in excitatory factors, decreases in inhibitory factors other than yABA, or both, may take place with the consequent restoration of normal sensitivity to electroshock in spite of the persistence of elevations in yABA content. With the abovd in mind it was of interest to examine the type of relationship between ),ABA content and protection against seizures during the first 1½ h after AOAA (Fig. 10). At the lower levels of yABA a linear relationship was found to exist between seizure susceptibility and brain yABA content with the curve approaching the point of complete protection asymptotically, as would be expected to occur if some saturatable inhibitory neuronal site were involved. A further look at Fig. 9 reveals the interesting fact that the inflection point of the first portion of the yABA curve occurred at the time when the curve of seizure susceptibility began to rise toward normal, and the inflection point of the latter curve just preceded the beginning of the second rise in

I00

.,.o,....-.-o-----o

/*

80

o" 60

40

20

/ t

I00

I10

i'

120

i

130

i

140

|

150

I

160

"Y-ABA (% OF CONTROL VALUE)

Fig. 10. Relationships between brain content of 7ABA and protection against seizures (75 mA current) during the first i½h-period after aminooxyacetic acid (25 mg/kg). Brain Research, 2 (1966) 117-166

THE SYNAPSE AS A MICRO.oCYBERNETIC UNIT

151

~,ABA content. This would be understandable in terms of a biochemical servo-mechanism in which a progressive, self-limited increase in amount of effective E.T. or a progressive, self-limited decrease in effective amount of one or more I.T.'s other than ),ABA would be triggered off at the time when the increase in 7ABA is occurring at the maximal rate (inflection point); likewise, at the time of maximal rate of change of the compensatory change a secondary rise in 7ABA could be induced through an increase in the rate of its formation or a decrease in the rate of utilization. Glutamic acid, a potentially important excitatory factor in the CNS, did not increase after AOAA. Work on acetylcholine content is in progress. 2. Elevation o f acetylcholine A well-studied case of a persistent elevation of acetylcholine content in brain is that of the ep strain of mice 64,6~,8z, in which susceptibility to convulsions after successive movements causing loss of postural equilibrium is inherited as a Mendelian dominant characteristic. The convulsive threshold to pentamethylenetetrazole is lower in the ep strain than in the normal strain studied. Spontaneous convulsions do not occur in the el) mice under ordinary cage conditions. A morphological basis for the defect could not be found on histological examination of these mice. Postural stimulation did not induce convulsions in animals younger than 7 weeks, and only brought on seizures in an incomplete form in animals between 7 and 10 weeks of age. Full-blown, but not lethal, convulsions could be produced in all animals of both sexes of the susceptible strain at all subsequent ages. The acetylcholine levels of the brains of ep mice were 40-60 % higher than those of the other strains studied. At 4, 5, 6, 7 weeks of age and in adults the levels for the el) strain were 2.09, 2.31, 2.54, 2.66, and 2.86/zg/g, respectively; for a control strain (gpe) the corresponding values were 1.47, 1.65, 1.94, 1.95, and 1.95/tg/g, respectively. The brain ~,ABA content in the ep mice also was 40-50 % higher than in the control strain, while both glutamine and glutamic acid levels were approximately 30 % lower. Thus, both aeetyicholine and ),ABA levels were found to be higher to approximately the same relative extent in the brains of the ep mice. The enzyme catalyzing the synthesis of acetylcholine from choline and acetyl CoA, choline aeetylase, was found to be 50 % higher in the ep mice than in the other 3 strains tested. The increased choline n..~,.,~. . . . . ,:.,;, . . . . . r,or.h, r , , . . , . ; ~ h , ~ the , ~ , , ~ . l ~ , . ~ , . r , , . , f,~,- th,~ higher value ,,f n~etylcholine in the brains of the ep mice, since the activities of the cholinesterase and the enzyme catalyzing the synthesis of acetyl CoA did not differ significantly between the el) mice and the control strains. In a study of the content of particle-bound acetylcholine in homogenates of brains of ep and normal mice it was found that the labile component (liberated by osmotic dilution) in the ep strain was twice that found in normal mice; the level of stable acetylcholine was approximately the same 65. The labile fraction of bound acetylcholine may be that fraction which is liberated during nerve activity, since decreases in this fraction were found to parallel decreases in total acetylcholine in anima!s that had been convulsed6L The above data tentatively may be placed into the scheme shown in Figs+ 3-6 as follows" For some, as yet unknown, genetic reason (failure of repression or too much Brain Research, 2 0966) 117-166

152

E. ROBERTS

derepression?) the activity of the enzyme which forms E.T. is greater than normal in the CNS of the ep mouse. This-resuRs in an increased level of labile, or physiologically available, E.T. in presynaptic storage sites of E.T. A given external stimulus will release more than normal amounts of E.T. at the synapses of the activated neuronal circuits. This would predispose to indiscriminate spread of impulses and convulsions, a tendency compensated for by higher levels of I.T. in physiologically available storage sites and an increased release of I.T. at synapses consequent to nerve activity. As a result, the animal could live out its life without visible handicap under ordinary laboratory conditions. The neuronal system poised at the above level would have less stability than the normal one, and, thus, intense stimulation would lead to incoordination in the least compensated circuits, which would be reflected behaviorally in a failure to adjust normally to certain types of environmental stresses. 3. Decreased sensitivity o[" neuronal membranes It is not unreasonable to assume from data in the literature that barbiturates may exert their inhibitory action at synapses by decreasing the excitability of both pro- and postsynaptic membranes. Reasoning from our synaptic model (Figs. 3-6), it could easily be suggested how daring the addiction to these drugs compensatory phenomena might take place which would cause more E.T. and less I.T. to be liberated per impulse, thus enabling essentially normal synaptic transmission to take place in spite of decreased sensitivity of the membranes. It has been reported that barbiturates cause an increase of bound acetylcholine in the cerebral cortex of the cat 77 and a decrease in total yABA content of the cerebral cortex of the rat 6. If the barbiturate should be withdrawn suddenly, the sensitivity of the membrane sites would return to normal probably shortly after the blood level had fallen, while it would be expected that the enzyme systems overproducing E.T. and underproducing I.T. would take longer to return to levels found in the predrug period. Therefore, hyperexcitability would be expected to result upon drug withdrawal. Indeed, convulsions and other neural disturbances occur in animals and man during barbiturate withdrawal, indicating a pathological predominance of excitatory influences. If the above general concept has some validity, then an elevation of 7ABA levels during the withdrawal of barbiturates in addicted organisms should decrease the severity of the symptoms. A pertinent experiment based on the above suggestion was performed in which dogs were addicted to barbital 37. After complete withdrawal of the drug a control group was given saline and the experimental group was given AOAA, which elevates ~,ABA levels. There was a dramatic difference in the behavior of the two groups. All the controls showecl convulsions, status epilepticus being attained in a number of instances, and a 56 °//o mortality occurred in the first 7 days after withdrawal. Far fewer seizures and deaths were found in the treated dogs, some of the animals showing no seizures at all during the 7-day period of administration of AOAA. When admir.istration of AOAA was termir, ated at 7 days after cessation of the barbiturate, several of the dogs showed convuls~or, s for the first time. The latter observation is in keeping with the suggestion of a slow return to normal of the compensatory changes developed during barbiturate addiction. Although not proved, it seems likely that AOAA exerts at least part of its Brain Research, 2 (1966) 117-166

THE SYNAPSE AS A MICRO-CYBERNETIC UNIT

153

inhibitory action in this instance through increasing the level of 7ABA in the CNS. Dilantin, acetazolamide, and pyridoxine failed to show anticonvulsant effects under these conditions.

IL Conjectures concerning possible rate-limiting processes in neuronal growth Degradative and synthetic enzymes are known to co-exist in neural structures. In any particular instance a growth limitation may be imposed by a relative deficiency of some enzyme activity involved in energy generation or in biosynthetic reactions. This may result from the insufficiency of the actual amount of a particular enzyme protein or of a cofactor necessary for its activity. A similar condition would result if the enzyme were present in an inactive form and a limitation would exist on the rate of its conversion to an active form. Growth also could be prevented by the lack (or inhibitory excess) of a number of precursors which are essential for protein, lipid, or R N A synthesis or even by an ionic balance which is unfavorable for the operation of a key reaction. Similar considerations to those above also might be applied to the glial end-feet which seem to be present ubiquitously at synapses. A few guesses can be made about the general metabolic areas in which one might look first. If during the course of the pre- and postsynaptic depolarization some material (transmitter, ammonia, CO2, ions, histamine, substance P, etc.) possessing marked effects on vascular permeability would be liberated, then the exchange with the blood would be accelerated at the time of greatest metabolic load*. Indeed, it has been shown with the qualitative inert radioactive gas technique together with autoradiography that photic stimulation of the retina in cats resulted in increased blood flow in cerebral structures associated with visual functions--lateral gyri, lateral geniculate ganglia, and superior colliculi !°°. Interestingly, the changes 'were so characteristic that autoradiographs from stimulated cats could be readily distinguished by simple inspection from those of control animals. The areas affected were, however, so discrete and so small a portion of the structures containing them that average blood flows in each of the structures as a whole were not significantly different in the control and experimental animals'. It may be presumed that if the degree of resolution had been sufficiently great, changes in blood flow could have been seen in the regions of individual synapses. In this latter connection, in addition to the entry of metabolites and exit of waste products, serious consideration also must be given to the possibility of the entry of specific nerve-growth promoting factors into the synaptic areas from the circulation during brief periods c,f increased vascular permeability. A potent protein factor, found in various mouse tissues and serum and which has been isolated in highly purified form from mouse submaxillary salivary glands, has been shown in catalytic * Although not directly applicable to nerve tissue, it is interesting in this connection that the action of estrogen on the uterus may be mediated by the local release of histamine, which produces vasodilatation. The hyperemia and increased capillary permeability then result in an early accumulation of water, electrolytes, plasma proteins, and possibly other substances and a later increase in mitotic activity in the liminal epithelium and an increase in glandular activity. Serotonin, although les effective than histamine, also can produce similar effects1°4,1°5,114.

Brain Research, 2 (1966) 117-166

i 54

E. ROBERTS

amounts to specifically promote the differentiation and growth of sympathetic ganglia ls,20'66-6s. The latter work opens the question as to whether or not such trophic factors may exist for other parts of the nervous system. Most reactions which occur in living systems may be considered to be potentially rate-limiting in terms of some cellular activity; but in a particular instance only one reaction actually is likely to play a decisive role. Although relatively little is known of the detailed quantitative chemistry of the synapse, one logical place to seek the ratelimiting reactions would be among the fundamental energy- and precursor-yielding reactions (glycolysis and respiration) which are common to all cells. In this connection it is of particularly great interest that a central regulatory role is being uncovered for adenosine-3',5'-phosphate (cyclic-3',5'-AMP) in a number of cellular functions 111 and that there is some evidence that some hormones and neuroactive substances (ACTH. gl~:cagon, thyroid stimulating hormone, vasopressin, the catecholamines, histamine, serotonin, and the methyl xanthines) may exert their action on specific target cells by increasing the amount of this substance16,59,70-72,81,86,112,ila. Cyclic3',5'-AMP has been observed to increase the amount of active phosphorylase and phosphofructokinase (PFK) in tissues, thus promoting glycogenolysis and glycolysis and in this manner opening the gateway for a more rapid flow of energy and carbon precursors to particular cell types, enabling them to perform their specialized physical and chemical functions as well as those related to general maintenance and growth. Several lines of evidence have shown that the activity of PFK, the enzyme which catalyzes the rezction between fructose-6-phosphate and adenosinetriphosphate (ATP) to form fructose diphosphate and adenosine-5-diphosphate (ADP), can be ratelimiting under certain conditions in such diverse biological systems as mammalian heart, diaphragm, and skeletal muscle; ascites tumor cells, granulation tissue, and brain; yeast cells, insect wing muscles, and intact and homogenized liver flukes70-7~, 88. The PFK can tze inhibited by ATP in the presence of fructose-6-phosphate (F-6-P) when these two substrates for the enzyme are present in concentrations ordinarily found in tissues. Under these conditions cyclic-3',5'-AMP can cause a marked increase in the activity of the ATP-inhibited enzyme. The inhibition also can be relieved less effectively than by cyclic-3',5'-AMP by ADP, adenosine-5'-phosphate (AMP), or by orthophosphate. At higher levels of F-6-P in in vitro test systems neither ATP inhibits nor does cyclic-3',5'-AMP activate the PFK 7i. fhere is an enzyme system, adenyl cyclase, in various tissues (including that of the central nervous system) which forms cycl,.'c-3',5'-AMP from ATP, and a phosphodiesterase is present which splits this substance to give AMp16,59,81,112A13. It is o[ great interest for the present discussion that the adenyl cyclase is stimulated by extremely low concentrations of catecholamines (10-7M to 10-6M) and recent evidence suggests that histamine also may stimulate this enzymeSL The methyl ~,anthines (theophylline, theobromine, and caffeine) inhibit the phosphodiesterase. Both of the latter groups of substances have been found to increase the levels in tissues of the cyclic-3',5'-AMP, which then accelerates glycolytic rates in these tissues through its action on enzymes, as described above. Perhaps the . . . 1, of an increase ofcyclic-3',5'-AMP in an intact organ. . . .most . . . . . .~f,;t.; . .e. .,~u,L ism has been observed in the liver fluke, the parasitic trematode, Faseiola hepatica 7o. Brain Research, 2 (1966) 117-166

THE SYNAPSE AS A MICRO-CYBERNETIC UNIT

155

Enhancement of motility produced in this organism by treatment with serotonin was attributable to an in~rease in glycolytic rate resulting from an initial increase in cyclic3',5'-AMP which then activated phosphorylase and PFK 72. In view of the lack of data at the present time, it is not possible to assign a major role, or any role at all, to cyclic-3',5'-AMP in regulation of activity at the synapse. Nonetheless, the location of its effect in a fundamentally important reaction sequence common to all cells and the action of some neurohormones upon it make this substance an important candidate for further consideration. In this connection it may be proposed that an inhibitory concentration of ATP could be reduced by the activation of the internal Na+-activated ATPase, believed to be intimately related to the Na+-pump mechanism, when permeabilization of membranes to ions takes place. Thus, some of the transmitters might h~ve a dual action in relieving a block on metabolism exerted by ATP, first by causing depolarization which activates an ATPase and second by increasing the amount of eyclic-3',5'-AMP which relieves ATP inhibition of the PFK reaction in glycolysis. Other points of control by ATP could lie among those reactions of the pyruvate oxidation sequence (Krebs cycle) and electron transport at which oxidative phosphorylation (conversion of ADP and ATP) is known to occur, if levels of ATP were to be maintained at such a high level that the concentration of the acceptor, ADP, would fall below that necessary for optimal activity then the rate of one of these reactions might fall to such an extent that the rate of the whole sequence could become negligible. However, from what is known of the sensitivity of the FPK reaction to ATP, the latter situation only would be of consequence if the substrate for oxidation were coming from some source other than glucose and if the multiple reactions which ordinarily utilize ATP were largely inoperative. The Krebs cycle and respiration, itse!f, could become inhibited as a result of limitation of suitable carbon substrate and/or oxygen. Both of these limitations could occur at the synapse if glycolysis were shut off and if the rate of exchange with the blood through the extraneuronal compartment were too slow. Here again the synoptic transmitters could play a role. If acetylcholine were the transmitter released from presynaptic endings, its hydrolysis by acetylcholinesterase would give acetate and choline. Coincident with the decrease in pro- and postsynaptic levels of ATP which would follow depolarization and which would release glycogenolysis and glycolysis from n n inhibited ~tate_ the small amounts of acetate could be . . . . . . . . "~ *o -"°*"~ coenzyme A and could condense with oxalacet,.te to give citrate and begin the operation of the Krebs cycle at key loci at intraneuronal synaptic sites and possibly also in the investing glial elements and the cellular elements of the blood vessels. Meanwhile, the negative feedback transmitter liberated postsynaptically also might be converted to a substance utilizable via the normal glycolytic and respiratory pathways. Important rate-limiting reactions may occur in the nietaboiic area dealing with RNA metabolism. It may be presumed that RNA may play a regulatory role in neuronal and glial growth (protein synthesis)in a manner similar to that envisioned for it in _:___,.:_1 ....... ,1..r~,~ ¢.,u .... ;. . . .I.l~.tv~-o-~-l~-'t" . . ,.,t~,,,, is from Ref. 90 in which suitable literature llllldlUOl~l ~lOWUlt. lllt~ lVllVV*Ill~ . . . . . . . . . . . citations are given" "The rate of protein synthesis is proportional to the numbers of BraOt Research, 2 (1966) 117-166

136

E. ROBERTS

ribosomes in the cell under steady-state conditions. Furthermore, the rate of RNA synthesis is highly sensitive to shifts in the medium. An increase in the richness of the medium, from glucose minimal medium to broth, for example, results in an immediate acceleration of the rate of RNA synthesis, later followed by increased rates of protein and DNA synthesis. Conversely, shifting to a poorer medium causes an immediate inhibition of RNA synthesis. These results lead to the conclusion that the amount of RNA determines the rate of protein synthesis, and the amount of RNA is regulated by environmental conditions so as to produce an optimal growth rate. For some years, it has been known that the synthesis of RNA requires the presence of all the common amino acids. Although the role of these amino acids in the mechanism of RNA synthesis is not known, it was early recognized that they provide a means of coordinating protein and RNA synthesis. Recently, this catalytic function of the amino acids in RNA synthesis has been further investigated. The factor which governs the rate of nucleic acid synthesis appears to be the intraceilular amino acid concentration. Excess amino acids permit more rapid RNA synthesis, while a shortage of amino acids reduces the rate of RNA synthesis. The amino acids are not thought to be components of the machinery for converting nucleotides into RNA (as the nucleotides are for activation of amino acids in the case of protein synthesis), but are thought to have a special regulatory role. It is suggested (,a:ithout direct evidence) that the amino acids act as inducers of RNA synthesis, acting to block the inhibitory action of repressors which may be the amino acid transport RNA molecules.' Many factors possibly could limit RNA metabolism, and through it, growth of pre- and postsynaptic connections and associated glial elements at a particular synapse. If amino acids were limiting, upon stimulation during the phase of increased permeability the limitation could be overcome by entry from the blood or by the entry oc activation of catheptic enzymes which could liberate the amino acids from proteins pre-existing in the nerve endings. A recent study employing high-resolution autoradiography together with microdensitometry showed that in exercised rats there was an increase in the incorporation of intraperitoneally administered tritiated leucine by individual neurons over that found in non-exercised controls 1. Although the highest relative increase was found in the motor structures-, increased incorporation of isotope was observed in neurones distributed over the entire brain. These results show clearly that, whatever the mechanism may be, activation of neural structures results in an acceleration in the rate of entry and incorporation of a circulating amino acid into neuronal proteins. The available data suggest that turnover of protein and RNA takes place rapidly in neuronal and glial elements and that the rate may be changed by neuronal activity and by drugs 50. It has not yet been possible to assess the physiological significance of net decreases or increases in the amounts of total protein or RNA, nor has it been possible to analyse events at individual synaptic endings to learn about the synchrony of these changes with activity. However, it is not surprising that chemical interference with RNA or protein metabolism, or for that matter any aspect of neuronal or glial metabolism, may lead to disturbed function in the organism2a,29,a8; or that when there are gross pathological processes taking place in the brain that the amounts or Brain Research, 2 (1966)117-166

THE SYNAPSEAS A MICRO-CYBERNETICUNIT

157

distributions of proteins and RNA may be altered. Little of functional significance can be concluded at present from a determination of overall changes in total contents of proteins, RNA, c-r other macromclecular censtituents and it has not yet been possible, generally, to study individual pure macromolecular constituents in nervous tissue. it is ~ot particularly revealing that purine or pyrimidine antimetabolites, various drugs, dietary deficiencies, etc., can interfere with learning processes, since all aspects of development of synaptic connectivity with use would require the properly coordinated function of many metabolic steps. In addition to the key role of RNA in protein synthesis, nucleotide cofactors are involved in numerous enzymatic activities in virtually all of the metabolically important reaction sequences whether it be glycolys~s and respiration or protein, RNA, polysaccharide, or phospholipid biosynthesis. Addition of uridine and cytidine to blood used in perfusion of the cat brain was shown to significantly prolong the maintenance of structural and functional integrity of the preparation4L in the latter category can be placed the effe~-ts on improvement of memory said to be observed upon administration of yeast RNA or ribonucleotides to aged patients with impaired cerebral circulation 17.

IlL Epigenetic influences on genetic expressivity with regard to properties o.f cell sur[aces One of the best documented examples of this phenomenon has been studied in Paramecium aureiia 7,34,~°2,i°3. When homologous anusc~um ~ ~uu~.u to t.~ ,v~. organisms they become immobilized. Qualitative and quantitative studies of the surface antigens have been made using such antisera. A particular stock of paramecia, consisting of a culture derived from a single organism, was found to contain individuals with a serie~ of different surface antigens, as many as twelve different serotypes being found in ene stock. Transformation from one serotype to another may be induced by temperature changes or exposure to homologous antiserum. From a study of genic and cytoplasmic variation it was concluded 7 'that every stock of P. aurelia can exist in a series of cytoplasmic states which act in such a way as to inhibit the expression of all the antigen-determining genes except those at one locus. By changing the environment, the cytoplasm can be made to change from one state to another, and this, in its turn, results in a switch in the manifestation of the genes which are effective in mgn molecular . ~"-~-qc ~,ntigenic materials are of '-'-'~ ,^.._.._,:..~ . v . u u . , , ~ ; .L^ m~ type of antigen "~o., . . .m . . ~-1' weight and have protein components. Although in a particular organism only one antigen is found, a mixture of two can be detected during the temporary period of transformation from one serotype to another. From an operational point of view it would be difficult to envision how one part of a neuron might have surface properties different from that of another, if all of the genetic information were to come from chromosomally contained genes. If only one pertinent gene locus were expressing itself at a time, as in paramecia, the entire cell would be expected to reflect this activity and have the same surface properties. Also, it would be difficult to envision how the rapid chemical experiences of the change occurring during excitation of a synapse fa_r away from the cell nucleus m,.'ght result in a rapid effect on the genic expression in the nucleus and how the continuous changes could occur that would be

Brain Research, 2 (1966) 1I7--166

158

E. ROBERTS

necessary to accommodate such experiences. However, the difficulties in thinking about such matters have been eased somewhat by recent experiments with the alga, Chlarnydomonas reinhardP 4-96, which have provided 'evidence for the existence of a class of genetic determinants which are nonchromosomal in location, stable in replication and transmission whether expressed or not, represented by alternative allelic states in wild-type and mutant cells, and influencing a wide range of cellular traits'. It is not known for certain where t_hes,~ nonchromosomal genes are located in the cell and whether they are composed of DNA, RNA, or both. A re=ent study showed that in isolated chin: c:plasts of the above alga there is present extranuclear DNA with a base ratio distinctively different from that of the total DNA of the organismgL D N A also has been detected in chloroplasts and mitochondria of Swiss chard 5s, in the cytoplasm of root cells of Nicotiana tabaeum s°, in mitochondria of chick embryos s3, as well as in other types of cells (see Ref. 58 for references). Whether or not this latter type of DNA is related to the nonchromosomal genes remains to be determined. Recent experiments have introduced the possibility that hormonal control of genetic expression can take place. The insect molting hormone, ecdysone, can cause visible puffing of specific areas (genes) on chromosomes in Chironomus larvae within 30 rain after administration, presumably activating the participation of these genes in synthesis of specific messenger RNA 56. Ecdysone treatment shifts the metabolism of tyrosine from pathways which convert it largely to p-hydroxyphenylacetic acid to those forming L~PA, dopanine, and ,r,.r-acety!-_r,OPA. Many different lines of evidence now are suggesting that hormonal control of genetic expressivity is a widespread phenomenon. In view of the above data it is not unreasonable now to propose that extranuclear genetic material (DNA and/or RNA) may be present at or close to the surfaces of all parts of neurons and that impingement of a variety of environmental influences might be able to turn these genes on and off. One cannot help but wonder whether the sympathetic nerve-growth factor discus.,ed previouslyla,Z0, 66-6a, and various other substances carried in the circulation of higher animals, may be acting as repressors and derepressors in nervous tissue on both chromosomal and nonchromosomal genetic material. In considering potential stable sources of information in living cells one cannot stop even with DNA and RNA. The possibility exists that phosphoiipoprotein aggregates, such as exist in membranes of neurons and other cells, may have roles in replication of cell properties. Support for the latter point of view comes fi'om work in experimental embryology. A region of the cell cortex of fertilized eggs of the South African clawed toad Xenopus laevis, the grey crescent, contains the information required for the formation of the future dorsal lip, which is all-important in gastrulatier and is the source of chemical inducers which lead to formation of a nervous system 23. Results of a series of ingenious grafting experiments 2~ led to the suggestion that 'the cortex carries a remarkable amount of the information required for building an amphibian embryo. The cortex provides a large amount of the spatial information required to determine where each part of the embryo forms, 'mapping it out', as it were . . . . The mechanism of cell division and the control of cell movement appear to be related in Dart to cortical urot~erties'. Since in some species the cell cortex of -

Brain Research, 2 (1966) 117-166

159

THE SYNAPSE AS A MICRO-CYBERNETIC UNIT

fertilized eggs consists of barely more than cell membrane material, it is suggested that the membrane, itself, may be the region wherein these special cortical properties reside. Since the cortex of Xenopus laevis embryos was found to have no nucleic acid, it also was suggested that the phospholipoprotein of the cell membrane might be carrying the cortical information. Preliminary results are cited which are in support of the possibility that this cortical region, from which the sex cells arise and which is divided between the two daughter cells at the first division, may represent a selfreplicating system. Defects have been found in eggs of normal-appearing animals which had arisen from eggs in which small defects were made in the grey crescent region. It still is not clear whether the cortical area involved produces effects on genes which regulate the building of a new cortex or whether independent replication takes place. IV. Does R N A code neural events at a molecular level?

In this section I will deal only with those experiments on RNA metabolism and function in neural tissue that might indicate whether or not this class of substances plays some specific role in the coding of neural events at a molecular level. The important question to be answered is whether or not, in the light of data presently available, it is necessary to assume that stimuli that affect neuronal and giiai elements must somehow leave permanent imprints in such a manner as to lead to the synthesis of messenger RNAs which are uniquely, different from those present. __~ .... at the..._ tima.. _ hr...stimulation i.,4.,aa,w. i,..wl.! from Lth..o~ ZL*J01~, ~TZltl~..,llt will be synthesized in response "" L ~ ' r,., I L J . LL,g.I.I,,, l . l | l t i l . t l | , C.lb.tL~J. I.'1.1 ~.o_ Ulh, ~ . a Lt,~t~,~ .~,h;oh . . . . .; ~,;.~,,1; .~.,a ,.. lieve that these unique RNAs will, in turn, guide the synthesis of new and uo_ique proteins. If the evidence for the latter point of view is not compelling, it would appear to be sufficient to adhere to the point of view for which there is biological precedent, viz. that the total populations of messenger RNAs in a cell and the quantitative distribution of the individual components therein reflect at any time the interaction of the genetic expressivity of a cell with a variety of intracellular and extracellular epigenetic influences, and that at all times in a normal cell the messenger RNAs carry only that information which is present in the genome. Elegant analytical studies dealing with RNA in neural tissue of animals in a .... •". . . . . ~ by Hyd6n and ~--'---:~' r gynazr", ralu~ulially learning situation have been oermrmeo n__.:_..,^_,.. per.

.

.

.

.

.

tinent to the present discussion were the findings that the total RNA was increased in cells of the vestibular nucleus of rats trained to obtain food while balancing on a steel wire. This increase over normal values was similar to that observed in rats stimulated ...... by rotation, but the ..-~:.~,~n,.,.,~-.n~mam ~ • -,- ~.uweu _L . . . . a change in base ratios in their nuclear BkIA lXt'qzTL.

...k:l. VVllg.i~

4.1. . . . . . . :.._1 .... :---.1_~.--.-i Ibill.Clm, [ . J g l t ~ a i ¥ ~ . , l . y ~tlblll|lJ.if.I.L1h~l.

__:__--1~ KLiiiiii(J,i~.,~

..1.'..1__ _ _ a . Th~ Ik.lltJ III..JL.

~l li~i ~; ~i. ,-.- L. .

111"** 1 .D.~. . I.~. . . . . l~l.111.)br-

Wi;lb

such as to suggest that more synthesis of chromosomal R N A was occurring in the nuclei of Deiters' cells in the trained rats than in the others. These observations may somehow reflect the fact that in the trained animals previously inactive neural connections were being activated and their connectivities strengthened, while in the passively stimulated animals ov,!y a reinforcement of already well-established path,.,.~jo

,,~

. . . . .

i,.,~.~

-V.tt~,'~.

r~'itllUU~ll

tlfg"

i~tttCl'l'ifl"ldl|l~b

i::ll'C

Of

gt~al i,teic~t

I i~ilU

,I ° tlll~ll

Brain Research, 2 (1966) 117-166

160

E. ROBERTS

significance merits detailed elucidation, they cannot be adduced as evidence for the formation of qualitatively different or uniquely new R N A molecules in the learning situation. Indeed, Hyd6n now appears to have adopted this view and believes as a result of recent studies 5s that" 'An acute learning situation may select parts o[ the genome which become activated. The primary gene products, in the case analyzed, adenosine-uracil-rich RNA, are followed by a ribosomal type of RNA which takes over the long-term synthesis of proteins necessary to sustain the neural function of the new behavior.' Also relevant to the present discussion are the experiments performed on learning in planaria 11,54. Two types of learning in planaria, light-shock conditioning 7a and habituation 11s to light have been shown to be transferred by cannibalization. The most direct experiments performed to date were those in which RNAs isolated from planaria which had been brought to criterion by a light-shock conditioning procedure or prep~ red from control worms were injected into the guts of two groups of untrained planariallL The animals given the R N A from the conditioned animals appeared to respond with turns and contractions more to the conditioned stimulus (light) than those which received the RNA from the unconditioned worms. These latter results and those on cannibalization should be considered in the light of the biology of the planarian 14. The wall of the gut, a sac with only one opening, is lined by large unciliated cells which engulf food particles and appear to degrade them in vacuoles. It is quite possible that at least a fraction of a population of macromolecules such as R N A and protein, particularly if isolated in undegraded form from planaria, could escape complete degradation in the planaria and could be reutilized as such. Between the ectoderm and the lining of the gut lies a mass of parenchymatous tissue which fills the interior of the body. The parenchyma, which has star-shaped cells with long irregular processes and much intercellular space, probably is involved in transport of materials within the organism and is the source of the undifferentiated cells used in repair of lost parts of the body. The central nervous system consists of a pair of cerebral ganglia and there is a nervous network throughout the body which consists of nerve cells and nerve fibers. The central nervous system appears to operate as a relay station for the stimuli from the special sense organs (eyes, rheotactic sensory and chemo-sensory cells, etc.) in which the stimuli are 'reinforced, often extended in time, and ,h . . . . . . . a +,. ,h . . . . . . . . . ,14, The nerve n,+ ~+~f ~o r,.~n,n~ir,1,, for th,~ r . n r r ~ la~icr~ of activity of the different p~rts of the musculature. It may be assumed that in the light-shock conditioning experiments mentioned above there were increases in connectivity in the central ganglia between the cells bringing signals from the visual system and those detecting the effects of passage of electric current, and that there also was an increase in connectivity between those neural elements which 'sense' the current and those neurons in the nerve net which regulate the activity of the musculature. The net result in the conditioned animals would be an increase in connectivity between the visual system and the musculature. It was suggested in a preceding section of this paper that one of the aspects of increase in connectivity at synapses with use might be a progressively increasing similarity of surfaces (and. therefore, adhesiveness) of elements in a synapse (pre- and postsynaptic endings and glial and endothelial LII~II

IJl[:l,~.,1~Ib, lki. LI.JF

I, l l ~

11~¢1

Vile

Brain Research, 2 (1966) 117-!66

lll[llCL

,

11~!.,

11~11~1¢11,

IO

l~l..~l.fVll~,.~llkJl~

m,l~

~ v t t ~

THE SYNAPSE AS A MICRO-CYBERNETIC UNIT

161

cells). This could result from an interchange between these elements of informationbearing molecules, possibly RNA, which would tend to increase the similarity in genetic expressivity of the cellular elements participating in the interchange. Because of the unique plasticity of the structure of the planarian it is possible to conceive that such molecules could be brought by pinocytotic mechanisms into areas of synapses with a low degree of connectivity and in a passive manner produce an increase in connectivity in these synapses by enhancing the degree of similarity of genetic expressivity in the contiguous cellular elements. This could account for the enhanced responsiveness of untrained planaria to light after receiving RNA prepared from conditioned animals. A recent failure to repeat the above experiments 9 does not necessarily invalidate the previously reported results. However, it does emphasize the inadequate state of the knowledge of all of the important variables in behavioral experiments with planaria and suggests that further extensive biological and biochemical studies with these organisms will be required before far~reaching conclusions can be reached with them about the biochemical correlates of learning. The data on planaria, like those on changes in RNA in learning in rats, create no necessity at ~P~e 7 ; r e ~ time either to postulate or to look for subtle unique molecular differenc~ between 'trained' or 'untrained' RNA. The problems appear to be only the general ones dealing with the nature of molecular coding in nucleic acids and the factors which control in cells the expression of their genetic potential. A recent report has appeared of the significant transfer of approach training in rats by intraperitoneal injection of RNA isolated from the brains of trained rats, but not by that from naive rats 5. The clarification of the meaning of this observation is awaited with interest. ACKNOWLEDGEMENTS

This investigation was supported in part by funds from the Max C. Fleischmann Foundation of Nevada, a grant from the National Association for Mental Health, and grant NB-01615 from the National Institute of Neurological Diseases and Blindness, National Institutes of Health. REFERENCES l ALTMAN, J., Differences in the utilization of tritiated leucine by single neurones in normal and exercfsed rats: an autoradiographic investigation with microdensitometry, Nature (Lond.), 199 (1963) 777-780. 2 ALTMAN,J., AND DAS, D. G., Autoradiographic study of the effects of enriched environment on the rate of g!ia! mu!tip!i¢-ation in the adult rat brain, Nature (Lond.), 204 (1964) 1161-1163. 3 AMOS, H., AND MOORE, M. O., Influence of bacterial ribonucleic acid-induced changes in mammalian cells, Proc. nat. Acad. Sci. (Wash.), 47 (1961) 1689-1700. 4 ARORA, H. L., AND SPERRY, R. W., Color discrimination after optic nerve regeneration in the fish Astronotus ocellatus, Develop. Biol., 7 (~963) 234-243. 5 BABICH, F. R., JACOBSON, A. L., BUBASH, S., AND 2ACOBSON,A., Transfer of a response to naive rats by injection of ribonucleic acid extracted from trained rats, Science, 149 (1965) 656-657. 6 BAXTFR, C. F., Personal communicate.on. In preparation. 7

B~.AL~,

G

•

H.,

. . . .

ar~L,

vv,L~J,,~aur~,

J.

•.,

~,,,,~,-,,,,-

,-,,,,

a.i

,.,,.a~,At

a

. . . . . . . . . . . . .

~ ..........

" ...........

Microbiol., 15 (1961) 263-296. Brain Research, 2 (1966) 117-166

162

E. ROBERTS

8 BELL, C., SIERRA, G., BUENDIA, N., AND SmUNDO, J. P., Sensory properties of neurons in the mesencephalic reticular formation, J. Neurophysiol., 27 (1964) 961-987. 9 BENNErr, E. L., AND CALVIN, M., Failure to train planarians reliably, Neurosciences Research Program Bulletin, Voi. 2, No. 4 (1964) 3-24. 10 BENNETT,E. L., DIAMOND, M. C., KRECH, D., AND ROSENZWEIG, M R., Chemical and anatomical plasticity of brain, Science, 146 0964) 610-619. I l BEST, J. B., Protopsychology, Sci. American, 208 (1963) 55-62. 12 BIRKS, R. I., The role of sodium ions in the metabolism of acetylcholine, Canad. J. Biochem., 41 (1963) 2573-2597. 13 BIRKS, R. I., AND MAC INTOSH, F. C., Acetylcholine metabolism of a sympathetic ganglion, Canad. J. Biochem., 39 (1961) 787-827. 14 BORRADAILE. L. A., EASTHAM, L. E. S., POTTS, F. A., AND SAUNDERS, J. T., The b~vertebrata, Cambridge University Press, London, 195!, pp. 198-213. 15 BUNGE, R, P., BUNGE, M. R., AND PETERSON, E. R., An electron microscope o,,...,o*,,avof cultured rat spinal cord, J. Cell Biol., 24 (1965) 163-191. 16 BUTCHER, R. W., AND SUTHERLAND, E. W., Adenosine-Y,5"-phosphate in biological material. I. Purification and properties of cyclic-3',5'-nucleotide phosphodiesterase and use of this enzyme to characterize adenosine-Y,5'-phosphate in human urine, J. Biol. Chem., 237 (1962,~ 1244-1250. 17 CAMERON, D. E., The processes of remembering, Brit. J. Psychiat., 10) (1963) 325-340. 18 CARAMIA,F., ANGELETH, P. U., AND LEvI-MONTALCIM, R., Experimental analyses of the mouse submaxillary salivary gland in relationship to its nerve-growth factor content, EndocrinMogy. 70 (1962) 915-922. 19 COGGESHALL, R. E., AND FAWCETT, O. W., The fine structure of the central nervous system of the leech Hirudo medicinalis, J. Neurophysioi., 27 (1964) 229-289. 20 COHEN, S., Purification of a nerve-growth-promoting protein from the mouse salivary gland and its neuro-cytotoxic antiserum, Proc. nat. Acad. Sci. (Wash.), 46 (1960) 302-311. 21 CRAIN, S. M., AND PEARSON, E. R.. Complex bioelectric activity in organized tissue cultures of spinal cord (human, rat and chick), J. cell comp. Physiol., 64 0964) 1-14. 22 CURTIS, A. S. G., Ceil contact and adhesion, Biol. Rev., 37 (i962) 82-i29. 23 C',R_TIS, A. S. G., Morphogenetic interactions before gastrulation in the amphibian, Xenopus laevis--the cortical field, J. Embryol. exp. Morph., i0 (1962) 410--422. 24 CURTIS, A. S. G., The cell cortex, Endcavour, 22 0963) 134--137. 25 DEUTSCH,J. A., Higher nervous function" the physiological bases of memory, Ann. Rev. Physiol., 24 0962) 259-286. 26 DIAMOND, M. C., KRECH, D., AND ROSENZWEIG, M. R., The effects of an enriched environment on the histology of the rat cerebral cortex, J. comp. Neurol., 123 (1964) I I l-! 19. 27 DIAMOND, M. C., KRECH, D., ROSENZWEIG, M. R., BENNETT, E. L., RHODES, H., LINDNEe; B., AND LAW, F., The anatomical effects of an enriched environment on the rat cerebral cortex. Methods and some preliminary results, Brain Chemistry and Behavior Research Project Newsletter, No. 1!, Feb. 19, 1965, Univ. of Calif., Berkeley. 28 DINGMAN, W., AND SPORN, M. B., The incorporation of 8-azaguanine into rat brain R N A and its effect on maze-learning by the rat: an inquiry into the biochemical basis of memory, J. psychiat. Res., ! (!96!) 1-!!. 29 DINGMAN, W., AND SPORN, M. B., Molecular theories of memory, Science, 144 (1964) 26-29. 30 DUDEL, J., AND KOFELER, S. W., Presynaptic inhibition at the crayfish neuromuscular junction, d. Physiol. (Lond.), 155 (1961) 543-562. 31 ECCLES,J. C., The Physiology of Synapses, Academic Press, New York, 1964. 32 EDSTROM, J. E., EICHNER, D., AND EDSTROM, A., The ribonucleic acid of axons and myelin sheaths from Mauthner neurons, Biochim. biophys. Acta, 61 (1962) 178-184. 33 EDWARDS,C., Physiology and pharmacology of the crayfish stretch receptor. In E. ROBERT~e, C. F. BAXTER, A. VAN HARREVELD, C. A. G. WiERSMA, W. R. ADEY AND K. F. K!LLAM (Eds.), l n h i b i tioga, i'a .'.he Nervo, t System and Gamma-Aminobutyric Acid, Pergamon Press, Oxford, 1960, pp. 386-408. 34 EHRET,C. F., AND POWERS, E. L., The cell surface of Paramechtm, Int. Rev. CytoL, 8(i ~'~'~'a'~ ! "~ 35 r:,~ ,,,~ r , L.,,E:._TL,N, E. M., AND COHEN, M. J., Learning in an isolated prothoracic insect ganglion, Anita. Behav., 13 (1965) 104-108. 36 EMMELOT, P., Bos, C. J., BENEDE'rTI, E. L., AND ROMKE, PH., Studies on plasma membranes. 1. Chemical composition and enzyme content of plasma membranes isolated from rat liver, ........... t - : . . t , , , ~ A,~ta. 90 (1964) 126-145. J ~ l l . l ~ . l l l l t l l

t l ~ o l . . . .

J

~

.

_

.

.

.

•

Brain Research, 2 (1966) 117-166

.

THE SYNAPSE A.S A MICRO-CYBERNETIC UNIT

163

37 EssIG, C. F., S nticonvulsant effect of aminooxyacetic acid during barbiturate withdrawal in the dog, Intern J. Neuropharmacol., 2 (1963) 199-204. 38 FLEXNER,J. B., FLEXNER,L. B., STELLAR,E. DE LA HABA,G., AND ROBERTS,R. B., Inhibition of protein synthesis in brain and learning and memory following puromycin, J. Neurochem., 9 (1962) 595-605. 39 FRANK, K., AND FUORTES, M. G. F., Excitation and conduction, Ann. Rev. Physiol., 23 (1961) 357-386. 40 FRIEDE, R. L., AND VAN HOUTEN, W. H., Neuronal extension and glial supply: functional significance of gila, Proc. nat. Acad. Sci. (Wash.), 48 (1962) 817-821. 41 FRlSCH,L., (Editor), Basic Mechanisms in Animal Viru:~.Biology, Cold Spring Harbor Symposia on Quantitative Biology, Voi. 27, Long Island Biological Association, 1962, 535 pp. 42 GEIGER, A., AND YAMASAKI,S., Cytidine and uridine requirement of the brain, J. Neurochem., 1 (i956) 93-100. 43 GLICKMAN,S. E., Perseverative neural processes and consolidation of the memory trace, Psychol. Bull., 58 (1961) 218-233. 44 HAMLYN,L. H., The fine structure of the mossy fibre endings in the hippocampus of the rabbit, J. Anat. (Lond.), 96 (1962) 112-120. 45 HEnri, D. O., The Organization of Behavior~ John Wiley, New York, 1949. 46 HERIOT, J. T., AND COLEMAN, P. D., The effect of electroconvulsive shock on retention of a modified 'one-trial' conditioned avoidance, J. comp. physiol. Psychol., 55 (1962) 10821084. 47 HORRIDGE,G. A., Learning of leg position by the ventral nerve cord in headless insects, Proc. roy. Soc. B, 157 (!962) 33-52. 48 HUBBARD, J. I., Repetitive stimulation at the mammalian neuromuscular junction, and the mobilization of transmitter, J. Physiol. (Lond.), 169 (1963) 641-662. 49 HUBBARD,J. I., AND SCHMIDT, R. F., An electrophysiolog.;cal investigation of mammalian motor nerve terminals, J. Physiol. (Lond.), 166 (1963) 145-167. 50 HYD~N, H., The neuron and its gliawa biochemical and functional unit, Endeavour, 21 (1962) 144-155. 51 HYDfi-N,H., AND EGYHAZI,E., Nuclear RNA changes of nerve cells during a learning experiment in rats, Proc. nat. Acad. Sci. (Wash.), 48 (1962) 1366-1373. 52 HYDi~N, H., AND LANGE, P. W., Rhythmic enzyme changes in neurons and gila during slcx:p, Science, 149 (1965) 654-656. 53 HYDI~N, H., AND LANGE, P. W., A differentiation in RNA response in neurons early and late during learning, Proc. nat. Acad. Sci. (Wash.), 53 (1965) ~46-952 54 JACOBSON,A. L., Learning in flatworms and annelids, P¢'chol. Bull.., 60 (1963) 74-94. 55 KAKIUCHI,S., AND RALL, T. W., Effects of noreT.irJephrine and histamine on levels of adenosine3',5'-phosphate (3,5-AMP) in brain ~lices, Fed. Proc., 24 (1965) 150. 56 KARLSON, P., Cher, fic~i and immunological aspects of hormones. On the chemistry and mode of action of insect hormones. Gen. comp. Endocr., Suppl. 1 (1962) 1-7. 57 KIMBLE,D. P. (Editor, Learning, Remembering, and Forgetting. Vol. 1: The Anatomy o f Memor.v, Science and Behavior Books, Inc., Palo Alto, 1965, 451 pp. The participants in this conference to whose contributions reference will be made are: J. V. MCCONNELL, E. R. JOHN, A. M. UTTLEY, H. YON FOERSTER,S. LEVINE. 58 KISLEV,N., SWIFT,H., AND BOGORAD, L., Nucleic acids of chloroplasts and mitochondria in Swiss chard, J. Cell Biol., 25 (1965) 327-344. 59 KLAINER,L. M., CHI, Y.-~'[., FREIBERG,S. L., RALL,T. W., ANDSUTHERLAND,E. W., Adenyl cyclase. IV. The effects of neurohormones on the formation of adenosine-3',5'..phosphate by preparations from brain and other tissues. J. Biol. Chem., 237 (1962) 1239-1243. 60 KRNJEVIC, K., Micro-iontophoretic studies on cortical neurons, Int. Rev. Neurobioi., 7 (1964) 41-98. 61 KUFFLER, S. W., AND POTTER, D. D., Gila in the leech central nervous system: physiological properties and neuron-gila relationship, J. Neurophysioi., 27 (1964) 290-320. 62 KURIYAMA, K., KAKEFUDA,T.~ AND ROBERTS, E., Unpublished observations. In preparation. 63 KURIYAMA,K., ROBERTS,E., AND RUBINSTEIN,M. K., Elevation of ~,-aminobutyric acid in brain -..:.~. ,,,,,;,,~,~,~,,~,.,.t;,. -.~:-, and susceptibility to convulsive seizures in mice. A quantitative reevaluation, Biochem. Pharmacol., In Press. 64 KUROKAWA, M., KATO, M., AND MACHIYAMA,Y., Choline acetylase activity in a convulsive strain of m3use, Biochim. biophys. Acta, 50 (1961) 385-386. Brai,t Research, 2 (1966) 117-166

164

E. ROBERTS

65 KUROKAWA, 1~t., MACHIYAMA, Y., AND KATO, M., Distribution of acetylcholine in the brain during various states of activity, J. Neurochem., 10 (1963) 341-348. 66 LEvI-MONTALON], R., ANt) ANOELETrl, P. U., Essential role of the nerve growth factor in the survival and maintenance of dissociated sensory and sympathetic embryonic nerve cells in vitro, Develop. Biol., 7 (1963) 653-659. 67 LEvI-MONTALCINI,R., AND BOOKER, B., Excessive growth of the sympathetic ganglia evoked by a protein isolated from mouse salivary glands, Proc. nat. Acad. Sci. (Wash.), 46 (1960) 373-384. 68 LEvI-MoNTALCIN.~,R., AND BOOKER, B., Destruction of the sympathetic ganglia in mammals by an antiserum to a nerve-growth protein, Proc. nat. Acad. Sci. (W.~h.), 46 11960) 384-391. 69 MADSEN, M. C., AND McGAUGH, J. L., The effect of ECS on one-trial avoidance learning, J. comp. physiol. PsychoL, 54 (1961) 522-523. 70 MANSOUR,T. E., Effect of serotonin on glycolysis in homogenates from the liver fluke Fasciola hepatica, J. Pitarmacol. exp. Ther., 135 (1962) 94-101. 71 MANSOUR,T. E., Studies on heart phosphofructokinase: purification, inhibition, and activation, J. Biol. Chem., 238 (1963) 2285-2292. 72 MANSOUR,T. E., AND MANSOUR, J. M., Effects of serotonin (5-hydroxytryptamine) and adenosine-3',5'-phosphate on phosphofructokinase from the liver fluke Fasciola hepatica, J. Biol. Chem., 237 (1962) 629-634. 73 McCONNELL, J. V., Memory transfer through cannibalism in planarians. J. Neuropsychiat., Suppl. 1, 3 (1962) 342-548. 74 MCGAUGa, J. L., Facilitative and disruptive effects of strychnine sulphate on maze learning, Psychol. Rev., 8 (1961) 99-104. 75 MCGAUGH, J. L., TaOMSON, C. W., WESTaROOK, W. H., AND HUDSPETH, W. J., A further study of learning facilitation with strychnine sulphate, Psychopharmacologia (Berl.), 3 (| 962) 352-360. "]6 McGAUGH, J. L., WESTBROOK, W., AND BURT, G., Strain differences in the facilitative effects of 5-7-diphenyl-l-3-diazadamantan-6-oL (17571.S.) on maze learning, J. comp. physiol. Psychoi., 54 (1961) 502-505. 77 MCLENNAN, H., AND ELLIOTT, K. A. C., Effects of convulsant and narcotic drugs on acetyicholine synthesis, J. Pharmacol., 103 (1951) 35-43. 78 MORRELL, F., Electrophysiological contributions to the neural basis of learning, Physiol. Rev., ~1 (1961) 443--494. 79 MOggELL, F., Lasting changes in synaptic organization produced by continuous neuronal bombardment. In J. F. DELAFRESNAYE(Ed.), Brain Mechanisms and Learning, Blackwell, Oxford, 1961, pp. 375-392. 80 MOURAD, E. B., Evidence for cytoplasmic DNA in root cells of Nicotiana, J. Cell Biol., 24 (1965) 267-276. 81 MURAD, F., CHI, Y.-M., RALL, T. W., .eND SUTHERLAND, E. W., Adenyl cyclase. III. The effect of catecholamines and choline esters o;, the formation of adenosine-3',5'-phosphate by preparations from cardiac muscle and liver, " Biol. Chem., 237 (1962) 1233-1238. 82 NARUSE, H., KATO, M., KUROKAWA, M., HABA, R., AND YABE, T., Metabolic defects in a convulsive strain of mouse, J. Neurochem., 5 (1960) 359-369. 83 NASS, S., AND NASS, M., Intramitochondrial fibers with D N A characteristics. II. Enzymatic and other hydrolytic treatments, J. Cell Biol., i9 (1963) 613-629. O,4 O~1" I'~IIUI"IULL~,J. iJ.,l" Ar~lJ lxul-rLIzl% S. Uvv~.,r:xtra~;vnumr" . . . . . . . ' ~ " - - space as a p a t h w a y for cx~;nange ot:twecn blood and neurons in the central nervous system of the leech: ionic composition of g!ia! cells and neurons, J. Neurophysiol., 27 (1964) 645-671. 85 NICHOLLS, J. G., AND KUFFLER, S. W., Na and K content of glial cells and neurons determined by flame photometry in the central nervous system of the l~ch, J. NeurophysioL, 28 (1965) 519525~ 86 PASSONNEAU,J. V., AND LOWRY, O. H., Phosphofructokinase and the Pasteur effect, Biochem. biophys. Res. Commun., 7 (1962) i0-i5. 87 PETERS6.'.'. E. R., CRA~N, S. M., AND MURRAY, M. R., Differentiation and prolonged maintenance of bioele~.tr'.'¢aily active spinal cord cultures (rat, chick and human), Z. Zellforsch., 66 (1965) 130--154. 88 PURPURA, D. P., SCHOFER, R. J., HOUSEPIAN, E M.. AND NOBACK, C. R., Electron microscopy of immature human and feline neocortex. In D. P. PURPURA Arid J. P. Sc'I4AD~ (Eds.), Growth Maturation of the Brain, Progress in Brain Research, Volume 4, Elsevier, Amsterdam, 1964, pp. 176-186. 89 PURPURA, D. P., SCHOFER, R. J., HOUSEPIAN, E. M., AND NOBACK, C. R., Compara,ive onto"liLT

.

.

.

.

.

.

.

.

.

.

.

.

~

.

.

Brain Research, 2 (1966) 117-166

.

.

.

.

.

.

.

~

.

.

.

.

.

.

.

.

.

.

t

L... -- ~ . . . . . .

THE SYNAPSE AS A MICRO-CYBERNETIC UNIT

165

genesis of structure-function relations in cerebral and cerebellar cortex. In D. P. PURPURAAND J. P. SCnAD~ (Eds.), Growth Maturation of the Brain, Progress in Brain Research, Volume 4, Elsevier, Amsterdam, 1964, pp. 187-221. 90 RaLE¢, M., AND PAgDE~, A. B., Gene expression: its specificity and regulation, Ann. Rev. Microbiol., 16 (1962) 1-34. 91 ROBERTS,E., The synapse as a cybernetic unit: a biochemist's fantasy, Neurosciences Research Program Bulletin, Volume 2, No. 1. 92 Ros~gTS, E., AND EIDELnEgG, E., Metabolic and neurophysiological roles of ~,-aminobutyric acid, Int. Rev. Neurobiol., 2 (1960) 279-332. 93 RoaFaTS, E., AND SANO, K., The specific occurrence of the 7-aminobutyric acid system in nervous tissue of animals. In D. MAZIA AND A. TYLER (Eds.), General Physiology of Cell Specialization, McGraw-Hill, New York, 1963, pp. 323-348. 94 SAGER,R., Streptomycin as a mutagen for nonchromosomal genes, Proc. nat. Acad. Sci. (Wash.), 48 (1962) 2018-2026. 95 SAGER, R., AND ISHIDA, M. R., Chloroplast DNA in Chlamydomonas, Proc. nat. Acad. ScL (Wash.), 50 (1963) 725-730. 96 SAGER, R., AND RAMANiS,Z., The particulate nature of nonchromosomal genes in Chlamydomonas, Proc. nat. Acad. Sci. (Wash.), 50 (1963) 260-266. 97 SCHAD~,J. P., VAN BACKER, H., AND COLON, E., Quantitative analysis of neuronal parameters in the maturing cerebral cortex. In D. P. PURPURA AND J. P. SCHAD~(Eds.), Growth and Maturation of the Brain, Progress in Brain Research, Volume 4, Elsevier, Amsterdam, 1964, pp. 150-175. 98 SCHWEET, R., BISHOP, J., AND MORRIS, A., Protein synthesis with particular reference to hemoglobin synthesis--a review. Lab. Invest., 10 (1961) 992-1011. 99 SHANES,A. M., Electrochemical aspects of physiological and pharmacological action m excitable cells, PharmacoL Rev., 10 (1958) 59-164. 100 SOKOLOFF, L., Local cerebral circulation at rest and during altered cerebral activity induced by anesthesia or visual stimulation. In S. S. KETY AND J. ELKES (Eds.), Regional Neurochemistry, Pergamon Press, Oxford, 1961, pp. 107-117. 101 SO~NEaORN, T. M., Developmental mechanisms in Paramecium, Growth Symp., 11 (1947) 291-307. 102 SO~EBORN, T. M., Genes, cytoplasm, and environment in Paramecium, Sci. Monthly, 67 (1948) 154-160. 103 SONNEaORN, T. M., AND LESUER, A., Antigenic characters in Paramecium aurelia (variety 4): determination, inheritance and induced mutations, Amer. Naturalist, 82 (1948) 69-78. 104 SPAZtANI, E., AND SZEGO, C. M., The influence of estradiol and cortisol on uterine histamine of the ovariectomized rat, Endocrinology, 63 (1958) 669-678. 105 SPAZIANI, E., AND SZEGO,C. M., Further evidence for mediation by histamine of estrogenic stimalation of the rat uterus, F.~docrinology, 64 0959)713-723. 106 SPEaRY, R. W., Regulative factors in the orderly growth of neural circuits, Growth, Suppl. to Vol. 15, 15 (1951) 63-87. 107 SPERRY, R. W., Physiological plasticity and brain circuit theory. In H. F. HARLOWAND C. N. WOOLSEY(Eds.), Biological and Biochemical Bases of Behavior, Univ. of Wisconsin Press, Madison, 1958, pp. 401-424. 108 STAMM,J. S., AND PgmRAM, K. H., Effects of epileptogenic lesions in frontal cortex on learning and retention in monkeys, J. NeurophysioL, 23 (1960) 552-563. 109 SrAMM, J. S., AND PRIaRAM, K. H., Effects of epileptogenic lesions in inferotemporal cortex on learning and retention in monkeys, J. romp. physiol. Psychol., 54 (1961) 614-618. 110 STAMM,J. S., AND WARgEN, A., Learning and retention by monkeys with epileptogenic implants in posterior parietal cortex, Epilepsia (Amst.), 2 (1961) 229-242. 111 SUTHEgLAND, E. W., AND RALI., T. W., The relation of adenosine-Y,5'-phosphate and phosphoryiase to the actions of catechoiamines and other hormones, Pharmacoi. Rev., i2 (i960) 265-299. 112 StJTHERLAND,E. W., A~D RALL, T. W., Adenyl cyclase. II. The enzymatically catalyzed formation of adenosine-3',5'-phosphate and inorganic pyrophosphate from adenosine triphosphate, J. BioL Chem., 237 (1962) 1228-1232. 113 StrrHERLAND,E. W., RALL, T. W., AND MENON, T., Adenyl cyclase. I. Distribution, preparation, ~nd nronerties. J. Biol. Chem. 227 (1962) 1220-1227. 114 SzEGo, (2. M., AND SLOAN,S. H., The influence of histamine and serotonin in promoting early uterine growth in the rat, Gen. romp. Endocr., 1 (1961) 295-305.

Brain Research, 2 (1966) 117-166

166

E. ROBERTS

115 VANDENHEUVEL, F. A., Studies of biological structure at the molecular level with stereomodel projections. I. The lipids in the myelin sheath of nerve, J. Amer. Od Chemists" Soc., 40 (1963) 455-471. 116 VANDENHEUVEL,F. A., Study of biological structure at the molecular level with stereomodel projections. II. The structure of myelin in relation to other membrane systems, J. Amer. Oil Chemists" Soc., 42 (1965) 481-492. 117 VAN DER LOOS, H., Fine structure of synapses in the cerebral cortex, Z. Zellforsch., 60 (1963) 815-825. 118 WESTERMAN, R. A., Somatic inheritance of habituation of responses to light in planarians, Science, 140 0963) 676-677. 119 Z~LMAN, A., KABOT, L., JACOnSON, R., AND _MCCONNELL, J. V., Transfer of training through in i¢ction of"conditioned" RNA into untrained planarians, Worm Runner's Digest, 5 (1963) 1421.

Brain Research, 2 (1966) 117-166