Neural correlates of the jamming avoidance response (JAR) in the weakly electric fish Eigenmannia

442

the simplest of cell systems. For example, the mechanism by which retinoids bring about the differentiation of teratocarcinoma cells in culture is not understood. In several systems, retinoids have been found to lead to changes in gene expression. For example, an early event in the differentiation of a neuroblastoma cell line treated with retinoids is the switching off of the myc-oncogene 34. One possibility is that retinoids may act directly on gene expression, and cytoplasmic binding proteins have been isolated that could transport retinoids to the nucleus3. However, an obligatory role for such binding proteins in the action of retinoids is not proven. Many workers have, for example, stressed instead the effects of retinoids on the cell surface. Of potential significance here is that retinoids can affect glycosylation35. It therefore remains to be established whether the effects on gene expression are direct or secondary. Selected references

17,~( ~, - ( ) c t o b e r

21 Maden, M. (1983) Dev. Biol. 98, 409-416 22 Maden, M., Keeble, S. and Cox, R. A. (1985) Wilhelm Roztx Arch. Develop. Biol. t 94, 228-235 23 Edwards, M. K S. and McBurney, M. W. (1983) Dev. Biol. 98, 187-191 24 Lewis, J. H. and Wolpert, L. (1976) J. Theor. Biol. 62,479-490 25 Meinhardt, H. (1978) Rev. Physiol. Biochem. Pharmacol. 80, 47-56 26 Summerbell, D. and Harvey, F. (1983) Prog. Clin. Biol. Res. IIOA, 109-118 27 Maden, M. (1984) in Pattern Formation. A Primer in Developmental Biology (Malacinski, G. M. and Bryant, S. V., eds) pp. 539-557, Macmillan, New York 28 Jelinek, R. and Kistler, A. (1981) Teratolology 23, 191-195 29 Kochhar, D. M. (1977) Birth Defects Orig. Artic. Ser. 13, 11-154

1~;'85

30 Hassell, J, R , PennypackcL J P. and Lev, is. C. A. (1978) Exp. Cell Res. 112, 409-417 31 Kapron-Bras, C. MI and rFraslcr. 1 ) G (1984) Teratolology 30, 14~-15(} 32 Hassell, J. R.. Greenberg, J . H . and Johnson. M.C. ( 1 9 7 7 ) / Embt3,ol. Exit. Morph. 39, 267-271 33 Tamarin, A., Crawley, A,, Lee, J. and Tickle, C. (1984) J. Embryol. Exp. Morph. 84. 105-123 34 Thiele, C. J., Reynolds, C. P. and Israel, M. A. (1985) Nature (London) 3131 404-406 35 Roberts, A. M. and Sporn, M, B. (1984) in The Retinoids (Sporn, M. B , Roberts, A. B. and Goodman, D., eds) vol. 2, pp. 211-289, Academic Press, New York C. Tickle is at the Department of Anatomy and Biology, Middlesex Hospital Medical School, London W I P 6DP, UK.

cor NNu of avoKlance reeponse

R)In the

weakly Walter Helligenberg and Gary Rose

1 Wolbach, S. B. and Howe, P. R. (1925) J. Exp. Med. 42, 753-778 Fish of the genus Eigenmannia generate weak electric fields by discharging their 2 Fell, H. B. and Mellanby, E. (1953) electric organ at rather stable, although individually different frequencies between J. Physiol. (London) 119, 470--488 250 and 700 Hz. Electroreceptors on the body surface monitor local current flow; 3 Lotan, R. (1980) Biochem. Biophys. Acta and primary afferents relay this information to somatotopically ordered, 605, 33-91 4 Sporn, M. B. and Roberts, A . B . (1983) laminated structures in the brain. An object that differs electrically from the surrounding water distorts the animal's electric field, and the ensuing pattern of Cancer Res. 43, 3034-3040 5 Siden, N. (1982) J. Natl Cancer Inst. 68, 589- alterations of current flow on the fish's body surface represents the electric image 593 of the object. This form of 'seeing' with the body surface is called etectrolocation 6 Wolpert, L. (1969) J. Theor. Biol. 25, 1-47 and enables the animal to operate in the dark and in waters of poor visibility. The 7 Summerbell, D., Lewis, J. H. and Wolpert, electrolocation of objects is impeded when electric signals of animals with similar L. (1973) Nature (London) 244, 492-496 8 Saunders, J. W. and Gasseling, M. T. (1968) discharge frequencies interfere. By shifting its signal frequency away from that of in Epithelial-mesenchymal Interactions its neighbor and thus increasing the magnitude of the difference frequency to (Fleischmajer, R. and Billingham, R . E . , values beyond 10 Hz, a fish can avoid the detrimental effects of jamming. eds), pp. 78--97, Williams and Wilkins, Eigenmannia has been found to discriminate the sign of difference frequencies of Baltimore less than 0.1 Hz, resolve differences in the timing of stimulus events as small as 9 Tickle, C., Summerbell, D. and Wolpert, L. 500 ns, and detect interfering stimuli of less than one thousandth of its own field (1975) Nature (London) 254, 199-202 intensity. The Jamming Avoidance Response (JAR) has served as a model system 10 Javois, L. (1984) in Pattern Formation. A Primer in Developmental Biology and behavioral assay for the study of central nervous processing of sensory (Malacinski, G. M. and Bryant, S. V., eds) information, lntracellular and light- and electron-microscopical studies have pp. 557-581, Macmillan, New York related morphological and functional properties of neurons in laminated 11 Honig, L. (1981) Nature (London) 291, 72-73 structures that process spatial and temporal patterns of sensory information 12 Tickle, C., Alberts, B., Wolpert, L. and Lee, relevant for the JAR. J. (1982) Nature (London) 296, 564-565 13 Summerbell, D. (1983) J. Embryol. Exp. Morph. 78, 269-289 The electrosensory system is very allowing continual monitoring of an 14 Eichele, G., Tickle, C. and Alberts, B. M. suitable for the study of mechanisms of intact behavioral output while record(1984) Anal. Biochem. t42,542-555 perception. Natural stimulus patterns ing intracellularly from neurons. 15 Tickle, C., Lee, J. and Eichele, G. (1985) have been explored in great detail and By mimicking and systematically Dev. Biol. 109, 82-95 can readily be mimicked and modified modifying the stimulus regimen that 16 Tickle, C. (1981) Nature (London) 289, 295electronically. The electric sense elicits the JAR, relevant stimulus 298 17 Bryant, S. V., French, V. and Bryant, P. J. shows a relatively simple neuroana- variables and the abstract computa(1982) Science 212, 993-1002 tomical organization although it shares tional rules used in their central 18 French, V., Bryant, P. J. and Bryant, S. V. basic design principles with more evaluation have been determined5,6, (1976) Science 193, 969-990 highly evolved sensory systems. MoreEigenmannia produces a continual, 19 Niazi, I. A. and Saxena, S. (1978) Folia Biol. over, simple behavioral responses, nearly sinusoidal electric organ dis(Krakow) 26, 3-11 such as the JAR, still function in charge (EOD) with individually differ20 Maden, M. (1982) Nature (London) 295,672675 neurophysiological preparations, thus ent but relatively stable fundamental ~) 1985,ElsevierSciencePublishersB.V.. Amsterdam 0378 5912/85/$ff2(S)

TINS - October I985

443

frequencies in the range from 250 to 700 Hz. The electric organ is driven by a pacemaker in the medulla which triggers each discharge cycle with a single pulse’. The continual, nearly sinusoidal wave form of the EOD is characterized by its momentary peakto-peak amplitude, measured in mV cm-l, and by the point in time when it passes through zero, and hence referred to as ‘timing of zero-

crossing’ or ‘phase’. Both aspects of the EOD, amplitude and phase, are monitored by electroreceptors on the animal’s body surface. Objects that differ from water in their ohmic and/or capacitive properties alter the current flow of the EOD most significantly in the area of body surface closest to the object, and these alterations represent the electric image of the object. An object with higher

s’%

ohmic resistance, such as a rock, diminishes the local amplitude of the current, while an object with significant capacitive properties, such as living membranes of animals and plants, also shift the phase of the local current: in the absence of objects, the EOD current, measured between inside and outside the skin, passes the body surface synchronously; the zerocrossings of the EOD are synchronous

area

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Fig. la. The addition of two sine waves, S, mimicking the animal’s electric organ discharge and S,, of similarfrequency and smaller amplintde, mimicking the electric organ discharge of a neighbor. The center shows an oscillosope display triggered by SI so that S, appears stationary. Simultaneous display of the added signals, S, + S,, yields II nearly sinusoidal signal whose momentary amplitude, 1.71,and phase, H, relative to S, are modulated at the difference frequency, Df = fi - f,. As ISI and H are modulated jointly, the peak of S,+S, is seen to rotate around the stationary peak of S, as indicated by the dashed circle. The graph at the bottom shows o Lissajous Figure display of ISIversus H in a two-dimensional state plane. The nearly circular graph rotates in the counterclockwise senseforpositive Dfs and in the clockwise sensefor negative Dfs, and the rate of rotation is the absolute value of Df. The mean value of ISIis the amplitude of S, which is considered unity. The meon value of H is zero which corresponds to the timing of the positive zero-crossing of S,. A posih.ve H implies that S, + S, lags with respect to S,. 2n denotes the period of the SI cycle, which is 2 ma if f, ia 500 Hz. Fig. lb. Znformarion aboutphase con be obtained by comparing areas of body surface with different amplitude ratios behveen the interfering signals, S, and &. Let the animal’s electric organ discharge or its substitute, S,, in areas A and B be contaminated by the foreign signal, S,, by 30% and IO%, respectively. The stimulus modulationgraphs, lSjA versus HA and ISI, versus HB, have radii equal to the localnmplihrde ratios between S, and S, and rotate synchronously and in the counterclockwise sense for positive difference frequencies (Dfs). By replacing the local phase modulations, HA and H,, by the differential phase modulations, HA-Hs and HBHA, elliptical graphs with opposite senses of rotation are obtained. While area A thus decelerates the pacemaker, area B accelerates the pacemaker, but to a lesser extent due to the smaller amplitude modulation in B (Ref. 7). The net effect of thispnirwise interaction would thus still be a deceleration of the pacemaker, in accordance with the positive sign of the difference frequency (Of). Note that the animal has no access to either H, or HB alone, but that it can only evaluate differences between such phase values by comparing the arrival times of spikes from T-type receptors.

444 in all areas of the body surface. The appearance of a capacitor, however, will alter the timing of zero-crossings in the nearest area, and such phase shifts can be detected by comparison of the timing of zero-crossings between different areas of the body surface. As will be explained in connection with Fig. la, the amplitude and phase of the animal's E O D can also be altered by interference with the E O D of a neighbor, and interfering EODs may therefore obscure the electric images of objects 2. When exposed to electric signals with a frequency close to the frequency of its own E O D , Eigenmannia will shift its own frequency to increase the difference frequency, Df. This J A R serves to space out E O D frequencies of near neigbors and thus to protect electrolocation abilities2-4. Magnitudes of Df between 2 and 6 Hz are most detrimental to electrolocation and also elicit the strongest JARs. Since the fish promptly lowers its frequency in response to a signal of a slightly higher frequency and raises its frequency in response to a signal of lower frequency it must be able to determine the sign of the difference frequency, Df. Extensive behavioral studies 3,5,6 have demonstrated that the fish can detect the sign of Df on the basis of modulations of amplitude and phase which characterize the pattern of interference between the EODs. Although the E O D is not purely sinusoidal (the presence of higher harmonics causes characteristic distortions) it can, without consequences for the JAR, be experimentally replaced by a pure sine wave 5. By use of pure sinewaves (labelled $1 for the E O D of the subject animal and $2 for its neighbor) and an oscilloscope one can readily demonstrate interference patterns and cues for the determination of the sign of the difference frequency (Df = f 2 - fl, see Fig. la). Since electroreceptors are stimulated more strongly by the animal's own signal, the amplitude of $1 is chosen larger than that of $2. The electronic addition of S~ and $2 simulates the addition of the two electric currents in the water. If the frequencies of $1 and $2 are similar the interference signal, $1 + $2, is nearly sinusoidal, and its instantaneous amplitude, IsI, and the timing of its zerocrossing, H, with reference to the zerocrossing of $1 alone are modulated at the difference frequency, Df = f2 - fl. In the case of an acoustic signal we perceive the modulation of ISI as the

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"beat ~ while we are insensitive to the modulaion of H. If the oscilloscope is triggered by S~, the peak of the waveform of S1 + $2 is seen to rotate in a counterclockwise sense around the stationary peak of S~ if the value of Df is positive. The opposite sense of rotation is obtained for a negative Df. This rotation can also be displayed by a Lissajous' figure (with H, the abscissa and Isl, the ordinate; bottom of Fig. la). By reading the sense of rotation of this figure the fish can immediately determine the sign of Df and thus the direction in which it must shift its own frequency in order to increase the magnitude of the difference, Df. Reading of the phase modulation, however, requires a phase reference, and since the animal's signal is normally contam-

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inated in all areas of its body surface ~t has no access to a pure S~ for a zerophase reference. As demonstrated ir~ Fig. lb, however, the animal can still solve the problem by comparing areas of body surface which differ in the amplitude ratio of the two signals. The animal is unable to perform a JAR if the amplitude ratio between $1 and $2 is identical in all parts of the body surface so that differential phase modulations are absent 6. The JAR mechanism displayed in Fig. lb represents a distributed system for the control of the pacemaker frequency. The instructions that the neuronal representation of any small area of body surface must follow are these: a rise in amplitude paired with a phase tag or a fall in amplitude paired with a phase lead (both components of a

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Fig. 2. The synaptic organization of P- and T-type electroreceptive primary afferents m the electro senaory lateral line lobe ( E L L L ) , identified by Maler9. T-type afferents (filled circles symbolize their samara) form electrical synapses upon spherical cells (sph ), which in turn project to lamina 6 of the ton,s. In addiK~n, c ~ of T-afferents synapse on targets originally assumed to be solely ¢orrtacted by P-type aflferents. P~type aflferents (open circles) form excitatory chemic~ synap~.s upon granule cells (g) and upon basilar dendrites of basilar pyramidal cells (bp). Basilar pyramidal celia also recetve inhibitory input from more distant granule cells, which in turn are excited by P-inputs from the pertphery of the receptive field. The total P-type input to the basilar pyramidal cell thus appears to have an excitatory center and inhibitory surround organization. Nonbasilar pyramidal cells (nhp) on the other hand receive inhibitory input from nearest granule cells and electrical synapses from more distant granule cells, with the latter inputs being inhibited by more centrally located granule cells. The total Pinput to a nonbasilar pyramidal cell thus appears to have an inhibitory center and an excitatory surround organization. Both types of pyramidal celiaproject to laminae 3, 5, 7, and 8 of the torus. Within the dorsal and ventral molecalar layers (DML and VML), the dorsal dendrites of pyramidalcells are contacted by parallel and vertical (V) fibars which originate in the Iobus caudalis of the cerebellum and in the nucleus p r a e e m i n ~ i s and provide descending recurrent electrosensory inputs. Polymorphic cells (pal) receive P-afferent input and project to the contralateral E L L L. Their function is still unknown. A L L G is the anterior lateral line nerve ganglion which houses the samara of primary afferents

T I N S - October 1985

445

counterclockwise rotation according to the Lissajous' figure representation) should lower the frequency in proportion to the size of the amplitude modulation. The opposite combination of changes in amplitude and phase should raise the frequency. Any area of body surface can be chosen as a phase reference point, and several representations of the same area of body surface could indeed choose different references. It would then depend upon the orientation of the interfering $2 field as to which pairs of areas yield strong phase modulation and thus contribute significantly to the JAR, and blind pooling of inputs from the whole body surface should always yield a correct JAR.

inside and the outside of the skin). The timing of the T-type receptor spike thus reflects the phase of the local stimulus, and by evaluating the difference in the timing of these spikes from two areas of the body surface, the animal can thus determine the differential phase (such as HA-Ha in Fig. lb). The depth of the differential phase modulation decreases with the relative intensity of the interfering stimulus, $2. At an amplitude ratio between Sz and S1 of 1/3 and an S1 frequency of 300 Hz, the maximal depth of the phase modulation is approximately 150 Ixs. At a ratio of 1/1000, which approaches the threshold for eliciting JARs, the maximal phase modulation is approximately 0.5 p.s. If nerve conduction speed were 10 m s-l, the difference in the arrival time of Treceptor spikes from the head and from a part of the tail, 10 cm away from the head, would be 10 ms, and a conduction speed 10 times faster would still result in a phase offset of 1 ms. The comparison of timings of Treceptor spikes would therefore suffer from systematic errors if the animal did not compensate for differences in conduction time of spikes originating at different distances from the brain.

The coding of stimulus amplitude and phase by electroreceptors Two types of electroreceptors have been distinguished7, P-type and Ttype, that are suitable to code amplitude and phase, respectively. P-type receptors fire intermittently and raise their rate of firing as stimulus amplitude rises. T-type receptors fire one spike per EOD cycle that is phaselocked to the zero-crossing of the stimulus (as measured between the a

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Fig. 3a. Responses o f a small cell in lamina 6 of the torus to modulations o f differential phase between head and tail. Top: Representation of stimulas regime with definition o f differential phase, ~, and latency, o, o f neuronal response (resp. ). The phase, z, is modulated by a triangular wave form, at a rate of 2 Hz and with a peak-to-peak excursion between + O.75 ms. A positive T represents a phase lag o f the signal at the head with reference to the unmodulated signal at the tail. Bottom: The occurrence of spikes is plotted as a function o f t and o in the scatter diagram at the left. The diagonal line represents the timing, ~, o f the zerocrossing of S A with respect to the cycle o f S a. The histogram to the right gives the rate o f firing per 10 ps bins. The notation underneath the histogram gives total number of spikes per total number o f phase modulation cycles. A camera lucida drawing of the lucifer-labelled cell is shown to the right (arrow). The soma of the nearest giant cell (g) is shown for comparison. Fig. 3b. Schematic representation of neuronal circuitry computing differential phase between two areas of body surface, a and b. (See text for explanation: Diagram provided by C. E. Carr.)

446

TINS October 1985 -

L. Maler who compared this structure to a simple cortex9 (Fig. 2). Afferents from P-type and T-type receptors project to three separate, somatotopically structured maps in the ELLL, each fiber sending collaterals to all three mapss. The significance of this triple representation of identical sensory information is still unknown. All three maps are similar in their architecture and projections. So-called spherical cells (Fig. 2) of the ELLL receive electrotonic input from small patches of T-type receptors. They then relay one spike per EOD cycle, phase-locked to the zerocrossing of the local stimulus, to the somatotopically ordered lamina 6 of the torus semicircularis of the midbrain1°. Synaptic inputs and spike initiation of spherical cells are structured so that they only fire in response to nearly synchronous arrival of several spikes9. As a consequence, spherical

cells should not relay single, stray spikes that could originate from an under-driven or defective receptor, and so the jitter in phase-locking of spherical cells is smaller than that in T-receptors 1~. The two types of pyramidal cells2 of the ELLL receive direct or indirect input from P-type receptors and respond in opposite ways to modulations in stimulus amplitude. Basilar pyramidal cells (or E-cells) receive direct P-type input via their ventral dendrites and are thus excited hy a rise in stimulus amplitude within their receptive field. Due to indirect P-input via inhibitory interneurons, non-basilar pyramidal cells (or I-cells) are excited by a fall in stimulus amplitude12-14. Both types of pyramidal cells project to laminae 3, 5, 7 and 8 of the torus~°. Eigenmannia thus has separate sets of cells to detect local upward and

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downward modulations in stimulus amplitude, respectively. What is further needed for the control of the JAR is the computation of differential phase and the ultimate integration of amplitude and phase information.

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Fig.4.A certaindifferencefrequency (D~-sign-selectiveceU of the tec~n discrintinatesthesign of Df ifthe interferingstimulus,S~ ispresentedlongitudinally (b ). Thiscellfa~ to respond if$2 ispn~ented transversely(a) or ifSt and $2 arepresentedthroughthesame pair of elecwodeJ (c),which abolbl~ modulations in diff~rential phase altogether. This neuron thus appears to be sensitive to diffc~naal phase modulations in the l o n ~ d i r e c a o n (b). Data are presented as histograma of spike frequency as a function of the beat cycle, with ~ , a, and pha~, p, modulations (relative to an unmodulaled $1) indicted underneath. With dif~rential stimulus field geometry (null ~ in line a and hiatosrama in line b), modulat~__ns of an~lintde and ~ phase result in circular graphs ~dloae aens¢ of rotation ~ the $ign of d~erevlce ~qiumlcy (D~. With identical atimulua ~ geometry (null ~ in line c), modulations in differential phase are absent, and the srimulns graph collapses into a vertical line. The numbers above each h i s ~ u m indicate total number of spikes per total number of modulation cycles. Three replications of the experiment are shown in (b). A camera luctda drawing of a Dr-sign-selective ceil is shown at the right. The neuron projeet~ to the reticular formation (RF), to lamina 9 of the torus (TS) and to the nucleus electrosensorius complex (NE). The soma is located at the boundary of the stratum album centtale and the stratum griseum centrale of the rectum.

T I N S - October 1985

lamina 6 and thus relay their phase information to the representations of many different areas of the body surface. Collaterals of these axons terminate upon somata of small cells, and only a single fiber appears to contact a given soma (Fig. 3b). Small cells thus receive information about the phase within the area of body surface corresponding to their somatotopic position in lamina 6 through their dendrites, and they receive a reference phase from another, apparently randomly chosen area of the body surface through giant cell input onto their soma. The firing rate of these cells is driven by the difference in arrival time of spikes at these two different locations and thus reflects differential phase between the corresponding areas of the body surface (Fig. 3a), No responses to modulation in amplitude have been observed, though this is to be expected, since there is an absence of amplitude-coding inputs to lamina 6. Approximately fifty cell types have been identified by Golgi studies 16, and approximately thirty of these have also been identified physiologically by intracellular recording and labelling15. We find a wide variety of cell types, driven either by amplitude or phase or by an additive combination of these two. A multipolar cell in lamina 8c, for example, may be excited by a rise in local amplitude as well as by a local phase lead with respect to some specific reference area, and this cell type will be excited most strongly at the moment that a rise in amplitude coincides with a lead in phase. The average firing rate of this neuron, however, does not depend upon the sign of Df. Many cell types project topographically to the rectum opticum and relay all information about amplitude and phase necessary for the final computation of the sign of Df.

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The role of the tectum in the JAR A small number of tectal cells has been identified which apparently combines phase and amplitude inputs by an 'and' operation 17. Fig. 4 shows an example of a cell that only responds to a rise in amplitude paired with a lead in phase and is otherwise silent. This form of modulation characterizes a clockwise rotation around phase zero in the amplitude-phase plane, i.e. a negative Df situation. That this particular cell discriminates the sign of Df only for the longitudinal orientation of the interfering stimulus field, $2,

Fig. 5. The computation of the sign of Df along an electrosensory pathway. Top portion of figure: Two beat patterns with opposite signs of difference frequency (Dr) and their modulations of amplitude and phase. Modulation graphs with opposite senses of rotation are obtained if amplitude is plotted against phase in each case. The spike patterns of E-units (basilar pyramidal cells) and I-units (non-basilar pyramidal cells) in the ELLL mark rises and falls, respectively, in stimulus amplitude. Their input originates from P-receptors. Higher-order E- and I-units of the torus relay amplitude information to the tectum. Phase-advance and phase-delay units of the torus code differential phase. This information is obtained by comparison of arrival times of T-receptor spikes relayed from different parts of the body surface via spherical cells of the electrosensory lateral linelobe (ELLL). Logical combinations ('and' gating) of amplitude and differential phase information at the level of the tectum yield neurons that only fire for ~ particular sign of Df (heavily framed). Higher-order neurons of this type are found in the nucleus electrosensorius complex which appears to form the link to the prepacemaker nucleus of the midbrain. This prepacemaker nucleus provides the only known input to the medullary pacemaker which controls the electric organ discharge (EOD) cycle by cycle. The classification of neurons of the torus has been simplified by ignoring neurons which combine phase and amplitude information in an additive manner.

TIN5 . October 1071.5

448 indicates that it receives information about phase comparisons between two areas of the body surface that experience maximal phase differences for this $2 orientation Ls'~7. Since this cell is only activated by oae sign of Df it is a suitable candidate for the control of the pacemaker frequency in the J A R . Moving objects elicit motor responses, such as swimming or tilting and bending of the body; and tectal neurons that are strongly driven by moving electrical and/or visual images appear to control such responses ~s. As mentioned initially, moving objects affect the amplitude and the phase of the animal's E O D in a similar manner a s do interfering EODs. Various types of toral and tectal neurons respond to moving objects as well as to jamming stimuli, and it appears that the animal's decision to produce a J A R or to respond with a physical movement does not so much depend upon the activity of individual neurons but rather upon the nature of the entire set of neurons recruited by a given stimulus regimen. The neuron morphologically in Fig. 4 is similar to tectal neurons which are strongly driven by moving objects and which project to motor nuclei in the reticular formation. It thus seems that the neuronal machinery of the J A R has emerged through gradual modification from a more general system controlling spatially oriented motor responses.

The f'mal output pathway Tectal cells that discriminate the sign of D f project to the region of the nucleus electrosensorins complex of the diencephalon. Within this complex, extraceUular recordings have identified neurons which discriminate the sign of Df in a similar manner w, H R P injections in the eleetrosensorius complex have revealed projections to the pre-pacemaker nucleus of the m i d b r a i n , the only known source of input to the medullary pacemaker that controls the E O D cycle by cycle. The role of the eleetrosensorius complex in the control of the J A R is further supported by the observation that electrical stimulations within this region produce changes in pacemaker' frequency indistinguishable in their time course from those shown in the J A R 19. Intracellular studies are still needed to identify the neuronal circuitry of these last neuronal elements in the control of the J A R .

General lessons we have learned from the study of the JAR

(a) Properties of a distributed neuronal organization The neuronal substrate of the J A R is characterized by its distributed organization. An area of skin surface less than l cm 2 can be sufficient to control a J A R . Furthermore, with the exception of the pacemaker nucleus at the motor end of the command structure, there is no center of 'pontifical' or decision neurons whose destruction would jeopardize the system. It is this principle of organization that makes this behavior so robust and so tolerant to loss of sensory inputs and even damage to central structures. A further benefit of a distributed organization and neuronal con.'ergence towards the motor output is an extreme behavioral sensitivity to stimulus features that is not observed at the level of even higher-order electrosensory neurons. The J A R can be driven by phase modulations as small as 500 ns peak-to-peak whereas the resolution of phase in the small neurons of lamina 6 appears to be on the order of tens of p.s (Ref. 15. Note that the histogram in Fig. 3a is based upon a sampling time of 17.5 s, while the latency for the J A R is less than 0.3 s). This, however, is not to say, that such high sensitivity never occurs at the level of any single neuron: each E O D cycle is driven by a discharge from the medullary pacemaker, a nucleus of electrotonically coupled cells 1. Recording from a single pacemaker cell, therefore, allows full monitoring of the E O D frequency and thus of the animal's JAR.

(b) The statistical emergence of stimulus-feature specificity Although the J A R is driven most strongly by absolute Df values in the range from 2 to 6 Hz (Ref. 3), this frequency is not the preferred modulation frequency of all neurons responding to amplitude modulations. However the majority of toral cells 20 and all electrosensorius complex celts w respond best within this frequency range. A sensory specificity displayed at the behavioral level thus emerges statistically from an ensemble of neurons tuned less uniformly.

(c) A seemingly unnecessary complexity o f organization Although the neuronal organization of the J A R appears to be relatively simple we still feel that it is unnecess-

arily complex. The computational rules of sensory information processing that govern this behavior are extremely simple, and these rules could be implemented by a neuronal network far simpler and transparent than what we find in reality 5.~. All that is needed are logical 'and' operations between either a stimulus amplitude rise or fall and a phase advance or delay; all neuronal elements for these operations are already available at the level of the torus. Why then should the tectum be involved as a further link in the chain? As was mentioned in the description of the role of the rectum, however, the J A R probably evolved from more general motor responses to moving objects, and the underlying neuronal hardware was thus likely obtained by modifications of. and additions to, an existing system that was already adapted to other functions. The design of this system, therefore, appears to be burdened by its history.

(d) The correlation between neuronal structure and function In his detailed anatomical studies of the electrosensory lateral line lobe (ELLL), Maler predicted physiological properties of certain cell types on the basis of their morphology 9. His predictions were borne out by subsequent intracellular studies l~.a4,JS, and a tight link between structural and functional properties could be established for the spherical cells and basilar and non-basilar pyramidal cells (Fig. 2). Carr found that neurons in lamina 6 of the torus can be divided into very distinct types on the basis of structural and functional properties. The pattern of the organization of lamina 6 (see Fig. 3b) suggests a simple set of probabilistic rules that guide the development of this structure so that: (1) axons of spherical cells of the ELLL should invade lamina 6 by maintaining their somatotopic neighborhood relations; (2) upon arrival in lamina 6. these axons should synapse upon local somata of giant cells and upon dendrites of small cells; (3) giant cells should send their axonic arborizations all over lamina 6 and synapse upon somata of small cells: and (4) only one synapse should fil onto a soma of a small cell. As a consequence of these rules, small cells would receive a randomly chosen phase reference input on their soma. a sufficient requirement for the control of the J A R .

T I N S - October 1985 Whereas structure-function relations are very obvious in lamina 6, they are less well understood in other laminae. Multipolar cells in lamina 8c, for example, display functional differences without any apparent morphologii~al differences, apparently showing random combinations of amplitude (E- or I-type) and phase (advance or delay-type) inputs 15. It is possible that these types of neurons can be distinguished by immunohistochemical means, but it can also not be ruled out that they are indeed identical in their design and that their functional differences are accidental, being determined by the nature of the type of neurons that happen to be first in occupying limited dendritic surfaces of their targets. Selected references 1 Bennett, M. V. L. (1971) in Fish Physiology

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5 6 7 8 9 10

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(Hoar, W. S. and Randall, D. J., eds), pp. 347-491, Academic Press, New York Heiligenberg, W. (1977) in Studies of Brain Function, Vol. 1, pp. 1--85, Springer Verlag, Berlin Bullock, T. H., Hamstra, R . H . and Scheich, H. (1972) J. Comp. Physiol. 77, 1-48 Scheich, H. and Bullock, T. H. (1974) in Handbook of sensory physiology Vol. III/3, (Fessard, A., ed.), pp. 201-256, Springer Verlag, Berlin Heiligenberg, W., Baker, C. and Matsubara, J. (1978) J. Comp. Physiol. 127, 267-286 Heiligenberg, W. and Bastian, J. (1980) J. Comp. Physiol. 136, 113-133 Scheich, H., Bullock, T. H. and Hamstra, R. H. (1973) J. Neurophysiol. 36, 39-60 Heiligenberg, W. and Dye, J. (1982) J. Comp. Physiol. 148, 287-296 Maler, L., Sas, E. and Rogers, J. (1981) J. Comp. Neurol. 195, 87-140 CarT, C. E., Maler, L., Heiligenberg, W. and Sas, E. (1981) J. Comp. NeuroL 203, 649-670 Cart, C. E. (1984) Structure and Function in

the Electric Fish Midbrain. (PhD Thesis) University of California, San Diego 12 Bastian, J. and Heiligenberg, W. (1980) J. Comp. PhysioL 136, 135-152 13 Bastian, J. (1981) J. Comp. Physiol. 144, 456-494 14 Saunders, J. and Bastian, J. (]984) J. Cornp. Physiol. 154, 199-209 15 Heiligenberg, W. and Rose, G. (1985) J. Neurosci. 5, 2, 515-531 16 Carr, C. E. and Maler, L. (1985) J. Cornp. Neurol. 235, 207-240 17 Rose, G. and Heiligenberg, W. (1984) Soc. Neurosci. Abstr. 10, 403 18 Bastian, J. (1982) J. Comp. Physiol. 147, 287-297 19 Bastian, J. and Yuthas, J. (1984) J. Comp. Physiol. 154, 895-908 20 Partridge, B. L., Heiligenberg, W. and Matsubara, J. (1981)J. Comp. Physiol. 145, 153-168

Walter Heiligenberg and Gary Rose are at the Scripps Institution of Oceanography, University of California, San Diego, LaJoUa, CA 92093, USA.

Current models of blood-brain transfer Joseph D. Fenstermacher The capillaries o f the CNS do not structurally resemble most other capillaries and greatly retard the blood-brain exchange of many substances, including some that are neuroactive or neurotoxic. Because they serve a protective function they are the anatomical correlate o f the blood-brain barrier (BBB). These capillaries also selectively facilitate the transfer o f solutes which are needed to support brain activity such as glucose. Over the past twenty years, blood-brain transfer has been measured by a number o f different methods. From such studies, the interaction o f blood flow and BBB permeability, the virtual lack of pores in brain capillaries, the significance o f endothelial cell enzyme systems, and the roles o f the luminal and abluminal membranes are emerging. In addition, regional and local differences in normal BBB function, which may be related to capillary density and local tissue activity, have now been described. The blood-brain barrier (BBB) of most vertebrates is formed by the capillary endothelium. These endothelial cells are devoid of fenestrae, contain very few (if any) microvesicles, and are joined together by continuous belts of tight intercellular junctions that block the paracellular passage of electron dense markers such as horseradish peroxidase and microperoxidase. Moreover, as many as one-half of the transversely and obliquely sectioned capillaries from eerebellar and cerebral cortex have no intercellular junctions or 'seams', suggesting that many - perhaps even most endothelial cells are tubular and completely ensheath the capillary lument.L Without a doubt, the 'tightness' of the BBB is correlated with some or all of these structural features of brain capillaries.

Methods

When investigating blood-brain transfer, the measurable parameters are the concentration of the test material in plasma and brain plus the sampling times. The resulting data are then generally used to calculate one of the following transfer constants: the extraction fraction (E), the influx constant (Ki; units = vol. mass -1 time-I), or the effiux constant (ko; units = time-l). When appropriately measured, these constants are indicators of unidirectional transfer or flux and depend not only on the permeability coefficient (P) and the surface area (S) of brain capillaries but also on the velocity of volume flow through brain capillaries (F) and the functional distribution volume of the test material in the blood or fluid passing through the lumen (Wf)3'4.

The extraction fraction is determined by injecting a bolus containing the test solute into the carotid artery and assessing brain uptake over the next several seconds: E is defined as: E = B/A (1) where B is the amount of test solute that is unidirectionally taken up by brain tissue and A is the total amount of extractable test material that has flowed into brain capillaries during the unidirectional uptake period 3,4. The following three methods are used to measure E: the indicator-diffusion technique; the brain-uptake index technique; and the external-registration technique; all three techniques yield useful evaluation of E when conducted over a short enough period of time to minimize backflux (usually less than 5 seconds) and the test material is moderately to highly permeable3. 4. The influx constant is measured in experiments where the test solute is intravenously administered by either continuous infusion or bolus injection and is defined as: Ki = B/foTCa(t)dt (2) where B is the amount that has passed across the BBB into the brain parenchyma as in Eqn 1; Ca(t) is activity or concentration of free or exchangeable test material in arterial plasma at any

~) 1985.ElsevierSciencePublishersB.V., Amsterdam (1378- 5912/85/$02.00