The properties of neurosecretory cells

GENERAL

AND

COMEUUTIVE

Supplement

ENDOCRIKOLOGY,

The Properties

1, 117-132

of Neurosecretory

HOWARD Department

of Zoology and its University of California,

Cancer Research Genetics Laboratory, Berkeley, California, U. S. A.

The generally accepted concept of the neurosecretory cell is that of a nerve cell possessing features associated with glandular activity in addition to ordinary neuronal characteristics. In these terms, the neurosecretory neuron can he considered also as an elongate gland cell. According to the picture associated with the names of Scharrer, Bargmann, HanstrSm, and others, the neurosecretory material is synthesized in the neuron cell body or perikaryon and is then transported distally into and down the axon to its point of discharge from the axonal termination, generally in contact with a blood vessel or vascular sinus. The aggregation of neurosecretion-bearing axonal bulbs or fine terminations in association with the vascular bed forms the neurohemal storage-release organ (Knowles and Carlisle, 1956). The present survey will attempt to assess the prevailing concept of the neurosecretory ceil by examining some of its properties on the cellular level. A more detailed consideration of the extensive literature upon which many of the statements made herein ‘are based will be found elsewhere (Bern and Hagadorn, 1962). The rapid accumulation of new data regarding the secretory activity of neurons makes periodic reconsideration of the phenomenon of neurosecretion highly desirable, if not indeed imperative. In the course of such a survey, unanswered questions should emerge. OF

Cells

A. BERN

INTRODUCTION

DEFINITION

(1962)

NEUROSECRETION

Currently it appears that the most useful definition of neurosecretion is a qualified one. The finding of stainable materials in neurons-cytoplasmic inclusions in the forms of droplets, granules or vacuoles117

regardless of the stain employed, only allows t,he conclusion that such neurons are possibly neurosecretory. If, on the other hand, evidence of a secretory cycle can be adduced, based on the same kind of cytologic criteria used in judging the secretory activity of an epithelial cell, then it is possible to conclude that such neurons are probably, but not necessarily, neurosecretory. Indications of stages in the elaboration and release of secretion, or changes in the cells at different times of the year, or alterations associated with specific physiologic activities of the organism or resulting from experimental manipulation of the organism, provide important support for the conclusion that neurons are probably neurosecretory. If the signs of secretory activity can be related to the production of chemical agents with measurable physiologic effects, agents definable as hormones, then it can be concluded that such cells are definitely neurosecretory. A modern conception of neurosecretion includes the attachment of functional significance to neurons possessing morphologic indicators of secretory activity. It must be admitted that in the case of a number of generally accepted neurosecretory systems, the acceptance at present is based on little more than the confidence that functional activities and specific hormonal agents will eventually be found (e.g., in many arachnid, myriapod, and mollusk groups; the caudal neurosecretory system of fishes). The occurrence of a restricted number of neurons, distinguishable from other neurons owing to particular staining properties, is almost universal among the Metazoa. The basic structural framework of a neurosecretory system consists of a cluster or nucleus of neurosecretory cell bodies

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HOWARD

giving rise to neurosecretion-bearing axons, which in turn are organized into a distinct tract or nerve terminating finally in a neurohemal organ. The existence of analogous systems in the majority of invertebrat,e groups and in the vertebrates has been discussed in detail previously (e.g., Hanstrom, 1947; B. Scharrer, 1959; E. Scharrer, 1959). The comparable organization shown by neurosecretory systems in a series of animal groups is a striking example of evolutionary convergence, apparently of major adaptive significance, at least in those organisms where the functions of the neurosecretory system have been determined. GLANDULAR PROPERTIES OF NEUROSECRETORY CELLS STRUCTURAL

NATURE OF NEUROSECRETORY MATERIAL

With the light microscope, the so-called Gomori stains (chrome hematoxylin, psraldehyde fuchsin) have proved to be of considerable value in demonstrating

A. BERN

masses of material interpretable as secretion both in cell bodies and in axons of specialized groups of neurons. However, the failure to stain with chrome hematoxylin or paraldehyde fuchsin does not eliminate the possibility of secretory activity in neurons. Stainability with the acid components of the LLGomori” staining mixtures or with other routine histologic stains can also be indicative of neurosecretory activity (Fig. 1). The caudal neurosecretory system of fishes, certain cells in the Xorgan of the crustacean eyestalk, and certain cells in the brain of insects are all examples of presumed neurosecretory cells which are “Gomori-negative.” A variety of inclusions present in neurons may mimic neurosecretory material, including neuromelanin (“wear-and-tear pigment,” often increasing with the age of the animal), other pigmented materials of unknown significance, and even stored metabolites (lipids, polysaccharides) . In teleosts, particularly in older animals, neurons of the brain contain variable amounts of pigmen& which are fuchsinophilic and

FIG. 1. Neurosecretory d-cell from the brain of Platynereia bicanaliculata (Polychaeta). Associated with “Gomori-positive” cell (gp) is a vacuole containing “Gomori-negative” droplets (gn). (gna), axons (?) containing “Gomori-negative” secretion droplets; (n), nucleus with nucleolus; (on), “ordinary” neurons. Paraldehyde fuchsin with counterstains. (Preparation of I. Hagadorn).

h-EUROSECRETORY

tend to accumulate toward the axon hillock, clearly imitating neurosecretion (cf. Stahl, 1957). In mollusks, several categories of inclusions in the cytoplasm of neurons have provoked considerable cytologic interest in the past, as possibly indicating secretory activity (e.g., Thomas, 1951) ; in the gastropod Cylichna, Lemche (1956) has described a large number of fuchsinophilic neurosecretory pathways which may, in fact, only reflect the presence of pigments in many neurons. The Purkinje cell of the cerebellum of vertebrates (Shanklin et al., 1957) also has been awarded secretory status owing to its content of fuchsinophilic material. As stated earlier, considerable caution needs to be taken to avoid premature conclusion about the secretory activity of neurons based upon staining images. A pair of secretory neurons in the subesophageal ganglion of the, cockroach (B. Scharrer, 1955) undergoes profound cytologic change following ovariectomy. The presumed alteration in secretory activity now appears to be rather an exaggerated instance of a generally altered metabolism consequent to ovariectomy. Inclusions similar to those seen in this special pair of neurons are seen in other neurons and in nonnervous tissues (B. Scharrer and von Harnack, 1960, 1961). It also seems probable that cell organelles themselves, and not just secretory products, may react with the stains which are ordinarily used to demonstrate secretory material. In some invertebrate neurons the dictyosomes have been described as secretory inclusion bodies. Miyawaki (1960) has described lLGomori-positive” structures in the t.horacic ganglion of crabs, which appear to be the invertebrate equivalent of the Golgi complex. Despite the fact that stains may be deceptive in indicating the existence of neurosecretion, the finding of groups of neurons with specialized staining characteristics among masses of nonstaining ordinary neurons should not be disregarded, even when the staining cells are found in areas where they are not “expected.” It has been known for many years that. the

CELLS

119

occurrence of neurosecretory cells is not restricted to the brain area. In Limulus, B. Scharrer (1941) early showed that the ventral ganglia contain a complement of neurosecretory cells indistinguishable from those present in the ganglia of the circumesophageal ring. In many teleost species, and in higher vertebrates as well, there are cranial nuclei other than those associated with the hypothalamus proper which may be truly neurosecretory. In the puffer, Arothron hispidus, we have encountered a particularly large area in the midbrain tegmentum, which includes decidedly secretory-appearing acidophilic cells (Fig. 2). Neurosecretory neurons are often larger than t,heir “ordinary” neighbors. However, it is not correct to extrapolate from this fact the conclusion that all giant cells within the nervous system are neurosecretory. The cells of the supramedullary ganglion of the puffer do not appear to be neurosecretory, nor do Mauthner’s neurons. The huge neurons in the visceral ganglion of Aplysia, studied by Taut (e.g., 1955) and others, appear t.o have less claim to neurosecretory status than do some of their relatively insignificant “ordinary” neighbors, inasmuch as the latter contain elementary neurosecretory granules (see below). Examination of neurohemal organs (the neurohypophysis and urohypophysis of vertebrates; the sinus gland, pericardial organ, and postcommissural organ of crustaceans; the corpus cardiacum of insects) with the electron microscope reveals the constant occurrence of at least one class of elementary neurosecretory granules, measuring between 1000 and 3OOOA in diameter. These granules are generally electron-dense, but occasionally have a vesicular appearance. They seem to be the basic components of the stainable masses evident on the light microscope level. Similar granules are present whether the neurosecretory material be “Gomori-positive” or “Gomori-negative.” Examination of cells containing inclusions which do not appear to be true neurosecretion, such as the large droplets evident in the giant neurons of Aplysia (Simpson and Bern,

unpublished) or the neuromelanin in vertebrate neurons, reveals osmiophilic droplets which may be considerably larger than 1 p in diameter. Unfortunately, the presence of electron-dense particles measuring 1000 t,o 3000A in diameter may not be a reliable ultrastructural criterion for true neurosecretory activity. These granules are defined largely on the basis of size, and they occur in places where neurosecretion

granules 1961). CHEMICAL

(cf.

Hagadorn

and

Nishioka,

NATURE OF NEUROSECRETORY MATERIAL

The earlier notion that vertebrate neurosecretory materials (Schiebler, 1952)) and possibly also invertebrate neurosecretory materials, were glycolipoprotein is no longer accepted. In the vertebrate hypo-

FIG. 2. Area of tegmentum of brain of old puffer (A~othron hispiclus, Teleostei). Note multinucleate cells with endocellular capillaries (c). Mass of “secretory” granules (s) is “Gomori-negative.” Paraldehyde fuchsin with counterstains. (Bern and Hagadorn, unpublished).

is not otherwise indicated. For example, they have been encountered in “ordinary” axons of insects (Hess, 1958) and in the presynaptic fibers of the myenteric plexus of the guinea pig (Hager and Tafuri, 1959). Ultrastructural studies of neurons in the leech Theromyzon rude, presently being conducted in our laboratory by Hagadorn and Nishioka, open up a major problem in the assessment of neurosecretory activity in neurons. Approximately 5% of the neurons of the leech brain are classifiable as secretory by ordinary staining reactions; however, with the electron microscope no neuron has yet been encountered in the leech which is completely devoid of presumed elementary neurosecretory

thalamus the neurosecretory material consistently shows the presence of proteinbound disulfide groups (Barrnett, 1954) ; insect neurosecretory material also shows cytochemical evidence of disulfide content (Sloper, 1957; Brousse et al., 1958). Certain vertebrates (especially amphibians) possess hypothalamic neurosecretory material which is regularly and intensely PAS-positive; others show little or no evidence of a carbohydrate moiety associated with the neurosecretory material (Gabe, 1960). Gabe has emphasized that there may be differences in the primarily proteinaceous material of which neurosecretion is constituted. It is ‘reasonable to postulate that there may be many kinds of

SEUROSECRETORT

materials (simple proteins, glycoproteins, lipoproteins, and conceivably even glycolipoproteins) which serve as carrier substances from which the active hormonal principles may be dissociated at the time of release. There does remain, however, a persistent controversy between those workers who consider the stainable neurosecretory material as a “Triigersubstanz” (a carrier protein of the act,ive hormones, such

of

FIQ. the

drion; and

8).

3. Golgi complexes (g) cockroach (Peripluneta (p), glial cell process.

or dictyosomes americana). Elementary

121

CELLS

a&the neurophysine of Acher and Fromageot, 1957) and those who consider the neurosecretory material to be a parent protein (the “prot&ne mbre” of Gabe) which is broken down to form active hormones of lower molecular weight. FORMATION

OF

NEUROSECRETORP

MATERIAL

Although the ultrastructure of neurohemal organs has been investigated thor-

from neurosecretory neurons (e), elementary neurosecretory granules are smaller in perikarya

of the pars intercerebralis granules; (m), mitochonthan in axons (cf. Figs.

5

122

HOWARD

oughly for the past several years, it was only recently that the perikaryon of neurosecretory cells-the presumed locus of synthesis of the secretory material-was subjected to investigation with the electron microscope. The studies of the Dahlgren cells of the teleost caudal neurosecretory system by Enami and Imai (1958) and by Sano and Knoop (1959) and of crustacean neurosecretory cells by Fingerman and Aoto (1959), are pioneering efforts. In our laboratory (Bern et al., 1961) we have considered the fine structure of a variety of neurosecretory cell types: from the brain of the leech Theromyzon rude, from the pars intercerebralis of the cockroach Periplaneta americana, from the preoptic nucleus of t,he frog Rana pipiens, as well as from the caudal neurosecretory systems of several teleost species. In all cases the elementary granules resemble those found in the neurohemal areas, although they may be significantly smaller in the perikaryon. We have found that the elementary neurosecretory granules constantly occur in association with systems of membranes or tubules to which the term Golgi apparatus can be applied. When most prominent, the Golgi complexes in these several neurosecretory areas consist of horseshoe-shaped groups of agranular paired membranes (Fig. 3). In the center of the curvilinear complex occur vesicles and granules of varying electron density. The greater the distance from the membrane system, the larger and denser the granules or vesicles appear. The association between the elementary neurosecretory granules and the Golgi membrane or tube is suggestive of a participation of the latter in the fashioning of the former. The smaller vesicles appear to arise both as a result of the vesiculation of the membranes or tubes and also as a result of “budidng” from the end of the structure (Fig. 4). These complexes are particularly well developed in the invertebrate cells examined, where it appears that they correspond to the so-called dictyosomes of the classical cytologist. In the vertebrate cells, the smooth Golgi membranes are less well organised into units; nevertheless, the nature of the association of granules and

A.

BERN

vesicles with the membrane system is similar to that seen among the invertebrates. A participation of the Golgi apparatus in the formation of the neurosecretory granules has been indicated by Sano and Knoop (1959) in the Dahlgren cells of the tenth, by Palay (1960) in preoptic nucleus cells of the goldfish, by Dalton (1960) in Helix neurons, by Nishiitsutsuji-Uwo (1960, 1961) in pars intercerebralis cells of three lepidopteran species, and by Scharrer

FIG. 4. Diagram to show possible elaboration neurosecretory granules from of elementary double membranes or tubes of Golgi complex. Granule formation appears to occur by vesiculation and by “budding.”

and Brown (1961a,b) in neurosecretory cells of the earthworm brain. An association of the Golgi complex with neurosecretion has also been reported by Miyawaki (1960) in crustacean thoracic ganglion cells. There are several suggestions in the literature to the effect that the mitochondria of neurosecretory cells, which are found not only in the perikaryon but also intermingled with elementary neurosecretory granules in the axon and its termination, are involved in the formation of elementary neurosecretory granules. The mitochondria may participate in the actual synthesis of the neurosecretory granules (Knowles, 1958), or they may transform into elementary neurosecretory granules (Green and Maxwell, 1959), or they may

KEUROSECRETORY

CELLS

123

F ‘ID. 5. Electron of micrograph of section through nerve fiber from tract leading to urohypophysis the goldfish (Carassius auratw). Note distinction between mitochondrion (m) and dense element2 w nem *osecretory granules. Methacrylate imbedded. F 'IQ. 6. Electron rlis micrograph of section through nerve fiber from tract adjacent to pars intercerebrr of t he cockroach (Petinlaneta americana). Note distinction between mitochondrion (m) and dense e!le;tary neurosecretory granules. Elementary granules are larger in this fiber than in urohypophy sis (cf. Fig. 5). Epon-imbcdt!rd ; Ph.I,,.-s: ninrtl.

124

HOWARD

fragment into neurosecretory granules (Nishiitsutsuji-Uwo, 1960, 1961). From our ultrastructural studies we have been unable to obtain any evidence for a direct association between mitochondria and the elementary granules referred to herein. The granules appear to be distinct from the mitochondria both in the cell body (Fig. 3) and in the axon (Figs, 5 and 6), although occasionally, of course, mitochondria occur in the region of the Golgi complex. In agreement with the conclusions of E. Scharrer (personal communication), a reasonable hypothesis can be presented which is consonant with the concept, of protein secretion formation in other secretory cells. The granular endoplasmic reticulum, present in great abundance in those neurosecretory cells not loaded with neurosecretory granules, is probably involved in the production of the basic protein of the neurosecretory material. The Golgi complex fashions this material into granules visible with the electron microscope. In other words, the supramolecular organization of the neurosecretory material may be accomplished by the Golgi complex. It has been suggested that the axon, as well as the perikaryon of the neurosecretory cell may participate in the synthesis of neurosecretory material (Green and Maxwell, 1959). If our hypothesis is correct, this is difficult to conceive, since there is no evidence for endoplasmic reticulum (or RNA, cf. Hyden, 1960) nor for Golgi membranes in the axon. However, there are data from several studies which suggest that neurosecretory granules may “mature” en route from the cell body to the point of discharge (e.g., Fingerman and Aoto, 1959; Gerechenfeld et al., 1960). The elementary neuroaecretory granule or vesicle may itself be a cell organelle which is able to maintain minimum synthetic activity as it is conveyed awsy from the Golgi membranes and down the axon. TRANSPORT AND RELEASE OF NEUROSECRETORY MATERIAL

The conveyance of neurosecretory material from cell body to axonal bulb is con-

A. BERN

sidered to be a consequence of intra-axonal (axoplasmic) flow as described by Weiss (1955). The rate of 1 to 2 mm of movement per day corresponds to some estimates of the movement, of hormonal material from hypothalamus to neurohypophysis (cf. Sloper, 1958). The electron microscopists can find no good evidence for the existence of special tubes or canals in the axons to guide the distad movement of the neurosecretory granules. However, recently Wigglesworth (1960) has suggested that the dictyosomes in ordinary insect neurons may be concerned with the formation of tubules, approximately 1 p in diameter, which would provide excellent channels for the flow of neurosecretory granules. Most of the elementary neurosecretory granules described do not appear to be in vacuoles such as are produced by the Golgi apparatus around formed secretions in other secretory cells, and in most instances the elementary granules appear to be transported considerable distance before release of secretion occurs. In the case of neurosecretion, the secretory material does not appear to enter the Golgi apparatus in order then t.o be ejected through the cell membrane. When the neurosecretory granules reach the axonal bulb, several possibilities exist as to the mode of release of their associated hormone. It is possible that upon the receipt of appropriate stimulation the neurosecretory granules release their contained hormone within the axonal bulb (cf. Gerschenfeld et al., 1960) ; this material can then pass through the bulb membrane to enter the vascular channel. It. is also possible that the limiting membrane of the neurosecretory granule or vesicle may fuse with the bulb membrane when it contacts it, thus allowing discharge of its contents into the tissue space. At the present time t.here is no evidence to support release of the formed granule itself from the bulb, although such discharge of secretory granules occurs in other kinds of cells (Farquhar, 1961). Elementary neurosecretory granules and mitochondria are not the only organized bodies detectable in the axonal endings of

SEUROSECRETORY

neurosecretion-bearing fibers. Structures which are indistinguishable from the synaptic vesicles characteristic of ordinary nerve fiber terminations are also a common occurrence (Palay, 1957; Bargmann et aI., 1957; Gerschenfeld et al., 1960). The concept of the neurosecretory cell includes the fact. that their axons are not in synapse with other neurons and do not innervate effector cells (cf. Knowles and Carlisle, 1956). These vesicles may be concerned with the release of hormones from elementary neurosecretory granules, or with the alteration of the axonal bulb membrane to permit ready passage of the released hormone. Another possible explanation of their presence in neurosecretion-bearing fibers arises from the relationship of these fibers to glial cells and the so-called intrinsic cells characteristic of neurohemal organs such as the neurohypophysis and corpus cardiacum, respectively. Conceivably the neurosecretion-bearing fibers innervat,e these cells. Since synaptic vesicles are found in concentration only at the axon termination, they may serve as a ready marker of a neurohemal area where neurosecretory axons terminate diffusely and are not organized into a distinct organ. Accordingly, the posterior surface of the dorsal commissure of the leech brain, indicated as being possibly neurohemal on the light microscope level, appears to be definitely so on the ultrastructural level, owing to the occurrence of axon terminations containing both elementary neurosecretory granules and appreciable numbers of particles resembling synaptic vesicles (Hagadorn, unpublished). NEURONAL PROPERTIES OF NEUROSECRETORY CELLS

In addition to qualifying fully as secretory cells, neurosecretory neurons are expected to show many if not all of the characteristics associated with ordinary neurons. Insofar as neurites are concerned, there is no doubt about the existence of axons in at least most neurosecretory cells, The Dahlgren cells of elasmobranchs are exceptional in possessing processes which

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125

are short and blunt; it is debatable whether or not a true axon exists (Bern and Hagadorn, 1959; Fridberg, 1959). For somewhat inexplicable reasons, students of neurosecretion have tended to assume that dendrites may often be absent from neurosccretory cells. It appears, however, that t.hc possession of dendrites has only been questioned because in most cases they have not been looked for. Maynard (1961) has established conclusively that the neurosecretory cells of the thoracic ganglion of crabs possess definite dendritic processes which, in contrast to the axon, do not appear to contain granules. Bipolar neurons can be found in hypothalamic areas, especially in amphibians. Here, however, the dendritic process often contains stainable neurosecretory material presumably moving away from the cell body by “dendritoplasmic” flow (e.g., Wilson et al., 1957). In addition, some neurosecretory cells send a dendrite-like process between ependymal cells to terminate in the third ventricle itself. Ventricular secretion by putat,ive neurosecretory cells is a common cytologic observation, again especially in :tn~phibians. There seems little reason to doubt the existence of Nissl substance or its equivalent in neurosecretory neurons. Cytochemitally these cells show the presence of considerable ribonucleic acid, as one would expect in a cell actively synthaizing a protein secretory material. With the electron microscope a prominent endoplasmic reticulum can be demonstrated, as mentioned above. Silver-staining technics reveal neurofibrils in hypothalamic neurons (e.g., Sano et aZ., 1957) and also in neurosecretory neurons of the leech (Hagadorn, 1958). In the leech the argyrophilic neurofibrillar network surrounding the nucleus and extending into the axon is represented in electron micrographs as densely packed osmiophilic fibrils (Fig. 7). Careful examination of the axons of neurosecretory neurons of the cockroach also reveals delicate neurofilaments indistinguishable from those encountered in ordinary axons (Fig. 8). Whether these different entities are

126

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A.

BERK

micrograph of part of neurosecretory cell from supraesophageal ganglion of the FIG. 7. Electron leech (Theromyzon rude). (e), elementary neurosecretory granules; (f), neurofibrillar mass; Cm), mitochondrion; (n), nucleus; (r), endoplasmic reticu!um.

both to be viewed as neurofibrils is open to debate. Neurosecretory fibers appear to enjoy a relatively constant association with glial elements. In many vertebrates the glial cells are especially evident as the parenchymatous pituicytes of the pars nervosa. Pituicyte-like cells similar to those in the oars nervosa occur in the median eminence of the parakeet (Kobayashi and Bern, unpublished), and such cells are also evident in the urohypophysis of the caudal

neurosecretory system. Rennels and Drager (1955) have commented upon the apparent termination of neurosecretion-bearing fibers upon the glial elements themselves in the rat pars nervosa, and it is possible that glia play a significant role in the release and conveyance of hormones of neurosecretory origin into the vascular channels. The relation between neurosecretorv neurons and the prominent glial e1ement.s often associated with them is worthy of more detailed analysis. Galambos ( 1961) has

NEUROSECRETORY

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127

FIG. 8. Electron micrograph of longitudinal section of nerve fibers adjacent to pars intercerebralis Note delicate microfibrils resembling neurofilaments (f) in axon containing numerous of Peripheta. elementary neurosecretory granules. Epon-imbedded ; PbAc&ained.

recently raised the possibility that glia may be involved in the nonneuronal conduction of information in the nervous systern generally. The discussion above makes it apparent that neurosecretory cells are good neurons on a morphologic level. Left unconsidered, however, is the fundamental question dealing with possible conduction by neurosecretory cells and the nature of electrical activity associated with such cells. The explanation of neuroendocrine reflexes, in which neurosecret.ory cells participate by the rapid discharge of preformed secretion from their axonal bulbs, is tied up with the question of impulse conduction. Figure 9 presents some of the

possible physiologic solutions of the problem of rapid nervous mediation of secretion release. Most neurosecretory tracts or nerves hitherto investigated contain nonneurosecretion-bearing fibers along with neurosecretion-bearing fibers. Accordingly, the recording of electrical activity from such mixed tracts or nerves does not allow one to conclude that the neurosecretory fibers are themselves capable of conducting impulses. Auxiliary neurons may innervate the axonal bulb regions; impulses conducted by them would then be responsible for the release of hormone. Figures 9A and B represent what is physiologically t,he most “economical” solution to the problem of nervous control

128

HOWARD

POSSIBLE RELEASE

OF

NERVOUS

SECRETION

FROM

A.

BERK

CONTROL

OF

NEUROSECRETORY

CELLS

9 9. Diagram to show possible methods of nervous control of release of secretion from neurosecretory cells. A, cell as in vertebrate hypothalamus to show conduction by neurosecretory axon itself; B, typical invertebrate neurosecretory neuron to show conduction by neurosecretory axon itself (influence on secretory activity of the perikaryon is also a possibility) ; C; neurosecretory neuron has parallel ordinary neuron influencing release of secretion from axonal bulb; D, release of secretion is conditioned by glial cell envelope of axonal bulb, the glial cell itself being innervated by an ordinary “secretomotor” neuron. FIQ.

release. Table 1 sumneurohormone marizes the evidence for the possible conduction of impulses by neurosecretory neurons. Examination of this table reveals

over

that

there

is a paucity

of data

to support

a definitive conclusion. The release of hormones as a result of electrical stimulation of neurosecretory centers, the electrical activity of neurohemal organs, the electrical activity of neurosecret.0i-y centers, and the recording from neurosecretory tracts or nerves which also include nonneurosecretory fibers, are all open to the criticism that the electrical activity may be due to cells other than the neurosecretory neurons themselves. The important studies of Cross and Green (1959) involve recording from single cells in the hypothalamus of rabbits subjected to osmotic manipulations. Unfortunately, here again it is impossible to decide finally whether the responsive cells are true neurosecretory cells. Most recently, Morita et al. (1961) have suc-

ceeded in recording from individual neurosecretory neurons of the caudal neurosecretory system of the eel. Stimulation of the spinal cord anterior to the location of the penetrated Dahlgren cells results in evidence of a true action potential. From this admirable study it is possible to conclude t.hat at least some neurosecretory neurons have the ability to conduct impulses as a result of presynaptic stimulation. COlUCLUSIONS

This brief consideration of the cellular properties of neurosecretory cells presents evidence in support of the concept of the neurosecretory cell as a modified neuron with neuronal properties, which also possesses the accepted characteristics of secretory cells. Evidence indicates that the synthesis of the neurosecretory material must occur at least largely in the perikaryon. Important questions remaining incompletely answered deal with the inter-

XEUROSECRETORY

TABLE EVIDENCE

FOR CONDUCTION

129

CELLS

1

BY NEUROSECRETORY

NEURONS

Examples

Neuro-endocrine

reflexes

Suckling and milk let-down. Crustacean pigmentation changes. etc.

Release of neurohormones as a result of electrical stimulation of neurosecretory centers.

From neurohypophysis.

Harris

From corpus cardiacum.

Hodgson and Geldiay

Electrical activity of neurohemal organ.

Sinus gland

Milburn

Electrical activity of neurosecretory centers

Cecropia brain

van der Kloot

Hypothalamus

Intracellular recording from neurosecretory neurons

(single cells)

Lop&s

hypophyseal

Limulus

lateral rudimentary

Eel Dahlgren

relation of cell organelles in the manufacture of neurosecretory material, the mechanics of transportation of neurosecretory granules to the point of discharge, the nature of the changes undergone during distal transport, the relation of the active hormonal principle to the granules, and the method of release of hormonal agents from the granules and the axons. The next international symposium should find many of these questions definitively answered. ACKNOWLEDGMENTS I am indebted greatly to Richard Nishioka for the preparation of the electron micrographs and to Irvine Hagadorn for his helpful discussion of some of the material presented. The work from our laboratory reported herein was supported by Grant G-8805 from the National Science Foundation REFERENCES R., AND FROMAGEOT, C. (1957). The relationship of oxytocin and vasopressin to active proteins of posterior pituitary origin. Studies concerning the existence or nonexistence of a single neurohypophysial hormone. In “The Neurohypophysis” (H. Heller, ed.), pp. 39-50. Butterworths, London.

ACHER,

(1955, 1958)

(1955)

Cross and Green (1959)

stalk

cells

(1959)

(1956)

Nakayama

Hypothalamus Recording from neurosecretory tracts or nerves

(1947)

Potter and Loewenstein Carlisle (1958) eye

Waterman Morita

(1955)

and Enami (1954)

et al. (1961)

W., KNOOP, A., AND THIEL, A. (1957). Elektronenmikroskopische Studie an der Neurohypophyse von Tropidonotus natrix (mit Beriicksichtigung der Pars intermedia). Z. Zellforsch. 47, 114-126. BARRNETT, R. J. (1954). Histochemical demonstration of disulfide groups in the neurohypophysis under normal and experimental conditions. Endocrinology 55, 484-501. BERN, H. A., AND HAGADORN, I. R. (1959). A comment on the elasmobranch caudal neurosecretory system. In “Comparative Endocrinology” (A. Gorbman, ed.), pp. 725-727. Wiley, New York. BERN, H. A., AND HAGADORN, I. R. (1962). Neurosecretion. In “The Structure and Function of the Nervous System of Invertebrates” (T. H. Bullock and G. A. Horridge). W. H. Freeman, San Francisco. BERN, H. A., NISRIOKA, R. S., AND HAGADORN, I. R. (1961). Association of elementary neurosecretory granules with the Golgi complex. J. Ultrastruct. Research 5, 31l-&%?O. BROUSSE, P., IDELMAN, S., AND ZAQURY, D. (1958). Mise en 6vidence de lipoprot&nes B groupements-SH au niveau des grains de s&&ion des cellules neuro-s&r&rices de la Blatte, Blabera fusca Br. Compt. rend. acad. sci. 246, 3106-3108. CARLISLE, D. B. (1958). Neurosecretory transport in the pituitary stalk of Lophius piscatorizrs. BARGMANN,

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In Zweites Neurosekretion

pp. 18-19. B. A., of single effect of

CROSS,

Physiol. DALTON, A.

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132

HOWARD

DISCUSSION What is the relationship between the amount of Nissl substance and the apparent rate of neurosecretory material formation7 FARNER:

BERN: We have no information on this question from our own work, other than the observation of a greater prominence of the endoplasmic reticulum in leech cells which contain fewer granules. Claims of an inverse relation between amount of stainable neurosecretory material and Nissl substance or RNA-protein are difficult to assess. The more secretory material there is in a cell, the more dispersed the granular membranes may be, thus influencing both the cytochemical reaction and the ultrastructural picture. GREEN : (1) The problem for me is to identify the light microscopy picture with the electron microscope findings. We have found that there are at least three basic types (100-200 rnp, some about one quarter of that size and some of 200 to 500A without internal structure) but our findings are confined to the vertebrate neurohypophysis. We, in fact, need several types of granules to explain the many activities attributed to the neurohypophysis. (2) I would also like to ask shortly your view on why it is that the granules seen by the electron microscope do not disappear but only change density following dehydration in contrast to light microscope findings. BERN: I have dealt in this paper with the generally accepted “elementary neurosecretory granule,” but I wish to emphasize that I do not consider this granule as necessarily diagnostic of neurosecretion on the ultrastructural level. We have also encountered a variety of other granules and vesicles in our material. Additional granule types exist in the teleost caudal neurosecretory system (as they do in the preoptic nucleus of the goldfish, according to Palay). Hagadorn and Nishioka in my laboratory are defining on the ultrastructural level a series of distinct granule types (and hence cell types) among leech neurons. Kobayashi and collaborators find a spectrum of granule types in the avian median eminence. (2) Gerschenfeld et al. describe a reduction in the number of granules in axonal bulbs in the depleted pars nervosa of the toad. The decreased electron density of granules after dehydration may mean loss of contents and be reflected as decreased staining intensity on the light microscope level. VAN

First a comment and then An excellent place to study a neuro-

DER KLOOT:

a question.

A.

BERN

secretory reflex is in the blood-sucking bug, Rhodnius. As Wigglesworth showed, the brain hormone is released when the bug ingests a blood meal-the stimulus is the swelling of the abdomen. Electrophysiological studies show that each abdominal segment contains a receptor which begins to discharge impulses when the abdomen is distended. These stretch receptors are very slowly adapting and continue to fire at a steady rate during a maintained stretch. The activity of the stretch receptor causes a reflex activation of axons in the nerve running from the brain to the corpora cardiaca. It seems that each stretch receptor drives a single neuron in the nerve to the cardiaca, so the neurosecretory reflex is organized quite simply. As yet, we do not know whether the impulses are in axons from the neurosecretory cells themselves. It is certain, however, that electrical activity goes on at the same time as hormone release. Now for the question. I was happy with your emphasis on the difllculties in being certain that a neurosecretory cell-defined cytologically-is a hormone source. I propose that it would be best to leave the neurosecretory cell defined cytologically, and call neurosecretory cells which have been demonstrated to release a true hormone neuro-endocrine cells. This would avoid a rather prevalent confusion between cytological and physiological evidence. After all, physiologists have good evidence that many neurons secrete chemicals whether or not they show signs of secretion to the light microscopist, even if the circulation of the substances is strictly limited. It would also reassure those who are beginning to worry that some animals have so many neurosecretory cells that they have nothing left to think with. BERN: I am disturbed by the use of neurosecretion indiscriminately to describe cells with inclusions that may not be secreted (released) from the cell. A definite neurosecretory cell should be one that releases a hormone into the vascular system. The requirement for evidence of hormone release would eliminate neurons with deceptive staining images and also neurons releasing only transmitter substances. If the possession by neurosecretory cells of the ability to receive and conduct impulses is conceded, it should not worry physiologists if all neurons in some animals should prove to be secretory. Neurons may be able to conduct their nervous business without being much influenced by their concurrent secretory activity.