The Organization and Biochemical Specificity of Afferent Projections to the Paraventricular and Supraoptic Nuclei

The Neurohypophysis :Structure, Function und Control, Progress in Bruin Resourch, Vol. 60, edited by B.A. Cross und G. Leng

0I983 Elsevier Science Publishers B . V .

The Organization and Biochemical Specificity of Afferent Projections to the Paraventricular and Supraoptic Nuclei P.E. SAWCHENKO* and L.W. SWANSON The Sulk Institute for Biological Studies and the Clayton FOUndUtiOnfor Research-California Division, Lu Jollu, CA 42037 ( U . S . A . )

INTRODUCTION The major stimuli that release oxytocin and vasopressin have been known for many years. Similarly, the final common path for hormone release, the paraventriculo-supraoptico-neurohypophysial system, is the most thoroughly characterized peptidergic system in the brain, both from the electrophysiological and the anatomical points of view. Until recently, however, little was known about the organization of the neural inputs which relay sensory information from the periphery to the paraventricular (PVN) and supraoptic (SON) nuclei, and which integrate activity in the magnocellular neurosecretory system with other, complementary, modes of neuroendocrine and autonomic regulation. In an insightful survey of the literature in 1974, Cross and Dyball concluded that no clearly defined system of afferent fibres to these nuclei had at that time been revealed by neuroanatomical methods. The development of sensitive tracing techniques based on the axonal transport of various marker molecules, and of immunohistochemical methods for localizing biochemically-defined neuronal systems have changed this situation dramatically, and it is now possible to outline the organization and biochemical specificity of inputs to the PVN and SON. Here we shall summarize the results of experiments carried out in the rat, and concentrate on systems that appear to influence the magnocellular system directly. THE CYTOLOGICAL AND FUNCTIONAL ORGANIZATION OF THE PARAVENTRICULAR AND SUPRAOPTIC NUCLEI To view the neural inputs to the PVN and SON in a functionat context, it is necessary to begin with a summary of the cellular architecture of these nuclei (for more detailed treatment see Swanson and Sawchenko, 1983). The cells that project to the posterior pituitary are concentrated in the SON, and in the compact cell masses that form the magnocellular division of the PVN (Sherlock et al., I975 ;Swanson and Kuypers, 1980). In the PVN, these cell groups are topographically segregated from populations of smaller, parvocellular neurones (some of which contain oxytocin and vasopressin; Sawchenko and Swanson, 1982a), that project to the external lamina of the median eminence or to autonomic centres in the brainstem and spinal

* Address for correspondence: The Salk Institute, P.O. Box “91

85800, San Diego, CA 92138, U.S.A.

20 cord (Swanson and Kuypers, 1980; Kelly and Swanson, 1980). In addition, smallerclusters of “accessory” magnocellular neurosecretory neurones, and cells scattered throughout the parvocellular division of the PVN and various basal forebrain nuclei, also project to the neural lobe (Sherlock et al., 1975; Swanson and Kuypers, 1980; Kelly and Swanson, 1980). Both the SON and the magnocellular division of the PVN can be further subdivided on the basis of the distribution of oxytocin- and vasopressin-immunoreactive neurones. In the PVN, the anterior and medial parts of the magnocellular division (see Swanson and Kuypers, 1980) consist almost exclusively of oxytocin neurones ;in the posterior magnocellular part, oxytocin cells are concentrated anteroventromedially, and vasopressin cells posterodorsolaterally, corresponding to the “medial” and “lateral” subdivisions recognized by Hatton et al. (1976) on cytoarchitectoniccriteria alone (modes et al., 1981;Sawchenko and Swanson, 1982a). In the SON oxytocin neurones are concentrated anterodorsally, while vasopressin cells are concentrated posteroventrally. This topographic arrangement has enabled us to suggest which cell type is associated with each particular afferent fibre system. Finally, recent GoIgi studies have shown that the dendrites of cells within the PVN and SON are mainly confined within the morphological boundaries of the nuclei (e.g. vanden Pol, 1982; Armstrong et al., 1982), thus facilitating the identification of projections that may influence the magnocellular neurones. The dendritic trees of neurones in the SON, and in both divisions of the PVN, are simple, and so we have attempted to draw functional inferences based on a light microscopic analysis of the distribution of fibre systems within each subdivision of the PVN and SON that has been recognized using cytoarchitectonic or immunohistochemical criteria. AFFERENT CONTROL OF VASOPRESSIN SECRETION Shortly after the advent of histofluorescencemethods for the demonstrationof catecholaminergic neurones, it was recognized that dense noradrenergic terminal fields lie within the PVN and SON (Carlsson et al., 1962). More recently, comparisons between the distribution of noradrenergic varicosities, stained with an antiserum against dopamine-/3-hydroxylase(DBH ; a marker for adrenergic and noradrenergic neurones), and the distribution of oxytocin- and vasopressin-immunoreactive cells in the PVN and SON established that DBH-stained fibres are concentrated in areas rich in vasopressin cell bodies (Swanson et al., 1981; see also McNeill and Sladek, 1980). Adrenergic fibres, demonstrated with an antiserum against phenylethanolamine-N-methyltransferase, are restricted almost entirely to the parvocellular division of the PVN (Swanson et al., 1981). To resolve a long-standing controversy as to which noradrenergic cells in the brainstem give rise to these inputs, we used a method that allows the concurrent localization within single cells of an antigen (DBH) and a retrogradely transported fluorescent dye (True Blue), after injections of the tracer in the PVN (Sawchenko and Swanson, 1981a). In these experiments (Sawchenko and Swanson, 1981b, 1982b) retrogradely labelled neurones were found to be associated with three noradrenergic cell groups (see Dahlstrom and Fuxe, 1964, for nomenclature). The greatest number (400-600 per brain) was found in the region of the A1 cell group in the ventrolateral medulla, while fewer (100-200 per brain) were found in the A2 cell group of the dorsal medulla, and in the locus coeruleus (30-60 per brain) or A6 cell group. The vast majority (over 80%) of retrogradely labelled cells were DBH-positive, indicating that the projection from each region is primarily noradrenergic. We then injected [3H]aminoacids into the A1 ,A2 and A6 regions (in separate experiments), and used the autoradiographicmethod to trace the course of each pathway to the PVN and SON

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as well as the distribution of labelled fibres in each nucleus. The results confirmed that the A1 , A2 and A6 cell groups each project to the parvocellular division of the PVN, where each input ends preferentially in a different subset of the cytoarchitectonically-and functionally-defined parts of the nucleus (see also Jones and Moore, 1977 ; Ricardo and Koh, 1978 ;Loewy et al., 1981 ;McKellar and Loewy, 1981). Interestingly, only the A1 cell group was found to project substantially to the SON, and to the magnocellular division of the PVN. As with the distribution of noradrenergic varicosities observed immunohistochemically in the magnocellular system, the projection from the Al region was concentrated over regions in which vasopressin cell bodies predominate (Fig. 1). The autoradiographic and retrograde transportimmunohistochemical experiments also suggested that a complex series of pathways interconnect the A l , A2 and A6 regions.

Fig. I . These photomicrographs show sections through roughly the same level of the PVN and illustrate the distribution of cells stained with an antiserum against vasopressin (A), of fibres stained with an antiserum against dopamine$-hydroxylase (B), and silver grains after an injection of [3H]amino acids centred in the A1 catecholamine cell group of the ventral medulla (C). The magnocellular division of the PVN is outlined in C. Noradrenergic varicosities are concentrated in regions rich in vasopressin neurones, and the only noradrenergic neurones that appear to project to the magnocellular division of the PVN (and to the SON) are those of the A1 cell group. Original magnification x 100.

The possible functional significance of these ascending noradrenergic pathways was brought into focus by the finding that the nucleus of the solitary tract (NTS) projects quite heavily to the A1 region (see also Norgren, 1978; Ricardo and Koh, 1978). Subsequent double-labelling experiments, in which True Blue was injected into the A1 region and the tissue was subsequently counterstained with an antiserum against DBH, indicated that this pathway is essentially non-noradrenergic. Important stimuli for vasopressin release are provided by sensory receptors that monitor the volume and composition of the blood. Atrial stretch receptors, carotid sinus baroreceptors, and carotid body chernoreceptors all influence the activity of neurones in the PVN and SON (e.g. Koizumi and Yamashita, 1978;Harris, 1979), and information supplied by these receptors is conveyed by the vagus and glossopharyngeal nerves to the nucleus of the solitary tract, the major central recipient of first-order visceral afferents. From the seminal experiments of Ricardo and Koh (1978), it has been generally assumed that cardiovascular stimuli influence the secretion of vasopressin by a direct projection from the nucleus of the solitary tract to the PVN, or by a disynaptic pathway involving the parabrachial nucleus, which receives an input from the nucleus of the solitary tract (Norgren, 1978)and projects to the PVN (Saper and Loewy, 1980).Careful analysis indicates, however, that neither input ends directly in the SON or in the magnocellular division of the PVN (McKellar and Loewy, 1981 ;Sawchenko and Swanson, 1981b, 1982b). The results suggest instead that visceral afferent information influences vasopressin secretion by way of the direct,

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Fig. 2. A drawing of a sagittal section through the rat brain to show the organization of inputs to vasopressin cell groups in the PVN (PVH is the figure). The SON, which also receives inputs from each cell group indicated, is not shown for the sake of clarity. Sensory inputs from receptors in the cardiovascular system are conveyed by the vagus (X) and glossopharyngeal (IX) nerves to the nucleus of the solitary tract (NTS). Non-noradrenergic cells in the NTS project to the region of the A1 cell group which in turn projects selectively to vasopressin-containing parts of the magnocellular neurosecretory system. We have suggested this as a likely route by which visceral afferent inputs influence vasopressin (VAS) secretion. Other abbreviations : SFO, subfornical organ ; MePO, median preoptic nucleus; ne, noradrenergic pathway.

primarily non-noradrenergic,pathway from the NTS to the A1 region, which in turn projects to vasopressin parts of the magnocellular neurosecretory system (Fig. 2 ) . Although detailed ultrastructural studies are needed to confirm this suggestion, it is supported by functional evidence. For example, in the cat, single neurones in the region of the A1 cell group respond to stimulation of the carotid sinus nerve, and to selective baroreceptor or chemoreceptor activation (Ciriello and Calaresu, 1977 ;Thomas et al., 1977), while lesions of this region attenuate the pressor response to stimulation of the carotid sinus nerve (Ciriello and Calaresu, 1977). Thus, while it remains to be shown that noradrenergic cells of the A1 group receive viseroceptive inputs, at least some cells in this region do so, and are involved in the mediation of pressor responses. That vasopressin is involved in such responses is indicated by the recent demonstration that discrete lesions of the A1 cell group produce increases in blood pressure that are mediated predominantly by increased vasopressin release (Blessing et al., 1982). Apart from the A1 cell group, the only additional sites that have been shown to project preferentially to vasopressin parts of the PVN and SON are the subfornical organ and the median preoptic nucleus. An input from the subfornical organ to the PVN and SON was first described by Miselis (1981), and we have confirmed this observation with both retrograde transport and autoradiographictechniques (Sawchenko and Swanson, 1983).The projection is distributed over all parts of the magnocellularneurosecretory system, but is considerably more dense over vasopressin regions. In agreement with this observation, Renaud et al. (198 1) have provided electrophysiological evidence that stimulation of the subfornical organ most commonly excites phasically-firing (presumably vasopressin) cells in the SON. The pathway from the median preoptic nucleus, which receives an input from the subfornical organ (Miselis, 1981), also appears to reach both cell types, but its terminal distribution appears somewhat more uniform with respect to regions in which oxytocin- and vasopressin-neuronesare located (Sawchenko and Swanson, 1983). The subfornical organ is a richly vascularized circurnventricularstructure that lies outside the blood-brain barrier and is therefore well situated to monitor the concentration of substances

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in the blood, such as hormones and ions. It now seems likely that the subfornical organ contains receptors that mediate both the drinking and pressor responses to circulating angiotensin I1 (see Simpson, 1981 , for a review). The projection from this region to the PVN and SON is, therefore, likely to play an important role in the integration of visceral responses that maintain fluid balance and blood pressure homeostasis. PROJECTIONS TO OXYTOCIN CELL GROUPS The best known stimulus for oxytocin release (in the female) is suckling, and until the function of oxytocin in the male is clarified, the milk ejection reflex will continue to provide the paradigm to study the sensory control of oxytocin secretion. Afferents from the mammary gland ascend in the spinal cord (Ingelbrecht, 1935), but the precise trajectories of relevant pathways, and their terminal distributions, remain poorly understood (see Cross and Dyball, 1974). Studies of the afferent control of the PVN and SON that have utilized modem neuroanatomical techniques have provided few insights into what pathways relay primary afferent information from the spinal cord to magnocellular neurosecretory neurones. The only cell groups in the brainstem that have been shown to project to oxytocin parts of the PVN and SON are a number of serotonergic raphe nuclei. Retrograde transport studies have suggested that cells in the dorsal and median raphe nuclei project to the PVN (Berk and Finkelstein, 198I ;Tribollet and Dreifuss, 198l), and preliminary double-labelling studies (Sawchenko and Swanson, 1981 c) with an antiserum against serotonin indicate that these neurones are primarily serotonergic and arise from the B7, B8 and B9 cell groups of Dahlstrom and Fuxe (1964). It was notpossible to confirm (Loewy et al., 1981) that the ventral medullary (B 1, B2, and B3) serotonergic cell groups also project to the PVN. These results must be viewed cautiously, however, because recent immunohistochemical studies (Steinbusch, 1981) have shown that the density of serotonergic varicosities in the neuropil surrounding the PVN and SON is greater than in the nuclei themselves, and markers injected in the region of the PVN may have been taken up primarily by nearby fibres-of-passage or terminals. Nevertheless, autoradiographic studies have shown light projections from the dorsal and median raphe nuclei to the PVN and SON in the rat (e.g. Azmitia and Segal, 1978; Moore et al., 1978). A recent examination of the distribution of serotonin-immunoreactive varicosities in the PVN and SON (Sawchenko and Swanson, 1981c) indicates that the innervation of the magnocellular neurosecretory system is sparse, but is concentrated over regions containing oxytocin cell bodies. Based on their density alone, it would seem unlikely that serotonergic inputs are critically involved in the control of oxytocin secretion, a view that is supported by reports that microiontophoretic application of serotonin inconsistently affects (inhibits) the electrical activity of neurosecretory neurones (Barker et al., 1971; Moss et al., 1972). Few other cell groups have been shown to project specifically to areas rich in oxytocin neurones (Fig. 3). A substantial projection to the PVN from a group of ACTHI,,-stained neurones centred in the ventral part of the arcuate nucleus of the hypothalamus has been identified using the combined retrograde transport-immunofluorescence method, and while ACTH-stained varicosities in the PVN are concentrated in the parvocellular division of the nucleus, a clear and preferential input to oxytocin parts of the PVN and SON was identified (Sawchenko et al., 1982). ACTH has been shown to coexist withp-endorphin in single arcuate neurones (e.g. Bugnon et al., 1979), and thusp-endorphin and perhaps other pro-opiomelano-

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Fig. 3. A drawing to illustrate the organization of projections to oxytocin-containing parts of the magnocellular neurosecretory system. The bestknownperipheral stimulusforoxytocin (OXY) release is suckling, and sensory inputs from the mammary glands reach the spinal cord by way of the dorsal root ganglia (DRG). The course and terminal distribution of these pathways are poorly understood. A number of cell groups, located primarily in the forebrain, are now known to project to oxytocin-containing parts of the PVN (PVH in the figure) and the SON. What role, if any, these might play in the primary sensory control of oxytocin secretion remains to be determined. Other abbreviations : ARH, arcuate nucleus; BST, bed nucleus of the stria terminalis; DMH, dorsomedial nucleus of the hypothalamus; DR, dorsal raphe nucleus; MR, median raphe nucleus ; 5-ht, serotonergic pathway; acth, ACTH1-39-containing projection, and see Fig. 2.

cortin-derived peptides may be contained within the projections to magnocellular cell groups. The double-labelling experiments indicate that fewer than half of the retrogradely labelled neurones in the arcuate nucleus were also ACTHI-,,-positive, and this suggests that neurones in this region which contain other neuroactive substances, such as dopamine and acetylcholine (see Renaud, 1979, for a review), may also project to the PVN and SON. In view of the role of the arcuate nucleus in the control of anterior pituitary function, and of electrophysiological evidence indicating that some arcuate neurones project both to the median eminence and to the PVN (Renaud, 1979), this pathway is well situated to coordinate endocrine responses involving both the anterior and the posterior lobes of the pituitary. Additional projections to oxytocin cell groups have been found to arise from the median preoptic nucleus, the subfornical organ (see above), and the dorsomedial nucleus of the hypothalamus (Sawchenko and Swanson, 1983). The functional importance of each of these is quite obscure. Finally, although we have emphasized that noradrenergic projections to the magnocellular neurosecretory system appear to mainly innervate the vasopressin cell groups, some DBH-stained varicosities are found within predominantly oxytocin parts of the PVN and SON (McNeill and Sladek, 1980; Swanson et al., 1981). As ventrally-directed dendrites of both oxytocin- and vasopressin-stained neurones aggregate along the ventral (pial) surface of the SON (Armstrong et al., 1982), a region replete with noradrenergic terminals, the distal dendrites of oxytocin cells in the PVN and SON may also receive an input from the A1 cell group. LIMBIC REGION MODULATION OF NEUROSECRETORY NEURONS There is little evidence for significant neocortical influences on oxytocin or vasopressin secretion. In contrast, it has been known for some time that the limbic region of the telencephalon can influence (generally by inhibition) the electrical activity of neurosecretory neurones (e.g. Pittman et al., 1981;Poulain et al., 1980), and hormone release (see Cross and

25 Dyball, 1974). Retrograde transport studies of the afferent connections of the PVN have found relatively large numbers of retrogradely labelled cells in parts of the septum, amygdala and hippocampal formation (specifically, the ventral part of the subiculum) (Berk and Finkelstein, 1981; Silverman et al., 1981; Tribollet andDreifuss, 1981; Sawchenko and Swanson, 1983). These results, however, contrast sharply with autoradiographicstudies of these regions, which have failed to document significant projections to either the PVN or the SON (Krettek and Price, 1978 ; Swanson and Cowan, 1977, 1979). We have re-examined much of this autoradiographic material and have found that while no inputs to either the PVN or the SON could be identified, projections from each of these regions either end in or traverse regions immediately adjacent to one or both magnocellular nuclei (Sawchenko and Swanson, 1983). For example, the ventral part of the subiculum projects heavily to the nucleus reuniens of the thalamus, and to (or through) the anterior hypothalamic area in a way that outlines (but does not end within) the morphological boundaries of the PVN. Fasciculated fibres from the ventral subiculum and caudal parts of the amygdala traverse the anterior parvocellular part of the PVN in the medial corticohypothalamic tract, however, and may give rise to synapses en passant in the PVN. Despite this possible exception it is clear that the amygdala, the septum and the hippocampal formation do not project in a substantial way to magnocellular cell groups. The results of the retrograde transport studies mentioned above are probably the result of uptake and transport of injected markers by nearby terminals, or by fibres-of-passage. How then, might limbic areas influence the PVN and SON? The bed nucleus of the stria terminalis receives a massive input from the amygdala (Krettek and Price, 1978) and a smaller, but substantial, projection from the ventral subiculum (Swanson and Cowan, 1977). Autoradiographicexperiments have shown that the bed nucleus projects heavily to all parts of the parvocellular division of the PVN, and moderately to the magnocellular division of the PVN and SON (Swanson and Cowan, 1977; Sawchenko and Swanson, 1983). Regions of the magnocellular system in which oxytocin cells predominate appear to be preferentially innervated. Thus, the bed nucleus of the stria terminalis provides a likely route through which information from the limbic system is funneled to reach the PVN and SON. INTRANUCLEAR INTERACTIONS

As dsscribed above, only a handful of cell groups have been shown to project directly to the magnocellular neurosecretory system. However, retrograde transport studies (Berk and Finkelstein, 1981;Silvermanet al., 1981;Tribollet andDreifuss, 1981; Sawchenko andswanson, 1982b, 1983) have suggested that many additional cell groups project to the PVN. A recent analysis of autoradiographic experiments has confirmed the existence of many of these projections and has shown that they end in various parts of the parvocellular division of the PVN (Sawchenko and Swanson, 1983). Among the structures that project primarily to the parvocellular division of the PVN are each recognized cell group (or area) in the hypothalamus and the preoptic region (with the exception of the SON, the mammillary nuclei and the magnocellular preoptic nucleus), the parabrachial nucleus, the locus coeruleus, and the nucleus of the solitary tract (Sawchenko and Swanson, 1981b, 1982b and c, 1983). A careful study of Golgi-impregnated neurones suggests that the axons of parvocellular neurones in the PVN ramify extensively in both the magnocellular and parvocellular parts of the nucleus (van den Pol, 1982). If this can be substantiated, it could broaden considerably the number of cell groups with the potential to influence the magnocellular neurones of the PVN.

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It should be emphasized that, from a light microscopic analysis of autoradiographic material, we cannot exclude the possibility that the cell groups listed above may project, in a minor way, directly to magnocellular neurosecretory cell groups. Furthermore, while more massive pathways are likely to exert a greater influence on a target cell population, mounting evidence for labile ephaptic interactions between neurosecretory neurones (e.g. Andrew et al., 198 1) suggests that under certain physiological conditions it may not be necessary to activate a large number of afferent fibres in order to influence the activity of entire clusters of neurosecretory neurones. DISCUSSION AND CONCLUSIONS The experimental results just described lead to several conclusions about the organization of central pathways that regulate the magnocellular neurosecretory system. First, while many cell groups project to the PVN, relatively few provide inputs that end in the magnocellular division, or in the SON, which does not contain a distinct parvocellular division. Pathways that do reach the magnocellular cell groups are summarized in Figs. 2 and 3. Second, with the possible exception of a projection from the median preoptic nucleus, inputs to the magnocellular neurosecretory system end preferentially, though not necessarily exclusively, in areas that are rich either in oxytocin- or in vasopressin-neurones. This is consistent with the view that secretion of the two hormones is largely independently controlled, although individual neurones may influence both cell types by way of axon collaterals or the diffusion of released neurotransmitter over relatively long distances through the extracellular fluid. Third, inputs that end in magnocellular cell groups display a similar distribution, with respect to regions in which oxytocin or vasopressin cells are concentrated, in both the SON and the magnocellular division of the PVN. This suggests that, regardless of their location within the hypothalamus, the magnocellular neurosecretory cells that produce oxytocin (or vasopressin) may be considered as a functional unit. The major feature that distinguishes the PVN and SON is the association of an elaborately organized parvocellular division with the former, and recent evidence suggests that cells in the parvocellular division project to nearby magnocellular neurones (van den Pol, 1982). The most obvious functional role for such connections would be to integrate the activity of neurosecretory neurones in the PVN with the outputs of the parvocellular division that influence complementary neuroendocrine (by way of the anterior pituitary) and autonomic regulatory mechanisms. Clearly then, circuitry involved in the afferent control of hormone producing neurones in PVN is more complex than in the SON. Even at the light microscopic level of analysis current knowledge of the origins of afferent projections to the magnocellular neurosecretory system is by no means complete. Projections from a number of cell groups to the PVN have been suggested on the basis of retrograde transport studies, and the relationship of projections from these regions to magnocellular cell groups await further analysis. The mesencephalic central grey is the most important example of this, as many functional studies suggest that it influences neurohypophysial hormone secretion (see Cross and Dyball, 1974). A recent survey of the literature (Swanson and Sawchenko, 1983) indicates that over 30 possible neurotransmitters are found in the PVN and SON, and the functional significance of this diversity is only vaguely understood. Ten of these neuroactive substances have been described within cell bodies in the PVN, and there is now evidence that both oxytocin and corticotropin releasing factor are found in some magnocellular neurones, while vasopressin

27 and dynorphin are found in others. Although the functional significance of the co-existence of peptides within neurosecretory neurones is not clear, it is tempting to speculate that inputs from different sources, which release different neuroactive substances, differentially affect the synthesis and (or) release of one or another of the peptides in a particular cell type in the PVN. Whether or not such mechanisms play a role in modulating the output of the PVN and SON, recent pharmacological studies indicate that multiple receptor subtypes for neuroactive substances exist and may mediate different responses (see Swanson and Sawchenko, 1983). Thus, it seems likely that new levels of complexity in the way in which the activity of the magnocellular neurosecretory system is controlled remain to be clarified. ACKNOWLEDGEMENTS The studies summarized here that were carried out in our laboratory were supported by Grant NS-16686 from the National Institutes of Health, a Grant-in-Aid from the American Heart Association - California Affiliate, and by the Clayton Foundation for Research-California Division. The authors are Clayton Foundation Investigators. We are grateful to Ms. Pat Thomas and Mr. Kris Trulock for assistance in the preparation of this manuscript. REFERENCES Andrew, R.D., MacVicar, B.A., Dudek, F.E. and Hatton, G.I. (1981) Dye transfer through gap junctions between neuroendocrine cells of rat hypothalamus. Science, 21 1 : 1187-1 189. Armstrong, W.E., Scholer, J. and McNeill, T.H. (1982) Immunocytochemical, Golgi and electron microscopic characterization of putative dendrites in the ventral glial lamina of the rat supraoptic nucleus. Nec~roscieirt.e,7 : 679-694. Azmitia, E.C. and Segal, M. (1978) An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat. J . comp. Neurol., 179: 641-668. Barker, J.L., Crayton, J.W. and Nicoll, R. (1971) Noradrenaline and acetylcholine responses of supra-optic neurosecretory cells. J . Physiol. (Lond.), 21 8 : 19-32. Berk, M.L. and Finkelstein, J.A. (1981) Afferent projections to the preoptic area and hypothalamic regions in the rat brain. Neuroscience, 6 : 1601-1 624. Blessing, W.W., Sved, A.F. and Reis, D.J. ( I 982) Destruction of noradrenergic neurons in rabbit brainstem elevates plasma vasopressin, causing hypertension. Scienw, 2 17 : 661-663. Bugnon, C., Bloch, B ., Lenys, D., Gouget, A . and Fellman, D. (1979) Comparative study of the neuronal populations containingp-endorphin, corticotropin and dopamine in the arcuate nucleus of the rat hypothalamus. Neuvosci. Lett., 14: 4 3 4 8 . Carisson, A., Falck, B. and Hillarp, N.-W. (1962) Cellular localization of brain monoamines. Actuplzysiul. scand., 56, Suppl. 196: 1-27. Ciriello, J. and Calaresu, F.R. (1977) Lateral reticular nucleus: a site of somatic and cardiovascular integration in the cat. Amer. J . Physiol., 233: RlOGR109. Cross, B.A. and Dyball, R.E.J. (1974) Central pathways for neurohypophyskal hormone release. In Handbook of Physiology, Sect. 7, Val. IV, American Physiological Society, Washington, DC, pp. 269-285. Dahlstrom, A. and Fuxe, K. (1964) Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons. Actu phvsiol. scund., 62, suppl. 232: 1-80. Harris, M.C. (1979) The effect of chemoreceptor and baroreceptor stimulation on the discharge of hypothalamic supraoptic neurones in rats. J . Endocr. 82: 115-125. Hatton, G.I., Hutton, U.E., Hoblitzell, E.R. and Armstrong, W.E. (1976) Morphological evidence for two populations of magnocellular elements in the rat paraventricular nucleus. Bruit1 Res., 108: 187-193. Ingelbrecht, P. (1935) Influence du systkme nerveux central sur la mammelle lactante chez le rat blanc. C.R. SOC. Biol., 120: 1369-1371. Jones, B.J. and Moore, R.Y. (1977) Ascending projections of the locus coeruleus in the rat. 11. Autoradiographic study. Bruin Res., 127: 23-53.

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