A mammalian neural tissue opsin (Opsin 5) is a deep brain photoreceptor in birds.

It has been known for many decades that nonmammalian vertebrates detect light by deep brain photoreceptors that lie outside the retina and pineal organ to regulate seasonal cycle of reproduction. However, the identity of these photoreceptors has so far remained unclear. Here we report that Opsin 5 is a deep brain photoreceptive molecule in the quail brain. Expression analysis of members of the opsin superfamily identified as Opsin 5 (OPN5; also known as Gpr136, Neuropsin, PGR12, and TMEM13) mRNA in the paraventricular organ (PVO), an area long believed to be capable of phototransduction. Immunohistochemistry identified Opsin 5 in neurons that contact the cerebrospinal fluid in the PVO, as well as fibers extending to the external zone of the median eminence adjacent to the pars tuberalis of the pituitary gland, which translates photoperiodic information into neuroendocrine responses. Heterologous expression of Opsin 5 in Xenopus oocytes resulted in light-dependent activation of membrane currents, the action spectrum of which showed peak sensitivity (lambda(max)) at approximately 420 nm. We also found that short-wavelength light, i.e., between UV-B and blue light, induced photoperiodic responses in eye-patched, pinealectomized quail. Thus, Opsin 5 appears to be one of the deep brain photoreceptive molecules that regulates seasonal reproduction in birds.

Distribution of somatostatin immunoreactive neurons and fibres in the central nervous system of a chondrostean, the Siberian sturgeon (Acipenser baeri).

Somatostatin (SOM) is a neuropeptide that is widely distributed in the central nervous system of vertebrates. Two isoforms of somatostatin (SS1 and SS2) have been characterized in sturgeon and in situ hybridisation studies in the sturgeon brain have demonstrated that mRNAs of the two somatostatin precursors (PSS1 and PSS2) are differentially expressed in neurons [Trabucchi, M., Tostivint, H., Lihrmann, I., Sollars, C., Vallarino, M., Dores, R.M., Vaudry, H., 2002. Polygenic expression of somatostatin in the sturgeon Acipenser transmontanus: molecular cloning and distribution of the mRNAs encoding two somatostatin precursors. J. Comp. Neurol. 443, 332-345.]. However, neither the morphology of somatostatinergic neurons nor the patterns of innervation have yet been characterized. To gain further insight into the evolution of this system in primitive bony fishes, we studied the distribution of somatostatin-immunoreactive (SOM-ir) cells and fibres in the brain of the Siberian sturgeon (Acipenser baeri). Most SOM-ir cells were found in the preoptic area and hypothalamus and abundant SOM-ir fibres coursed along the hypothalamic floor towards the median eminence, suggesting a hypophysiotrophic role for SOM in sturgeon. In addition, SOM-ir cells and fibres were observed in extrahypothalamic regions such as the telencephalon thalamus, rhombencephalon and spinal cord, which also suggests neuromodulatory and/or neurotransmitter functions for this peptide. Overall there was a good correlation between the distribution of SOM-ir neurons throughout the brain of A. baeri and that of PSS1 mRNA in Acipenser transmontanus. Comparative analysis of the results with those obtained in other groups of fishes and tetrapods indicates that widespread distribution of this peptide in the brain is shared by early vertebrate lines and that the general organization of the somatostatinergic systems has been well-conserved during evolution.

A molecular census of arcuate hypothalamus and median eminence cell types.

The hypothalamic arcuate-median eminence complex (Arc-ME) controls energy balance, fertility and growth through molecularly distinct cell types, many of which remain unknown. To catalog cell types in an unbiased way, we profiled gene expression in 20,921 individual cells in and around the adult mouse Arc-ME using Drop-seq. We identify 50 transcriptionally distinct Arc-ME cell populations, including a rare tanycyte population at the Arc-ME diffusion barrier, a new leptin-sensing neuron population, multiple agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) subtypes, and an orexigenic somatostatin neuron population. We extended Drop-seq to detect dynamic expression changes across relevant physiological perturbations, revealing cell type-specific responses to energy status, including distinct responses in AgRP and POMC neuron subtypes. Finally, integrating our data with human genome-wide association study data implicates two previously unknown neuron populations in the genetic control of obesity. This resource will accelerate biological discovery by providing insights into molecular and cell type diversity from which function can be inferred.

Sexual dimorphism in the organization of the rat hypothalamic infundibular area.

The hypothalamic infundibular area is located outside the blood-brain barrier and includes, the ventromedial arcuate nucleus (vmARC) sensing circulating substances, and the median eminence (ME) where neurohormones are released into the hypothalamo-hypophysial vasculature. This integrated functional unit, pivotal in endocrine control, adjusts neuroendocrine output to feedback information. Despite a differing physiology in males and females, this functional unit has not appeared differently organized between sexes. Using immunocytochemistry, we describe here for the first time in adult rats, a conspicuous sex-difference in its axonal wiring by intrinsic glutamatergic neurons containing the neuropeptides neurokinin B (NKB) and dynorphin. In the male, NKB neurons send axons to capillary vessels of the vmARC and of the ME (only where gonadotropin-releasing hormone (GnRH) axons terminate). Electron microscopy revealed that NKB axons target the barrier of tanycytes around fenestrated capillary vessels (in addition to GnRH axons), suggesting a control of regional bidirectional permeability. In the female, NKB neurons send axons to the neuropile of the vmARC, suggesting a direct control of its sensor neurons. The other projections of NKB neurons, studied by surgical isolation of the ARC-ME complex and confocal microscopy, are not sexually dimorphic and target both integrative and neuroendocrine centers controlling reproduction and metabolism, suggesting a broad influence over endocrine function. These observations demonstrate that the mechanisms subserving hypothalamic permeability and sensitivity to feedback information are sexually dimorphic, making the infundibular area a privileged site of generation of the male-to-female differences in the adult pattern of pulsatile hormonal secretions.

Brain-derived neurotrophic factor in the brain of Xenopus laevis may act as a pituitary neurohormone together with mesotocin.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, occurs abundantly in the brain, where it exerts a variety of neural functions. We previously demonstrated that BDNF also exists in the endocrine melanotroph cells in the intermediate lobe of the pituitary gland of the amphibian Xenopus laevis, suggesting that BDNF, in addition to its neural actions within the brain, can act as a hormone. In the present study, we tested whether BDNF, in addition to its neural and hormonal roles, can be released as a neurohormone from the neural pituitary lobe of X. laevis. By light immunocytochemistry, we show that BDNF is present in perikarya, in ventrolaterally projecting axons of the hypothalamic magnocellular nucleus and in the neural lobe of the pituitary gland, and that it coexists in these structures with the amphibian neurohormone, mesotocin. The neural lobe was studied in detail at the ultrastructural level. Two types of neurohaemal axon terminals were observed, occurring intermingled and in similar numbers. Type A is filled with round, moderately electron-dense secretory granules with a mean diameter of approximately 145 nm. Type B terminals contain electron-dense and smaller, ellipsoid granules (long and short diameter approximately 140 and 100 nm, respectively). BDNF is exclusively present in secretory granules of type A axon terminals. Double gold-immunolabelling revealed that BDNF coexists in these granules with mesotocin. Furthermore, we demonstrate in an superfusion study performed in vitro that mesotocin stimulates peptide release from the endocrine melanotroph cells. On the basis of these data, we propose that BDNF can act on these cells as a neurohormone.

Role of androgens and glucocorticoids in the regulation of diazepam-binding inhibitor mRNA levels in male mouse hypothalamus.

In peripheral organs, gonadal and adrenal steroids regulate diazepam-binding inhibitor (DBI) mRNA expression. In order to further investigate the involvement of peripheral steroid hormones in the modulation of brain DBI mRNA expression, we studied by semiquantitative in situ hybridization the effect of adrenalectomy (ADX) and castration (CX) and short-term replacement therapy on DBI mRNA levels in the male mouse hypothalamus. Cells expressing DBI mRNA were mostly observed in the arcuate nucleus, the median eminence and the ependyma bordering the third ventricle. In the median eminence and the ependyma bordering the third ventricule, the DBI gene expression was decreased in ADX rats and a single injection of corticosterone to ADX rats induced a significant increase in DBI gene expression at 3 and 12 h time intervals without completely restoring the basal DBI mRNA expression observed in intact mice. In the arcuate nucleus, ADX and corticosterone administration did not modify DBI mRNA expression. CX down-regulated DBI gene expression in the ependyma bordering the third ventricle. The administration of dihydrotestosterone (3-24 h) completely reversed the inhibitory effect of CX. In the median eminence and arcuate nucleus, neither CX or dihydrotestosterone administration modified DBI mRNA levels. These results suggest that the effects of glucocorticoids on the hypothalamo-pituitary-adrenocortical axis and androgens on the hypothalamo-pituitary-gonadal axis are mediated by DBI.

Co-expression of vasopressin and androgen-binding protein in the rat hypothalamus.

In previous studies we have observed the expression of androgen binding protein (ABP) in the rat hypothalamo-neurohypophysial system. With immunocytochemical double staining we found partial co-localization with oxytocin. In the present study we used antibodies to the anti-diuretic hormone arginine vasopressin (AVP) for co-localization with ABP in the rat hypothalamus. Both antigens were seen in the magnocellular paraventricular and supraoptic nuclei. Dense fiber networks with varicosities containing both AVP and ABP immunoreactivity were visible throughout the hypothalamus, the median eminence and in the posterior pituitary lobe. Double immunostaining revealed also co-existence in the parvocellular portion of the paraventricular nucleus and in the suprachiasmatic nucleus. ABP immunoreactive neurons in the preoptic region were devoid of AVP staining, AVP neurons in the bed nucleus of the stria terminalis stained only occasionally for ABP. We conclude that both the magnocellular and the parvocellular hypothalamic vasopressin systems are capable of expressing the steroid binding globulin, which is probably subject to axonal transport, along with the peptide hormone. Intrahypothalamic expression of ABP may be among the mechanisms necessary for rapid actions of steroids on hypothalamic neuroendocrine systems.

A novel stereotaxic approach to the hypothalamus for the use of push-pull perfusion cannula in Holstein calves.

To determine secretory patterns of growth hormone-releasing hormone (GHRH) and somatostatin (SS) and their roles in the regulation of growth hormone (GH) secretion, a method for collecting hypothalamic perfusates, a push-pull perfusion method was developed in calves. With the use of the stereotaxic apparatus for cattle, a cannula was implanted into the hypothalamus of four male calves based upon cerebral ventriculography. Push-pull perfusates were collected at 10 min intervals for 6h and GHRH and SS concentrations in perfusates and plasma GH concentration were determined by EIAs and RIA, respectively. A cannula was implanted into the hypothalamus based on the image of the third ventricle and maintained for 1 month. GHRH and SS showed pulsatile secretion and the pulses for GHRH and SS were irregular in conscious animals. Neither GHRH nor SS secretion had a clear relationship with GH secretion. In the present study, we thus (1) established a stereotaxic technique for approaching the hypothalamus using cerebral ventriculography for calves, and (2) demonstrated that GHRH and SS secretion were pulsatile but not closely related to GH profile in conscious calves. The technique is useful for the study of the functions of the hypothalamus in the control of pituitary hormones in cattle.

The course of thyrotropin-releasing hormone fibers to the median eminence in rats.

The topographical distribution of fibers containing TRH that run to the median eminence has been investigated by RIA of TRH after hypothalamic surgical transections in rats. Anterolateral or lateral cuts bilaterally transecting fibers at the caudal edge of the optic chiasm (lateral retrochiasmatic area) diminish TRH levels in the median eminence by 95% and 73%, respectively. Paramedian cuts are ineffective. These results suggest that TRH fibers reach the medial basal hypothalamus from an anterolateral direction just as several other classes of peptide containing fibers do.

Neurosecretory pathways in the mallard duck (Anas platyrhynchos) brain: localization by aldehyde fuchsin and immunoperoxidase techniques for neurophysin (NP) and gonadotrophin releasing hormone (Gn-RH).

Localization studies of the hypothalamohypophysial and tuberoinfundibular neurosecretory systems were performed in the adult male mallard duck with an immunoperoxidase techinque for the demonstration of neurophysin (NP) and gonado-tropin-releasing hormone (Gn-RH) and with aldehyde fuchsin for the staining of neuosecretory material (NSM). A comparison was made between the distribution of NSM stained with aldehyde fuchsin and NP seen by immunocytochemistry. The magnocellular perikarya of the supraoptic (SON) and paraventricular (PVN) nuclei, the zona externa of the anterior median eminence (ME), the fiber layer of both the anterior and posterior ME, and small neurons in the tractus quintofrontalis were stained by both the immunoperoxidase method for NP and by the aldehyde fuchsin stain. In contrast, the parvocellular neurons of the PVN, extra-hypothalamic neurosecretory fibers dorsal to the anterior commissure in the septal region and tanycytes lining the ventral 1/3 of the third ventricle at the level of the anterior ME, were stained only by the immunocytochemical procedure for NP. These observations indicate that immunocytochemistry is more sensitive than aldehyde fuchsin staining for detecting low concentrations of NP in cells and tissues, but the two techniques produce comparable results where the concentration of the NP is relatively high. Two populations of beaded axons containing Gn-RH were distributed throughout the zone externa of both the anterior and posterior ME. One group of fibers paralleled the hypothalamo-hypophysial neurosecretory tract whereas the other was distributed in the contact zone of the ME. Immunoreactive Gn-RH was found in the cytoplasm of a sparse population of cell bodies in the dorsolateral portion of the arcuate nucleus as well as in the axons that project from this nucleus ventrally towards the ME.

Brainstem afferents to the thalamus in a lizard, Varanus exanthematicus.

HRP was injected into various thalamic nuclei in order to investigate the brainstem projections to the thalamus in the lizard Varanus exanthematicus. Nucleus dorsomedialis receives afferents from the septal area, nucleus entopeduncularis anterior, nucleus periventricularis hypothalami, area triangularis, nucleus raphes superior, nucleus reticularis inferior, and locus coeruleus. Nucleus dorsolateralis receives afferents from septal area, nucleus dorsomedialis, nucleus entopeduncularis anterior, nucleus periventricularis hypothalami, and the torus semicircularis. Nucleus rotundus receives an input from the tectum mesencephali, the pretectal area, and from the mesencephalic reticular formation. Nucleus intermedius dorsalis receives afferents from the dorsal column nuclei and nucleus periventricularis hypothalami. Nucleus ventrolateralis receives afferents from the dorsal column nuclei, the trigeminal complex, locus coeruleus, and the reticular formation. Nucleus ventromedialis also receives afferents from the trigeminal complex and the reticular formation. Afferents to the habenula have been demonstrated from the septal area, nucleus entopeduncularis anterior, triangular area, nucleus periventricularis hypothalami, nucleus interpeduncularis, nucleus raphes superior, locus coeruleus, nucleus isthmi, nucleus dorsalis motorius nervi vagi, and the mesencephalic tegmentum. The laminar part of the torus semicicularis projects to nucleus medialis.

Ultrastructure and synaptic organization of luteinizing hormone-releasing hormone (LHRH) neurons in the anestrous ewe.

Electron microscopic immunocytochemistry was employed to examine the ultrastructure of luteinizing hormone-releasing hormone (LHRH) neurons and their projections to the median eminence in the sheep brain. LHRH perikarya in the preoptic area of anestrous ewes are less innervated than nonimmunoreactive cells in the same sections, but still receive numerous synaptic inputs, primarily onto distal dendrites and small somatic protuberances. Axon terminals synapsing upon LHRH cells contain a combination of clear spherical vesicles and larger dense-core vesicles. Interestingly, LHRH cell bodies and dendrites are almost entirely surrounded by glial processes. These processes intervene between immunoreactive elements that at a light microscopic level appear to be in contact with each other. Thus no evidence was obtained at the ultrastructural level for contacts among adjacent LHRH cells or dendrites in the preoptic area. Synaptic inputs onto LHRH cell bodies and dendrites appear to penetrate this glial sheath. In contrast to the absence of contacts among LHRH cells in the preoptic area, individual LHRH terminals in the median eminence are often clustered in direct plasma membrane contact. Comparisons between animals of differing reproductive status are needed to determine whether alterations in synaptic inputs, glial ensheathment, or LHRH-LHRH appositions, may underlie seasonal changes in the activity of LHRH neurons.

CRF-containing neuron systems in the rat hypothalamus: retrograde tracing and immunohistochemical studies.

By employing a combination of the immunohistochemistry for rat corticotropin-releasing factor (rCRF) and retrograde tracing with biotinylated wheat germ agglutinin (b-WGA) injected into the posterior pituitary (group 1) or into the middle portion of the median eminence (group 2), functionally different populations of CRF neurons were identified in the rat hypothalamus. In the group 1 animals, WGA-labeling was seen not only in the posterior lobe but also in the intermediate lobe, in which CRF fibers exist. In these animals, WGA-labeling occurred for almost all large neurons in the anterior commissural, paraventricular (PV), and supraoptic (SO) nuclei, some of the neurons showing a slight immunoreactivity for anti-rCRF. Conversely, CRF positive neurons appeared in large numbers, some being labeled with WGA, in the caudal periventricular region (CPR), and in the dorsomedial to lateral hypothalamic area (DLH), especially in the latter. In the group 2 animals, WGA was disparsed throughout the subependymal, internal, and external layers of the medial portion of the median eminence, and was taken up by many small cells in the PV, almost half of the cells being immunoreactive for CRF. Slight WGA-labeling further occurred in some large neurons of the PV and SO, and in some cells of the DLH. It is concluded that hypophysiotropic CRF existing in the external layer of the median eminence originates from small neurons located in the PV, whereas CRF distributed in the posterior and intermediate pituitary originates from the magnocellular PV and SO, and from some neurons in the DLH and CPR.

Habenular asymmetry and the central connections of the parietal eye of the lizard.

Histochemical and autoradiographic analyses of the axonal transport of horseradish peroxidase and tritiated amino acids were employed to study the central connectivity of the lizard parietal eye. Somata and processes of centrifugal fibers to the parietal eye were localized in tissue of the dorsal sac and in the leptomeningeal sheath of the pineal gland. Analyses of series of transverse sections of the brain showed the left medial habenular nucleus to be subdivided into pars dorsolateralis and pars ventromedialis, and the right medial habenular nucleus not to be so subdivided. Centripetal fibers of parietal eye ganglion cells project to only the pars dorsolateralis of the left medial habenular nucleus and terminate there in two distinct fields. The asymmetry of the lizard habenula may be a specialization associated with the unilateral projection from the parietal eye.

Vasoactive intestinal peptide in the hypothalamohypophysial system of the Mongolian gerbil.

By use of the indirect peroxidase-antiperoxidase immunohistochemical technique the location of perikarya and fibers exhibiting vasoactive intestinal peptide (VIP)-like immunoreactivity was investigated in the hypothalamus and the posterior pituitary of the Mongolian gerbil (Meriones unguiculatus), because of the involvement of VIP in several neuroendocrine functions. In the hypothalamus, a large number of VIP-immunoreactive perikarya were seen in the ventromedial part of the suprachiasmatic nucleus. Few VIP-positive perikarya were present in the periventricular, paraventricular, and supraoptic nuclei and in the medial preoptic area close to the third ventricle. The perikarya in the paraventricular nucleus projected fibers in the direction of the median eminence. In the median eminence VIP-immunoreactive fibers were present especially in the external layer, concentrated in the perivascular spaces surrounding the portal vessels. Scattered VIP-immunoreactive fibers were also located in the internal layer of the median eminence as well as in the posterior pituitary lobe. In the latter, large VIP-positive Herring-like bodies were observed. With receptor autoradiography a large number of grains were demonstrated in the anterior pituitary lobe in contrast to the neural lobe. Many VIP-positive fibers and some perikarya were observed within the ependyma covering the rostroventral part of the third ventricle. Finally, fibers exhibiting VIP immunoreactivity were also seen in the organum vasculosum laminae terminalis (OVLT). These results support the concept that VIP is released into the portal vascular system and plays a role in the regulation of the activity of the anterior pituitary. In addition, VIP might be secreted into the cerebrospinal fluid of the third ventricle.

Organization of afferent connections of the feline lateral habenular nucleus.

The organization of afferent projections to the lateral habenular nucleus (LHB) was studied in adult cats. In autoradiographic experiments, tritiated leucine injections were made into regions shown in retrograde transport studies to project to LHB (lateral hypothalamus, preoptic region, entopeduncular nucleus, and midbrain raphe) to determine the locations of axonal trajectories and terminal zones. The distribution of silver grains in the ipsilateral LHB differed according to the injection site. Entopeduncular nucleus projected to the ventrolateral part, raphe to the dorsomedial part, preoptic region to the central part, anterior hypothalamus to all but the ventrolateral part, and posterior hypothalamus to all but the dorsomedial part. The lateral hypothalamus, preoptic region, and anterior portion of the entopeduncular nucleus projected primarily through the inferior thalamic peduncle and stria medullaris, while the posterior portion of the entopeduncular nucleus projected more diffusely through thalamus to enter LHB from its ventral aspect. Raphe axons reached LHB through the fasciculus retroflexus. Entopeduncular and lateral hypothalamic axons passed through the habenular commissure to the contralateral stria medullaris to reach the contralateral LHB. Silver grains in LHB were distributed similarly bilaterally, but were much less dense contralateral to the injection. The major afferent projections to the feline LHB are therefore topographically organized. The significance of this organization in relation to the possible role of the LHB in integrating limbic and extrapyramidal motor systems is discussed.

Afferent connections of the interpeduncular nucleus and the topographic organization of the habenulo-interpeduncular pathway: an HRP study in the rat.

The HRP tracing method was employed to investigate the organization and afferent connections of the interpeduncular nucleus (IPN) in the rat. To study the topographical features of the different projections, a method was devised for obtaining HRP placements of limited size in different areas of the IPN. The main afferent connection of the IPN is a topographically organized projection from the medial habenula (Hb). This projection follows a reversed caudorostral pattern, terminating throughout all but the caudalmost part of the IPN. The dorsal part of the IPN receives a sparse innervation arising mainly from a narrow lateral and ventrolateral area of the medial Hb. The ventral two thirds of the IPN receives a much heavier projection, as follows: A large ventrolateral area of the medial Hb projects to the lateral part of the IPN in a completely bilateral way. An additional projection, which is predominantly ipsilateral, arises from the rostral half of the dorsolateral part of the medial Hb and terminates in the caudal IPN. The medial part of the medial Hb projects preferentially to central areas of the IPN. The projection from the lateral Hb is quantitatively much smaller but appears to be distributed to the entire length of the IPN, following a nonreversed caudorostral arrangement, with the ipsilateral projection predominating. The projections from the medial and lateral Hb to the IPN were confirmed by tracing anterogradely transported HRP as well. No reciprocal connection from the IPN to the Hb could be demonstrated. A sparse projection to the IPN with a strong ipsilateral predominance arises from the horizontal limbs of the nucleus of the diagonal band of Broca. This was the only projection observed from the septal region. Sparse projections from the premammillary and supramammillary nuclei were also demonstrated. Confirmatory data and some details of organization were also obtained for projections to the IPN from other areas, including the medial and dorsal raphe nuclei, the dorsal tegmental nucleus of Gudden, and the adjacent dorsolateral tegmental nucleus. Very small projections from the ventral tegmental nucleus and the locus coeruleus were also found.

Cells of origin of the afferent fibers to the median eminence in the rat.

The cell bodies of origin of axons terminating in the median eminence have been identified by retrograde axonal transport of horseradish peroxidase (HRP) or 125I-wheat germ agglutinin (WGA). The tracers were injected into the median eminence by pressure and under direct visual control, using a ventral surgical approach. The retrogradely labeled cells are exclusively located within the hypothalamus. The most heavily labeled cells are parvocellular neurons in the arcuate nucleus, the periventricular area, the medial part of the paraventricular nucleus, and the rostral paraventricular nucleus; a few cells are also located in the rostral part of the preoptic area immediately lateral and dorsal to the organum vasculosum of the lamina terminalis (OVLT). Less heavily located cells are found in the magnocellular neurosecretory nuclei, including the lateral parts of the paraventricular and rostral paraventricular nuclei, the supraoptic nucleus, and the accessory magnocellular nuclei. Retrogradely labeled cells are not found in the ventromedial hypothalamic nucleus, except for a few lightly labeled cells in the posterior division of the nucleus. However, if the injected tracer spreads into the arcuate nucleus, labeled cells are present throughout the ventromedial nucleus. Labeled cells are not found in other parts of the hypothalamus, including lateral and posterior portions of the preoptic area, the anterior hypothalamic area, and the suprachiasmatic nucleus, or in catecholaminergic cell groups of the midbrain, pons, and medulla. Control injections of HRP into the posterior pituitary and the ventromedial nucleus produce patterns of cell labeling which are very distinct from that seen with injections into the median eminence. Following injections into the posterior pituitary, the cells of the magnocellular neurosecretory nuclei are all heavily labeled, but small cells in the parvocellular neuronal groups are not labeled. Direct injections into the ventromedial nucleus resulted in labeled cells in widespread parts of the hypothalamus, as well as in the bed nucleus of the stria terminalis and the lateral septum, in parts of the amygdaloid complex and the subiculum, and in several cell groups in the midbrain, pons, and medulla.

The subnuclear organization of the rat interpeduncular nucleus: a light and electron microscopic study.

The subnuclear organization of rat interpeduncular nucleus (IPN) has been examined by light microscopy following staining with Nissl and Holmes methods, 3H-leucine autoradiography, acetylcholinesterase (AChE), and cytochrome oxidase histochemistry on plastic sections stained with toluidine blue, and by electron microscopy. Three unpaired and four paired subnuclei are recognized. The rostral subnucleus is heavily stained for AChE, which clearly delineates its borders. It is distinguished ultrastructurally by two types of synapses on dendrites, and two on perikarya. Of the former, one type is formed by presynaptic processes which contain spherical and dense-cored vesicles and make asymmetrical contacts. Dense-cored vesicles are observed in many of the postsynaptic dendrites. A second type has presynaptic processes containing small, pleomorphic vesicles which make symmetrical contacts. Synapses on perikarya are found in the rostral, central, intermediate, lateral, and interstitial subnuclei. The dorsal subnucleus is continuous with the serotonin-containing B8 cells. The central subnucleus is distinguished by longitudinally oriented medial habenular axons separating palisades of cell bodies. These axons, which also traverse the intermediate subnuclei, form en passant S synapses with small dendrites of the central subnucleus. The intermediate subnuclei react faintly for AChE and intensely for cytochrome oxidase. They contain crest synapses formed by two habenular afferents, one from each medial habenula, which contact a narrow dendritic process en passant. The lateral subnuclei react intensely for AChE and have ultrastructural features similar to the rostral subnuclei. The interstitial subnuclei lie within each fasciculus retroflexus as it enters IPN. The small dorsal lateral subnuclei are evident by light microscopy.

Distribution and projections of cholecystokinin-immunoreactive neurons in the hypothalamic paraventricular nucleus of rat.

Analysis of coronal sections from colchicine-treated rat brains reveals that CCK-immunoreactivity (CCK-ir) is present in two distinguishable neuronal systems in the paraventricular nucleus (PVN). More than 60% of these cells were found to be typical parvicellular neurons; the remainder were magnocellular neurons. The magnocellular CCK-ir neurons were concentrated in the medial magnocellular subdivision, while more caudally they formed a ring around a zone of unstained magnocellular neurons. Immunostained parvicellular neurons predominate in medial and periventricular parvicellular subdivisions. The efferent projections of CCK-ir neurons were investigated by looking for retrograde accumulation of CCK-ir in cell bodies after selective knife cuts. A parasagittal cut of the lateral retrochiasmatic area as well as transection of the rostral median eminence resulted in an accumulation of CCK-ir material in a large number of both parvi- and magnocellular neurons. After pituitary stalk lesions, however, increased staining was only seen in magnocellular neurons. It is inferred that the magnocellular (presumed oxytocin-CCK) cells send their axons to the pituitary, whereas axons of CCK-ir parvicellular neurons appear to terminate in the median eminence. After transection of the medial forebrain bundle (MFB), immunostaining increased in a small number of scattered transected fibers proximal to the knife cut and in a few perikarya in the PVN, indicating that very few CCK cells may send descending fibers to the lower brainstem.