Light and electron microscopic immunocytochemistry of neurotensin-like immunoreactive neurons in the rat hypothalamus

Neurotensin-like immunoreactive neuronal perikarya, fibers and terminals in the rat hypothalamus, particularly in the arcuate nucleus, the paraventricular nucleus and the median eminence, were investigated by light and electron microscopic immunocytochemistry. The main distributional areas of immunoreactive neuronal perikarya were found to be the arcuate nucleus, the periventricular nucleus and the paraventricular nucleus by light microscopic immunocytochemistry. Immunoreactive neuronal perikarya showed a characteristic distributional pattern in the arcuate nucleus. In the paraventricular nucleus they were distributed in both the magnocellular and parvocellular portions. A large number of immunoreactive terminals were observed throughout the external layer of the median eminence, particularly its lateral portion. A moderate number of immunoreactive terminals were also observed in the internal layer of the median eminence. By electron microscopic immunocytochemistry immunoreactive neuronal perikarya both in the arcuate and paraventricular nuclei showed generally well-developed cell organelles such as mitochondria, r-ER, and Golgi complex. In addition, immunoreactive dense granules were dispersed throughout the perikarya. A large number of immunoreactive terminals containing immunoreactive dense granules, clear vesicles and mitochondria were observed in the vicinity of pericapillary spaces of the external layer of the median eminence. This observation strongly suggests that neurotensin-like immunoreactive substance is released into the portal capillaries.     

Light and electron microscopic immunocytochemistry of β-endorphin/β-LPH-like immunoreactive neurons in the arcuate nucleus and surrounding areas of the rat hypothalamus

β-Endorphin/β-LPH-like immunoreactive neurons in the hypothalamic arcuate nucleus and its surrounding areas were visualized by light and electron microscopic immunocytochemistry. Immunoreactive processes were found in the vicinity of the pia mater, in the lateral part of the external layer of the median eminence and near the lateral wall of the third ventricle. Neuronal perikarya contained immunoreactive dense granules as well as developed cell organellae. They received neuronal inputs from other neurons through axoplasmic and axodendritic synapses. Immunoreactive neuronal processes containing dense granules and mitochondria were found as preterminal elements on non-immunoreactive neuronal soma and dendrites. Immunoreactive processes also make intimate contact with capillaries in the arcuate nucleus near the median eminence.     

Some cellular characteristics of somatostatin neurons and terminals in the periventricular nucleus of the rat hypothalamus and median eminence. Electron microscopic immunohistochemistry

Somatostatin neuronal perikarya and their processes, presumably dendrites, in the periventricular nucleus of the rat hypothalamus and terminals in the median eminence were observed by electron microscopic immunohistochemistry. Neuronal perikarya and processes contained immunoreactive dense granules (100-120 nm in diameter) and other cellular components such as polysomes, rER membranes occasionally showed high electron density. Few axo-somatic terminals were found on the somatostatin neurons, but we could detect a number of preterminal axons on immunoreactive processes, presumably dendrites. Therefore, we considered that somatostatin neurons receive mainly neuronal input through axo-dendritic synapses rather than through axo-somatic ones. In the somatostatin terminals in the external layer of the median eminence immunoreactivity was completely restricted on the granules.     

Immunocytochemical demonstration of dynorphin(PH-8P)-like immunoreactive elements in the human hypothalamus

PH-8P (dynorphin[1-8])-like immunoreactive neuronal perikarya, processes, and terminals located within the human hypothalamus were investigated by the avidin-biotin peroxidase complex (ABC) immunocytochemical procedure. Immunopositive neurons were distributed throughout the hypothalamus. The distributional pattern was found to be similar to that in other mammalian species by the use of antisera against dynorphin. A large number of immunoreactive neuronal perikarya were detected in the supraoptic nucleus (SON) and the magnocellular portion of the paraventricular nucleus (PVN). Their processes appeared to project to the posterior pituitary via the internal layer of the median eminence and their distribution seemed to be less dense than in other mammalian species. PH-8P and vasopressin were colocalized in the neuronal perikarya in the human SON unlike the colocalization of these peptides in the rat SON and PVN. There were a few immunoreactive terminals in the external layer of the median eminence; their immunoreactive substances may be released into the portal veins to act on anterior pituitary cells. In addition, PH-8P-like immunoreactive neurons in the human hypothalamus may project to the extrahypothalamic area.     

Immunohistochemical mapping of neuropeptides in the premamillary region of the hypothalamus in rats

The topographical distribution of neuropeptide-containing cell bodies, fibers and terminals was studied in the premamillary region of the rat hypothalamus using light microscopic immunohistochemistry. Alternate coronal sections through the posterior third of the hypothalamus of normal and colchicine-treated male rats were immunostained for 19 different neuropeptides and their distributions were mapped throughout the following structures: the ventral and dorsal premamillary, the supramamillary, the tuberomamillary and the posterior hypothalamic nuclei, as well as the premamillary portion of the arcuate nucleus and the postinfundibular median eminence. Seventeen of the investigated neuropeptides were present in neuronal perikarya, nerve fibers and terminals while the gonadotropin associated peptide and vasopressin occurred only in fibers andn terminals. Growth hormone-releasing hormone-, somatostatin-, α-melanocyte stimulating hormone-, adrenocorticotropin, β-endorphin- and neuropeptide Y-immunoreactive neurons were seen exclusively in the premamillary portion of the arcuate nucleus. Thyrotropin-releasing hormone-, dynorphin A- and galanin-containing neurons were distributed mainly in the arcuate and the tuberomamillary nuclei. A high number of methionine- and leucine-enkephalin-immunoreactive cells were detected in the arcuate and dorsal premamillary nuclei, as well as in the area ventrolateral to the fornix. Substance P-immunoreactive perikarya were present in very high number within the entire region, in particular in the ventral and dorsal premamillary nuclei. Cell bodies labelled with cholecystokinin- and calcitonin gene-related peptide antisera were found predominantly in the supramamillary and the terete nuclei, respectively. Corticotropin-releasing hormone-, vasoactive intestinal polypeptide- and neurotensin-immunoreactive neurons were scattered randomly in low number, mostly in the arcuate and the ventral and dorsal premamillary nuclei. Peptidergic fibers were distributed unevenly throughout the whole region, with each peptide showing an individual distribution pattern. The highest density of immunoreactive fibers was presented in the ventral half of the region including the arcuate, the ventral premamillary and the tuberomamillary nuclei. The supramamillary nucleus showed moderately dense fiber networks, while the dorsal premamillary and the posterior hypothalamic nuclei were poor in peptidergic fibers.     

Immunohistochemical localization of avian pancreatic polypeptide-like immunoreactivity in the rat hypothalamus

The distribution of avian pancreatic polypeptide-like (APP) immunoreactivity within the rat hypothalamus was investigated with the indirect immunoperoxidase method. APP immunoreactive perikarya are found in largest numbers in the retrochiasmatic area, the arcuate nucleus, and the supracommissural portion of the interstitial nucleus of the stria terminalis. Small clusters of immunoreactive neurons are also consistently observed in the ventral aspect of the medial preoptic area and lateral hypothalamic area, immediately dorsolateral to the optic chiasm and tracts. These neurons are apparent in all animals but are more intensely strained and occur in larger numbers following colchicine pretreatment. Other immunoreactive neurons are visible only in colchine-treated rats and are scattered throughout the anterior and lateral hypothalamic areas and the supramammillary nucleus. Immunoreactive axons and terminal fields present an extensive and highly characteristic distribution throughout the hypothalamus, which in many instances exhibits differential distribution within specific subfields of hypothalamic nuclei and areas. The heaviest concentrations of APP immunoreactive axons are present in the periventricular nucleus throughout the rostrocaudal extent of the hypothalamus, the ventrolateral portion of the suprachiasmatic nucleus, the retrochiasmatic area, the parvocellular paraventricular nucleus, the ventral supraoptic nucleus, the perifornical nucleus, the ventral dorsomedial nucleus, and the arcuate nucleus. Moderate plexuses of immunoreactive fibers are also present in the medial preoptic area, the anterior and lateral hypothalamic areas, the nucleus circularis, the median eminence, and the ventral premammillary area. Other areas, such as the ventromedial nucleus, contain virtually no immunoreactive axons but are encapsulated by a dense plexus of immunoreactive terminals. The distribution of a major component of APP immunoreactive fibers exhibits a marked similarity to that of previously described norepinephrine-containing hypothalamic afferents. Other groups of APP immunoreactive perikarya and fibers appear to represent components of intrinsic diencephalic systems.     

Immunohistochemical evidence for synaptic connections between pro-opiomelanocortin-immunoreactive axons and LH-RH neurons in the preoptic area of the rat

Connections between adrenocorticotropic hormone (ACTH)-immunoreactive neurons in the arcuate nucleus and the preoptic area were studied in the female rat. ACTH-immunopositive terminals were observed in the medial preoptic area in contact with dendritic shafts, while in the ventrolateral preoptic area the majority of ACTH-immunoreactive synapses were found on dendritic spines. Double-label electron microscopic immunocytochemistry using peroxidase and avidin-ferritin as contrasting electron-dense markers revealed numerous synaptic contacts between ACTH-immunopositive boutons and luteinizing hormone-releasing hormone (LH-RH)-immunoreactive dendritic shafts in the medial preoptic area. Following injection of horseradish peroxidase (HRP) into the medial preoptic area, retrogradely HRP-labeled perikarya were observed throughout the arcuate nucleus. Double-staining experiments revealed that a proportion of these retrogradely labeled cells, in the ventromedial arcuate nucleus, are also immunoreactive for ACTH. These results suggest that pro-opiomelanocortin peptide-producing neurons in the ventromedial arcuate nucleus project to the medial preoptic area. Some of these neurons establish direct synaptic contacts with LH-RH-immunoreactive cells.     

Light and electron microscopic study of tyrosine hydroxylase-immunoreactive neurons within the developing rat arcuate nucleus

The topography, fine structure, and patterns of connections of tyrosine hydroxylase (TH)-immunoreactive tubero-infundibular dopaminergic (TIDA) neurons were examined by light and electron microscopic immunocytochemistry in the arcuate nucleus of 2-, 15- and 30-day-old female Wistar rats. In 2-day-old animals, TH-immunoreactive perikarya were mainly located in the ventrolateral portion of the arcuate nucleus. In 15-day-old rats numerous TH-positive cell bodies were still present ventrolaterally, but a cluster of labeled cells was also apparent in the mediodorsal segment of the nucleus. In the 30-day-old rats, most TH-immunoreactive neurons were concentrated mediodorsally, as seen in the adult. At the ultrastructural level, TH-immunoreactive somata exhibited, in all age groups, a large nucleus surrounded by a thin rim of cytoplasm containing mitochondria, Golgi apparatus, endoplasmic reticulum, multivesicular bodies and lysosomes. These labeled somata were synaptically contacted by unlabeled axon terminals and often laid adjacent to either labeled or unlabeled dendrites. Similarly, in all age groups, labeled dendrites were synaptically contacted by unlabeled axon terminals and were often directly apposed to either labeled or unlabeled perikarya and dendrites, or to tanycytic processes. These results indicate that TIDA neurons establish extensive connections early in development, and that their pattern of intercellular relationships remains qualitatively unchanged from 2 days to adulthood. It is suggested that TIDA neurons may be already functional at birth, and could therefore, influence the maturation of other arcuate neuronal populations.     

The fine structures of the VIP-Like immunoreactive neurons in the cat hypothalamus

The fine structures of the VIP-like immunoreactive neurons in the suprachiasmatic nucleus (SCN) and the arcuate nucleus ( ARN ) of the cat hypothalamus were investigated by electron microscopic immunocytochemistry. The VIP-like immunoreactive soma and fibers could be successfully visualized by a modified PAP method. VIP-like immunoreactive neurons in both nuclei contained immunoreactive rER, Golgi complexes and many immunoreactive granules, as well as well developed mitochondria. VIP-like immunoreactive synaptic endings with synaptic membrane specialization of Gray's type I and II were found in the SCN. Moreover VIP-like immunoreactive preterminal elements that made synaptic contact with VIP-like immunoreactive neuronal soma were also detected. On the other hand, it was difficult to detect typical preterminal endings with immunoreactivity in the ARN ; however, VIP-like immunoreactive processes in contact with the basement membrane of the capillaries were observed. These observations indicate that VIP-like immunoreactive neurons in the SCN act as intrinsic neurons and are involved in neuroendocrine function in ARN .     

GRF immunoreactive neurons in the paraventricular nucleus of the rat: an immunohistochemical study with monoclonal and polyclonal antibodies

Our study demonstrates a complex GRF neuronal system within the rat hypothalamus. Using both high affinity polyclonal and high specificity monoclonal antibodies to rat (r) GRF, we have substantiated evidence for immunoreactive GRF (GRF-i) perikarya in the parvocellular portion of the paraventricular nucleus. Other hypothalamic areas containing rGRF-positive perikarya include the lateral arcuate nucleus, lateral hypothalamus, perifornical area and dorsomedial nucleus. GRF-i neuronal terminals were seen in the external zone of the median eminence, more rostrally in the periventricular nucleus, and near the suprachiasmatic nucleus and more caudally in the dorsomedial nucleus and ventral premammillary nucleus.     

Light and electron microscopic study of somatostatin-like immunoreactive neurons in rat hippocampus

The distribution and fine structure of somatostatin-like immunoreactive neurons in rat hippocampus and gyrus dentatus were investigated by light and electron microscopic immunocytochemistry. Somatostatin-like immunoreactive neuronal perikarya and fibers could be visualized by using modified PAP immunocytochemistry. Immunoreactive neurons were selectively localized in the stratum orience of the hippocampus and hilar region of the gyrus dentatus, however, immunoreactive neurons were also sparsely observed throughout hippocampal formation. Somatostatin-like immunoreactive varicosities were abundantly distributed in the stratum pyramidale and some of them were considered to terminate on the pyramidal cells. Somatostatin-like immunoreactive neurons in the hippocampal formation generally contained many mitochondria, well-developed rough surfaced endoplasmic reticulum (rER), polysomes and some dense granules. Immunoreactivity was observed especially in the dense granules and membrane of rER. Pre- and post-synaptic elements of somatostatin-like immunoreactive neurons were detected throughout the hippocampal formation.     

Luteinizing Hormone-Releasing Hormone Neurons which are R-etrogradely Labeled After Peripheral Fluoro-Gold Administration in the Male Ferret

This study identified luteinizing hormone-releasing hormone (LHRH)-producing neurons which have access to fenestrated capillaries in prepubertal male European ferrets. Fluoro-Gold was injected intraperitoneally to retrogradely label neurons with terminals outside the blood-brain barrier. LHRH neurons were identified by immunofluorescence using a secondary antibody tagged with tetramethylrhodamine isothiocyanate. Cell bodies which demonstrated both tetramethylrhodamine isothiocyanate and Fluoro-Gold fluorescence were defined as LHRH-producing neurons with axon terminals in regions containing fenestrated capillaries. The total number and neuroanatomical distribution of immunopositive (LHRH +) cells concurred with previous studies in the ferret in which cell bodies were diffusely distributed from rostral forebrain through caudal diencephalon, with approximately 70% of the LHRH + cell bodies located in retrochiasmatic hypothalamus. In the present study, an average of 59.8% of all LHRH+ neuronal perikarya also contained Fluoro-Gold. The majority of Fluoro-Gold filled LHRH+ neurons demonstrated only faint to moderate amounts of Fluoro-Gold when compared to other Fluoro-Gold filled neurosecretory neurons. This limited uptake of Fluoro-Gold may be due to a relative inactivity of LHRH neurons projecting outside the blood-brain barrier. Double-labeled LHRH + neurons were dispersed throughout the entire population of LHRH+ cell bodies and no apparent nuclear groups of double-labeled neurons were found. This observation suggests that the LHRH+ neurons responsible for neurosecretion into the median eminence coexist with the LHRH+ neurons responsible for intracerebral neurotransmission or neuromodulation. One distinguishable population of LHRH + neurons was consistently observed in all the brains. Only 26% of total LHRH+ perikarya within the caudal arcuate nucleus contained Fluoro-Gold, while at least 50% of LHRH+ neurons in other structures, including the rostral arcuate nucleus, contained Fluoro-Gold. Thus, in the prepubertal male ferret, the majority of LHRH cell bodies located in the caudal arcuate nucleus may be differentially regulated and/or involved in non-neuroendocrine functions.     

Distribution of immunoreactive growth hormone releasing factor (1–44)NH2 in the tuberoinfundibular system of the rhesus monkey

Using an antiserum which reacts with the carboxyl terminus of GRF(1-44)NH2, the distribution of immunoreactive growth hormone releasing factor (GRF) in the rhesus monkey hypothalamus was delineated by peroxidase immunocytochemistry. Immunoreactive material was present in dense terminal fields in the median eminence closely associated with portal capillaries but in a location distinct from that noted for immunoreactive thyrotropin-releasing hormone (TRH) or somatostatin. GRF-immunoreactive cell bodies were identified in the arcuate nucleus and ventromedial nucleus. These studies provide evidence for the presence of GRF(1-44)NH2 in the primate brain and demonstrate that in the hypothalamus it is localized exclusively in cells and fibers corresponding to the tuberoinfundibular system.     

Neurotensin in the rat median eminence: the possible sources of neurotensin-like fibers and varicosities in the external layer

The possible sources of neurotensin-like immunoreactive axons in the median eminence were studied after several experimental surgical approaches including unilateral lateral retrochiasmatic area transection, midsagittal knife cut through the median eminence, complete surgical isolation of the medial basal hypothalamus and bilateral paraventricular nucleus lesions. Both immunohistochemical and radioimmunoassay data demonstrate that neurotensin-containing neuronal located in the hypothalamic arcuate nuclei represent the main source of neurotensin occurring in the external zone of the median eminence of the rat: (1) neither the complete isolation of the medial basal hypothalamus nor the transection of the major neuronal input channel to the median eminence in the lateral retrochiasmatic area altered neurotensin-like immunoreactivity in the median eminence; (2) bilateral lesioning of the paraventricular nucleus resulted in insignificant changes of neurotensin level in the median eminence; and (3) two days after lesioning the median eminence an increased amount of retrogradely accumulated neurotensin-like immunoreactivity was found in several perikarya of the arcuate nuclei due to the blockage of axonal transport in the transected fibers. Retrograde accumulation of neurotensin-like material in other cells scattered in the anterior hypothalamus (in the paraventricular, paraventricular and anterior hypothalamic nuclei) indicates that in addition to the arcuate neurons these neurons may also participate in the neurotensin innervation of the median eminence.     

Immunocytochemical distribution of [Met]enkephalin-Arg-Gly-Leu immunoreactivity in the rat diencephalon

Recently, [Met]Enkephalin-Arg-Gly-Leu (MEAGL) was isolated from bovine adrenal glands, and it was found to be derived exclusively from proenkephalin. Therefore, we investigated the distribution of MEAGL-like immunoreactive neuronal perikarya and fibers in the rat diencephalon pretreated with colchicine by PAP immunocytochemistry. In the thalamus MEAGL immunoreactive neuronal perikarya were distributed in the paraventricular nucleus and the ventral part of the lateral geniculate nucleus. Immunoreactive fibers were found in the paraventricular, paracentral, anteroventral, reuniens and rhomboid nuclei. In addition, immunoreactive fibers were also noted in the anterior pretectal nucleus. In the hypothalamus, immunoreactive neuronal perikarya were observed in the medial preoptic area, anterior and lateral hypothalamic nuclei, perifornical region, parvocellular and postero-magnocellular regions of paraventricular nucleus, ventromedial nucleus, dorsomedial nucleus, arcuate nucleus, premammillary, medial mammillary and lateral mammillary nuclei. The distribution of immunoreactive fibers was similar to that of neuronal perikarya. However, immunoreactive fibers were also observed in the supraoptic and suprachiasmatic nuclei where no immunoreactive neuronal perikarya were detected. Numerous immunoreactive fibers were detected in the external layer of the median eminence, but there were few in the internal layer. The similarity and difference in the distribution between MEAGL and other proenkephalin peptides such as [Met]enkephalin were also discussed.     

Substance P-like immunoreactive neurons in the arcuate nucleus project to the median eminence in rat

Substance P-containing nerve fibers in the median eminence of rat arise in cells located in the arcuate nucleus. Two days following surgical lesioning of the median eminence immunoreactive substance P accumulated in neuronal perikarya in the middle part (rostrocaudally) of the arcuate nucleus, mainly in its ventromedial portion. Substance P-immunostained cells appeared nowhere else in the hypothalamus following surgical lesion of the median eminence while they were found in several hypothalamic and extrahypothalamic cell groups after colchicine treatment.     

Electron microscopic immunocytochemical evidence for the existence of bidirectional synaptic connections between growth hormone-releasing hormone- and somatostatin-containing neurons in the hypothalamus of the rat

Synaptic contacts between growth hormone-releasing hormone (GHRH)- and somatostatin-containing neurons were demonstrated in the rat hypothalamus by a double-staining immunocytochemical method at the electron microscopic level. Somatostatin-immunoreactive nerve terminals synapse on GHRH-positive dendrites and cell bodies in the arcuate nucleus. A fine network of GHRH-immunopositive nerve terminals was observed at the light microscopic level in the rostral part of the periventricular nucleus and in the dorsal part of the arcuate nucleus around somatostatin-containing neuronal elements. With the electron microscope synaptic contact between GHRH-containing nerve terminals and somatostatin-containing dendrites are demonstrated. The reciprocal innervation between GHRH- and somatostatin-containing neurons that project to the median eminence and regulate growth hormone secretion must allow them to coordinate their activities.     

Distribution of neuropeptide Y-like immunoreactivity in the hypothalamus of the adult golden hamster

The distribution of neuropeptide Y (NPY)-like immunoreactivity within the hypothalamus of the adult golden hamster was investigated with conventional immunohistochemical techniques. Neuropeptide Y immunoreactive cell bodies were found in greatest numbers in the arcuate nucleus while a few stained perikarya were seen in the internal and subependymal zones of the median eminence. Isolated perikarya were observed in the anterior commissure and supracommissural portion of the interstitial nucleus of the stria terminalis. Immunoreactive axons were located throughout the hypothalamus with the highest concentrations in the subependymal and internal zones of the median eminence, the interstitial nucleus of the stria terminalis, the medial preoptic area, and in the following nuclei: periventricular, suprachiasmatic, paraventricular, perifornical, median preoptic, and arcuate. Moderate to dense plexuses of immunoreactive fibers were observed in the anterior, lateral, and posterior hypothalamic areas and in the infundibular stalk. The supraoptic nucleus and lateral preoptic area displayed a small number of labeled axons whereas the ventromedial nucleus contained only a few fibers. NPY immunoreactive fibers were present in the optic tract and in the dorsomedial aspect of the optic chiasm. Labeled fibers penetrated the ependymal lining of the third ventricle throughout the ventral aspect of the periventricular zone. Additional fibers were observed in the pia lining the ventral aspect of the hypothalamus. This systematic analysis of hypothalamic NPY immunoreactivity in the adult golden hamster suggests that a portion of the labeled fibers display a distribution that is similar to previously described noradrenergic fibers in the hypothalamus.     

Distribution of GABA immunoreactive systems in the forebrain and midbrain of the chameleon

An immunocytochemical method, using glutaraldehyde fixation and an antiserum developed against a GABA-glutaraldehyde protein conjugate, permitted direct visualization of GABAergic structures in the brain of a reptile (chameleon). GABA immunoreactive cell bodies and nerve terminals were observed to be evenly distributed throughout the forebrain and midbrain. In the forebrain, GABA-positive perikarya were shown in all cortical areas, the septal area, the striatum, the dorsal ventricular ridge, and in the nucleus accumbens. In the midbrain, the optic tectum contained a dense and laminar distribution of GABA neurons. These neurons were also observed in the lateral geniculate nucleus, nucleus profundus mesencephali, nucleus opticus tegmenti and substantia nigra. Immunoreactive nerve fibers and terminals were observed in the same structures and, additionally, in the tractus septo-hypothalamicus, habenula complex, median eminence, intermediate lobe of the pituitary, basal part of the subcommissural organ, torus semicircularis and nucleus reticularis isthmi. These results provide a framework for a further electron microscopic analysis of the GABAergic innervation of some encephalic areas involved in physiological regulations particular to this species especially the visual system.     

Coexpression of dynorphin and neurokinin B immunoreactivity in the rat hypothalamus: Morphologic evidence of interrelated function within the arcuate nucleus

Considerable evidence suggests that dynorphin and neurokinin B (NKB) neurons in the hypothalamic arcuate nucleus participate in the sex-steroid regulation of reproduction. In the present study, we used dual-label immunofluorescence to explore the distribution of prodynorphin and proNKB immunoreactivity in the rat hypothalamus. Additionally, we investigated whether arcuate prodynorphin-ir (immunoreactive) neurons expressed the neurokinin 3 receptor (NK3R) or nuclear estrogen receptor-alpha (ERalpha). We found that the majority of prodynorphin-ir neurons in the rat arcuate nucleus expressed proNKB, whereas nearly all (99%) of the proNKB neurons were immunoreactive for prodynorphin. The arcuate nucleus was the only site in the hypothalamus where neuronal somata coexpressing prodynorphin and proNKB-immunoreactivity were identified. A dense plexus of double-labeled prodynorphin/proNKB-ir fibers was found within the arcuate nucleus extending to the median eminence and throughout the periventricular zone of the hypothalamus. Prodynorphin/proNKB fibers were also identified in the paraventricular nucleus, anterior hypothalamic area, medial preoptic area, median preoptic nucleus, anteroventral periventricular nucleus, and bed nucleus of the stria terminalis in a distribution consistent with previously described arcuate nucleus projections. Interestingly, the majority of prodynorphin-ir neurons in the arcuate nucleus expressed NK3R, and nearly 100% of the prodynorphin-ir neurons contained nuclear ERalpha. Our results suggest that there is a close functional relationship between dynorphin and NKB peptides within the arcuate nucleus of the rat, which may include an autofeedback loop mediated through NK3R. The diverse hypothalamic projections of fibers expressing both prodynorphin and proNKB provide evidence that these neurons may participate in a variety of homeostatic and neuroendocrine processes.