Development of the hypothalamic luteinizing hormone-releasing hormone-containing neuron system in the rat: in vivo and in transplantation studies

The development of the hypothalamic LHRH-containing neuron system was immunohistochemically investigated in vivo and in tissue transplantation using rat embryos aged from 12.5 to 17.5 days of gestation. The sera used were generated against rat gonadotropic hormone-releasing hormone-associated peptide (28-56) (rGAP) and LHRH. Immunoreaction for rGAP was first found in cells migrated from and in the vomeronasal organ on Days 13.5 and 14.5 of gestation. Immunoreactive cells seem to ascend along the terminal nerves, reaching the medial surface of the forebrain vesicles. Subsequently the cells occurred in the septum and further into their final position in the septopreoptic-diagonal band area on Days 16.5-17.5 of gestation; during this traverse the cells become secretory neurons after changes in morphology and in behavior. Intraventricular transplantation revealed that nasal epithelia of Day 12.5 embryos raised only a few cells immunoreactive both for LHRH and rGAP, but a great number of immunoreactive cells and fibers in the presence of the medial basal hypothalamus (MBH). The fibers formed a median eminence-like structure together with dense capillary plexus that had grown in the cografted MBH. The same phenomenon was apparently observed in the grafts obtained from older embryos of gestation, but not in the combined grafts of the anterior septum and the nasal epithelium or the MBH. We conclude that hypothalamic LHRH neurons originate from the nasal placode and acquire secretory behavior in the presence of the MBH.     

Development of the hypothalamic luteinizing hormone-releasing hormone-containing neuron system in the rat: In vivo and in transplantation studies

The development of the hypothalamic LHRH-containing neuron system was immunohistochemically investigated in vivo and in tissue transplantation using rat embryos aged from 12.5 to 17.5 days of gestation. The sera used were generated against rat gonadotropic hormone-releasing hormone-associated peptide (28–56) (rGAP) and LHRH. Immunoreaction for rGAP was first found in cells migrated from and in the vomeronasal organ on Days 13.5 and 14.5 of gestation. Immunoreactive cells seem to ascend along the terminal nerves, reaching the medial surface of the forebrain vesicles. Subsequently the cells occurred in the septum and further into their final position in the septopreoptic-diagonal band area on Days 16.5–17.5 of gestation; during this traverse the cells become secretory neurons after changes in morphology and in behavior. Intraventricular transplantation revealed that nasal epithelia of Day 12.5 embryos raised only a few cells immunoreactive both for LHRH and rGAP, but a great number of immunoreactive cells and fibers in the presence of the medial basal hypothalamus (MBH). The fibers formed a median eminence-like structure together with dense capillary plexus that had grown in the cografted MBH. The same phenomenon was apparently observed in the grafts obtained from older embryos of gestation, but not in the combined grafts of the anterior septum and the nasal epithelium or the MBH. We conclude that hypothalamic LHRH neurons originate from the nasal placode and acquire secretory behavior in the presence of the MBH.     

Localization of immunoreactive lamprey gonadotropin-releasing hormone in the rat brain

A highly specific antiserum against lamprey gonadotropin-releasing hormone (GnRH) was used to localize 1-GnRH in areas of the rat brain associated with reproductive function. Immunoreactive 1-GnRH-like neurons were observed in the ventromedial preoptic area (POA), the region of the diagonal band of Broca and the organum vasculosum lamina terminalis, with fiber projections to the rostral wall of the third ventricle and the organum vasculosum lamina terminalis. Another population of 1-GnRH-like neurons was localized in the dorsomedial and lateral POA, with nerve fibers projecting caudally and ventrally to terminate in the external layer of the median eminence. Other fibers apparently projected caudally and circumventrically to terminate around the cerebral aqueduct in the mid-brain central gray. By using a highly specific antiserum directed against mammalian luteinizing hormone-releasing hormone (m-LHRH), the localization of the LHRH neuronal system was compared to that of the 1-GnRH system. There were no LHRH neurons in the dorsomedial or the lateral region of the POA that contained the 1-GnRH neurons. As expected, there was a large population of LHRH neurons in the ventromedial POA associated with the diagonal band of Broca and organum vasculosum lamina terminalis. In both of these regions, there were many more LHRH neurons than 1-GnRH neurons and the LHRH neurons extended more dorsally and laterally than the 1-GnRH neurons. The LHRH neurons seemed to project to the median eminence in the same areas as those that were innervated by the 1-GnRH neurons. Absorption studies indicated that 1-GnRH cell bodies were eliminated by adding 1 microg of either 1-GnRH-I or 1-GnRH-III, but not m-LHRH to the antiserum before use. Fibers were largely eliminated by the addition of 1 microg 1-GnRH-III to the antiserum. No chicken GnRH-II neurons or nerve fibers could be visualized by immunostaining. Because the antiserum recognized GnRH-I and GnRH-III equally, we have visualized an 1-GnRH system in rat brain. The results are consistent with the presence of either one or both of these peptides within the rat hypothalamus. Because 1-GnRH-I has only weak nonselective gonadotropin-releasing activity, whereas 1-GnRH-III is a highly selective releaser of follicle-stimulating hormone, and because 1-GnRH neurons are located in areas known to control follicle-stimulating hormone release selectively, our results support the hypothesis that 1-GnRH-III, or a closely related peptide, may be mammalian follicle-stimulating hormone-releasing factor.     

Localization of immunoreactive lamprey gonadotropin-releasing hormone in the rat brain

A highly specific antiserum against lamprey gonadotropin-releasing hormone (GnRH) was used to localize 1-GnRH in areas of the rat brain associated with reproductive function. Immunoreactive 1-GnRH-like neurons were observed in the ventromedial preoptic area (POA), the region of the diagonal band of Broca and the organum vasculosum lamina terminalis, with fiber projections to the rostral wall of the third ventricle and the organum vasculosum lamina terminalis. Another population of 1-GnRH-like neurons was localized in the dorsomedial and lateral POA, with nerve fibers projecting caudally and ventrally to terminate in the external layer of the median eminence. Other fibers apparently projected caudally and circumventrically to terminate around the cerebral aqueduct in the mid-brain central gray. By using a highly specific antiserum directed against mammalian luteinizing hormone-releasing hormone (m-LHRH), the localization of the LHRH neuronal system was compared to that of the 1-GnRH system. There were no LHRH neurons in the dorsomedial or the lateral region of the POA that contained the 1-GnRH neurons. As expected, there was a large population of LHRH neurons in the ventromedial POA associated with the diagonal band of Broca and organum vasculosum lamina terminalis. In both of these regions, there were many more LHRH neurons than 1-GnRH neurons and the LHRH neurons extended more dorsally and laterally than the 1-GnRH neurons. The LHRH neurons seemed to project to the median eminence in the same areas as those that were innervated by the 1-GnRH neurons. Absorption studies indicated that 1-GnRH cell bodies were eliminated by adding 1 microg of either 1-GnRH-I or 1-GnRH-III, but not m-LHRH to the antiserum before use. Fibers were largely eliminated by the addition of 1 microg 1-GnRH-III to the antiserum. No chicken GnRH-II neurons or nerve fibers could be visualized by immunostaining. Because the antiserum recognized GnRH-I and GnRH-III equally, we have visualized an 1-GnRH system in rat brain. The results are consistent with the presence of either one or both of these peptides within the rat hypothalamus. Because 1-GnRH-I has only weak nonselective gonadotropin-releasing activity, whereas 1-GnRH-III is a highly selective releaser of follicle-stimulating hormone, and because 1-GnRH neurons are located in areas known to control follicle-stimulating hormone release selectively, our results support the hypothesis that 1-GnRH-III, or a closely related peptide, may be mammalian follicle-stimulating hormone-releasing factor.     

Effects of intrahypothalamic testosterone implants on LHRH levels in the preoptic area and the medial basal hypothalamus.

Following castration LHRH levels in the MBH but not in the POA decreased. Testosterone implants in the medial POA following castration failed to alter the LHRH activity either locally in the POA or in remote sites in the MBH. On the contrary, similar T implants in the MBH blocked castration-induced depletion of MBH LHRH stores without affecting either the POA LHRH content or the post-castration hypersecretion of pituitary LH. These findings identify the MBH as the focal site of T action in the regulation of hypothalamic LHRH activity.     

Transplantation of fetal growth hormone-releasing factor-immunoreactive neurons into the ventricular system of adult MSG-treated rats.

Fetal (17-18 days of gestation) mediobasal hypothalamic tissue (MBH) was transplanted into the third ventricle of adult, male rats which had been treated neonatally with monosodium glutamate (MSG). MSG treatment caused a marked reduction of growth hormone-releasing factor-like-immunoreactive (GRF-i) perikarya in the arcuate nucleus and GRF-i fibers in the median eminence (ME), as compared to littermate controls. When normal fetal MBH was transplanted into the third ventricle of MSG recipients, numerous GRF-i perikarya were located within the graft four weeks following surgery. GRF-i fibers in the ME of MSG-treated rats were enhanced when MBH grafts were in close contact with the ME, but not when transplants were located dorsally or rostrally in the third ventricle without making contact with the recipient's ME. Fetal cerebral cortex, which was grafted as a control tissue, did not contain GRF-i neurons. These immunohistochemical results suggest that grafted fetal GRF-i perikarya may contact the recipient's ME to increase the content of GRF previously depleted by exposure to MSG.     

Slice cultures of LHRH neurons in the presence and absence of brainstem and pituitary

Luteinizing hormone releasing hormone (LHRH) neurons from the preoptic area (POA)/hypothalamus of the postnatal rat were cultured for up to 7 weeks using a slice explant roller culture technique. The slices thinned to quasi-monolayers, but maintained organotypic distributions of large numbers of immunocytochemically identifiable LHRH, neurotensin, tyrosine hydroxylase, neurophysin and corticotropin releasing hormone-containing neurons. The distribution, survival and morphology of LHRH cells in co-cultures with brainstem and anterior pituitary was quantitated, and found to be similar to that observed in single cultures. LHRH fibers grew into either pituitary or brainstem tissue, however when all three tissues were co-cultured, LHRH fibers preferentially invaded the pituitary. LH immunoreactive anterior pituitary gonadotropes were maintained only in co-cultures containing POA/hypothalamic slices, and addition of an LHRH antagonist in such cultures, inhibited LH immunoreactivity in the gonadotropes. This slice explant roller culture method effectively maintains the cyto- and chemoarchitecture and functional properties of the LHRH system for long periods in vitro and should provide excellent models for studying the interactive and molecular characteristics of postnatal LHRH neurons.     

Light- and electron-microscopic evidence of costoring of immunoreactive enkephalins and substance P in dorsal horn neurons of rat

Summary The topographical localization of substance P (SP) and methionine-enkephalin-octapeptide (Enk-8) was examined immunohistochemically in the surface layer of the dorsal horn of rat cervical spinal cord. Although a few neurons were immunoreactive for Enk-8 in the intact animals, after an intracisternal administration of colchicine, immunoreactive Enk-8 neurons were numerous, and half of them indicated immunoreactivity also for SP. Some immunoreactive SP neurons appeared to show no immunoreactivity for Enk-8. Immuno-reactive nerve fibers, on the other hand, were numerous, and many of them contained both peptides. Electron-microscopic examination of the nerve fibers in tissue prepared by a freeze-drying procedure and stained by a postembedding procedure, revealed the costoring of both peptides in the same cored vesicles. The physiological significance of this costoring is discussed.     

Ontogenesis of hypothalamic immunoreactive ACTH cells in vivo and in vitro: role of Rathke's pouch

The ontogenesis of immunoreactive (ir) ACTH cells and ir alpha-MSH cells in rat hypothalamus was studied in vivo and in vitro. Ir ACTH cells first appeared in the neuroepithelial cell layer lining the floor of the third ventricle on Day 13.5 of gestation, whereas ir alpha-MSH first appeared in the cytoplasm of several ir ACTH cells in the basal part of the arcuate nucleus of the hypothalamus on Day 19.5. When the medial-basal hypothalamus of 12.5-day embryos was cultured alone, a few ir ACTH cells were found after culture for 10 days, but not 3 days, and no ir alpha-MSH cells were observed in the cultures. When the hypothalamus was cultured with Rathke's pouch (intact or without the intermediate lobe anlage), ir ACTH cells appeared within 3 days. In these cultures on Days 6 and 10, long beaded fibers were seen projecting from cells in the neuronal tissue, and some cells showed immunolabeling for alpha-MSH. When the hypothalamus was cocultured with oral epithelium instead of Rathke's pouch, the appearance of neuronal ir ACTH cells was like that in cultures of hypothalamus alone. These in vitro findings suggest that stimulus from the anterior lobe anlage of the pituitary is necessary for normal development of ir ACTH/alpha-MSH cells in the hypothalamus.     

In-vitro pulsatile luteinizing hormone-releasing hormone output is dependent on hypothalamic region and the stage of the estrous cycle

In the following experiments, the role of the preoptic-suprachiasmatic area (POA-SCN) in the control of luteinizing hormone-releasing (LHRH) release was examined by in vitro superfusion of either mediobasal hypothalamus (MBH) or MBH-POA-SCN fragments obtained from cycling rats killed on various days of the estrous cycle. The lowest level of LHRH output occurred during estrus, highest levels during diestrus, and intermediate levels on proestrus in the MBH-POA-SCN preparation. As expected, significant decreases in LHRH output from the MBH alone occurred during both days of diestrus and on proestrus, as compared to output from the MBH-POA-SCN tissue, since this structure contains most of the LHRH perikarya. However, similar LHRH secretion patterns were detected in estrus from both preparations. The average period of the LHRH pulses for the estrous cycle in the MBH-POA-SCN was 30.9 +/- 1.2 min compared to 97.7 +/- 25.1 min in the MBH, with significant differences occurring on diestrus 2. The increase and extreme variability of the period of LHRH pulses in the MBH region, compared to the MBH-POA-SCN region, suggests that it is the latter region that contains the neural circuits that control the LHRH pulse generator. The LHRH pulse amplitudes from both hypothalamic regions were similar during all phases of the estrous cycle, except diestrus 2, when the LHRH pulse amplitude from the MBH region was significantly lower than the LHRH pulse amplitude from MBH-POA-SCN. The percentage of LHRH released in the MBH did not vary with the estrous cycle, however, in the MBH-POA-SCN significant changes were observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ascorbic acid enhances the release of luteinizing hormone-releasing hormone from the mediobasal hypothalamus in vitro

Ascorbic acid is frequently used in in vitro studies of neurotransmitter-evoked release of luteinizing hormone-releasing hormone (LHRH) from hypothalamic fragments. Although it is assumed that ascorbate merely prevents the oxidative degradation of catecholamines, we have discovered that ascorbic acid itself produces significant increases in the release of LHRH. Our studies showed that ascorbic acid, at concentrations below 1 mM, produced a dose-dependent release of LHRH from incubated rat mediobasal hypothalamus (MBH). The magnitude of the ascorbate-induced release was in the range of 100-200% above controls; significant amounts of LHRH were released only if the MBH were incubated with ascorbate for time periods longer than 30 minutes. We also found that ascorbate-induced increases in LHRH were equivalent to those produced by another LHRH secretagogue, naloxone, and that the combined effects of the two substances were additive in nature. Although the mechanisms underlying this effect are not fully understood, nonspecific chemical reduction is probably not a factor since sodium metabisulfite did not induce the release of LHRH. It seems probable that ascorbate may enhance the activity of endogenous norepinephrine in the MBH and, thereby, lead to increased release of LHRH.     

Disruption of the olfactory placode and brain conditioned medium increase the number of LHRH immunostained neurons in explants

The olfactory placode gives rise to both olfactory receptor neurons, which remain as a component of the peripheral nervous system, and to luteinizing hormone-releasing hormone (LHRH) neurons, which migrate to the central nervous system. In this study, we used chick olfactory placode explants to ask several questions regarding LHRH neuronal differentiation. We found that explants of ectoderm from the fronto-nasal region of embryos as early as Hamilton & Hamburger (HH) stage 12 gave rise to LHRH neurons, that explants from all regions of the olfactory placode were able to generate LHRH neurons, that both brain conditioned medium and disruption of the olfactory placode increase the number of LHRH neurons observed in explants, and that the combination of these two manipulations results in the production of more LHRH neurons than either treatment alone. We conclude that LHRH neurons originate in the olfactory epithelium and that some of the same factors which influence olfactory receptor neuron development also affect LHRH neuronal development.     

Appearence of LH-Immunoreactive Cells in the Rathke's Pouch of the Chicken Embryo

The differentiation of the pituitary of the chicken embryo was studied by means of an immunohistochemical technique using antisera to turkey and chicken pituitary hormones. Immunoreactive LH-cells are detected in 4-day embryos (stage 23 of Hamburger and Hamilton) when the primordium of the anterior pituitary, the Rathke's pouch is only composed of a single-layer epithelium lined with an undifferentiated mesenchyme. A few immunoreactive cells are observed grouped on the posterior aspect of the pouch. As development proceeds, a strip of positive cells is detected encircling the Rathke's pouch. Prolactin-, growth hormone-, and ACTH-immunoreactive cells are detected in 6- and 7-day embryos, only after the pituitary has acquired its characteristic structure with cords in which different cell types become progressively recognizable. The early appearance of immunoreactive LH-cells following a precise distribution shows that secretory properties and differentiation capacities are acquired simultaneously in the epithelium of the Rathke's pouch and may be induced by the same stimulus.     

Immunocytological study on the differentiation of chick and quail adenohypophysis epithelial rudiments, grafted or cultivated in vitro: evidence for polypeptidic hormones

Epithelial rudiments of adenohypohysis were removed from chick and quail embryos between days 3 and 5 of development. Chick rudiments were grafted for 11--13 days onto the chorioallantoic membrane of decapitated chick embryo hosts. Quail rudiments were cultivated in vitro for 6 days. Both grafted and cultivated Rathke's pouches differentiated into adenohypophyseal tissue. The adenohypophyseal tissue cultured on chorio-allantoic membrane exhibited cells reacting with the following immune sera: anti-beta-(1--24)ACTH, anti-alpha-(17--39)-ACTH, anti-alpha-endorphin, anti-beta-endorphin and anti-beta-LPH, which also gave a positive reaction when applied to adenohypophysis of corresponding age which had differentiated in situ. In situ, corticotrophs were located exclusively in the cephalic lobe of adenohypophysis. Therefore, the differentiation of corticotrophs in the whole graft, i.e., from both cephalic and caudal lobes of Rathke's pouch, showed that the cells of the caudal lobe, or at least some of them, were uncommitted when the rudiment was removed. In vitro, tissue derived from Rathke's pouch contained cells reacting with antibodies to beta-(1--24)-ACTH, alpha-(17--39)-ACTH, and beta-LPH, as did adenohypophysis from quail embryos of corresponding age (9--10 days), differentiated in situ. The differentiation of quail Rathke's pouch in vitro corroborates that differentiation can occur without influence from hypothalamus and, moreover, shows that at least some kinds of cells can differentiate without influence exerted by any other encephalic factors, and in the absence of mesenchyme. The question arises whether fibroblastic cells derived from Rathke's pouch cells act as feeder-cells and/or secrete some factors promoting differentiation.     

Appearance of LH-immunoreactive cells in the Rathke's pouch of the chicken embryo

The differentiation of the pituitary of the chicken embryo was studied by means of an immunohistochemical technique using antisera to turkey and chicken pituitary hormones. Immunoreactive LH-cells are detected in 4-day embryos (stage 23 of Hamburger and Hamilton) when the primordium of the anterior pituitary, the Rathke's pouch is only composed of a single-layer epithelium lined with an undifferentiated mesenchyme. A few immunoreactive cells are observed grouped on the posterior aspect of the pouch. As development proceeds, a strip of positive cells is detected encircling the Rathke's pouch. Prolactin-, growth hormone-, and ACTH-immunoreactive cells are detected in 6- and 7-day embryos, only after the pituitary has acquired its characteristic structure with cords in which different cell types become progressively recognizable. The early appearance of immunoreactive LH-cells following a precise distribution shows that secretory properties and differentiation capacities are acquired simultaneously in the epithelium of the Rathke's pouch and may be induced by the same stimulus.     

The differentiation of prolactin cells in an organ culture of chick embryo adenohypophysis

We have studied differentiation of prolactin cells in explants of cephalic and caudal parts of Rathke's pouch of 4.5 day and 5.5 day old chick embryos after their incubation in vitro lasting for 7-8 days. Indirect immunofluorescence using an antiserum against bovine prolactin was used to detect prolactin cells in the cultures. Differentiation of prolactin cells was detected regularly in explants of the cephalic lobe of the adenohypophysis anlage in 5.5 day old embryos; under certain growth conditions prolactin cells were found in explants of the same lobe in 4.5 day old embryos. Prolactin cells were either absent or found in small numbers in cultures of the caudal part of adenohypophysis of 5.5 day old embryos. Our results provide evidence for the appearance of the committed precursors of prolactin cells in the Rathke's pouch at late stages of its formation and for their regional localization in the cephalic part of the anlage. This localization is in correspondence with the distribution of differentiated cells of this type in definitive adenohypophysis.     

GABAergic Neurons in the Embryonic Olfactory Pit/Vomeronasal Organ: Maintenance of Functional GABAergic Synapses in Olfactory Explants

In previous work, we showed a robust γ-aminobutyric acid (GABAergic) synaptic input onto embryonic luteinizing hormone-releasing hormone (LHRH) neurons maintained in olfactory explants. In this study, we identify GABAergic neurons in olfactory pit (OP) of embryonic micein vivoand study, using patch-pipet whole-cell current and voltage clamp techniques, synaptic interactions of these neurons in explant cultures.In vivo,glutamate decarboxylase (GAD, the enzyme which synthesizes GABA) mRNA was first detected in nasal regions on Embryonic Day (E) 11.5. From E12.5 to E13.5, robust GAD expression was localized to cells primarily in the ventral aspect of the OP. GAD mRNA was not detected over dorsally located cells in olfactory sensory or respiratory epithelium. In addition, GAD mRNA was not observed in cells along olfactory axons. GAD mRNA was dramatically reduced in the OP/vomeronasal organ by E16.5. Using antibodies against both GABA and GAD, immunopositive axonal-like tracts were detected in the nasal septum on E12.5. GABAergic staining decreased by E13.5. To examine synaptic interactions of these GABAergic cells, embryonic olfactory explants were generated and maintained in serum-free media. As explants spread, neuron-like cells migrated into the periphery, sometimes forming ganglion-like clusters. Cells were recorded, marked intracellularly with Lucifer Yellow and post-fixation, immunocytochemically examined. Forty-six cells, typically multipolar, were GABAergic, had resting potentials around −50 mV, and exhibited spontaneous action potentials which were generated by spontaneous depolarizing GABAergic (GABA_A) synaptic activity. OP neurons depolarized in response to GABA by increasing Cl⁻conductance. The biophysical properties of OP-derived GABAergic neurons were distinct from those reported for olfactory receptor neurons but similar to embryonic LHRH neurons. However, unlike LHRH neurons, GABAergic neurons did not migrate large distances in olfactory explants or appear to leave the olfactory pitin vivo.     

Hypothalamic LHRH and plasma LH levels after electrical stimulation of the deafferented medial basal hypothalamus

The medial basal hypothalamus (MBH) of female rats was surgically isolated on the morning of proestrus and luteinizing hormone (LH) levels in the blood were determined before and after electrochemical stimulation (ECS) of the disconnected arcuate-median eminence (ARC-ME) region either on the same afternoon (Group 1) or on the 5th postoperative day (Group 2). Animals of Groups 3 and 4 were stimulated and sampled for LH on the 5th or 10th postoperative day, respectively, these rats having been primed with 5 micrograms estradiol injected daily throughout the experimental period. ECS resulted in a significant rise of plasma LH level in Group 1 and caused full ovulation as evaluated by the presence of ova found the next morning in the Fallopian tubes. Rats of Groups 2-4 failed to show any changes in plasma LH, and no ovulation was observed following ECS. Immunohistochemical examination of the brains revealed that axons staining for the luteinizing hormone-releasing hormone (LHRH) in the ARC-ME region remained at control levels 3 days after deafferentation (e.g. Group 1), whereas a marked decrease or complete absence of these structures was observed 8 or 13 days following surgical isolation of the MBH (e.g. Groups 2-4). These studies strongly support the view that no LHRH synthesizing perikarya are located within the MBH of the rat.     

Ultrastructural localization of immunolabeled substance P and methionine-enkephalin-octapeptide in the surface layer of the dorsal horn of rat spinal cord

Summary A preembedding dual immunolabeling technique and electron microscopy were utilized to demonstrate the localization of immunoreactive substance P and methionine-enkephalin-octapeptide (Enk-8) in ultrathin sections of the surface layer (laminae I and II) of rat spinal dorsal horn. The immunoreaction of Enk-8 was visualized as goldtoned silver particles and that of substance P as diaminobenzidine reaction products. Axonal terminals with immunoreactive substance P, and also unlabeled axonal terminals, formed synaptic junctions with the perikarya and dendritic processes of Enk-8-containing neurons. Dendritic profiles immunolabeled for substance P were synaptically linked with unlabeled axons but not with Enk-8-positive ones. Furthermore, it was found that Enk-8 axons and substance P axons terminated synaptically in juxtaposition to one another on the same immunonegative dendrites. Among the Enk-8-containing neurons axonal profiles also appeared to be synaptically associated with immunoreactive Enk-8 dendritic processes.