In vivo biosynthesis and transport of oxytocin, vasopressin and neurophysin from the hypothalamus to the spinal cord

The biosynthesis of oxytocin, vasopressin and their associated neurophysins were studied in the projection from the paraventricular nucleus of the hypothalamus to the spinal cord in individual freely-moving adult male rats. Neuropeptide biosynthesis was studied in vivo by the delivery of [35S]cysteine through stereotaxically implanted indwelling cannulae using an osmotic minipump delivery system. Following the appropriate chase times, the neural lobe and spinal cord segments T1-T4 and T12-L2 were removed from fresh tissue; in addition, the nucleus of the solitary tract was punched from frozen coronal sections. The radiolabeled peptides were purified from the tissue homogenates by sequential linear and exponential gradient elution from reverse-phase high performance liquid chromatography columns. This approach has allowed us to purify radiolabeled oxytocin and vasopressin from both the upper and lower spinal cord. However, the kinetics of oxytocin and vasopressin biosynthesis appeared to be remarkably different, as judged by their differential labeling with different pulse and chase times. Additionally, the use of different chase periods following the pulse of radiolabel has allowed us to determine that oxytocin reaches the spinal cord via the fast component of axonal transport (greater than 8 mm h-1). Using immunoprecipitation and purification by high performance liquid chromatography, we were also able to purify radiolabeled neurophysins from spinal cord tissue homogenates. These results lend further support to a role for oxytocin and vasopressin in the modulation of autonomic nervous system function and to the role of the paraventricular nucleus as an integration center for endocrine and autonomic function.     

Co-existence of unrelated peptides in oxytocin and vasopressin terminals of rat neurohypophyses: Immunoreactive methionine5-enkephalin-, leucine5- enkephalin- and cholecystokinin-like substances

In neurohypophyses of normal rats and of vasopressin-deficient Brattleboro rats, applying immunocytochemical methods, we have found (1) in oxytocin terminals co-existing methionine-enkephalin-, cholecystokinin- and possibly leucine-enkephalin-like substances and (2) in vasopressin terminals, co-existing leucine-enkephalin-like material. The correspondence between the enkephalin immunoreactivity patterns and the oxytocin or vasopressin immunoreactivity pattern in serial 0.5 μ thick sections was so close that occurrence of enkephalin in separate nerves appears unlikely. At a subcellular level the enkephalin- and the cholecystokinin-like immunoreactivities were localized mainly in the neurosecretory granules that also contain oxytocin or vasopressin; there was no evidence for separate enkephalingranule populations. The intensity and frequency of leucine-enkephalin immunostaining in vasopressin terminals was much enhanced by treatment of the deplasticized sections with trypsin prior to incubation with antibodies. This suggests incorporation of the leucine-enkephalin sequence into longer peptide chains, presumably dynorphin and/or α-neo-endorphin. In oxytocin endings tryptic cleavage enhanced cholecystokinin-like immunoreactivity, while methionine-enkephalin immunostaining was as intense without enzyme treatment.The opioid peptide forms of vasopressin neurons appear to be elongated chains containing exclusively the leucine-enkephalin sequence, whereas in oxytocin neurons methionine-enkephalin as free pentapeptide seems to prevail. Association of enkephalins with vasopressin and oxytocin was also indicated when methionine- and leucine-enkephalin contents of neurointermediate lobes of homozygous Brattleboro rats were compared with peptide stores in heterozygous controls in a high pressure liquid chromatography system and highly specific leucine- and methionine-enkephalin radioimmunoassays. We assume that the opioid peptides in the oxytocin system and in the vasopressin system are derived from different opioid precursor molecules.     

Galanin lacks binding sites in the porcine pituitary and has no detectable effect on oxytocin and vasopressin release from rat neurosecretory endings.

High concentrations of immunoreactive galanin-like material in rat hypothalamus, median eminence and neurohypophysis have been reported in the literature suggesting a regulatory role of galanin on hormone release from the anterior and posterior lobe of the pituitary. We studied binding of iodinated galanin to crude membrane preparations from porcine anterior hypothalamus, anterior and neurointermediate lobe of the hypophysis. In contrast to the hypothalamus where specific binding of 125I-galanin was found, there was no displaceable galanin binding in membranes of the anterior or neurointermediate lobe of porcine pituitaries. Effects of galanin on oxytocin and vasopressin release were investigated using isolated neurosecretory endings from rat neurohypophyses. Galanin had no detectable effect on the release of oxytocin or vasopressin.     

Mediation of changes in paraventricular vasopressin and oxytocin mRNA content to the medullary vagal complex and spinal cord of the rat

Retrograde tracing was combined with in situ hybridization to demonstrate that a small percentage of neurons of the paraventricular hypothalamic nuclei (PVN) which project to the spinal cord or the medullary vagal complex contain the mRNA to produce the peptides vasopressin or oxytocin. These projection neurons respond to salt loading with an upregulation of mRNA for these peptides. The present study provides an anatomical basis for a direct regulatory influence of PVN neurons on preganglionic neurons in the spinal cord and medulla.     

The role of oxytocin and the paraventricular nucleus in the sexual behaviour of male mammals

The paraventricular nucleus of the hypothalamus contains the cell bodies of a group of oxytocinergic neurons projecting to extrahypothalamic brain areas and to the spinal cord, which are involved in the control of erectile function and copulation. In male rats, these neurons can be activated by dopamine, excitatory amino acids, nitric oxide (NO), hexarelin analogue peptides and oxytocin itself to induce penile erection and facilitate copulation, while their inhibition by gamma-aminobutyric acid (GABA) and GABA agonists and by opioid peptides and opiate-like drugs inhibits sexual responses. The activation of paraventricular oxytocinergic neurons by dopamine, oxytocin, excitatory amino acids and hexarelin analogue peptides is apparently mediated by the activation of nitric oxide (NO) synthase. NO in turn activates, by a mechanism that is as yet unidentified, the release of oxytocin from oxytocinergic neurons in extrahypothalamic brain areas. Paraventricular oxytocinergic neurons and mechanisms similar to those reported above are also involved in the expression of penile erection in physiological contexts, namely, when penile erection is induced in the male by the presence of an inaccessible receptive female, which is considered a model for psychogenic impotence in man, as well as during copulation. These findings show that paraventricular oxytocinergic neurons projecting to extrahypothalamic brain areas and to the spinal cord and the paraventricular nucleus play an important role in the control of erectile function and male sexual behaviour in mammals.     

Quantitative autoradiographic mapping of neurohypophysial hormone binding sites in the rat forebrain and pituitary gland--I. Characterization of different types of binding sites and their distribution in the Long-Evans strain

Oxytocin and vasopressin binding sites were localized and characterized by quantitative autoradiography on consecutive sections of Long-Evans rat forebrains and pituitary glands, incubated in the presence of 5 nM [3H]oxytocin or 5 nM [3H]vasopressin. In the forebrain, two types of neurohypophysial hormone binding sites were thus defined. (1) Oxytocin/vasopressin sites with similar nanomolar-range affinities for [3H]oxytocin and [3H]vasopressin; both tritiated peptides were displaced from these sites in the presence of 10 microM of either oxytocin or vasopressin. The main areas bearing such sites were the ventral subiculum, several nuclei of the amygdala, the ventromedial hypothalamic nucleus, the bed nucleus of the stria terminalis and the olfactory tubercle. (2) Selective vasopressin sites, binding [3H]vasopressin with nanomolar-range affinity and [3H]oxytocin with a much lower affinity; these sites were not labelled in the presence of 5 nM [3H]oxytocin, and 10 microM oxytocin displaced [3H]vasopressin binding by 80%. Such sites occurred in several thalamic nuclei, in the dopaminergic A13 cell group of the zona incerta, the suprachiasmatic nucleus, the fundus striati and the lateral septal nucleus. No selective oxytocin sites were detected. Different oxytocin and vasopressin binding characteristics were found in the hypothalamo-neurohypophysial system. In the paraventricular and supraoptic nuclei and in the pituitary neural lobe the [3H]vasopressin binding density was twice that of [3H]oxytocin; vasopressin was always more potent than oxytocin in displacing both [3H]vasopressin and [3H]oxytocin binding from those sites. Interaction of the tritiated peptides with neurophysins cannot be completely ruled out in these locations. The present data are discussed in correlation with the functional roles of the neurohypophysial peptides in the brain and the pharmacological characteristics of their receptors.     

Anatomical and ontogenetic studies of the human paraventriculo-infundibular corticoliberin system

In human fetus, newborn, infant and adult hypothalami, antibodies to ovine corticoliberin-41 stain a paraventriculo-infundibular neuroglandular pathway. The perikarya are located in the paraventricular nucleus, they mainly project to the ventral and lateral areas of the median eminence. Eminential corticoliberin-positive fibres appear during the 16th week of fetal life, and increase in number during the following weeks. Perikarya were first revealed in the 19th week. In some areas of the median eminence, corticoliberin-, vasopressin- or [Met]enkephalin-immunoreactive terminals are similarly distributed. Sequential stainings or staining comparison of contiguous semi-thin sections failed to prove the coexpression of corticoliberin and [Met]enkephalin immunoreactivities in fibres, but indicated that corticoliberin and vasopressin immunoreactivities may be coexpressed in a few fibres. Those methods enabled us to observe, in the paraventricular nucleus, perikarya revealed by corticoliberin and vasopressin antisera. Our results suggest a possible release of corticoliberin in portal vessels of the median eminence beginning in the 16th week of fetal life, i.e. 8 weeks later than appearance of the corticotrophs in the pituitary. Establishment of a corticoliberin hypothalamic control of pituitary corticotrophs at mid gestation agrees with previous physiological and teratological studies. Abundance, as well as immunostaining intensity of the corticoliberin processes, in the infant and adult median eminence attest to the physiological importance of this system. Close vicinity of corticoliberin, vasopressin and [Met]enkephalin fibres, in some eminential areas and coexpression of corticoliberin and vasopressin immunoreactivities in some neurons, are morphological correlates of functional relations which were reported.     

Vasopressin- and neurophysin-immunoreactive neurons in the septal region, medial amygdala and locus coeruleus in colchicine-treated rats

The distribution and morphology of neurons containing vasopressin, oxytocin and their associated neurophysins were examined immunohistochemically in rats given intracerebroventricular injections of colchicine. Under these conditions, numerous neurons containing vasopressin and neurophysin were found in several brain areas in addition to those previously described in the hypothalamus. Individual parvocellular vasopressin neurons were scattered in the medial and lateral septum and vertical limb of the nucleus of the diagonal band, while a large number of such neurons were found throughout both the bed nucleus of the stria terminals and the dorsal portion of the medial amygdala. In addition a small cluster of parvocellular vasopressin neurons was present adjacent to the top of the third ventricle in the posterior dorsal hypothalamic area and a number of such neurons were found in the ventral locus coeruleus and sub coeruleus. The mean diameters of these parvocellular vasopressin neurons ranged from 16.6 to 19.8 micron in the different regions, in contrast to the 25.4 micron mean diameter of hypothalamic magnocellular vasopressin neurons, or the 13.7 micron mean diameter of parvocellular vasopressin neurons in the suprachiasmatic nucleus. No vasopressin neurons were found in other brain and spinal cord regions under the conditions used in this study, although all regions were examined. No oxytocin neurons other than those previously described in the hypothalamus and immediately contiguous regions were found. Measurement of the mean diameter of oxytocin neurons showed that neurons in the caudal paraventricular nucleus were clearly smaller (18.9 micron) than magnocellular oxytocin neurons (24.8 micron) in other parts of the hypothalamus. These parvocellular oxytocin neurons with experimentally documented central connections were similar in both size and appearance to the parvocellular vasopressin neurons seen after colchicine treatment, which are potential sources of certain central vasopressin pathways. These findings indicate that there are at least two types of oxytocin neurons in the hypothalamus and several types of vasopressin neurons in a variety of different areas in the brain, many of which are outside of the hypothalamus.     

A re-examination of the localization of immunoreactive dynorphin(1–8), [Leu]enkephalin and [Met]enkephalin in the rat neurohypophysis

We addressed in this study, with immunocytochemical methods, the following questions:

(1) are immunoreactive enkephalins in the rat neurohypophysis stored in nerves distinct from neurosecretory nerves;

(2) where is [Met]enkephalin immunoreaction localized;

(3) does immunoreactive [Leu]enkephalin coexist with pro-enkephalin or with pro-dynorphin fragments; and

(4) are the interpretations of localization studies influenced by the choice of pre-embedding or post-embedding immunocytochemical techniques? We compared immunoreactions due to antibodies which had been used by others in previous studies, examined both lyophilized and conventionally fixed specimens, and applied pre- and post-embedding protocols. Both pre- and post-embedding stainings confirmed co-storage of immunoreactive dynorphin(1–8)-like materials with vasopressin. Immunoreactive [Met]enkephalin-like material always coexisted with oxytocin. Most of the immunoreactive [Leu]enkephalin-like material appeared to occur in oxytocin nerves; only in larger vasopressin varicosites was there some dot-like [Leu]enkephalin immunoreaction. This indicates that neural lobe [Leu]enkephalin predominantly is cleaved from a precursor which also contains [Met]enkephalin.

When pre-embedding methods were modified in order to block diffusion and to enhance penetration of antibodies, enkephalin immunoreactivity was always found in typical neurosecretory varicosities with large granules. Structures previously interpreted as enkephalinergic nerve terminals contacting pituicytes most likely are neurosecretory varicosities.     

Naloxone potentiates the release of oxytocin induced by systemic administration of cholecystokinin without enhancing the electrical activity of supraoptic oxytocin neurones

Summary Studies performed in conscious female rats confirmed that iv injection of cholecystokinin octapeptide (CCK; 20µ/kg) increased the circulating concentration of oxytocin but not that of vasopressin, and confirmed that the stimulation of oxytocin release was markedly facilitated after iv administration of naloxone (1mg/kg), indicating attenuation of oxytocin release by endogenous opioids. To investigate the site of action of the endogenous opioids, the electrical activity of putative oxytocin neurones in the supraoptic nucleus was recorded in urethaneanaesthetised female rats. Oxytocin neurones responded to CCK injection with an increase in firing rate lasting 5–15 min, but this response was not facilitated by prior injection of naloxone. The results suggest that the opioid influence upon CCK-induced oxytocin release operates at the level of the neurosecretory terminals in the neurohypophysis rather than centrally. Since CCK does not elevate vasopressin release, it appears unlikely that dynorphin, the opioid peptide co-existing with vasopressin, is responsible in these circumstances for the cross-inhibition of oxytocin release. It is suggested that products of proenkephalin A, the met-enkephalin precursor present in the supraoptic nucleus and in the neurohypophysis itself, may be active in the regulation of oxytocin release.     

The kappa opioid receptor and dynorphin co-localize in vasopressin magnocellular neurosecretory neurons in guinea-pig hypothalamus

The relationship between the cloned kappa opioid receptor, dynorphin, and the neurohypophysial hormones vasopressin and oxytocin was analysed in the guinea-pig hypothalamic magnocellular neurosecretory neurons. This analysis was performed in order to understand better which population of neuroendocrine neurons in the guinea-pig is modulated by kappa opioid receptors and its endogenous ligand dynorphin. Extensive co-localization was observed between kappa opioid receptor immunoreactivity and preprodynorphin immunoreactivity in neuronal cell bodies in the paraventricular and supraoptic nuclei. Cells positive for either the kappa opioid receptor or both the kappa opioid receptor and preprodynorphin were restricted to the vasopressin expressing neuronal population and not found in the oxytocin expressing neuronal population. The kappa opioid receptor and dynorphin were examined in the posterior pituitary and both were found to be extensively distributed. Staining for the kappa opioid receptor and dynorphin B co-localized in posterior pituitary. In addition, immunogold electron microscopy confirmed that kappa opioid receptor and dynorphin B immunoreactivity were found in the same nerve terminals. Ultrastructural analysis also revealed that kappa opioid receptor immunoreactivity was associated with both nerve terminals and pituicytes. Within nerve terminals, kappa opioid receptor immunoreactivity was often associated with large secretory vesicles and rarely associated with the plasma membrane.Our data suggest that the cloned kappa opioid receptor may directly modulate the release of vasopressin but not oxytocin in guinea-pig hypothalamic magnocellular neurosecretory neurons and posterior pituitary. Furthermore, we propose that this receptor is an autoreceptor in this system because our results demonstrate a high degree of co-localization between kappa opioid receptor and dynorphin peptide immunoreactivity in magnocellular nerve terminals.     

Coexistence of salusin and vasopressin in the rat hypothalamo-hypophyseal system

Salusins are two newly discovered TOR-related peptides consisting of 28 and 20 amino acids and designated salusin-alpha and salusin-beta, respectively. Using immunohistochemistry techniques, salusin-like immunoreactivity was detected in the rat hypothalamo-neurohypophyseal tract and immunopositive cells were distributed in the suprachiasmatic, supraoptic and paraventricular nucleus. In the paraventricular nucleus, salusin-like immunoreactivity was observed both in parvocellular and magnocellular neurons. Many salusin-positive nerve fibers and their terminals were identified in the internal layer of the median eminence and posterior pituitary. Less intense salusin-positive staining of fibers and terminals was found in the suprachiasmatic nucleus and external layer of the median eminence. Dual immunostaining was performed to determine if salusin coexisted with vasopressin or oxytocin in the hypothalamus. Most of the salusin-like immunoreactivity was detected in vasopressin- but not in oxytocin-containing neurons in these nuclei. The functional significance of the coexistence of salusin with vasopressin is discussed, including the possibility that salusin participates in the regulation of blood pressure.     

Distribution of some peptides (substance P, [Leu]enkephalin, [Met]enkephalin) in the brain stem and spinal cord of a lizard, Varanus exanthematicus

The distribution of substance P-like and [Leu]- and [Met]enkephalin-immunoreactive cell bodies, fibers and terminal structures in the brain stem and spinal cord of a lizard, Varanus exanthematicus, was studied with the indirect immunofluorescence technique, using antibodies to these peptides. Substance P-like immunoreactive cell bodies were found in the hypothalamus, in a periventricular cell group in the rostral mesencephalon, in the interpeduncular nucleus, in and ventral to the descending nucleus of the trigeminal nerve, in and directly ventral to the nucleus of the solitary tract, scattered in the brain stem reticular formation and in the trigeminal and spinal ganglia. A rather widespread distribution of substance P-like immunoreactivity was found in the brain stem and spinal cord, mainly concentrated in striatotegmental projections related to visceral and/or taste information (nucleus of the solitary tract, parabrachial region), in the descending nucleus of the trigeminal nerve and in the dorsal horn of the spinal cord (areas I and II). In the spinal cord also around the central canal (area X and adjacent parts of area V-VI) a distinct substance P innervation was found. The ventral horn receives only a very sparse substance P innervation. The distribution of [Leu]- and [Met]enkephalin in the brain stem and spinal cord of Varanus exanthematicus is less impressive than that of substance P. Enkephalinergic cell bodies were found particularly in the caudal hypothalamus. Small populations of enkephalinergic cell bodies were found in the vestibular nuclear complex, in the nucleus of the solitary tract, in and around the descending nucleus of the trigeminal nerve and throughout the rhombencephalic reticular formation. Enkephalins are likely to be present in efferent projections of the striatum, in projections related to taste and/or visceral information (nucleus of the solitary tract, parabrachial region) and in descending pathways to the spinal cord. Enkephalinergic fibers are present in the lateral funiculus and enkephalin-immunoreactive cell bodies are found in the reticular formation, particularly the inferior reticular nucleus which is known to project to the spinal cord. In the spinal cord enkephalinergic terminal structures were found especially in the superficial layer of the dorsal horn (areas I and II) and around the central canal. The ventral horn including the motoneuron area receives only a relatively sparse enkephalinergic innervation.     

Lack of response in the release of oxytocin and vasopressin from isolated neurohypophyses to dopamine,met-enkephalin and leu-enkephalin

Summary We investigated the effects of dopamine, met-enkephalin and leu-enkephalin on basal and ouabain-stimulated release of oxytocin and vasopressin from isolated neurointermediate lobes. The present study revealed that neurohypophyseal hormone release was not affected by dopamine, neither from lobes of untreated rats nor from those of rats with dopamine-deficiency (pretreated with -methyl-p-tyrosine-methylester). Likewise, metoclopramide, a dopamine antagonist, was unable to alter the neurohypophyseal hormone release. Our results also indicate that the opioid peptides met-enkephalin and leu-enkephalin do not influence spontaneous or ouabain-stimulated oxytocin and vasopressin release, which is in accordance with our findings that naloxone under our experimental conditions is also ineffective.     

Increased [Met]enkephalin and decreased substance P in spinal cord following thermal injury to one limb.

Thermal injury to one hind limb of rats was induced by immersion into water at 62 degrees C. Both a mild (15 s) or severe (30 s) lesion caused inflammation of the limb when observed 24 h later; but at this time the animals used the injured limb when they walked. Animals with a severe lesion of the injured limb subsequently withdrew it from use when walking. Limb withdrawal did not occur following a mild lesion. At 24 h following the lesion, lumbar spinal cord levels of [Met]enkephalin, as measured by radioimmunoassay, were elevated (70%) bilaterally in both hemisegments, ipsi- and contralateral to the lesion. At seven days following either mild or severe hind limb lesion [Met]enkephalin levels were elevated only in the ipsilateral lumbar hemisegment. At that time no changes in thoracic [Met]enkephalin levels were observed. Substance P levels were decreased (20-25%) bilaterally in the lumbar cord 24 h following a severe limb lesion, but no change was observed at seven days in any cord segment following a mild or severe lesion. Changes in spinal cord [Met]enkephalin content occur in response to thermal injury to one hind limb. However, the changes do not appear to be related to the withdrawal of the damaged limb from use following a severe lesion. Peptide changes in the spinal cord may reflect pain or injury to the damaged limb following a thermal lesion. In contrast, limb withdrawal may be a physiological rest mechanism related to altered basal ganglia peptide function.     

Enkephalin innervation of the paraventricular nucleus of the hypothalamus: distribution of fibers and origins of input

The opioid peptide enkephalin emerges as a major neuromodulator in the regulation and integration of the physiologic response in stressful conditions. The paraventricular nucleus of the hypothalamus is a coordinating center of neuroendocrine and autonomic functions. However, the detailed distribution of the enkephalin fibers and terminals in the paraventricular nucleus and the sources of enkephalinergic innervation are not well defined. In the present study, we used immunocytochemistry for the proenkephalin-derived octapeptide met-arg⁶-gly⁷-leu⁸ enkephalin to determine the distribution of enkephalin-immunoreactive fibers and somata within paraventricular nucleus. Without colchicine pretreatment, enkephalinergic fibers were prominent mainly in the ventromedial part of the parvicellular subdivision of the paraventricular nucleus, appearing in coronal sections as a dense collection of short segments of enkephalin-immunoreactive fibers. In the periventricular portion of the paraventricular nucleus, enkephalin-immunoreactive fibers produced a moderate plexus of short enkephalin-immunoreactive fibers dorsoventrally oriented. With colchicine treatment, a dense cluster of enkephalin-immunoreactive cell bodies was located in the dorsomedial and the dorsal parts of the parvicellular subdivisions. These enkephalin-immunoreactive neurons were small (< 10 μm) to medium sized (10–15 μm), with round and elongated shapes. Retrograde transport of wheat germ-conjugated gold particles. WGA-apoHRP-Au, from the paraventricular nucleus, combined with immunocytochemistry for enkephalin revealed that the major sources of extrahypothalamic enkephalin afferents to the paraventricular nucleus are provided by enkephalin neurons neurons in the lateral reticular nucleus and the paragigantocellularis reticular nucleus of the medulla (20% of retrogradely labeled neurons within this nucleus were double labeled) and in the nucleus solitary tract (10% of retrogradely labeled neurons within thisnucleus were double labeled). Retrogradely labeled enkephalin neurons were also observed in the medial preoptic area, median preoptic nucleus, dorsomedial hypothalamic nucleus, lateral septum and hypothalamic arcuate nucleus. These enkephalinergic pathways from the medulla and the forebrain could represent an anatomical substrate underlying the opioid effects on paraventricular neurons during physiological processes, such as a cardiovascular regulation, feeding or stress responses.     

Ontogeny and regional distribution of proenkephalin- and prodynorphin-derived peptides and opioid receptors in rat hippocampus

Levels of prodynorphin- and proenkephalin-derived peptides were determined in whole hippocampus of prenatal and early postnatal rats and in five regions of the hippocampus of the adult rat. Using autoradiography, opioid receptor subtypes were localized in coronal sections of adult hippocampus. The opioid peptides are present in very low concentrations in prenatal hippocampus, with only dynorphin B and alpha-neo-endorphin being present in significant amounts. The main increase in concentrations of the opioid peptides occur between day 7 and 14 postnatally, when dynorphin A, dynorphin A-(1-8), dynorphin B and alpha-neo-endorphin reach their adult levels. beta-Neo-endorphin and [Met]enkephalyl-Arg-Gly-Leu do not reach their maximal level until later in development. There is a distinct differential distribution of the opioid peptides in the subregions of the hippocampus; the subiculum and CA1 are relatively poor in prodynorphin-derived peptides but do contain significant amounts of [Met]enkephalin and [Leu]enkephalin. Very high concentrations of dynorphin B and alpha-neo-endorphin are present in region CA4. Dynorphin A-(1-8) and [Met]enkephalin have their highest concentrations in the dentate gyrus. There is a 5-fold higher concentration of [Met]enkephalin in the ventral hippocampus compared to the dorsal hippocampus. A similar trend is seen with dynorphin A-(1-8) but not with the other opioid peptides. The most abundant opioid receptor population in the hippocampus is of the mu type and it is densest in and around stratum pyramidale of the region CA3. There are relatively few kappa opioid receptors in the rat hippocampus. These results indicate the presence of at least two independent opioid neuronal systems (enkephalin and dynorphin) in rat hippocampus and the presence of mu-, delta- and kappa-opioid receptor subtypes.     

The immunocytochemical distribution of seven peptides in the spinal cord and dorsal root ganglia of horse and pig

Summary The distribution of calcitonin gene-related peptide (CGRP), enkephalin, galanin, neuropeptide Y (NPY), somatostatin, tachykinins and vasoactive intestinal polypeptide (VIP) was compared in cervical, thoracic, lumbar and sacral segmental levels of spinal cord and dorsal root ganglia of horse and pig.In both species, immunoreactivity for the peptides under study was observed at all segmental levels of the spinal cord. Peptide-immunoreactive fibres were generally concentrated in laminae I–III, the region around the central canal, and in the autonomic nuclei. A general increase in the number of immunoreactive nerve fibres was noted in the lumbosacral segments of the spinal cord, which was particularly exaggerated in the case of VIP immunoreactivity. In the horse, some CGRP-, somatostatin- or tachykinin-immunoreactive cell bodies were present in the dorsal horn. In the pig, cells immunoreactive for somatostatin, enkephalin or NPY were noted in a similar location.In the ventral horn most motoneurones were CGRP-immunoreactive in both species. However, in pig many other cell types were CGRP-immunoreactive not only in the ventral horn, but also in laminae V–VI of the dorsal horn.With the exception of enkephalin and NPY immunoreactivity, which was not seen in pig dorsal root ganglia, all peptides studied were localised to neuronal cell bodies and/or fibres in the dorsal root ganglia. In both species, immunolabelled cell bodies were observed in ganglia from cervical, thoracic, lumbar and sacral levels, with the exception of VIP-immunoreactive cells that were detected only in the lumbosacral ganglia. Numerous CGRP- and tachykinin-immunoreactive cell bodies were visualised in both species, while the cells immunolabelled with other peptide antisera were much lower in number.In both species, immunostaining of serial sections revealed that a subset of CGRP-immunoreactive cells co-expressed tachykinin, galanin or somatostatin immunoreactivity. In the horse some enkephalin-immunoreactive cells were also CGRP positive and occasionally combinations of three peptides, e.g. CGRP, tachykinin and galanin or CGRP, tachykinin and enkephalin were identified.The results obtained suggest that the overall pattern of distribution of peptide immunoreactivities is in general agreement with that so far described in other mammals, although some species variations have been observed, particularly regarding the presence of immunoreactive cell bodies in the dorsal horn of the spinal cord.     

Immunohistochemical studies on the effect of capsaicin on spinal and medullary peptide and monoamine neurons using antisera to substance P, gastrin/CCK, somatostatin, VIP, enkephalin, neurotensin and 5-hydroxytryptamine

Summary After neonatal treatment of rats with capsaicin, the spinal cord, the spinal trigeminal nucleus and spinal and trigeminal ganglia were analysed with immunohistochemistry using antisera to several peptides and 5-hydroxytryptamine. A marked decrease was observed in substance P-, cholecystokinin-, somatostatin- and VIP-like immunoreactivity present in the central branches of primary sensory neurons in the spinal cord and in substance P- and somatostatin-like immunoreactivity in sensory ganglion cells. No definite depleting effect of capsaicin could be established on 5-hydroxytryptamine and peptides, such as enkephalin and neurotensin, present in centrally originating fibres in the dorsal horn of the spinal cord. The results demonstrate that the effects of capsaicin are not confined to substance P immunoreactive primary sensory neurons. The possibility is discussed that capsaicin effects specifically functioning rather than chemically specific primary sensory neurons.