Localization of chemical messengers in magnocellular neurons of the hypothalamic supraoptic and paraventricular nuclei: an immunohistochemical study using experimental manipulations.

Indirect immunofluorescence histochemistry was used to investigate the distribution and extent of co-localization of chemical messengers in magnocellular neurons of the supraoptic and paraventricular nuclei. In order to increase the number of neurons immunoreactive to the antisera used, experimental manipulations were employed. The homozygous Brattleboro (diabetes insipidus) rat was also investigated. In untreated rats, only vasopressin- and oxytocin-like immunoreactivities could be observed. Colchicine treatment alone resulted in appearance of galanin-, dynorphin-, cholecystokinin-, [Leu]enkephalin- and thyrotropin-releasing hormone-positive cells. In hypophysectomized rats, all these markers, except tyrosine hydroxylase, showed substantial further increases. In addition, peptide histidine-isoleucine-immunoreactive cell bodies could now be seen. After salt-loading alone, tyrosine hydroxylase-like immunoreactivity was markedly increased, whereas vasopressin- and oxytocin-like immunoreactivity were very weak or undetectable. When salt-loaded rats received colchicine, corticotropin-releasing factor- and peptide histidine-isoleucine-like immunoreactivity in addition increased, whereas galanin- and dynorphin-like immunoreactivity markedly decreased. The Brattleboro rats resembled untreated rats, except their lack of vasopressin-like immunoreactivity, the marked increase in tyrosine hydroxylase-like immunoreactivity, and smaller increase in galanin- and dynorphin-like immunoreactivity. Addition of colchicine to Brattleboro rats resulted in some distinct further changes in that dynorphin-like immunoreactivity decreased in some neurons and that [Leu]enkephalin-, corticotropin-releasing factor- and peptide histidine-isoleucine-like immunoreactivity increased substantially. Several similarities could be observed between the salt-loaded and Brattleboro rats, with or without colchicine. However, a marked difference in immunoreactive [Leu]enkephalin levels was observed with no difference in dynorphin-like immunoreactivity, and opposite changes in galanin-like immunoreactivity. The results confirm the traditional view that hypothalamic magnocellular neurons in the supraoptic and paraventricular nuclei contain two separate cell populations, characterized by vasopressin and oxytocin, respectively, and that they contain additional messenger molecules in specific patterns. Vasopressin-containing neurons primarily express tyrosine hydroxylase, galanin, dynorphin, [Leu]enkephalin and peptide histidine-isoleucine, and to a minor extent cholecystokinin and thyrotropin-releasing hormone. Oxytocin-containing neurons mainly have cholecystokinin and corticotropin-releasing factor, and to a minor extent galanin, dynorphin, [Leu]enkephalin and thyrotropin-releasing hormone. Furthermore, our results detail individual co-existence situations among these putative messenger molecules. Thus, magnocellular neurons respond in a differential way to various stimuli and they store multiple bioactive substances in specific combinations.     

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.     

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.     

Immuno-electron microscopic evidence for two different types of partial somatic repair of the mutant Brattleboro vasopressin gene.

In homozygous Brattleboro rats a frame-shift mutation in the vasopressin gene prevents secretion of vasopressin by magnocellular neurosecretory neurons and thus causes diabetes insipidus. Whereas most "vasopressin" neurons in Brattleboro homozygotes apparently lack vasopressin and its associated neurophysin and glycopeptide, some isolated cells overcome the mutation and "revert" to producing readily detectable amounts of vasopressin. We describe here two morphologically and immunocytochemically distinct subsets of such "revertant" cells. One subset contain, in their rough endoplasmic reticulum cisterns, electron-dense aggregates immunoreactive for vasopressin, for parts of oxytocin-neurophysin, and for CP14 (a peptide with a sequence deduced from the mutated precursor), but not for vasopressin-associated glycopeptide ("glycopeptide") or vasopressin-neurophysin. In Brattleboro heterozygotes, which have one mutant and one normal copy of the vasopressin gene, morphologically similar revertant cells exist; the aggregates in the rough endoplasmic reticulum of these cells do not immuno-label for CP14, but the cells do produce 160-nm neurosecretory granules immunoreactive for vasopressin, vasopressin-neurophysin and glycopeptide. In Brattleboro homozygotes, the second, more abundant subset of neurons which recover vasopressin immunoreactivity also express vasopressin-associated glycopeptide and CP14 but not oxytocin-neurophysin; both glycopeptide and CP14 are restricted to the rough endoplasmic reticulum but do not form aggregates. We conclude that two different somatic repairs of the Brattleboro mutation can occur. We propose that, in aggregate-containing neurons, exons B and C have been exchanged between the vasopressin and oxytocin genes; glycopeptide-immunoreactive neurons have either undergone mismatch repair or exchanged exon B.     

Solitary magnocellular neurons in the homozygous Brattleboro rat have vasopressin and glycopeptide immunoreactivity

A small but distinctive population (about 1 in 600) of magnocellular neurosecretory neurons in homozygous Brattleboro rats are immunoreactive for vasopressin, and a similar number for the carboxy-terminal glycopeptide of the vasopressin prohormone. These solitary cells are found in all animals and in all parts of the magnocellular system, but not in the suprachiasmatic or other hypothalamic nuclei. The majority of the solitary cells do not differ morphologically from the remainder of the magnocellular neurons. The immunoreactivity is markedly denser in the Nissl bodies than in the Golgi region. Serial sections show that the vasopressin and glycopeptide immunoreactive material is co-localized in the same cells, and that these cells are not immunoreactive for oxytocin. A published sequence for the Brattleboro vasopression gene mutation indicates a base-deletion upstream from the glycopeptide-encoding portion, and implies a frameshift that would cause translation of incorrect protein continuing into the poly-A tail of the mRNA. Although this could apply to the majority of the Brattleboro presumptive vasopressin neurons, the co-localization in our solitary cells of material immunoreactive with antibodies to both the amino- and carboxy-terminals of the vasopressin prohormone suggest that in these cases an additional mechanism may be operating.     

Enkephalin fibers synapse on cholinergic neurons in the rat sacral intermediolateral nucleus: a double-immunostaining at the light and electron microscopic levels

Relationships between leucine-enkephalin fibers and cholinergic neurons in the rat sacral intermediolateral nucleus were examined by light and electron microscopy using double-immunostaining method. Cholinergic neurons in the sacral intermediolateral nucleus were labeled by a rat-mouse monoclonal antibody to choline acetyltransferase and stained bluish green with 5-bromo-4-chloro-3-indolyl-beta-D- galactoside reaction products using beta-galactosidase as a marker. On the same sections, leucine-enkephalin fibers were labeled by a rabbit polyclonal antiserum to leucine-enkephalin and stained brown by diaminobenzidine reaction products using peroxidase as a marker. After embedding in Epon, the sections were examined in light and electron microscopes. In the light microscope, choline acetyltransferase-like immunoreactive cells were seen in the sacral intermediolateral nucleus. In the same region, leucine-enkephalin-like immunoreactive cells. In the electron microscope, 5-bromo-4-chloro-3-indolyl-beta-D-galactoside reaction products were in the form of coarse electron dense deposits in the choline acetyltransferase-like immunoreactive structures and could be distinguished from the much finer grained diaminobenzidine reaction products. Choline acetyltransferase-like immunoreactive neurons received synaptic inputs from leucine-enkephalin fibers-like immunoreactive terminals. These findings suggest that leucine-enkephalin fibers may affect the activity of cholinergic parasympathetic preganglionic neurons.     

Met-enkephaline-like immunoreactivity in a cephalopod neurohemal organ.

Using a highly specific antibody we found Met-enkephalin-like immunoreactivity in large granules of numerous distinct cells that are embedded in a layer of secretory terminals inside the octopus vena cava. Application of antibodies against fragments of the mammalian pro-opiocortin precursor -- alpha-MSH, ACTH, beta-endorphin and 16k-fragment -- and against growth hormone did not produce immunostaining in the octopus enkephalin cells. The vena cava neuropil may represent a favourable system for the examination of the physiological role of the opioid peptide enkephalin.     

A quantitative analysis of the interrelationships between subpopulations of rat sensory neurons containing arginine vasopressin or oxytocin and those containing substance P, fluoride-resistant acid phosphatase or neurofilament protein

In rat L5 dorsal root ganglia 50% of neurons contained arginine vasopressin-like immunoreactivity and 38% oxytocin-like immunoreactivity, the oxytocin entirely coexisting with the arginine vasopressin. Staining of alternate mirror-image sections with RT97 (an antibody to neurofilament protein, and a marker for large light neurons) and with arginine vasopressin antiserum showed that the two were entirely complementary, thus establishing arginine vasopressin as a marker for all small dark neurons. Mirror-image staining also showed that neurons containing substance P-like immunoreactivity and those containing fluoride-resistant acid phosphatase activity were each contained within the arginine vasopressin-positive population. Arginine vasopressin-like immunoreactivity was axonally transported in the dorsal root and (in greater quantity) in sciatic nerve. Arginine vasopressin-like immunoreactivity was present also in laminae I and II of the dorsal horn of the spinal cord and this reactivity was absent in animals which had been treated neonatally with capsaicin, suggesting that it was contained in primary afferent terminals. These results are discussed in terms of their implications for the classification of primary afferent neurons and of a possible physiological role for arginine vasopressin in these neurons.     

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.     

Galanin coexists with vasopressin in the normal rat hypothalamus and galanin's synthesis is increased in the Brattleboro (diabetes insipidus) rat

Galanin is a peptide containing 29 amino acid residues, that is present in the median eminence, in the magnocellular neurons of the supraoptic (SON) and paraventricular nuclei (PVN) of the rat hypothalamus and in the posterior pituitary. We report here that: (1) immunoreactivity for galanin (GAL) and vasopressin coexist in the SON of normal rats, (2) levels of mRNA encoding preprogalanin are markedly elevated in the PVN and SON of Brattleboro (diabetes insipidus) rats, as determined by in situ hybridization histochemistry but (3) levels of GAL-like immunoreactivity (GAL-LI) are significantly reduced in the posterior pituitary of these rats, as determined by radioimmunoassay. We suggest that production and possibly secretion of the peptide GAL may be increased in the Brattleboro rat.     

Quantitative autoradiographic mapping of neurohypophysial hormone binding sites in the rat forebrain and pituitary gland--II. Comparative study on the Long-Evans and Brattleboro strains

The anatomical distribution and pharmacological characteristics of the different types of neurohypophysial hormone binding sites were compared in the forebrains and pituitary glands of Long-Evans rats and its mutant Brattleboro strain, genetically deficient in vasopressin. Quantitative autoradiography on sections incubated in the presence of 5 nM of either [3H]oxytocin or [3H]vasopressin revealed the presence of the same types of sites in the brains of both strains but noticeable variations in their densities were found in several areas. In the forebrain, oxytocin/vasopressin sites, which bind both peptides with similar high nanomolar affinities, had the same locations and densities in the ventral subiculum, in several nuclei of the amygdala, the bed nucleus of the stria terminalis and the olfactory tubercle. The density of such sites was, in contrast, lower in the ventromedial hypothalamic nucleus of the Brattleboro rat. Selective vasopressin sites which bind [3H]vasopressin with a nanomolar-range affinity and [3H]oxytocin with a much lower affinity showed more variations. They were not found in the Brattleboro rat thalamus but were highly concentrated in several thalamic nuclei in the Long-Evans rat. Conversely, their densities were higher in the dopaminergic A13 cell group of the zona incerta and the suprachiasmatic nucleus of the Brattleboro rat. Their densities were similar in the lateral septal nucleus and in the fundus striati of both strains. In the hypothalamo-neurohypophysial system, [3H]oxytocin and [3H]vasopressin binding occurred in the Long-Evans rat with characteristics different from those found in other brain areas. In the Brattleboro rat, no [3H]vasopressin binding and only low [3H]oxytocin binding, restricted to the magnocellular nuclei, were found.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opioid synapses on vasopressin neurons in the paraventricular and supraoptic nuclei of juvenile monkeys.

Opioid peptide- as well as vasopressin-containing neurons synapse on gonadotropin releasing hormone neurons in juvenile macaques. In this study we performed double-label immunostaining for opioid and vasopressin neurons in the paraventricular and supraoptic nuclei in order to assess their interrelationships. Neuroendocrine neurons in the hypothalamus were prelabeled by microinjection of electron-dense retrograde tracer into the median eminence, and were easily identified in frontal Vibratome sections. Sections through the paraventricular and supraoptic nuclei were immunostained for vasopressin with the peroxidase-antiperoxidase technique, and for opioids using the indirect immunogold method. By light microscopy, opioid-immunoreactive inputs appeared to innervate an average of 39% of the vasopressin neurons in the paraventricular nucleus and 33% in the supraoptic nucleus, and were more prevalent anteriorly. Clusters of opioid afferents formed cup-like calices around major processes of many vasopressin neurons, especially in the paraventricular nucleus. Electron microscopy revealed that these groups of opioid axon terminals made frequent symmetrical and fewer asymmetrical synapses on both neuroendocrine and non-neuroendocrine vasopressinergic cell bodies and dendrites. Our study did not reveal vasopressin-opioid synapses in these hypothalamic nuclei, but this does not preclude the possibility of their existence elsewhere. These results indicate that opioid afferents modulate vasopressin neuronal activity in the monkey paraventricular and supraoptic nuclei. Previous results have suggested that corticotropin releasing hormone acts via vasopressinergic neurons to stimulate opioid neuronal activity and to inhibit gonadotropin releasing hormone release. Taken together, the data suggest that stressful stimuli could initiate a series of neuropeptidergic interactions which ultimately alter pulsatile gonadotropin releasing hormone secretion and thus gonadotropin secretion in primates.     

Calcium-containing mitochondrial granules in neurohypophysial axon terminals disappear following vasopressin treatment of Brattleboro rats

The presence of intramitochondrial calcium-containing electron-dense granules was demonstrated in axon terminals of chronically hyperactive neurosecretory neurons of untreated homozygous Brattleboro rats. Following vasopressin treatment for 30 days, which has been shown to attenuate this neuronal hyperactivity, calcium-containing deposits could not be detected in mitochondria. It is concluded that the presence of intramitochondrial calcium-containing dense deposits is connected with the functional state of neurosecretory neurons.     

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.     

Neuropeptide gene expression in hypothalamic magnocellular neurons of normal and hypophysectomized rats: a combined immunohistochemical and in situ hybridization study.

Hypothalamic magnocellular neurons of the paraventricular and supraoptic nuclei contain several peptides and non-peptide putative neurotransmitters co-existing with vasopressin and oxytocin. However, the functional role of these substances is still unknown. In the present paper the temporal course of changes in the expression of vasopressin, oxytocin, galanin, cholecystokinin, dynorphin and tyrosine hydroxylase in magnocellular hypothalamic neurons of rats subjected to hypophysectomy was examined. Following different survival times the animals were processed either for immunohistochemistry with antibodies against the above mentioned peptides or for in situ hybridization with synthetic oligonucleotide probes complementary to the mRNAs encoding for the peptides. The results obtained showed a marked rise in vasopressin mRNA levels at two days followed by a decrease up to 36 days of survival. Oxytocin mRNA responded to the lesion with a transient decrease, with its lowest values between five and seven days. This was followed by a recovery which almost reached normal values at 36 days of survival. The results also showed a marked, transient activation of the synthetic pathway for galanin and cholecystokinin. The numbers of cells expressing these peptides were maximal between five and seven days, and the respective mRNA levels were significantly increased at these survival times. This was followed by a decrease in the amount of galanin- and cholecystokinin-like immunoreactivity as well as in the levels of their respective mRNAs. Dynorphin-like immunoreactivity showed a course similar to that of galanin and cholecystokinin in operated animals. However, the amounts of dynorphin mRNA were significantly increased at two days, but were followed by a reduction at five days and remained low throughout the different survival times tested. The experiments performed with the tyrosine hydroxylase antibodies and probe showed undetectable levels of the enzyme and its mRNA in normal and hypophysectomized animals. These results demonstrate that, in magnocellular hypothalamic neurons, expression of several peptides occur in differential ways after hypophysectomy. The possibility is discussed that these changes represent part of the mechanisms underlying the process of degeneration and regeneration known to occur in magnocellular hypothalamic neurons after hypophysectomy.     

Met]- and [Leu]enkephalin-like immunoreactive cell bodies and nerve fibres in the coeliac ganglion of the cat

The occurrence and distribution of methionine- and leucine-enkephalin-like immunoreactivity were investigated in the cat coeliac ganglion using either the indirect immunoperoxidase method or the peroxidase-antiperoxidase technique. Several antisera raised to methionine- and leucine-enkephalin were used. Their specificity was assessed by incubating sections of the coeliac ganglion with increasing dilutions of antisera and with antisera saturated with their respective antigen. The present study was performed both in untreated and in colchicine-treated cats. Immunoreactive methionine- and leucine-enkephalin-like cell bodies were only visualized in colchicine-treated cats. Two types of labeled cells were observed. The first type had a size similar to that of unlabeled principal ganglion cells. These labeled cells were numerous and scattered throughout the ganglion; they probably represented enkephalin-containing ganglion cells. The second type of immunoreactive cells were of a much smaller size. They were always gathered in small clusters of about 5-15 cells and were not numerous; they presumably represented enkephalin-containing small intensely fluorescent cells. Immunoreactive nerve fibres were mainly observed in untreated cats and accessorily in colchicine-treated cats. In untreated animals dense networks of methionine- and leucine-enkephalin-like immunoreactive fibres were found in the coeliac ganglion. These fibres had numerous varicosities which often closely surrounded unlabeled principal ganglion cells. In colchicine-treated cats some immunoreactive fibres surrounded labeled principal ganglion cell bodies. The present results establish for the first time the presence of enkephalin-like immunoreactive principal ganglion cells in a mammalian sympathetic prevertebral ganglion. The presence of enkephalin-containing principal ganglion cells, small intensely fluorescent cells and nerve terminals, supports an important role of enkephalins in the integrative synaptic activities of cat coeliac ganglion cells.     

Immunohistochemical study on the distribution of neuropeptides within the pontine tegmentum--particularly the parabrachial nuclei and the locus coeruleus of the human brain.

The topographical distribution of neuropeptide-containing cell bodies, fibers and terminals was studied in human parabrachial nuclei and the pontine tegmentum with immunohistochemical stainings. Brains of seven adult human subjects of 35-72 years were fixed within 2 h post mortem. Serial sections were immunostained by antisera of 14 different neuropeptides--oxytocin, vasopressin, thyrotropin-releasing hormone, angiotensin II, calcitonin gene-related peptide, beta-endorphin, dynorphin A, dynorphin B, leucine-enkephalin, alpha-melanocyte stimulating hormone, substance P, neuropeptide Y, cholecystokinin and galanin--alternately. All of these peptides were found to be present in nerve fibers and terminals, but only two, angiotensin II and dynorphin B, in cell bodies of the parabrachial nuclei. Calcitonin gene-related peptide-, neuropeptide Y-, cholecystokinin- and galanin-immunoreactive cells were present in other areas of the pontine tegmentum, like the motor trigeminal nucleus, locus coeruleus, periventricular gray matter but not in the parabrachial nuclei. Peptidergic fibers were distributed unevenly throughout the pontine tegmentum having unique, individual distribution patterns. In the parabrachial nuclei, substance P, neuropeptide Y, cholecystokinin and galanin showed the highest density of immunoreactive neuronal networks. Moderate to low concentrations of immunoreactive processes were detected by calcitonin gene-related peptide, alpha-melanocyte stimulating hormone, dynorphin B, thyrotropin releasing hormone, leucine-enkephalin, dynorphin A, angiotensin II, beta-endorphin, vasopressin and oxytocin antisera, respectively. Other pontine tegmental areas, like the locus coeruleus, dorsal tegmental, pontine raphe and motor trigeminal nuclei as well as the central gray of the tegmental region exhibited a varying assortment of neuropeptides with distinct, individual localization patterns. Their detailed topographical distributions are mapped and given in coronal sections.     

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.     

Ultrastructural localization of enkephalin-like immunoreactivity in developing rat cerebellum

Methionine enkephalin, an endogenous opioid peptide, participates in the regulation of growth in the developing brain. In the present study, enkephalin-like immunoreactivity was localized in the cerebellum of developing and adult rats by immunoelectron microscopy. In 10-day-old animals, enkephalin-like immunoreactivity was found in the somata of proliferating, migrating and differentiating neural cells, and was associated with the plasma membrane, microtubules, filaments, mitochondria, endoplasmic reticulum and nuclear envelope. Both neurons and glia in the cerebellum of the preweaning rat displayed a similar profile of immunoreactivity. Reaction product was also detected in the dendrites and dendritic spines of Purkinje cells where it was concentrated in postsynaptic densities. The majority of internal granule neurons in 10-day-old animals were not immunoreactive, nor were axons, glial processes and postsynaptic elements (with the exception of mossy fiber terminals). At weaning (Day 21), enkephalin-like immunoreactivity was confined primarily to the somata of Purkinje, basket and stellate neurons, and to Purkinje cell dendrites and synaptic spines. Adult rats (day 75) exhibited no enkephalin-like immunoreactivity. These results establish that enkephalin or an enkephalin-like substance can be detected during the ontogeny of both neurons and glia in the cerebellar cortex, and appears to be associated with certain structural elements.     

Effect of morphine on cholecystokinin and mu-opioid receptor-like immunoreactivities in rat spinal dorsal horn neurons after peripheral axotomy and inflammation

In order to further investigate the interaction between the octapeptide cholecystokinin and opioid analgesia in the spinal cord we used double-colour immunofluorescence to examine the anatomical distribution of cholecystokinin and mu-opioid receptors in the dorsal horn, as well as the effect of morphine on cholecystokinin- and mu-opioid receptor-like immunoreactivities following peripheral nerve injury and inflammation. Mu-opioid receptor-like immunoreactivity was present in 65.6% of cholecystokinin-positive neurons in laminae I and II of rat spinal cord. Conversely, 40.4% of mu-opioid receptor-positive neurons contained cholecystokinin-like immunoreactivity. Systemic application of morphine (1, 3 or 10 mg/kg; i.v.) after sciatic nerve section significantly, but reversibly, decreased mu-Opioid receptor-like immunoreactivity in the medial half of lamina II in segment L5 of the ipsilateral dorsal horn, and cholecystokinin-like immunoreactivity was also markedly reduced in the same region. These effects were dose- and time-dependent and could be prevented by naloxone preadministration. In contrast, no significant change in the pattern of distribution or intensity of mu-opioid receptor- and cholecystokinin-like immunoreactivities was observed in intact rats or during peripheral inflammation. These results provide a cellular basis for the interaction of mu-opioid receptors and cholecystokinin at the spinal level by showing a high degree of co-existence of these two molecules in local interneurons, and also show that morphine can induce rapid and short lasting effects on mu-opioid receptors after peripheral nerve injury. The results contribute to our understanding of how endogenous cholecystokinin reduces the analgesic effect of morphine.