Statistical methods for in situ hybridization: identification of autoradiographically labelled cells and structures.

In situ hybridization experiments frequently use autoradiography to identify labelled structures. Ideally, labelled cells will be overlain with a dense accumulation of particles, allowing one to discriminate them from unlabelled cells easily. However, if noise is high or the density of labelling is low, it can be difficult to distinguish bona fide labelling 'by eye'. In such situations, labelled cells could be overlooked. This paper evaluates two statistical solutions to this problem: (1) a parametric method proposed by Hashimoto and co-workers and (2) Wang & Wessendorf's non-parametric method using contingency testing (i.e. the chi-square or Fisher's exact tests). The Hashimoto method determines the mean and standard deviation of the density of background labelling, using sense-strand controls as the source of background levels. Cells labelled at densities greater than two standard deviations above the mean (P < 0.0455) are defined as significantly labelled. Contingency testing determines whether the grain density over a cell is significantly higher than that over the remainder of the image. When compared, the two methods gave similar results. The Hashimoto method may be more sensitive if most cells are labelled but contingency testing requires no assumptions about the uniformity of non-specific labelling.

Mu- and delta-opioid receptor mRNAs are expressed in periaqueductal gray neurons projecting to the rostral ventromedial medulla.

Opioid antinociception appears to be mediated at least in part by a pathway that projects from the periaqueductal gray (PAG) to the rostral ventromedial medulla (RVM), but the relationship between opioid receptors and PAG-RVM projection neurons is unclear. Previous electrophysiological studies have suggested that opioids act directly on some PAG neurons projecting to the RVM. However, immunoreactivity for neither the cloned mu-opioid receptor (MOR1) nor the cloned delta-opioid receptor (DOR1) has been observed in PAG cells retrogradely labeled from the RVM. In the present study, we examined the expression of DOR1 and MOR1 mRNAs in PAG neurons projecting to RVM using quantitative in situ hybridization and retrograde tract-tracing. Mesencephalic neurons were labeled in three male Sprague-Dawley rats by microinjection of Fluoro-Gold into the RVM. Five micrometer cryostat sections were cut and in situ hybridization was performed using full-length cRNA probes labeled with 35S-UTP. Retrogradely labeled neurons that were also labeled for MOR1 or DOR1 mRNA were observed in the dorsomedial, lateral, and ventrolateral portions of the PAG. Quantification was performed in the dorsomedial and ventrolateral PAG using the physical disector. We found that of 219 retrogradely labeled neurons, 50 +/- 14% expressed DOR1 mRNA. In a second set of 120 Fluoro-Gold-labeled neurons, 27 +/- 8% expressed MOR1 mRNA. Significantly more PAG-RVM projection neurons were labeled for MOR1 mRNA in the ventrolateral subregion of the PAG than in the dorsomedial subregion. However, no significant difference was observed in the proportions of retrogradely labeled neurons labeled for DOR1 mRNA in the ventrolateral subregion compared to the dorsomedial subregion. We conclude that opioids are likely to exert direct effects on PAG-RVM projection neurons through both delta- and mu-opioid receptors. In addition, direct effects on PAG-RVM projection neurons from activation of MOR1 appear more likely to be exerted in the ventrolateral PAG than in the dorsomedial PAG.

Coexistence of kappa- and delta-opioid receptors in rat spinal cord axons.

It has been found that heterodimers of kappa- and delta-opioid receptors can occur in vitro, but it has been unclear whether they also occur in intact animals. In the present study we examined whether kappa-delta heterodimers might occur in vivo by staining for these receptors with two-color fluorescence immunocytochemistry. Sections of rat spinal cord were double-stained using rabbit anti-kappa opioid receptor combined with rat anti-delta-opioid receptor. It was found that axons in the superficial dorsal horn of the spinal cord were double-labeled. In addition, structures within axonal varicosities were sometimes double-labeled. We conclude that single axons, and single structures within axons, express both kappa- and delta-opioid receptors. These observations are consistent with heterodimers of these receptors existing in vivo.

Equal proportions of small and large DRG neurons express opioid receptor mRNAs.

Previous studies have reported that the mRNAs encoding the cloned mu-opioid receptor (MOR1) and the cloned delta-opioid receptor (DOR1) are expressed in the dorsal root ganglia (DRG) of rats. In the present study, we determined the sizes of DRG neurons expressing DOR1 and MOR1 mRNAs and examined whether or not DRG neurons were likely to be the source of the DOR1 and MOR1 immunoreactivity previously observed in the spinal dorsal horn. DRG neurons were labeled in five male Sprague-Dawley rats by applying Fluoro-Gold (FG) topically to the dorsal root entry zone. Five-micrometer cryostat sections were cut, and in situ hybridization was performed using full-length cRNA probes labeled with 35S-UTP. The distribution of sizes of DRG neuronal profiles (1372 neuronal profiles were evaluated) ranged from 98 to 2081 microm(2) and was similar to those found in previous reports. Of 583 retrogradely labeled neuronal profiles in DRGs, 246 (40 +/- 14%, mean +/- SD, n = 5) expressed MOR1 mRNA. Of 789 DRG cell profiles from sections that were hybridized for DOR1 mRNA, 687 (85 +/- 18%, mean +/- SD, n = 5) were labeled for DOR1. The proportion of DRG cell profiles expressing DOR1 mRNA was significantly higher than that expressing MOR1 mRNA (P < 0.0001, chi-square test). No significant differences were observed between small (less than or = 700 microm(2)) and large (> 700 microm(2)) FG-labeled neurons in the proportions labeled for either MOR1 mRNA (202/497 vs. 44/86, P > 0.2, chi-square test) or DOR1 mRNA (555/651 vs. 132/138, P > 0.3, chi-square test). Most FG-labeled neurons that expressed either MOR1 mRNA or DOR1 mRNA (82.1 and 80.8%, respectively) were smaller than 700 microm(2). In addition to cells expressing a single opioid receptor, individual DRG neurons were observed that expressed both MOR1 and DOR1. In a sample of 25 DRG neurons expressing MOR1-mRNA, 23 also expressed DOR1 mRNA. Within the spinal cord itself, DOR1 and MOR1 mRNAs had different patterns of expression. Both were expressed in the dorsal horn, but of the two, only MOR1 message was expressed in the superficial dorsal horn. We conclude that both small and large DRG neurons express DOR1 and MOR1 mRNAs, but most cells expressing these mRNAs are small. In addition, some DRG neurons express both MOR1 and DOR1 mRNAs. Finally, both DOR1 and MOR1 in the spinal dorsal horn originate, at least in part, from DRG neurons.

Opioid- and GABA(A)-receptors are co-expressed by neurons in rat brain.

Pharmacological data suggest that opioids exert their excitatory action in brain indirectly, by inhibiting release of the inhibitory neurotransmitter GABA. However, it is also possible that single neuron may interact with both opioids and GABA. In the present study, we investigated whether neurons in rat midbrain and medulla express both opioid and GABA(A) receptors. Coronal sections through rat brain were double-stained using antibodies against the alpha 1 subunit of GABA(A) receptor that were combined with antibodies either against the cloned mu-opioid receptor (MOR1) or the cloned kappa-opioid receptor (KOR1). Neurons double-labeled for GABA(A) receptors and either MOR1 or KOR1 were found in many brain regions including inferior colliculus, mesencephalic trigeminal nuclei, pontine reticular nuclei and raphe interpositus nucleus. Neurons double-labeled for GABA(A) and MOR1 were observed less frequently than those labeled for GABA(A) and KOR1. Our findings provide anatomical evidence that GABAergic and opioidergic systems are closely linked and activity of the same neuron may be regulated directly by both GABA and opioids.

Immunocytochemical mapping of endomorphin-2-immunoreactivity in rat brain.

Endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2)) is a novel endogenous opioid with high affinity and selectivity for the mu-opioid receptor. Immunocytochemical studies have located this peptide in spinal cord, brainstem and selected brain regions. However, there are disagreements regarding its distribution between published reports. Furthermore, the distributions reported for the endomorphins resemble that of neuropeptide FF, suggesting that some of the previous findings might be due to cross-reactivity with the latter substance. In the present study, the distribution of endomorphin-2-immunoreactivity (ir) was examined throughout the entire rat brain using an affinity-purified antiserum that appeared not to cross-react with neuropeptide FF. Endomorphin-2-ir cell somata were most prominent in the hypothalamus and the nucleus of the solitary tract (NTS). Endomorphin-2-ir varicose fibers were observed in such areas as the bed nucleus of the stria terminalis, the septal nuclei, the periaqueductal gray, the locus coeruleus, the lateral parabrachial nucleus, the NTS, and the substantia gelatinosa of the medulla. More modest immunoreactivity was seen in substantia nigra, nucleus raphe magnus, the ventral tegmental area, the pontine nuclei and the amygdala. Fibers were also observed in the ventral cerebellum. Of note was the negligible immunoreactivity in the striatum, a region known to express high levels of mu-opioid receptors. Thus, endomorphin-2-ir was widely, but not uniformly, distributed throughout the central nervous system and was associated largely, but not exclusively, with regions expressing mu-opioid receptors. Based on its distribution, it may have a role in the control of neuroendocrine, cardiovascular and respiratory functions, and mood, feeding, sexual behavior and pain.

Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue.

The fluorescent pigment lipofuscin accumulates with age in the cytoplasm of cells of the CNS. Because of its broad excitation and emission spectra, the presence of lipofuscin-like autofluorescence complicates the use of fluorescence microscopy (e.g., fluorescent retrograde tract tracing and fluorescence immunocytochemistry). In this study we examined several chemical treatments of tissue sections for their ability to reduce or eliminate lipofuscin-like autofluorescence without adversely affecting other fluorescent labels. We found that 1-10 mM CuSO4 in 50 mM ammonium acetate buffer (pH 5) or 1% Sudan Black B (SB) in 70% ethanol reduced or eliminated lipofuscin autofluorescence in sections of monkey, human, or rat neural tissue. These treatments also slightly reduced the intensity of immunofluorescent labeling and fluorescent retrograde tract tracers. However, the reduction of these fluorophores was far less dramatic than that for the lipofuscin-like compound. We conclude that treatment of tissue with CuSO4 or SB provides a reasonable compromise between reduction of lipofuscin-like fluorescence and maintenance of specific fluorescent labels.

Serotonergic and GABAergic neurons in the medial rostral ventral medulla express kappa-opioid receptor immunoreactivity.

Activation of kappa-opioid receptors in the rostral ventral medulla has been reported to attenuate analgesia induced by activation of mu-opioid receptors in the periaqueductal gray matter. Previous studies have suggested that the cells associated with this effect might contain serotonin. In the present study, we investigated the relationship of the cloned kappa-opioid receptor to spinally projecting neurons immunoreactive for serotonin or GABA. This was done by employing two-color immunofluorescence in combination with retrograde tract-tracing using Fluoro-Gold. In the rostral ventral medulla, neurons triple-labeled for the cloned kappa-opioid receptor, serotonin and Fluoro-Gold were observed; neurons double-labeled for the cloned kappa-opioid receptor and serotonin, or single-labeled for the cloned kappa-opioid receptor or for serotonin were also observed. In addition, cloned kappa-opioid receptor immunoreactivity was expressed in some cell profiles immunoreactive for GABA. The expression of the cloned kappa-opioid receptor in the spinal cord dorsal horn was not associated with processes immunoreactive for serotonin. Our findings suggest that kappa-opioid receptors in the rostral ventral medulla are positioned to directly control the activity of at least some serotonergic neurons projecting to the dorsal spinal cord. Thus, it appears possible that the anti-analgesic action resulting from microinjection of kappa-opioid agonists into the rostral ventral medulla is mediated, at least in part, by these neurons.

Mu- and delta-opioid receptor mRNAs are expressed in spinally projecting serotonergic and nonserotonergic neurons of the rostral ventromedial medulla.

The rostral ventromedial medulla (RVM) is an important mediator of the supraspinal component of opioid antinociception. Previous studies have suggested that activation of the cloned mu- and delta-opioid receptors (MOR1 and DOR1 respectively) in the RVM produces the antinociception mediated by spinally projecting neurons. In the present study, we investigated the expression of mRNA encoding either MOR1 or DOR1 in the RVM of rats. In addition, we examined quantitatively the expression of MOR1 and DOR1 mRNAs in spinally projecting RVM neurons including serotonergic (5HT) cells by using in situ hybridization, immunocytochemistry, retrograde tract-tracing, and the physical disector. Brainstem neurons were labeled in 14 male Sprague-Dawley rats by applying Fluoro-Gold (FG) topically to the dorsal surface of the lumbosacral spinal cord. Five-micrometer-thick cryostat sections were cut and in situ hybridization was performed by using full-length cRNA probes labeled with 35S-UTP. We found that 43% of RVM projection neurons expressed MOR1 mRNA and 83% of RVM projection neurons expressed DOR1 mRNA. Of 192 retrogradely labeled cells in the RVM, 51 cells (27%) were immunoreactive for 5HT. Of this population, half appeared to be labeled for the mRNA encoding MOR1 and over three-fourths appeared to be labeled for the mRNA encoding DOR1. Thus, we conclude that bulbospinal neurons express MOR1 and DOR1; moreover, MOR1 and DOR1 are expressed by significant proportions of 5HT neurons projecting to or through the dorsal spinal cord.

Immunoreactivity for endomorphin-2 occurs in primary afferents in rats and monkey.

Antisera were raised against endomorphin-2, a recently isolated endogenous opioid peptide that binds potently and selectively to the mu-opioid receptor. When sections of spinal cord were stained immunocytochemically, a dense plexus of fibres and varicosities was visualized in the superficial dorsal horn of rats and one monkey. Following unilateral multiple dorsal rhizotomy, labeling for endomorphin-2 was markedly reduced ipsilateral to the lesion. In sections stained for both endomorphin-2 and CGRP, double-labeling was observed. Taken together, these data suggest that endomorphin-2 occurs in small diameter primary afferent fibres in rodents and primates. It appears possible that the release of neurotransmitters from nociceptive primary afferents might be regulated by release of endomorphin-2 from primary afferent terminals.

Relationship of mu- and delta-opioid receptors to GABAergic neurons in the central nervous system, including antinociceptive brainstem circuits.

Inhibition of neurons containing gamma-aminobutyric acid (GABA) may underlie some of the excitatory effects of opioids in the central nervous system (CNS). In the present study, we examined the relationship of the cloned mu- and delta-opioid receptors (MOR1 and DOR1, respectively) to GABAergic neurons in brain and spinal cord. This was done by combining immunofluorescent staining for MOR1 or DOR1 with that for GABA or glutamic acid decarboxylase (GAD); fluorescent retrograde tract-tracing was used in some cases to identify neurons with particular projections. In rats, cells double labeled for GABA and MOR1 were observed in layers II-VI of the parietal cortex and in layers II-IV of the piriform cortex. In the hippocampus, double labeling was observed in the dentate gyrus and in regions CA1 and CA3. Double labeling was very prominent in the striatum and in the reticular nucleus of the thalamus; it was also observed in other portions of the diencephalon. However, double labeling for GABA and MOR1 was never observed in the cerebellar cortex. Cells double labeled for GABA and MOR1 were common in the periaqueductal gray (PAG) and the medial rostral ventral medulla (RVM) of both rats and monkeys, suggesting that involvement of GABAergic neurons with supraspinal opioid antinociception may extend to primates. In the RVM of rats, many of those double-labeled neurons were retrogradely labeled from the dorsal spinal cord. In contrast, double-labeled neurons in the PAG were almost never retrogradely labeled from the RVM. No unequivocal examples of double labeling for DOR1 and GAD were found in any region of the CNS that we examined in either rats or monkeys. However, GABAergic neurons were often apposed by DOR1 immunoreactive varicosities. Our findings suggest that activation of mu-opioid receptors directly modulates the activity of GABAergic neurons throughout the CNS, including neurons involved in the supraspinal component of opioid analgesia. In contrast, delta-opioid receptors appear to be positioned to modulate the activity of GABAergic neurons indirectly.

CNS GABA neurons express the mu-opioid receptor: immunocytochemical studies.

It has been proposed that mu-opioid receptors excite neurons in hippocampus and nucleus raphe dorsalis (NRD) by decreasing GABAergic tone. In the present study, we examined whether immunocytochemical evidence of interaction between GABAergic neurons and the mu-opioid receptor could be found in the CNS. Portions of rat brain were sectioned and stained for GABA and for the cloned mu-opioid receptor (MOR1) using two-color immunofluorescence. Neurons double-labeled for GABA and MOR1 were present in hippocampus and NRD, as well as in olfactory bulb, dorsal lateral periaqueductal gray matter, nucleus raphe medianis, nucleus raphe obscurus, and the spinal trigeminal nucleus and tract. We conclude that expression of the mu-opioid receptor by GABAergic neurons is common in the rat CNS.

Spinal analgesic actions of the new endogenous opioid peptides endomorphin-1 and -2.

Two highly-selective mu-opioid receptor agonists, endomorphin-1 and -2, were recently purified from bovine brain and are postulated to be endogenous mu-opioid receptor ligands. We sought to determine the effects of these ligands at the spinal level in mice. Endomorphin-1 and -2 produced short acting, naloxone-sensitive antinociception in the tail flick test and inhibited the behavior elicited by intrathecally injected substance P. Both endomorphin-1 and -2 were anti-allodynic in the dynorphin-induced allodynia model. Although acute tolerance against both endomorphins developed rapidly, endomorphin-1 required a longer pretreatment time before tolerance was observed. We conclude that the endomorphins are potent spinal antinociceptive and anti-allodynic agents and that they or related compounds may prove therapeutically useful as spinal analgesics.

A subset of ventral tegmental area neurons is inhibited by dopamine, 5-hydroxytryptamine and opioids.

Neurons originating in the ventral tegmental area are thought to play a key role in the formation of addictive behaviors, particularly in response to drugs such as cocaine and opioids. In this study we identified different populations of ventral tegmental area neurons by the pharmacology of their evoked synaptic potentials and their response to dopamine, 5-hydroxytryptamine and opioids. Intracellular recordings were made from ventral tegmental area neurons in horizontal slices of guinea-pig brain and electrical stimulation was used to evoke synaptic potentials. The majority of cells (61.3%) hyperpolarized in response to dopamine, depolarized to 5-hydroxytryptamine, failed to respond to [Met]5enkephalin and exhibited a slow GABAB-mediated inhibitory postsynaptic potential. A smaller proportion of cells (11.3%) hyperpolarized in response to [Met]5enkephalin, depolarized to 5-hydroxytryptamine, failed to respond to dopamine and did not exhibit a slow inhibitory postsynaptic potential. These two groups of cells corresponded to previously described "principal" and "secondary" cells, respectively. A further group of cells (27.4%) was identified that like the principal cells, hyperpolarized to dopamine. However, these "tertiary cells" also hyperpolarized to both 5-hydroxytryptamine and [Met]5enkephalin and exhibited a slow, cocaine-sensitive 5-hydroxytryptamine(1A)-mediated inhibitory postsynaptic potential. When principal and tertiary cells were investigated immunohistochemically, 82% of the principal cells were positive for tyrosine hydroxylase compared with only 29% of the tertiary cells. The 5-hydroxytryptamine innervation of both these cell types was investigated and a similar density of putative contacts was observed near the somata and dendrites in both groups. This latter finding suggests that the existence of a 5-hydroxytryptamine-mediated inhibitory postsynaptic potential in the tertiary cells may be determined by the selective expression of 5-hydroxytryptamine receptors, rather than the distribution or density of the 5-hydroxytryptamine innervation. We conclude that tertiary cells are a distinct subset of ventral tegmental area neurons where cocaine and mu-opioids both mediate inhibition.

mu-Opioid and delta-opioid receptors are expressed in brainstem antinociceptive circuits: studies using immunocytochemistry and retrograde tract-tracing.

Opioid-produced antinociception in mammals seems to be mediated in part by pathways originating in the periaqueductal gray (PAG) and the rostroventral medulla (RVM), and these pathways may include serotonergic neurons. In the present study, we examined the relationship of the cloned mu- and delta-receptors (MOR1 and DOR1, respectively) to PAG neurons projecting to the RVM, and RVM neurons projecting to the dorsal spinal cord. This was carried out by combining immunocytochemical staining for MOR1, DOR1, and serotonin with fluorescent retrograde tract-tracing. Of 133 retrogradely labeled cells in the RVM, 31% were immunoreactive for MOR1. Of the double-labeled cells, 41% also were immunoreactive for 5HT. Fifty-three percent of retrogradely labeled cells were apposed by DOR1-ir varicosities; 29% of the apposed cells were immunoreactive for 5HT. In the mesencephalon, cells retrogradely labeled from the RVM were usually surrounded by MOR1-ir structures; however, retrogradely labeled cells were never observed to be immunoreactive for MOR1. Similarly, retrogradely labeled cells in the caudal midbrain were seldom, if ever, labeled for DOR1; however, they frequently were apposed by DOR1-ir varicosities. Of 156 retrogradely labeled profiles from three rats, 52 (33%) were apposed by DOR1-ir varicosities. We conclude that both mu- and delta-opioid receptors could be involved in the antinociception mediated by the PAG-RVM-spinal cord circuit. In addition, opioids seem likely to have both direct and indirect effects on spinally projecting RVM cells in general, and on serotonergic RVM cells in particular.

Modulation of retinoblastoma and retinoblastoma-related proteins in regenerating rat liver and primary hepatocytes.

Protein expression of the retinoblastoma (Rb) tumor suppressor gene product was examined by immunoblot analysis of nuclei isolated from regenerating rat liver after 70% partial hepatectomy (PH). Levels were almost undetectable in quiescent 0-h livers but increased 15- to 60-fold 3 to 24 h post-PH, 105-fold at 30 h, and 20- to 50-fold at 60 to 72 h post-PH. Expression returned to near baseline levels at 18, 42, and 48 h post-PH. A similar pattern of Rb protein expression in the regenerating liver was observed by indirect immunofluorescence microscopy, with peak nuclear expression at 30 h post-PH. Rb-related proteins with apparent molecular masses of 300, 156, and 74 kDa were detected in regenerating liver using mAbs to the Rb protein. Their expression increased 6- to 8-fold during regeneration, and only p156 returned to baseline levels at 60 h post-PH. Rb and its related proteins were detected in cultured primary hepatocytes, and although total protein levels did not change appreciably, there was a dramatic shift from cytosol into nuclei through 96 h. The half-life of the Rb protein was determined to be 1.9 h in regenerating liver and 2.2 h in cultured primary hepatocytes. Rb protein abundance in synchronized HuH-7 human hepatoma cells was cell cycle dependent and exhibited peak nuclear expression during S phase. Rb protein was detected primarily in its hyperphosphorylated state during liver regeneration and through the cell cycle of the HuH-7 cells. In vivo administration of transforming growth factor beta 1, an inhibitor of DNA synthesis in regenerating liver, resulted in reduced expression of Rb as well as its protein partners, cell cycle-dependent kinase 4 and cyclin E. The results suggest that in the regenerating rat liver and in synchronized HuH-7 cells, expression of Rb protein is modulated in a cell cycle-dependent fashion, remains primarily in a hyperphosphorylated state, and exhibits a relatively short half-life. The inhibition of Rb protein expression by transforming growth factor beta 1 may be linked to its simultaneous suppression of cell cycle-dependent kinase 4 and cyclin E protein levels.

Transneuronal labeling of CNS neuropeptide and monoamine neurons after pseudorabies virus injections into the stellate ganglion.

The viral transneuronal labeling method was used in combination with immunohistochemical procedures to identify CNS neuropeptide and monoamine neurons that innervate the sympathetic preganglionic neurons (SPNs) which project to the stellate ganglion--the principal source of the sympathetic supply to the heart. Transneuronal labeling was found at three CNS levels: spinal cord, brainstem, and hypothalamus. In the thoracic spinal cord, apart from the pseudorabies virus (PRV)-labeled stellate SPNs, PRV-labeled neurons were localized in laminae I/II, IV, V, VII, and X as well as in the lateral spinal nucleus and lateral funiculus. In the C1-C4 spinal segments, labeled neurons were found in the lateral funiculus as well as laminae V and VII of the spinal gray matter. PRV-labeled cells were identified in lamina V and the dorsolateral funiculus of the lumbar spinal cord. Three medullary areas were consistently labeled: rostral ventromedial medulla (RVMM), rostral ventrolateral medulla (RVLM), and caudal raphe nuclei. The greatest concentration of labeling was found in the RVMM. This projection arose from adrenergic, serotonergic (5-HT), thyrotropin releasing hormone (TRH), substance P, somatostatin, enkephalin, and vasoactive intestinal peptide (VIP) immunoreactive neurons. The RVLM projection originated mainly from C1 adrenergic neurons, some of which contained immunoreactive neuropeptide Y (NPY). C3 adrenergic-NPY neurons lying near the floor of the 4th ventricle were also labeled. Enkephalin-, somatostatin- and VIP-immunoreactive RVLM neurons also contributed to this projection. 5-HT neurons of the caudal raphe nuclei (raphe pallidus, raphe obscurus, and raphe magnus) were labeled; some of these contained substance P or TRH-immunoreactivity with an occasional neuron staining for all three putative neurotransmitters. In the pons, catecholamine neurons in the A5 cell group, subcoeruleus and Kolliker-Fuse nuclei were labeled. The midbrain contained relatively few infected cells, but some were present in the Edinger-Westphal and precommissural nuclei. Forebrain labeling was concentrated in the paraventricular hypothalamic nucleus (PVN) with lesser amounts in the lateral hypothalamic area (LHA) and the perifornical region. In the PVN, oxytocin-immunoreactive neurons accounted for the greatest chemically-defined projection while corticotrophin releasing factor (CRF), vasopressin-, and angiotensin II-immunoreactive neurons provided successively lesser inputs. In the LHA, angiotensin II-immunoreactive neurons were labeled. In summary, this study provides the first detailed map of the chemically-coded CNS neurons involved in the control of the cardiosympathetic outflow.

Distribution and targeting of a mu-opioid receptor (MOR1) in brain and spinal cord.

Opioid receptors regulate neuronal activity by both pre- and postsynaptic mechanisms. We recently reported that the cloned delta-opioid receptor (DOR1) is primarily targeted to axons, suggesting a presynaptic role. In the present study we have studied the distribution and targeting of another opioid receptor, the mu-opioid receptor (MOR1), by raising anti-peptide antisera to the C-terminal peptide of MOR1. The specificity of the antisera was determined by analysis of transfected cells, Western blots, and immunoisolation studies. Immunohistochemistry showed that MOR1 immunoreactivity was enriched in many brain areas including cerebral cortex, striatum, hippocampus, locus coeruleus, and the superficial laminae of the dorsal horn. Moreover, MOR1-expressing neurons seem to target this receptor preferentially to their somatodendritic domain as determined by double-labeling experiments with MAP2. However, discrete populations of neurons target MOR1 to their axons, including some primary afferent neurons that express DOR1. In many regions enkephalin-containing axons were complementary to MOR1, suggesting by their proximity that enkephalins may be physiologically relevant ligands for this receptor. Thus, these results provide a morphological basis for understanding pre- and postsynaptic functions mediated by MOR1.

The kappa-opioid receptor is primarily postsynaptic: combined immunohistochemical localization of the receptor and endogenous opioids.

Antisera were raised against a synthetic peptide corresponding to the carboxyl terminus of the kappa-opioid receptor (KOR1). Specificity of the antisera was verified by staining of COS-7 cells transfected with KOR1 and epitope-tagged KOR1 cDNAs, by recognition by the antisera of proteins on Western blots of both transfected cells and brain tissue, by the absence of staining of both brain tissue and transfected cells after preabsorption of the antisera with the cognate peptide, and on the strong correlation between the distribution of KOR1 immunoreactivity and that of earlier ligand binding and in situ hybridization studies. Results indicate that KOR1 in neurons is targeted into both the axonal and somatodendritic compartments, but the majority of immunostaining was seen in the somatodendritic compartment. In sections from rat and guinea pig brain, prominent KOR1 staining was seen in the ventral forebrain, hypothalamus, thalamus, posterior pituitary, and midbrain. While the staining pattern was similar in both species, distinct differences were also observed. The distribution of preprodynorphin and KOR1 immunoreactivity was complementary in many brain regions, suggesting that KOR1 is poised to mediate the physiological actions of dynorphin. However, the distribution of KOR1 and enkephalin immunoreactivity was complementary in some regions as well. These results suggest that the KOR1 protein is primarily, but not exclusively, deployed to postsynaptic membranes where it mediates the effects of products of preprodynorphin and possibly preproenkephalin.