Impact of 6-hydroxydopamine lesions and cocaine exposure on mu-opioid receptor expression and regulation of cholinergic transmission in the limbic-prefrontal territory of the rat dorsal striatum

Information processing within the striatum is regulated by local circuits involving dopamine, cholinergic interneurons and neuropeptides released by recurrent collaterals of striatal output neurons. In the limbic-prefrontal territory of the dorsal striatum, enkephalin inhibits the NMDA-evoked release of acetylcholine directly through micro-opioid receptors (MORs) located on cholinergic interneurons and indirectly through MORs of output neurons of striosomes. In this territory, we investigated the consequence of changes in dopamine transmission, bilateral 6-hydroxydopamine-induced degeneration of striatal dopaminergic innervation or cocaine (acute and chronic) exposure on (i) MOR expression in both cholinergic interneurons and output neurons of striosomes, and (ii) the direct and indirect enkephalin-MOR regulations of the NMDA-evoked release of acetylcholine. Expression of MORs in cholinergic interneurons was preserved after 6-hydroxydopamine and down-regulated after cocaine treatments. Accordingly, the direct enkephalin-MOR control of acetylcholine release was preserved after 6-hydroxydopamine treatment and lost after cocaine exposure. Expression of MORs in output neurons of striosomes was down-regulated in the 6-hydroxydopamine situation and either preserved or up-regulated after acute or chronic cocaine exposure, respectively. Accordingly, the indirect enkephalin-MOR control of acetylcholine release disappeared in the 6-hydroxydopamine situation but surprisingly, despite preservation of MORs in striosomes, disappeared after cocaine treatment. Showing that MORs of striosomes are still functional in this situation, the MOR agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin inhibited the NMDA-evoked release of acetylcholine after cocaine exposure. Therefore, alteration in the regulation of cholinergic transmission by the enkephalin-MOR system might play a major role in the motivational and cognitive disorders associated with dopamine dysfunctions in fronto-cortico-basal ganglia circuits.     

Functional and anatomical localization of mu opioid receptors in the striatum, amygdala, and extended amygdala of the nonhuman primate

The subregional distribution of mu opioid receptors and corresponding G-protein activation were examined in the striatum, amygdala, and extended amygdala of cynomolgus monkeys. The topography of mu binding sites was defined using autoradiography with [(3)H]DAMGO, a selective mu ligand. In adjacent sections, the distribution of receptor-activated G proteins was identified with DAMGO-stimulated guanylyl 5'(gamma-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding. Within the striatum, the distribution of [(3)H]DAMGO binding sites was characterized by a distinct dorsal-ventral gradient with a higher concentration of binding sites at more rostral levels of the striatum. [(3)H]DAMGO binding was further distinguished by the presence of patch-like aggregations within the caudate, as well as smaller areas of very dense receptor binding sites, previously identified in human striatum as neurochemically unique domains of the accumbens and putamen (NUDAPs). The amygdala contained the highest concentration of [(3)H]DAMGO binding sites measured in this study, with the densest levels of binding noted within the basal, accessory basal, paralaminar, and medial nuclei. In the striatum and amygdala, the distribution of DAMGO-stimulated G-protein activation largely corresponded with the distribution of mu binding sites. The central and medial nuclei of the amygdala, however, were notable exceptions. Whereas the concentration of [(3)H]DAMGO binding sites in the central nucleus of the amygdala was very low, the concentration of DAMGO-stimulated G-protein activation in this nucleus, as measured with [(35)S]GTPgammaS binding, was relatively high compared to other portions of the amygdala containing much higher concentrations of [(3)H]DAMGO binding sites. The converse was true in the medial nucleus, where high concentrations of binding sites were associated with lower levels of DAMGO-stimulated G-protein activation. Finally, [(3)H]DAMGO and [(35)S]GTPgammaS binding within the amygdala, particularly the medial nucleus, formed a continuum with the substantia innominata and bed nucleus of the stria terminalis, supporting the concept of the extended amygdala in primates.     

Localization of dopamine D2 receptors on cholinergic interneurons of the dorsal striatum and nucleus accumbens of the rat

Striatal cholinergic interneurons located in the dorsal striatum and nucleus accumbens are amenable to influences of the dopaminergic mesolimbic pathway, which is a pathway involved in reward and reinforcement and targeted by several drugs of abuse. Dopamine and acetylcholine neurotransmission and their interactions are essential to striatal function, and disruptions to these systems lead to a variety of clinical disorders. Dopamine regulates acetylcholine release through dopamine receptors that are localized directly on striatal cholinergic interneurons. The dopamine D2 receptor, which attenuates acetylcholine release, has been implicated in drug relapse and is targeted by therapeutic drugs that are used to treat a variety of neurological disorders including Tourette Syndrome, Parkinson's disease and schizophrenia. The present study provides the first direct evidence for the localization of dopamine D2 receptors on striatal cholinergic interneurons of the rat brain using dual labeling immunocytochemistry procedures. Using light microscopy, dopamine D2 receptors were localized on the cell somata and dendritic and axonal processes of striatal cholinergic interneurons in the dorsal striatum and nucleus accumbens of the rat brain. These findings provide a foundation for understanding the specific roles that cholinergic neuronal network systems and interacting dopaminergic signaling pathways play in striatal function and in a variety of clinical disorders including drug abuse and addiction.     

Localization of delta opioid receptor immunoreactivity in interneurons and pyramidal cells in the rat hippocampus

To study possible cellular targets and subcellular sites of action of opioid ligands in the rat hippocampus, we examined the distribution of the delta opioid receptor (DOR) by immunocytochemistry. By light microscopy, numerous interneurons, or non-principal cells, were intensely labeled for DOR, whereas the CA1 and CA3 pyramidal cells were lightly labeled. DOR-immunoreactive interneurons were found throughout the hippocampus but were particularly concentrated in stratum oriens of the CA1 region. Double labeling immunofluorescence revealed that DOR-immunoreactivity was found in a subpopulation of γ-aminobutyric acid (GABA)-containing interneurons, which included most somatostatin-immunoreactive cells. Electron microscopic analysis of sections singly labeled for DOR revealed that DOR-immunoreactive profiles were abundant and widespread throughout all hippocampal lamina and had a similar distribution in CA1 and CA3. DOR-immunoreactivity was sometimes found in dendrites, which corresponded in morphology to those of interneurons. In addition, DOR-labeling was found in the shafts and spines of many dendrites, which exhibited the morphology of pyramidal cell dendrites. Within dendrites, dense DOR-immunoreactivity was associated with the plasmalemmal surface at or near the postsynaptic density, usually of asymmetric synapses. In addition, DOR labeling was present in a heterogeneous population of axon terminals, as well as in astrocytic profiles. At mossy fiber synapses, DOR labeling was occasionally found at both pre-and post-synaptic sites. These studies demonstrate that DOR is present at multiple sites on diverse cell types where it may function to regulate neuronal activity in the hippocampus. J. Comp. Neurol. 373-387, 1997. © 1997 Wiley-Liss, Inc.     

Decreased number of parvalbumin and cholinergic interneurons in the striatum of individuals with Tourette syndrome

Corticobasal ganglia neuronal ensembles bring automatic motor skills into voluntary control and integrate them into ongoing motor behavior. A 5% decrease in caudate (Cd) nucleus volume is the most consistent structural finding in the brain of patients with Tourette syndrome (TS), but the cellular abnormalities that underlie this decrease in volume are unclear. In this study the density of different types of interneurons and medium spiny neurons (MSNs) in the striatum was assessed in the postmortem brains of 5 TS subjects as compared with normal controls (NC) by unbiased stereological analyses. TS patients demonstrated a 50%–60% decrease of both parvalbumin (PV)+ and choline acetyltransferase (ChAT)+ cholinergic interneurons in the Cd and the putamen (Pt). Cholinergic interneurons were decreased in TS patients in the associative and sensorimotor regions but not in the limbic regions of the striatum, such that the normal gradient in density of cholinergic cells (highest in associative regions, intermediate in sensorimotor and lowest in limbic regions) was abolished. No significant difference was present in the densities of medium‐sized calretinin (CR)+ interneurons, MSNs, and total neurons. The selective deficit of PV+ and cholinergic striatal interneurons in TS subjects may result in an impaired cortico/thalamic control of striatal neuron firing in TS. J. Comp. Neurol. 518:277–291, 2010. © 2009 Wiley‐Liss, Inc.     

Limbic thalamus in rabbit: architecture, projections to cingulate cortex and distribution of muscarinic acetylcholine, GABAA, and opioid receptors.

Nuclei of the thalamus that project to cingulate cortex have been implicated in responses to noxious stimuli, cholinergic and motor functions. The rabbit limbic thalamus may play an important role in these functions, but has not been studied extensively in terms of its cytoarchitecture, the topographical organization of its cortical projections, and differential transmitter regulation of its subnuclei. Therefore, the architecture, projections to cingulate cortex, and radioligand binding were investigated in the anterior, ventral, lateral, and midline nuclei of rabbit thalamus. The anterior nuclei are highly differentiated because both the dorsal and ventral nuclei have parvicellular and magnocellular divisions. Fluorescent dyes were injected into cingulate cortex to evaluate limbic thalamocortical connections. The anterior medial, submedial, and parafascicular nuclei project primarily to anterior cingulate cortex, while they have small or no projections to posterior areas. The ventral anterior and ventral lateral nuclei have a significant projection to dorsal cingulate cortex, including areas 24b and 29d. Projections of the anterior ventral nucleus are topographically organized, since medial parts of the parvicellular division project to rostral area 29, and lateral parts project to caudal area 29. The lateral nuclei and the parvicellular and magnocellular divisions of the anterior dorsal nucleus project with progressively higher densities in the rostrocaudal plane of area 29. Finally, the magnocellular division of the anterior ventral nucleus projects almost exclusively to caudal and ventral area 29, i.e., granular retrosplenial cortex. Ligand binding studies employed coverslip autoradiography and single grain counting techniques. Muscarinic receptor binding was moderate for both pirenzepine and oxotremorine-M in the parvicellular anterior ventral nucleus, while in other nuclei, there was an inverse relationship in the binding for these ligands. Most notably, the anterior dorsal nucleus, which receives no cholinergic input, had very high oxotremorine-M and low pirenzepine binding, while the anterior medial nucleus, which receives a moderate cholinergic input, had the highest pirenzepine binding and very low oxotremorine-M binding. Muscimol binding to GABAA receptors was highest in the anterior ventral nucleus, while it was at moderate levels in the anterior dorsal and lateral nuclei. The binding of Tyr-D-Ala-Gly-MePhe-Gly-ol to mu opioid receptors and 2-D-penicillamine-5-D-penicillamine-enkephalin to delta opioid receptors were both high in the parvicellular and low in the magnocellular divisions of the anterior dorsal nucleus. The magnocellular division of the anterior ventral, the lateral dorsal, and the parafascicular nuclei had high mu opioid binding, while the lateral dorsal and lateral magnocellular nuclei had low levels of delta opioid binding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Morphological and functional characterization of cholinergic interneurons in the dorsal horn of the mouse spinal cord

Endogenous acetylcholine is an important modulator of sensory processing, especially at the spinal level, where nociceptive (pain-related) stimuli enter the central nervous system and are integrated before being relayed to the brain. To decipher the organization of the local cholinergic circuitry in the spinal dorsal horn, we used transgenic mice expressing enchanced green fluorescent protein specifically in cholinergic neurons (ChAT::EGFP) and characterized the morphology, neurochemistry, and firing properties of the sparse population of cholinergic interneurons in this area. Three-dimensional reconstruction of lamina III ChAT::EGFP neurons based either on their intrinsic fluorescence or on intracellular labeling in live tissue demonstrated that these neurons have long and thin processes that grow preferentially in the dorsal direction. Their dendrites and axon are highly elongated in the rostrocaudal direction, beyond the limits of a single spinal segment. These unique morphological features suggest that dorsal horn cholinergic interneurons are the main contributors to the plexus of cholinergic processes located in lamina IIi, just dorsal to their cell bodies. In addition, immunostainings demonstrated that dorsal horn cholinergic interneurons in the mouse are γ-aminobutyric acidergic and express nitric oxide synthase, as in rats. Finally, electrophysiological recordings from these neurons in spinal cord slices demonstrate that two-thirds of them have a repetitive spiking pattern with frequent rebound spikes following hyperpolarization. Altogether our results indicate that, although they are rare, the morphological and functional features of cholinergic neurons enable them to collect segmental information in superficial layers of the dorsal horn and to modulate it over several segments.     

Analysis of the influence of diurnal variation in maternal movements on fetal heart rate acceleration

To determine the influence of diurnal variation in maternal movements (MM) on fetal heart rate (FHR) accelerations, 24-hour maternal heart rates (MHR), MM and FHR recordings were made simultaneously on nine healthy pregnant women at 36-39 weeks of gestation. Correlations between the diurnal variations in maternal factors and FHR parameters; that is, baseline FHR, amplitude, duration and hourly percentage of time spent in accelerations, were examined. Only a few cases showed a significant correlation between acceleration parameters and the diurnal variation of MHR or MM, while most cases indicated a high correlation between the diurnal variation in baseline FHR and that of maternal factors. In conclusion, the occurrence and form of accelerations might be independent of maternal activity.     

Ionotropic glutamate receptors are expressed in GABAergic terminals in the rat superficial dorsal horn

Ionotropic glutamate receptors (IGR), including NMDA, AMPA, and kainate receptors, are expressed in terminals with varied morphology in the superficial laminae (I-III) of the dorsal horn of the spinal cord. Some of these terminals can be identified as endings of primary afferents, whereas others establish symmetric synapses, suggesting that they may be gamma-aminobutyric acid (GABA)-ergic. In the present study, we used confocal and electron microscopy of double immunostaining for GAD65, a marker for GABAergic terminals, and for subunits of IGRs to test directly whether IGRs are expressed in GABAergic terminals in laminae I-III of the dorsal horn. Although colocalization is hard to detect with confocal microscopy, electron microscopy reveals a substantial number of terminals immunoreactive for GAD65 also stained for IGRs. Among all GAD65-immunoreactive terminals counted, 37% express the NMDA receptor subunit NR1; 28% are immunopositive using an antibody for the GluR2/4 subunits of the AMPA receptor; and 20-35% are immunopositive using antibodies for the kainate receptor subunits GluR5, GluR6/7, KA1, or KA2. Terminals immunoreactive for IGR subunits and GAD65 establish symmetric synapses onto dendrites and perikarya and can be presynaptic to primary afferent terminals within both type 1 and type 2 synaptic glomeruli. Activation of presynaptic IGR may reduce neurotransmitter release. As autoreceptors in terminals of Adelta and C afferent fibers in laminae I-III, presynaptic IGRs may play a role in inhibiting nociception. As heteroreceptors in GABAergic terminals in the same laminae, on the other hand, presynaptic IGRs may have an opposite role and even contribute to central sensitization and hyperalgesia.     

Changes in cholinergic and opioid receptors in the rat spinal cord,dorsal root and sciatic nerve after ventral and dorsal root lesion

Summary Changes in the distribution of³H-quinuclidinylbenzilate (³ H-QNB),³ H-acetylcholine (³ H-ACh) and³ H-alpha-bungarotoxin (alpha-BTx) binding sites were studied with the use of quantitative in vitro autoradiography in the L4–L6 segments of rats 7 days after ventral L4–L6-rhizotomies and 24 hours after ligation of the dorsal roots L4–L6. The changes in the binding sites of these ligands and of³ H-etorphine binding sites were also studied in the dorsal roots of the rats operated with dorsal root ligation and in the sciatic nerves (around a ligature) in the rats operated with ventral rhizotomy. After ventral rhizotomy³ H-QNB binding sites in the ipsilateral motor neuron area were decreased by about 25% from 100±5 to 73±5 fmol/mg wet weight. After dorsal root ligation³ H-QNB binding sites in the ipsilateral posterior horn were reduced by about 30% from 91±5 to 64±7 fmol/mg wet weight. No significant changes in the binding of the other cholinergic ligands in the spinal cords were observed after the operations. In the dorsal root³ H-alpha-Btx and³ H-etorphine binding sites were higher on the distal side of the ligation (3.5±0.8 and 14±4 fmol/mg wet weight, respectively) than on the proximal side (0.7±0.5 and 2.4±1.2 fmol/mg wet weight, respectively).The same level of³ H-ACh (total, muscarinic and nicotinic) binding was observed on both sides of the ligation. In the sciatic nerve³ H-QNB and total, muscarinic and nicotinic ACh binding sites were higher on the proximal side of the ligation than on the distal side. Except for a small emergence of muscarinic-ACh binding distally to the ligation there were no changes in the number of binding sites in the sciatic nerve after the ventral rhizotomy.Muscarinic antagonist binding sites are probably located on the perikarya of the motor neurons and presynaptically on the primary afferents in the posterior horn and in the dorsal root. Cholinergic agonist binding sites in the spinal cord seem less sensitive to axonal damage than antagonist binding sites. Cholinergic and opioid receptors in peripheral nerves are transported in both anterograde and retrograde directions and their origin seems to be the dorsal root ganglion.     

Morphine can produce analgesia via spinal kappa opioid receptors in the absence of mu opioid receptors

Previous studies have demonstrated the virtual lack of analgesia in mu opioid receptor knockout mice after systemic administration of morphine. Thus, it has been suggested that analgesic actions of morphine are produced via the mu opioid receptor, despite its ability to bind to kappa and delta receptors in vitro. However, it is not clear whether the results of these studies reflect the effect of morphine in the spinal cord. In the present study, we report study of the analgesic actions of spinally-administered morphine and other opioid receptor agonists in mu opioid receptor knockout and wild type mice. Morphine produced a dose-dependent antinociceptive effect in the tail flick test in the knockout mice, although higher doses were needed to produce antinociception than in wild type mice. The antinociceptive effect of morphine was completely blocked by naloxone (a non-selective opioid antagonist) and nor-binaltorphimine (nor-BNI, a selective kappa-opioid receptor antagonist), but not by naltrindole (a selective delta-opioid receptor antagonist). U-50,488H (a selective kappa-opioid receptor agonist) also produced a dose-dependent antinociceptive effect in knockout mice but presented lower analgesic potency in knockout mice than in wild type mice. Analgesic effects of [d-Pen2,d-Pen5]enkephalin (DPDPE, a selective delta-opioid receptor agonist) were observed in wild type mice but abolished in knockout mice. SNC80 (a selective delta-opioid receptor agonist) was not antinociceptive even in wild type mice. The present study demonstrated that morphine can produce thermal antinociception via the kappa opioid receptor in the spinal cord in the absence of the mu opioid receptor. Lower potency of U50,488H in mu opioid receptor knockout mice suggests interaction between kappa and mu opioid receptors at the spinal level.     

Serotonin 5-HT2A receptors are expressed on pyramidal cells and interneurons in the rat cortex

The distribution of 5-HT_2A receptors in rat cortex was evaluated using newly developed antibodies. Each of three antibodies tested identified an identical pattern of 5-HT_2A-like immunoreactivity (5-HT_2A-li) in rat cortex with 5-HT_2A-li showing a widespread distribution. The majority of 5-HT_2A-li cells displayed a pyramidal morphology. While a minority, some cortical neurons with a bipolar morphology displayed 5-HT_2A-li as well. Dual-label fluorescence confocal microscopic studies with a 5-HT_2A antibody and a mouse monoclonal antibody to parvalbumin, a marker of a subset of gamma aminobutyric acid (GABA)ergic interneurons in the cortex, demonstrated that although some cells expressing 5-HT_2A-li were interneurons, most were not. Synapse 27:79–82, 1997. © 1997 Wiley-Liss, Inc.     

Time-related decreases in mu and delta opioid receptors in the superficial dorsal horn of the rat spinal cord following a large unilateral dorsal rhizotomy.

The aim of the present study was to measure the time-related modifications of mu and delta opioid binding sites in the superficial layers of the dorsal horn of the rat spinal cord after a C4-T2 unilateral dorsal rhizotomy. Using specific ligands, namely [3H]DAMGO for mu sites and [3H]DTLET for delta sites, and a quantitative autoradiographic analysis, we have observed: (a) a decrease in binding on the ipsilateral side to the lesion as early as the first day postrhizotomy, the maximal loss being attained at 8 days postlesion, (b) after 8 days postlesion, the residual binding remains stable over the period of analysis (90 days), (c) the loss of mu receptors (71-74%) is significantly more pronounced than the loss of delta receptors (57-62%) and (d) affinities of postsynaptic mu and delta receptors are similar to those of the total receptor population in the superficial layers of the dorsal horn. Comparison of these results with the degeneration of primary afferent fibers reported in literature favors the localization of the majority of mu and delta opioid binding sites on fine diameter primary afferent fibers.     

Comparative analysis of cholinergic innervation in the dorsal hippocampus of adult mouse and rat: a quantitative immunocytochemical study

To obtain quantitative data on the distribution of the acetylcholine (ACh) innervation in the dorsal hippocampus of adult mouse (C57/B6) and rat (Sprague-Dawley), a semicomputerized method was used to measure the length of immunostained axons in hippocampal sections processed for light microscopic immunocytochemistry with a highly sensitive antibody against choline acetyltransferase (ChAT). The results could be expressed in density of axons (meters per mm3) for the different layers and regions of dorsal hippocampus (CA1, CA3, DG), and also in density of axon varicosities (millions per mm3), after having determined the average number of varicosities per unit length of ChAT-immunostained axon (4 varicosities/10 microm). In mouse, the mean regional densities of ACh innervation were thus measured at 13.9, 16.1, and 15.8 m of axons, for 5.6, 6.4, and 6.3 million varicosities per mm3 of tissue, in CA1, CA3, and DG, respectively. The values were comparable in rat, except for CA1, in which the densities were lower than in mouse by 40% in the stratum lacunosum, and 20% in the stratum radiatum. Otherwise, the laminar patterns of innervation were similar in the two species, the highest densities being found in the stratum lacunosum moleculare of CA3, pyramidale of both CA1 and CA3, and moleculare of DG. These quantitative data will be of particular interest to evaluate changes in mutant mice, or mice and rats subjected to experimental conditions affecting the cholinergic phenotype.     

Kappa opioid receptor is expressed by somatostatin- and neuropeptide Y-containing interneurons in the rat hippocampus

In our previous studies (J. Chem. Neuroanat. 2000;19:233-241), kappa opioid receptors were immunocytochemically identified in inhibitory interneurons of the dentate hilus and CA1 area of the rat hippocampus. From among the known interneuron subtypes, somatostatin- (SOM) and neuropeptide Y- (NPY) immunoreactive (IR) hippocampal interneurons show morphology and distribution similar to the kappa opioid receptor (KOR) immunopositive cells. In the present study, with the help of double immunocytochemical labelling, we provide direct evidence that the majority of the interneurons immunoreactive for SOM and/or NPY also express the kappa opioid receptor. The receptor was localized on the perikaryal and proximal dendritic region of the SOM- and NPY-immunopositive neurons in the dentate hilus and the CA1 region. From among the SOM-immunoreactive cells, 77% in the dentate hilus and 51% in the CA1 stratum oriens was double labelled. In the case of NPY-immunoreactive neurons this proportion was 56 and 65%, respectively. The co-expression of KOR and SOM/NPY suggests that hippocampal interneurons can selectively be activated by the different opioids under different physiological circumstances.     

Regional circadian variation of acetylcholine muscarinic receptors in the rat brain

The level of binding of a labeled acetylcholine muscarinic antagonist (quinuclidinyl benzilate) to different cerebral membranes has been measured. Of the regions examined, circadian rhythmicity of binding could only be detected significantly in the hippocampus and the hypothalamus and not in the cerebral cortex, striatum, or cerebellum.     

Cellular interactions between axon terminals containing endogenous opioid peptides or corticotropin-releasing factor in the rat locus coeruleus and surrounding dorsal pontine tegmentum

Recent evidence suggests that certain stressors release both endogenous opioids and corticotropin-releasing factor (CRF) to modulate activity of the locus coeruleus (LC)-norepinephrine (NE) system. In ultrastructural studies, axon terminals containing methionine(5)-enkephalin (ENK) or CRF have been shown to target LC dendrites. These findings suggested the hypothesis that both neuropeptides may coexist in common axon terminals that are positioned to have an impact on the LC. This possibility was examined by using immunofluorescence and immunoelectron microscopic analysis of the rat LC and neighboring dorsal pontine tegmentum. Ultrastructural analysis indicated that CRF- and ENK-containing axon terminals were abundant in similar portions of the neuropil and that approximately 16% of the axon terminals containing ENK were also immunoreactive for CRF. Dually labeled terminals were more frequently encountered in the "core" of the LC vs. its extranuclear dendritic zone, which included the medial parabrachial nucleus (mPB). Triple labeling for ENK, CRF, and tyrosine hydroxylase (TH) showed convergence of opioid and CRF axon terminals with noradrenergic dendrites as well as evidence for inputs to TH-labeled dendrites from dually labeled opioid/CRF axon terminals. One potential source of ENK and CRF in the dorsal pons is the central nucleus of the amygdala (CNA). To determine the relative contribution of ENK and CRF terminals from the CNA, the CNA was electrolytically lesioned. Light-level densitometry revealed robust decreases in CRF immunoreactivity in the LC and mPB on the side ipsilateral to the lesion but little or no change in ENK immunoreactivity, confirming previous studies of the mPB. Degenerating terminals from the CNA in lesioned rats were found to be in direct contact with TH-labeled dendrites. Together, these data indicate that ENK and CRF may be colocalized to a subset of individual axon terminals in the LC "core." The finding that the CNA provides, to dendrites in the area examined, a robust CRF innervation, but little or no opioid innervation, suggests that ENK and CRF axon terminals impacting LC neurons originate from distinct sources and that terminals that colocalize ENK and CRF are not from the CNA.     

Developmental expression of mu and delta opioid receptors in the rat brainstem: evidence for a postnatal switch in mu isoform expression

Opioid receptors are expressed in the brain during fetal and postnatal development, and the expression patterns vary with developmental age. To investigate the role of opioids in brain development, immunoblotting and immunohistochemical techniques were used to determine mu (MOR) and delta (DOR) opioid receptor expression levels and regional distributions in fetal, early postnatal and adult rat brainstem. Two immunoreactive bands were seen on Western blots of brainstem lysates for both MOR (50 and 70 kDa) and DOR (30 and 60 kDa). The expression levels of the isoforms changed dramatically between 6 and 15 days after birth. Total MOR protein was expressed at low levels in fetal and early postnatal animals with the 50-kDa band predominating. MOR expression then increased in the older animals and the 70-kDa isoform became dominant. Total DOR protein showed the opposite pattern, being high in the fetal and neonatal brainstem and low in the juvenile and adult. A postnatal switch in isoform expression for DOR was not evident in our study. In general, regional brainstem distributions in developing and adult animals were comparable to those reported in the literature, and both receptors were localized in the same areas where opioid receptor expression was high. It was concluded that MOR and DOR are developmentally regulated in the brainstem of the rat, that the isoform ratio switches postnatally from a fetal-neonatal pattern to a juvenile-adult pattern and that both receptors are generally expressed in the same brainstem regions from E16 to adult.     

Ultrastructural localization of high‐affinity choline transporter in the rat anteroventral thalamus and ventral tegmental area: Differences in axon morphology and transporter distribution

The high‐affinity choline transporter (CHT) is a protein integral to the function of cholinergic neurons in the central nervous system (CNS). We examined the ultrastructural distribution of CHT in axonal arborizations of the mesopontine tegmental cholinergic neurons, a cell group in which CHT expression has yet to be characterized at the electron microscopic level. By using silver‐enhanced immunogold detection, we compared the morphological characteristics of CHT‐immunoreactive axon varicosities specifically within the anteroventral thalamus (AVN) and the ventral tegmental area (VTA). We found that CHT‐immunoreactive axon varicosities in the AVN displayed a smaller cross‐sectional area and a lower frequency of synapse formation and dense‐cored vesicle content than CHT‐labeled profiles in the VTA. We further examined the subcellular distribution of CHT and observed that immunoreactivity for this protein was predominantly localized to synaptic vesicles and minimally to the plasma membrane of axons in both regions. This pattern is consistent with the subcellular distribution of CHT displayed in other cholinergic systems. Axons in the AVN showed significantly higher levels of CHT immunoreactivity than those in the VTA and correspondingly displayed a higher level of membrane CHT labeling. These novel findings have important implications for elucidating regional differences in cholinergic signaling within the thalamic and brainstem targets of the mesopontine cholinergic system. J. Comp. Neurol. 518:1908–1924, 2010. © 2010 Wiley‐Liss, Inc.     

Distribution of mu, delta, and kappa opioid receptors in the hypothalamus of the rat

The radioautographic distribution of mu, delta and kappa opioid binding sites was examined by in vitro radioautography in the rat hypothalamus using the highly selective ligands [125I]-FK 33-824, [125I]azidoDTLET and [125I]DPDYN, respectively. Levels of mu opioid binding sites varied considerably amongst hypothalamic nuclei. mu Opioid labeling was dense in the medial preoptic area, medial preoptic nucleus, suprachiasmatic nucleus and ventromedial nucleus, whilst the supraoptic nucleus, paraventricular nucleus, arcuate nucleus and dorsomedial nucleus were devoid of labeling. Delta opioid labeling was sparse throughout most of the hypothalamus; however, moderate binding densities were detected in the suprachiasmatic and ventromedial nucleus. kappa Opioid labeling was also scant throughout the hypothalamus with the exception of the suprachiasmatic nucleus which was very densely labeled. Our results indicate that the 3 opioid receptors types are differentially distributed within the hypothalamus, although a significant overlap exists. In general, the distribution of hypothalamic opioid receptors correlates well with that of opioid-containing terminal fibers and may represent the anatomical substrate for opioid involvement in the hypothalamic regulation of autonomic, behavioral and neuroendocrine functions.