Cholinergic axon terminals in the ventral tegmental area target a subpopulation of neurons expressing low levels of the dopamine transporter.

Cholinergic activation of dopaminergic neurons in the ventral tegmental area (VTA) is thought to play a major role in cognitive functions and reward. These dopaminergic neurons differentially project to cortical and limbic forebrain regions, where their terminals differ in levels of expression of the plasmalemmal dopamine transporter (DAT). This transporter selectively identifies dopaminergic neurons, whereas the vesicular acetylcholine transporter (VAchT) is present only in the neurons that store and release acetylcholine. We examined immunogold labeling for DAT and immunoperoxidase localization of VAchT antipeptide antisera in single sections of the rat VTA to determine whether dopaminergic somata and dendrites in this region differ in their levels of expression of DAT and/or input from cholinergic terminals. VAchT immunoreactivity was prominently localized to membranes of small synaptic vesicles in unmyelinated axons and axon terminals. VAchT-immunoreactive terminals formed almost exclusively asymmetric synapses with dendrites. Of 159 dendrites that were identified as cholinergic targets, 35% contained plasmalemmal DAT, and 65% were without detectable DAT immunoreactivity. The DAT-immunoreactive dendrites postsynaptic to VAchT-labeled terminals contained less than half the density of gold particles as seen in other dendrites receiving input only from unlabeled terminals. These results suggest selective targeting of cholinergic afferents in the VTA to non-dopaminergic neurons and a subpopulation of dopaminergic neurons that have a limited capacity for plasmalemmal reuptake of dopamine, a characteristic of those that project to the frontal cortex.     

Serotonergic synaptic input to cholinergic neurons in the rat mesopontine tegmentum

Serotonergic synaptic inputs to cholinergic neurons in the laterodorsal and pedunculopontine tegmental nuclei were examined with pre-embedding dual-label immunoelectron microscopy. Numerous serotonin-immunoreactive axon terminals visualized with a silver-enhanced immunogold method were present in both of these tegmental nuclei. Serotonergic terminals occasionally made synaptic contacts with the soma and proximal dendrites of cholinergic tegmental neurons labelled with a choline acetyltransferase-immunoreactive peroxidase-anti-peroxidase diaminobenzidine reaction product. In the rostralmost region of the laterodorsal tegmental nucleus, a few serotonergic neurons of the dorsal raphe nucleus were interspersed among cholinergic neurons. Some dendrites of these serotonergic neurons appeared to contain synaptic vesicles. Both myelinated and unmyelinated serotonergic axons were present in the mesopontine tegmentum. The presence of serotonergic synapses onto tegmental cholinergic neurons is consistent with previous behavioral and electrophysiological findings suggesting an inhibitory role of serotonin in the induction of rapid eye movement sleep and its phenomenology through an action on cholinergic neurons in the mesopontine tegmentum.     

Ascending projections from the pedunculopontine tegmental nucleus and the adjacent mesopontine tegmentum in the rat

Ascending projections from the pedunculopontine tegmental nucleus (PPT) and the surrounding mesopontine tegmentum to the forebrain in the rat are here examined by using both retrograde and anterograde tracing techniques combined with choline acetyltransferase (ChAT) immunohistochemistry. The anterogradely transported lectin Phaseolus vulgaris-leukoagglutinin (PHA-L) was iontophoretically injected into the PPT in 12 rats. Anterogradely labelled fibers and varicosities were observed in the thalamic nuclei, confirming the findings of our previous retrograde studies (Hallanger et al: J. Comp. Neurol. 262:105-124, '87). In addition, PHA-L-labelled fibers and varicosities suggestive of terminal fields were observed in the anterior, tuberal, and posterior lateral hypothalamic regions, the ventral pallidum in the region of the nucleus basalis of Meynert, the dorsal and intermediate lateral septal nuclei, and in the central and medial nuclei of the amygdala. To determine whether these were cholinergic projections, the retrograde tracer WGA-HRP was injected into terminal fields in the hypothalamus, septum, ventral pallidum, and amygdala. Numerous ChAT-immunoreactive neurons in the PPT and laterodorsal tegmental nucleus (LDT) were retrogradely labelled from the lateral hypothalamus. These cholinergic neurons constituted over 20% of those retrogradely labelled in the dorsolateral mesopontine tegmentum; the balance consisted of noncholinergic neurons of the central tegmental field, retrorubral field, and cuneiform nucleus. Following placement of WGA-HRP into dorsal and intermediate lateral septal regions, the vast majority (greater than 90%) of retrogradely labelled neurons were cholinergic neurons of the PPT and LDT, with few noncholinergic retrogradely labelled neurons in the adjacent tegmentum. In contrast, fewer cholinergic neurons were retrogradely labelled following placement of tracer into the nucleus basalis of Meynert or into the central, medial, and basolateral nuclei of the amygdala, while numerous noncholinergic neurons of the central tegmental field rostral to the PPT and of the retrorubral field adjacent to the PPT were retrogradely labelled in these cases. These anterograde and retrograde studies demonstrate that cholinergic PPT and LDT neurons provide a substantial proportion of mesopontine tegmental afferents to the hypothalamus and lateral septum, while projections to the nucleus basalis and the amygdala are minimal.     

Distribution of high affinity choline transporter immunoreactivity in the primate central nervous system

A mouse monoclonal antibody (clone 62-2E8) raised against a human recombinant high-affinity choline transporter (CHT)-glutathione-S-transferase fusion protein was used to determine the distribution of immunoreactive profiles containing this protein in the monkey central nervous system (CNS). Within the monkey telencephalon, CHT-immunoreactive perikarya were found in the striatum, nucleus accumbens, medial septum, vertical and horizontal limb nuclei of the diagonal band, nucleus basalis complex, and the bed nucleus of the stria terminalis. Dense fiber staining was observed within the islands of Calleja, olfactory tubercle, hippocampal complex, amygdala; moderate to light fiber staining was seen in iso- and limbic cortices. CHT-containing fibers were also present in sensory and limbic thalamic nuclei, preoptic and hypothalamic areas, and the floccular lobe of the cerebellum. In the brainstem, CHT-immunoreactive profiles were observed in the pedunculopontine and dorsolateral tegmental nuclei, the Edinger-Westphal, oculomotor, trochlear, trigeminal, abducens, facial, ambiguus, dorsal vagal motor, and hypoglossal nuclei. In the spinal cord, CHT-immunoreactive ventral horn motoneurons were seen in close apposition to intensely immunoreactive C-terminals at the level of the cervical spinal cord. CHT immunostaining revealed a similar distribution of labeled profiles in the aged human brain and spinal cord. Dual fluorescent confocal microscopy revealed that the majority of CHT immunoreactive neurons contained the specific cholinergic marker, choline acetyltransferase, at all levels of the monkey CNS. The present observations indicate that the present CHT antibody labels cholinergic structures within the primate CNS and provides an additional marker for the investigation of cholinergic neuronal function in aging and disease.     

Metabotropic glutamate receptor 4-immunopositive terminals of medium-sized spiny neurons selectively form synapses with cholinergic interneurons in the rat neostriatum

Metabotropic glutamate receptor 4 (mGluR4) is localized mainly to presynaptic membranes in the brain. Rat neostriatum has been reported to contain two types of mGluR4-immunoreactive axon varicosities: small, weakly immunoreactive varicosities that were distributed randomly (type 1) and large, intensely immunoreactive ones that were often aligned linearly (type 2). In the present study, most type 1 terminals formed asymmetric synapses on dendritic spines, whereas type 2 terminals made symmetric synapses on dendritic shafts, showing immunoreactivity for GABAergic markers. After depletion of neostriatal neurons, type 2 but not type 1 varicosities were largely decreased in the damaged region. When medium-sized spiny neurons (MSNs) were labeled with Sindbis virus expressing membrane-targeted green fluorescent protein, mGluR4 immunoreactivity was observed on some varicosities of their axon collaterals in immunofluorescence and immunoelectron microscopies. Furthermore, type 2 varicosities were often positive for substance P but mostly negative for striatal interneuron markers and preproenkephalin. Thus, striatonigral/striato-entopeduncular MSNs are likely to be the largest source of type 2 mGluR4-immunopositive axon terminals in the neostriatum. Next, in the double-immunofluorescence study, almost all choline acetyltransferase (ChAT)-immunopositive and 41% of NK1 receptor-positive dendrites were heavily associated with type 2 mGluR4-immunoreactive varicosities. Neuronal nitric oxide synthase (nNOS)-positive dendrites, in contrast, seemed associated with only a few type 2 varicosities. Conversely, almost all type 2 varicosities were closely apposed to NK1 receptor-positive dendrites that were known to be derived from cholinergic and nNOS-producing interneurons. These findings indicate that the mGluR4-positive terminals of MSN axon collaterals selectively form synapses with neostriatal cholinergic interneurons.     

Pedunculopontine nucleus in the squirrel monkey: Distribution of cholinergic and monoaminergic neurons in the mesopontine tegmentum with evidence for the presence of glutamate in cholinergic neurons

The topographical relationships between cholinergic neurons, identified by their immuno-reactivity for choline acetyltransferase (ChAT) or their staining for β-nicotinamide ademine dinucleotide phosphate (NADPH)-diaphorase, and dopaminergic, serotoninergic, Nonadrenergic, and glutamatergic neurons that occur in the mesopontine tegmentum, were studied in the squirrel monkey (Saimiri sciureus). The ChAT-positive neurons in the pedunculopontine nucleus (PPN) form two distinct subpopulations, one that corresponds to PPN pars compacta(PPNc) and the other to PPN pars dissipata (PPNd). The ChAT-positive neurons in PPNc are clustered along the dorsolateral border of the superior cerebellar peduncle (SP) at trochlear nucleus levels, whereas those in PPNd are scattered along the SP from midmesencephalic to midpontine levels. At levels caudal toe the trochlear nucleus, ChAT-positive neurons corresponding to the laterodorsal tegmental nucleus (LDT) lie within the periaqueductal gray and extend caudally as far as locus coeruleus levels. All ChAT-positive neurons in PPN and LDT stain for NADPH-diaphorase; the majority of large neurons in PPN and LDT are cholinergic, but some large neurons devoid of NADPH-diaphorase also occurnin these nuclei. Cholinergic neurons in the mesopontine tegmentum form clusters that are largely segregated from raphe serotonin immunoreactive neurons, as well as from nigral dopaminergic and coeruleal noradrenergic neurons, as revealed by tyrosine hydroxylase immunohistochemistry. Nevertheless, dendrites of cholinergic and noradrenergic neurons are clolinergic and noradrenergic neurons are closely intermingled, suggesting the possibility of dendrodendritic contacts. In addition, numerous large and medium-sized glutamate-immunoreactive neurons are intermingled among cholinergic neurons in PPN. Furthermore, at trochlear nucleus levels, about 40% of cholinergic neurons display glutamate immunoreactivity, whereas other neurons express glutamate or ChAT immunoreactivity only. This study demonstrates that (1) cholinergic neurons remain largely segregated from monoaminergic neurons throughout the mesopontine tegmentum and (2) PPN contains cholinergic and glutamatergic neurons as well as neurons coexpressing ChAT and Glutamate in primates. © 1994 Wiley-Liss, Inc.     

Aberrant trafficking of the high-affinity choline transporter in AP-3-deficient mice

The high-affinity choline transporter (CHT) is expressed in cholinergic neurons and efficiently transported to axon terminals where it controls the rate-limiting step in acetylcholine synthesis. Recent studies have shown that the majority of CHT is unexpectedly localized on synaptic vesicles (SV) rather than the presynaptic plasma membrane, establishing vesicular CHT trafficking as a basis for activity-dependent CHT regulation. Here, we analyse the intracellular distribution of CHT in the adaptor protein-3 (AP-3)-deficient mouse model mocha . In the mocha mouse, granular structures in cell bodies are intensely labelled with CHT antibody, indicating possible deficits in CHT trafficking from the cell body to the axon terminal. Western blot analyses reveal that CHT on SV in mocha mice is decreased by 30% compared with wild-type mice. However, no significant difference in synaptosomal choline uptake activity is detected, consistent with the existence of a large reservoir pool for CHT. To further characterize CHT trafficking, we established a PC12D-CHT cell line. In this line, CHT is found associated with a subpopulation of synaptophysin-positive synaptic-like microvesicles (SLMV). The amounts of CHT detected on SLMV are greatly reduced by treating the cell with agents that halt AP-dependent membrane trafficking. These results demonstrate that APs have important functions for CHT trafficking in neuronal cells.     

Ultrastructural localization of Leu5-enkephalin immunoreactivity in mesocortical neurons and their input terminals in rat ventral tegmental area

Enkephalin (ENK) immunoreactivity is widely distributed in the ventral tegmental area (VTA), where endogenous ENK and dynorphin opioid peptides are known to have opposing actions in reward, stress, cognition, and fear-related behaviors. Many neurons in the VTA give rise to mesocortical projections terminating in the medial prefrontal cortex (mPFC), and these projections have been implicated to varying extents in all these functions. To determine whether there is a synaptic basis for ENK and/or dynorphin modulation of cortically projecting neurons within the VTA, we combined retrograde tract-tracing from the mPFC with dual immunocytochemical-labeling electron microscopy in the rat VTA. The retrograde tracer Fluorogold (FG) was microinjected into mPFC. At optimal survival periods, sections through the VTA were processed for immunolabeling of anti-FG and a Leu(5)-ENK antibody recognizing both ENK and dynorphin peptides. Over 26% of the retrogradely labeled neuronal somatodendritic profiles (n = 177) were contacted by ENK-immunoreactive axonal profiles including small axons and axon terminals. The axon terminals varied in their subcellular distribution of ENK immunoreactivity and also differed in forming either inhibitory-type (symmetric) or excitatory-type (asymmetric) synapses. Many of the axonal profiles also were apposed to FG-labeled somata or dendrites without forming recognizable synapses. Approximately one-third of the mesocortical neuronal perikarya also showed sparsely distributed somatodendritic ENK-immunoreactivity. Our results provide ultrastructural evidence that ENK and possibly dynorphin in the rat VTA have distributions consistent with involvement in diverse physiological actions affecting the output of mesocortical neurons, some of which also contain one or both peptides.     

Intrinsic membrane plasticity via increased persistent sodium conductance of cholinergic neurons in the rat laterodorsal tegmental nucleus contributes to cocaine‐induced addictive behavior

The laterodorsal tegmental nucleus (LDT) is a brainstem nucleus implicated in reward processing and is one of the main sources of cholinergic afferents to the ventral tegmental area (VTA). Neuroplasticity in this structure may affect the excitability of VTA dopamine neurons and mesocorticolimbic circuitry. Here, we provide evidence that cocaine‐induced intrinsic membrane plasticity in LDT cholinergic neurons is involved in addictive behaviors. After repeated experimenter‐delivered cocaine exposure, ex vivo whole‐cell recordings obtained from LDT cholinergic neurons revealed an induction of intrinsic membrane plasticity in regular‐ but not burst‐type neurons, resulting in increased firing activity. Pharmacological examinations showed that increased riluzole‐sensitive persistent sodium currents, but not changes in Ca²⁺‐activated BK, SK or voltage‐dependent A‐type potassium conductance, mediated this plasticity. In addition, bilateral microinjection of riluzole into the LDT immediately before the test session in a cocaine‐induced conditioned place preference (CPP) paradigm inhibited the expression of cocaine‐induced CPP. These findings suggest that intrinsic membrane plasticity in LDT cholinergic neurons is causally involved in the development of cocaine‐induced addictive behaviors.     

Synapses between corticotropin-releasing factor-containing axon terminals and dopaminergic neurons in the ventral tegmental area are predominantly glutamatergic

Interactions between stress and the mesocorticolimbic dopamine (DA) system have been suggested from behavioral and electrophysiological studies. Because corticotropin-releasing factor (CRF) plays a role in stress responses, we investigated possible interactions between neurons containing CRF and those producing DA in the ventral tegmental area (VTA). We first investigated the cellular distribution of CRF in the VTA by immunolabeling VTA sections with anti-CRF antibodies and analyzing these sections by electron microscopy. We found CRF immunoreactivity present mostly in axon terminals establishing either symmetric or asymmetric synapses with VTA dendrites. We established that nearly all CRF asymmetric synapses are glutamatergic, insofar as the CRF-immunolabeled axon terminals in these synapses coexpressed the vesicular glutamate transporter 2, and that the majority of CRF symmetric synapses are GABAergic, insofar as the CRF-immunolabeled axon terminals in these synapses coexpressed glutamic acid decarboxylase, findings that are of functional importance. We then looked for synaptic interactions between CRF- and DA-containing neurons, by using antibodies against CRF and tyrosine hydroxylase (TH; a marker for DA neurons). We found that most synapses between CRF-immunoreactive axon terminals and TH neurons are asymmetric (in the majority likely to be glutamatergic) and suggest that glutamatergic neurons containing CRF may be part of the neuronal circuitry that mediates stress responses involving the mesocorticolimbic DA system. The presence of CRF synapses in the VTA offers a mechanism for interactions between the stress-associated neuropeptide CRF and the mesocorticolimbic DA system.     

Nocifensive reflex-related on- and off-cells in the pedunculopontine tegmental nucleus, cuneiform nucleus, and lateral dorsal tegmental nucleus

Cholinergic projections from the pedunculopontine tegmental nucleus (PPTg) to the rostral ventromedial medulla (RVM) have been implicated in nociceptive modulation. The goal of this study was to identify neurons with nocifensive reflex-related activity in the mesopontine tegmentum including the PPTg. This study used the same behavioral neurophysiological classification system to identify neurons as has been extensively described in the RVM. Extracellular microelectrode recording was conducted in lightly anesthetized rats. Changes in firing associated with the noxious heat-evoked tail flick reflex were used to classify neurons as "on-cells" (displayed a burst in neuronal activity associated with the reflex), "off-cells" (displayed a pause in activity), and neutral cells (showed no response). Of 188 neurons studied in 23 rats, 77 were classified as on-cells, 14 as off-cells, the remainder as neutral cells. Recordings during periods without noxious stimulation found that some of the on- and off-cells displayed spontaneous transitions between active and silent periods termed cell cycling. The distribution of on- and off-cells in the mesopontine tegmentum overlapped and included the cholinergic PPTg and lateral dorsal tegmental nucleus identified by NADPH diaphorase staining, as well as the cuneiform nucleus and periaqueductal gray. The mesopontine tegmentum thus contains nocifensive reflex-related neurons with neurophysiological characteristics similar to those reported in the RVM. Neurons showing reflex-related activity were frequently encountered in the cholinergic PPTg and LDTg. Further studies will be required to determine whether these neurons modulate nociception through a link to the RVM.     

The inhibitory influence of the lateral habenula on midbrain dopamine cells: ultrastructural evidence for indirect mediation via the rostromedial mesopontine tegmental nucleus

The lateral habenula (LHb) provides an important source of negative reinforcement signals to midbrain dopamine (DA) cells in the substantia nigra and ventral tegmental area (VTA). This profound and consistent inhibitory influence involves a disynaptic connection from glutamate neurons in the LHb to some population of γ-aminobutyric acid (GABA) cells that, in turn, innervates DA neurons. Previous studies demonstrated that the GABA cells intrinsic to the VTA receive insufficient synaptic input from the LHb to serve as the primary source of this intermediate connection. In this investigation, we sought ultrastructural evidence supporting the hypothesis that a newly identified region of the brainstem, the rostromedial mesopontine tegmental nucleus (RMTg), is a more likely candidate for inhibiting midbrain DA cells in response to LHb activation. Electron microscopic examination of rat brain sections containing dual immunoreactivity for an anterograde tracing agent and a phenotypic marker revealed that: 1) more than 55% of the synapses formed by LHb axons in the RMTg were onto GABA-labeled dendrites; 2) more than 80% of the synapses formed by RMTg axons in the VTA contacted dendrites immunoreactive for the DA synthetic enzyme tyrosine hydroxylase; and 3) nearly all RMTg axons formed symmetric synapses and contained postembedding immunoreactivity for GABA. These findings indicate that the newly identified RMTg region is an intermediate structure in a disynaptic pathway that connects the LHb to VTA DA neurons. The results have important implications for understanding mental disorders characterized by a dysregulation of reward circuitry involving LHb and DA cell populations.     

Colocalization of ionotropic glutamate receptor subunits with NADPH-diaphorase-containing neurons in the rat mesopontine tegmentum

Tegmental cholinergic neurons vary their discharge patterns across the sleep-wake cycle, and glutamate is suggested to play an important role in determining these firing patterns. Cholinergic and noncholinergic neurons in the mesopontine tegmentum have different susceptibilities to various excitotoxins, presumably because of heterogeneity in the expression of glutamate receptor subtypes in this area. By using a double-labeling procedure that combines nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) histochemistry and avidin-biotin-peroxidase immunocytochemistry with diaminobenzidine as the chromogen, we compared the colocalization of AMPA receptor subunits GluR1, GluR2/3, and GluR4, kainate receptor subunits GluR5/6/7, and an NMDA receptor subunit NMDAR1 on NADPH-diaphorase-positive (cholinergic) neurons in the mesopontine tegmentum. Throughout the brainstem, neurons immunoreactive for GluR2/3 and NMDAR1 were most numerous, whereas neurons labeled for GluR1, GluR4, and GluR5/6/7 were less common. Specifically within the mesopontine tegmentum, the proportion of double-labeled neurons in the diaphorase-containing cell population was highest with GluR1 (43%) and lowest with GluR5/6/7 (12%). Regardless of the receptor subunit type, the greatest numbers of double-labeled neurons were observed in the pedunculopontine tegmental nucleus pars compacta and the fewest in the dorsal aspect of the laterodorsal tegmental nucleus. In addition, there were regional differences in the relative expression of receptor subunits and diaphorase-positive neurons across the subdivisions of the tegmental cholinergic column. Because each ionotropic subunit confers distinctive properties to a receptor channel, the present results suggest that mesopontine cholinergic neurons have nonuniform responses to glutamate and are also discriminable from basal forebrain cholinergic neurons in terms of glutamate receptor configuration. © 1996 Wiley-Liss, Inc.     

Region-specific targeting of dopamine D2-receptors and somatodendritic vesicular monoamine transporter 2 (VMAT2) within ventral tegmental area subdivisions

Throughout the ventral tegmental area (VTA), dopamine is packaged within subcellular organelles by the vesicular monoamine transporter-2 (VMAT2). Somatodendritically released dopamine in this region binds to the D2 receptor (D2R) to modulate ongoing neurotransmission. Although autoregulation of mesocortical dopaminergic neurons in the parabrachial VTA (PB-VTA) is known to be less efficacious than that of mesolimbic dopaminergic neurons in the paranigral (PN-VTA), the cellular basis for this regional heterogeneity is not known. For this reason, we used electron microscopic immunocytochemistry to determine the subcellular localization of the dopamine storage vesicles (identified by the presence of VMAT2) in relation to the D2R in these VTA subdivisions. In both regions, D2R immunoreactivity was principally located on extrasynaptic dendritic plasma membranes near excitatory-type synapses. Equivalent percentages (72 and 74%) of the D2R-labeled dendrites in each region contained VMAT2-immunoreactive tubulovesicles. Of the total VMAT2-labeled dendrites, however, a significantly lower percentage in the PB-VTA (26%) than in the PN-VTA (38%) contained D2R labeling. In contrast, a significantly higher number of VMAT2 immunogold-silver deposits was seen within individual dendrites in the PB-VTA than in PN-VTA. In both regions, D2R immunoreactivity was also detected in VMAT2-negative axon terminals that formed synapses on dendrites containing VMAT2. Our results are the first to demonstrate that within VTA neurons and their afferents the D2R is strategically positioned for activation by dopamine released from dendritic storage vesicles. These findings also suggest that the potential for D2R activation may affect the expression levels of VMAT2 in VTA dendrites.     

Distribution of GABAAreceptor alpha 1 subunit-like immunoreactivity in comparision with that of enkephalin and substance P in the rat forebrain

The γ-aminobutyric acid-A receptor consists of several subunits. In this immunohistochemical study we investigated the regional distribution of the α1 subunit with an antibody directed against a specific amino acid sequence (1-9) of the α1 subunit. We compared the distribution pattern of the α1 subunit-like immunoreactivity with that of substance P- and enkephalin-like immunoreactivities in adjacent sections of the rat forebrain. α1 subunit-like immunoreactivity appeared in the form of varicosities and fibers. A band-like terminal staining pattern (woolly fibers) that has been shown by others for substance P- and enkephalin-like immunoreactivity is also observed for α1 subunit-like immunoreactivity. In contrast to substance P and enkephalin, numerous α1 subunit-like immunoreactive perikarya were found. The highest density of α1 subunit-like immunoreactive fibers and perikarya was found in the pallidal areas and the substantia nigra pars reticulata whereas the nucleus accumbens and the caudate putamen displayed a low density. α1 subunit-like immunoreactive neurons resembled typical pallidal neurons. Some of these neurons were pericellularly stained with enkephalin-like immunoreactive varicosities in the dorsal pallidum. The distribution pattern of α1 subunit-like immunoreactivity reflects a partial overlap with the substance P and enkephalin system although a differential distribution to each of these peptides was observed for cell bodies, fibers, and axon terminals. © 1995 Wiley-Liss, Inc.     

Mu-opioid receptors in the ventral tegmental area are targeted to presynaptically and directly modulate mesocortical projection neurons.

Mesocorticolimbic projections originating from dopaminergic and GABAergic neurons in the ventral tegmental area (VTA) play a critical role in opiate addiction. Activation of mu-opioid receptors (MOR), which are located mainly within inhibitory neurons in the VTA, results in enhanced dopaminergic transmission in target regions, including the medial prefrontal cortex (mPFC). We combined retrograde tract-tracing and electron microscopic immunocytochemistry to determine if neurons in the VTA that project to the mPFC contain MOR or receive input from MOR-containing terminals. Rats received unilateral injections of the retrograde tracer Fluoro-Gold (FG) into the mPFC. Tissue sections throughout the VTA were then processed for electron microscopic examination of FG and MOR. Immunoperoxidase labeling for FG was present in VTA cell bodies that contained immunogold-silver particles for MOR that often were contacted by profiles exclusively immunoreactive for MOR, including somata and axon terminals. The majority of dually labeled profiles were dendrites that received convergent input from unlabeled axon terminals forming either symmetric or asymmetric type synapses. Within retrogradely labeled cell bodies and proximal dendrites, MOR immunoreactivity was mainly sequestered within the cytoplasm. In contrast, distal retrogradely labeled dendrites contained MOR gold particles located along the plasma membranes. These data suggest that opiates active at MOR in the VTA modulate cortical activity through 1) presynaptic actions on MOR in terminals contacting mesocortical cell bodies, and 2) direct activation of MOR in distal dendrites of projection neurons.     

c‐Fos marking of identified midbrain neurons coactive after nicotine administration in‐vivo

Despite the reduced life expectancy and staggering financial burden of medical treatment associated with tobacco smoking, the molecular, cellular, and ensemble adaptations associated with chronic nicotine consumption remain poorly understood. Complex circuitry interconnecting dopaminergic and cholinergic regions of the midbrain and mesopontine tegmentum are critical for nicotine associated reward. Yet our knowledge of the nicotine activation of these regions is incomplete, in part due to their cell type diversity. We performed double immunohistochemistry for the immediate early gene and surrogate activity sensor, c‐Fos, and markers for either cholinergic, dopaminergic or GABAergic cell types in mice treated with nicotine. Both acute (0.5 mg/kg) and chronic (0.5 mg/kg/day for 7 days) nicotine strongly activated GABAergic neurons of the interpeduncular nucleus and medial terminal nucleus of the accessory optic tract (MT). Acute but not chronic nicotine also activated small percentages of dopaminergic and other neurons in the ventral tegmental area (VTA) as well as noncholinergic neurons in the pedunculotegmental and laterodorsal tegmental nuclei (PTg/LDTg). Twenty four hours of nicotine withdrawal after chronic nicotine treatment suppressed c‐Fos activation in the MT. In comparison to nicotine, a single dose of cocaine caused a similar activation in the PTg/LDTg but not the VTA where GABAergic cells were strongly activated but dopaminergic neurons were not affected. These results indicate the existence of drug of abuse specific ensembles. The loss of ensemble activation in the VTA and PTg/LDTg after chronic nicotine represents a molecular and cellular tolerance which may have implications for the mechanisms underlying nicotine dependence.     

Muscarinic control of rostromedial tegmental nucleus GABA neurons and morphine‐induced locomotion

Opioids induce rewarding and locomotor effects by inhibiting rostromedial tegmental GABA neurons that express μ‐opioid and nociceptin receptors. These GABA neurons then strongly inhibit dopamine neurons. Opioid‐induced reward, locomotion and dopamine release also depend on pedunculopontine and laterodorsal tegmental cholinergic and glutamate neurons, many of which project to and activate ventral tegmental area dopamine neurons. Here we show that laterodorsal tegmental and pedunculopontine cholinergic neurons project to both rostromedial tegmental nucleus and ventral tegmental area, and that M4 muscarinic receptors are co‐localized with μ‐opioid receptors associated with rostromedial tegmental GABA neurons. To inhibit or excite rostromedial tegmental GABA neurons, we utilized adeno‐associated viral vectors and DREADDs to express designed muscarinic receptors (M4D or M3D respectively) in GAD2::Cre mice. In M4D‐expressing mice, clozapine‐N‐oxide increased morphine‐induced, but not vehicle‐induced, locomotion. In M3D‐expressing mice, clozapine‐N‐oxide blocked morphine‐induced, but not vehicle‐induced, locomotion. We propose that cholinergic inhibition of rostromedial tegmental GABA neurons via M4 muscarinic receptors facilitates opioid inhibition of the same neurons. This model explains how mesopontine cholinergic systems and muscarinic receptors in the rostromedial tegmental nucleus and ventral tegmental area are important for dopamine‐dependent and dopamine‐independent opioid‐induced rewards and locomotion.     

Analysis of neuronal connections in the basal ganglia]

Morphological features indicating occurrence of two types of extrasynaptic chemical transmission were observed within rat basal ganglia. (1) Striatonigral neurons containing substance P (SP) sent many axon collaterals equipped with axonal varicosities to the striatum: the varicosities displayed synaptophysin-like immunoreactivity (-LI). However, only 15% of the varicosities appeared to be in close contact with structures showing SP receptor (SPR)-LI. Many of axon terminals of striatonigral neurons were confirmed electron microscopically not to be in synaptic contact with SPR-like immunoreactive structures within the striatum. SP released from the varicosities might, at least partly, diffuse to reach SPR at distance from the release sites. (2) Immunoreactivities for metabotropic glutamate receptors (mGluRs) 4 a, 7 a, 7 b and 8 were in axon terminals within the globus pallidus (external segment of the globus pallidus in primates). The immunoreactivities disappeared after destruction of the striatum, but not after destruction of the subthalamic nucleus. The immunoreactivity for mGluR 7 a was confirmed electron microscopically to be within axon terminals showing glutamic acid decarboxylase-LI. Glutamate released from glutamatergic subthalamopallidal neurons might partly spilled over from the synaptic sites to reach mGluRs on "nearby" axon terminals of GABAergic striatopallidal neurons. Functional significance of thalamostriatal and corticosubthalamic fibers was also discussed.     

Narcoleptic orexin receptor knockout mice express enhanced cholinergic properties in laterodorsal tegmental neurons

Pharmacological studies of narcoleptic canines indicate that exaggerated pontine cholinergic transmission promotes cataplexy. As disruption of orexin (hypocretin) signaling is a primary defect in narcolepsy with cataplexy, we investigated whether markers of cholinergic synaptic transmission might be altered in mice constitutively lacking orexin receptors (double receptor knockout; DKO). mRNA for Choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT) and the high‐affinity choline transporter (CHT1) but not acetylcholinesterase (AChE) was significantly higher in samples from DKO than wild‐type (WT) mice. This was region‐specific; levels were elevated in samples from the laterodorsal tegmental nucleus (LDT) and the fifth motor nucleus (Mo5) but not in whole brainstem samples. Consistent with region‐specific changes, we were unable to detect significant differences in Western blots for ChAT and CHT1 in isolates from brainstem, thalamus and cortex or in ChAT enzymatic activity in the pons. However, using ChAT immunocytochemistry, we found that while the number of cholinergic neurons in the LDT and Mo5 were not different, the intensity of somatic ChAT immunostaining was significantly greater in the LDT, but not Mo5, from DKO than from WT mice. We also found that ChAT activity was significantly reduced in cortical samples from DKO compared with WT mice. Collectively, these findings suggest that the orexins can regulate neurotransmitter expression and that the constitutive absence of orexin signaling results in an up‐regulation of the machinery necessary for cholinergic neurotransmission in a mesopontine population of neurons that have been associated with both normal rapid eye movement sleep and cataplexy.