Role of the ventral tegmental area in methamphetamine extinction: AMPA receptor-mediated neuroplasticity.

The molecular mechanisms underlying drug extinction remain largely unknown, although a role for medial prefrontal cortex (mPFC) glutamate neurons has been suggested. Considering that the mPFC sends glutamate efferents to the ventral tegmental area (VTA), we tested whether the VTA is involved in methamphetamine (METH) extinction via conditioned place preference (CPP). Among various METH-CPP stages, we found that the amount of phospho-GluR1/Ser845 increased in the VTA at behavioral extinction, but not the acquisition or withdrawal stage. Via surface biotinylation, we found that levels of membrane GluR1 were significantly increased during METH-CPP extinction, while no change was observed at the acquisition stage. Specifically, the number of dendritic spines in the VTA was increased at behavioral extinction, but not during acquisition. To validate the role of the mPFC in METH-CPP extinction, we lesioned the mPFC. Ibotenic acid lesioning of the mPFC did not affect METH-CPP acquisition, however, it abolished the extinction stage and reversed the enhanced phospho-GluR1/Ser845 levels as well as increases in VTA dendritic spines during METH-CPP extinction. Overall, this study demonstrates that the mPFC plays a critical role in METH-CPP extinction and identifies the VTA as an alternative target in mediating the extinction of drug conditioning.

Hypocretin1/orexinA-immunoreactive axons form few synaptic contacts on rat ventral tegmental area neurons that project to the medial prefrontal cortex.

BACKGROUND: Hypocretins/orexins (Hcrt/Ox) are hypothalamic neuropeptides involved in sleep-wakefulness regulation. Deficiency in Hcrt/Ox neurotransmission results in the sleep disorder narcolepsy, which is characterized by an inability to maintain wakefulness. The Hcrt/Ox neurons are maximally active during wakefulness and project widely to the ventral tegmental area (VTA). A dopamine-containing nucleus projecting extensively to the cerebral cortex, the VTA enhances wakefulness. In the present study, we used retrograde tracing from the medial prefrontal cortex (mPFC) to examine whether Hcrt1/OxA neurons target VTA neurons that could sustain behavioral wakefulness through their projections to mPFC. RESULTS: The retrograde tracer Fluorogold (FG) was injected into mPFC and, after an optimal survival period, sections through the VTA were processed for dual immunolabeling of anti-FG and either anti-Hcrt1/OxA or anti-TH antisera. Most VTA neurons projecting to the mPFC were located in the parabrachial nucleus of the ipsilateral VTA and were non-dopaminergic. Only axonal profiles showed Hcrt1/OxA-immunoreactivity in VTA. Hcrt1/OxA reactivity was observed in axonal boutons and many unmyelinated axons. The Hcrt1/OxA immunoreactivity was found filling axons but it was also observed in parts of the cytoplasm and dense-core vesicles. Hcrt1/OxA-labeled boutons frequently apposed FG-immunolabeled dendrites. However, Hcrt1/OxA-labeled boutons rarely established synapses, which, when they were established, were mainly asymmetric (excitatory-type), with either FG-labeled or unlabeled dendrites. CONCLUSIONS: Our results provide ultrastructural evidence that Hcrt1/OxA neurons may exert a direct synaptic influence on mesocortical neurons that would facilitate arousal and wakefulness. The paucity of synapses, however, suggest that the activity of VTA neurons with cortical projections might also be modulated by Hcrt1/OxA non-synaptic actions. In addition, Hcrt1/OxA could modulate the postsynaptic excitatory responses of VTA neurons with cortical projections to a co-released excitatory transmitter from Hcrt1/OxA axons. Our observation of Hcrt1/OxA targeting of mesocortical neurons supports Hcrt1/OxA wakefulness enhancement in the VTA and could help explain the characteristic hypersomnia present in narcoleptic patients.

Columnar distribution of catecholaminergic neurons in the ventrolateral periaqueductal gray and their relationship to efferent pathways.

The periaqueductal gray (PAG) is a critical brain region involved in opioid analgesia and provides efferents to descending pathways that modulate nociception. In addition, the PAG contains ascending pathways to regions involved in the regulation of reward, including the substantia nigra (SN) and the ventral tegmental area (VTA). SN and VTA contain dopaminergic neurons that are critical for the maintenance of positive reinforcement. Interestingly, the PAG is also reported to contain a population of dopaminergic neurons. In this study, the distribution of catecholaminergic neurons within the ventrolateral (vl) PAG was examined using immunocytochemical methods. In addition, the catecholaminergic PAG neurons were examined to determine whether these neurons are integrated into ascending (VTA, SN) and descending rostral ventral medulla (RVM) efferent pathways from this region. The immunocytochemical analysis determined that catecholaminergic neurons in the PAG are both dopaminergic and noradrenergic and these neurons have a distinct rostrocaudal distribution within the ventrolateral column of PAG. Dopaminergic neurons were concentrated rostrally and were significantly smaller than noradrenergic neurons. Combined immunocytochemistry and tract tracing methods revealed that catecholaminergic neurons are distinct from, but closely associated with, both ascending and descending efferent projection neurons. Finally, by electron microscopy, catecholaminergic neurons showed close dendritic appositions with other neurons in PAG, suggesting a possible nonsynaptic mechanism for regulation of PAG output by these neurons. In conclusion, our data indicate that there are two populations of catecholaminergic neurons in the vlPAG that form dendritic associations with both ascending and descending efferents suggesting a possible nonsynaptic modulation of vlPAG neurons.

Glutamatergic and nonglutamatergic neurons of the ventral tegmental area establish local synaptic contacts with dopaminergic and nondopaminergic neurons.

The ventral tegmental area (VTA) contributes to reward and motivation signaling. In addition to the well established populations of dopamine (DA) or GABA VTA neurons, glutamatergic neurons were recently discovered in the VTA. These glutamatergic neurons express the vesicular glutamate transporter 2, VGluT2. To investigate whether VTA glutamatergic neurons establish local synapses, we tagged axon terminals from resident VTA neurons by intra-VTA injection of Phaseolus vulgaris leucoagglutinin (PHA-L) or an adeno-associated virus encoding wheat germ agglutinin (WGA) and by immunoelectron microscopy determined the presence of VGluT2 in PHA-L- or WGA-positive terminals. We found that PHA-L- or WGA-positive terminals from tagged VTA cells made asymmetric or symmetric synapses within the VTA. VGluT2 immunoreactivity was detected in the vast majority of PHA-L- or WGA-positive terminals forming asymmetric synapses. These results indicate that both VTA glutamatergic and nonglutamatergic (likely GABAergic) neurons establish local synapses. To examine the possible DAergic nature of postsynaptic targets of VTA glutamatergic neurons, we did triple immunolabeling with antibodies against VGluT2, tyrosine hydroxylase (TH), and PHA-L. From triple-labeled tissue, we found that double-labeled PHA-L (+)/VGluT2 (+) axon terminals formed synaptic contacts on dendrites of both TH-positive and TH-negative cells. Consistent with these anatomical observations, in whole-cell slice recordings of VTA neurons we observed that blocking action potential activity significantly decreased the frequency of synaptic glutamatergic events in DAergic and non-DAergic neurons. These observations indicate that resident VTA glutamatergic neurons are likely to affect both DAergic and non-DAergic neurotransmission arising from the VTA.

Drugs of abuse and stress impair LTP at inhibitory synapses in the ventral tegmental area.

Synaptic plasticity in the ventral tegmental area (VTA) is modulated by drugs of abuse and stress and is hypothesized to contribute to specific aspects of addiction. Both excitatory and inhibitory synapses on dopamine neurons in the VTA are capable of undergoing long-term changes in synaptic strength. While the strengthening or weakening of excitatory synapses in the VTA has been widely examined, the role of inhibitory synaptic plasticity in brain reward circuitry is less established. Here, we investigated the effects of drugs of abuse, as well as acute stress, on long-term potentiation of GABAergic synapses onto VTA dopamine neurons (LTP(GABA)). Morphine (10 mg/kg i.p.) reduced the ability of inhibitory synapses in midbrain slices to express LTP(GABA) both at 2 and 24 h after drug exposure but not after 5 days. Cocaine (15 mg/kg i.p.) impaired LTP(GABA) 24 h after exposure, but not at 2 h. Nicotine (0.5 mg/kg i.p.) impaired LTP(GABA) 2 h after exposure, but not after 24 h. Furthermore, LTP(GABA) was completely blocked 24 h following brief exposure to a stressful stimulus, a forced swim task. Our data suggest that drugs of abuse and stress trigger a common modification to inhibitory plasticity, synergizing with their collective effect at excitatory synapses. Together, the net effect of addictive substances or stress is expected to increase excitability of VTA dopamine neurons, potentially contributing to the early stages of addiction.

Periaqueductal gray afferents synapse onto dopamine and GABA neurons in the rat ventral tegmental area.

The midbrain central gray (periaqueductal gray; PAG) mediates defensive behaviors and is implicated in the rewarding effects of opiate drugs. Projections from the PAG to the ventral tegmental area (VTA) suggest that this region might also regulate behaviors involving motivation and cognition. However, studies have not yet examined the morphological features of PAG axons in the VTA or whether they synapse onto dopamine (DA) or GABA neurons. In this study, we injected anterograde tracers into the rat PAG and used immunoperoxidase to visualize the projections to the VTA. Immunogold-silver labeling for tyrosine hydroxylase (TH) or GABA was then used to identify the phenotype of innervated cells. Electron microscopic examination of the VTA revealed axons labeled anterogradely from the PAG, including myelinated and unmyelinated fibers and axon varicosities, some of which formed identifiable synapses. Approximately 55% of these synaptic contacts were of the symmetric (presumably inhibitory) type; the rest were asymmetric (presumably excitatory). These findings are consistent with the presence of both GABA and glutamate projection neurons in the PAG. Some PAG axons contained dense-cored vesicles indicating the presence of neuropeptides in addition to classical neurotransmitters. PAG projections synapsed onto both DA and GABA cells with no obvious selectivity, providing the first anatomical evidence for these direct connections. The results suggest a diverse nature of PAG physiological actions on midbrain neurons. Moreover, as both the VTA and PAG are implicated in the reinforcing actions of opiates, our findings provide a potential substrate for some of the rewarding effects of these drugs.

VTA Glutamatergic Neurons Mediate Innate Defensive Behaviors.

The ventral tegmental area (VTA) has dopamine, GABA, and glutamate neurons, which have been implicated in reward and aversion. Here, we determined whether VTA-glutamate or -GABA neurons play a role in innate defensive behavior. By VTA cell-type-specific genetic ablation, we found that ablation of glutamate, but not GABA, neurons abolishes escape behavior in response to threatening stimuli. We found that escape behavior is also decreased by chemogenetic inhibition of VTA-glutamate neurons and detected increases in activity in VTA-glutamate neurons in response to the threatening stimuli. By ultrastructural and electrophysiological analysis, we established that VTA-glutamate neurons receive a major monosynaptic glutamatergic input from the lateral hypothalamic area (LHA) and found that photoinhibition of this input decreases escape responses to threatening stimuli. These findings indicate that VTA-glutamate neurons are activated by and required for innate defensive responses and that information on threatening stimuli to VTA-glutamate neurons is relayed by LHA-glutamate neurons.

Lateral habenula projections to dopamine and GABA neurons in the rat ventral tegmental area.

Ventral tegmental area (VTA) dopamine (DA) neurons and their forebrain projections are critically involved in reward processing and cognitive functions. Descending projections from the lateral habenula (LHb) play a central role in inhibiting DA cell activity in response to the absence of expected rewards. As LHb efferents are reportedly glutamatergic, their ability to inhibit DA cells would theoretically require a disynaptic connection involving VTA GABA neurons and their local collateral inputs to DA cells. We therefore used anterograde tract-tracing from the LHb to investigate the relative selectivity of LHb synapses onto GABA versus DA VTA neurons. LHb axons were visualized using immunoperoxidase, and DA and GABA cells were marked by immunogold-silver labeling for tyrosine hydroxylase (TH) or GABA, respectively. By ultrastructural analysis, 16% of LHb axons were observed to form synaptic contacts in the VTA, and most of these were of an intermediate morphological type that did not exhibit definitive asymmetric or symmetric character. LHb axons synaptically targeted TH- and GABA-labeled dendrites to a comparable extent (45 and 52% observed incidence, respectively). Pre-embedding immunogold labeling for the vesicular glutamate transporter type 2 and post-embedding immunogold staining for GABA confirmed that approximately 85% of LHb terminals were glutamatergic and not GABAergic. These results suggest that the robust inhibition of DA cells evoked by the LHb is unlikely to arise from a selective innervation of VTA GABA neurons. Moreover, the LHb may mediate a direct excitation of DA cells that is over-ridden by indirect inhibition originating from an extrinsic source.

Behavioral sensitization to cocaine is associated with increased glutamate receptor trafficking to the postsynaptic density after extended withdrawal period.

Glutamatergic signaling plays an important role in the behavioral and molecular plasticity observed in behavioral sensitization to cocaine. Redistribution of the glutamate receptors in the synaptosomal membrane fraction was investigated in the nucleus accumbens, dorsolateral striatum, and ventral tegmental area at 1 or 21 days of withdrawal in behaviorally sensitized rats. At 1 day of withdrawal, there were no changes in either tissue level or redistribution of glutamate receptors in nucleus accumbens core and shell and ventral tegmental area. At 21 days of withdrawal, there was a decrease in the expression of mGluR2/3 protein in core and shell, an increase in GluR1 and a decrease in Homer1b/c proteins in the nucleus accumbens core tissue. In dorsolateral striatum, the tissue level of NMDAR2B protein was increased. Moreover, there was an augmented presence of AMPA (GluR1, GluR2), NMDA (NMDAR1, 2A, 2B), and group 1 metabotropic glutamate receptor (mGluR5) proteins in the synaptosomal fraction in core and shell of the nucleus accumbens. There was also an increase in synaptosomal mGluR2/3 protein in nucleus accumbens core. The redistribution of glutamate receptors was selective for nucleus accumbens since no changes were observed in the dorsolateral striatum and ventral tegmental area. While the tissue level of the Homer1b/c protein was selectively reduced in nucleus accumbens core, that of PSD95, PICK1, and actin was not changed in any of the brain regions examined. However, the synaptosomal membrane fraction level of Homer1b/c and PSD95 was increased in nucleus accumbens core and shell, with no changes in PICK1, and a decrease in actin protein. These observations suggest that significant redistribution of glutamate receptors and postsynaptic scaffolding proteins into synaptosomal membrane fraction is associated with withdrawal from behavioral sensitization to cocaine.

Ventral Tegmental Area Projection Regulates Glutamatergic Transmission in Nucleus Accumbens.

The ventral tegmental area (VTA) projection to the nucleus accumbens shell (NAcSh) regulates NAcSh-mediated motivated behaviors in part by modulating the glutamatergic inputs. This modulation is likely to be mediated by multiple substances released from VTA axons, whose phenotypic diversity is illustrated here by ultrastructural examination. Furthermore, we show in mouse brain slices that a brief optogenetic stimulation of VTA-to-NAc projection induced a transient inhibition of excitatory postsynaptic currents (EPSCs) in NAcSh principal medium spiny neurons (MSNs). This inhibition was not accompanied by detectable alterations in presynaptic release properties of electrically-evoked EPSCs, suggesting a postsynaptic mechanism. The VTA projection to the NAcSh releases dopamine, GABA and glutamate, and induces the release of other neuronal substrates that are capable of regulating synaptic transmission. However, pharmacological inhibition of dopamine D1 or D2 receptors, GABAA or GABAB receptors, NMDA receptors, P2Y1 ATP receptors, metabotropic glutamate receptor 5, and TRP channels did not prevent this short-term inhibition. These results suggest that an unknown mechanism mediates this form of short-term plasticity induced by the VTA-to-NAc projection.

Decreased vesicular somatodendritic dopamine stores in leptin-deficient mice.

An increasing number of studies indicate that leptin can regulate the activity of the mesolimbic dopamine system. The objective of this study was to examine the regulation of the activity of dopamine neurons by leptin. This was accomplished by examining the dopamine D2 receptor-mediated synaptic current that resulted from somatodendritic release of dopamine in brain slices taken from mice that lacked leptin (Lep(ob/ob) mice). Under control conditions, the amplitude and kinetics of the IPSC in wild-type and Lep(ob/ob) mice were not different. However, in the presence of forskolin or cocaine, the facilitation of the dopamine IPSC was significantly reduced in Lep(ob/ob) mice. The application of L-3,4-dihydroxyphenylalanine (L-DOPA) increased the IPSC in Lep(ob/ob) mice significantly more than in wild-type animals and fully restored the responses to both forskolin and cocaine. Treatment of Lep(ob/ob) mice with leptin in vivo fully restored the cocaine-induced increase in the IPSC to wild-type levels. These results suggest that there is a decrease in the content of somatodendritic vesicular dopamine in the Lep(ob/ob) mice. The release of dopamine from terminals may be less affected in the Lep(ob/ob) mice, because the cocaine-induced rise in dopamine in the ventral striatum was not statistically different between wild-type and Lep(ob/ob) mice. In addition, the relative increase in cocaine-induced locomotion was similar for wild-type and Lep(ob/ob) mice. These results indicate that, although basal release is not altered, the amount of dopamine that can be released is reduced in Lep(ob/ob) mice.

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.

Predominant surface distribution of neurokinin-3 receptors in non-dopaminergic dendrites in the rat substantia nigra and ventral tegmental area.

Neurokinin-3 (NK(3)) receptors are prevalent within the substantia nigra (SN) and ventral tegmental area (VTA), where their activation can affect motor and motivational behaviors as well as cardiovascular function and stress responses. These actions are mediated, in part, by dopaminergic neurons in each region. To determine the relevant sites for activation of these receptors, we examined the electron microscopic localization of NK(3) receptors and tyrosine hydroxylase (TH), the catecholamine synthesizing enzyme in dopaminergic neurons in the SN and VTA of rat brain. In each region, immunogold-silver labeling for NK(3) receptors was detected in many somatodendritic profiles, some of which contained TH-immunoreactivity. NK(3)-immunogold particles were largely associated with endomembranes resembling smooth endoplasmic reticulum, and only occasionally located on the plasma membrane in TH-labeled dendrites. In comparison with these dendrites, non-TH immunoreactive dendrites contained significantly more total (VTA) and more plasmalemmal (VTA and SN) NK(3)-immunogold particles. In each region, NK(3) gold particles also were seen in axonal as well as glial profiles, some of which contacted TH-immunoreactive dendrites. The NK(3)-labeled axon terminals formed either symmetric or asymmetric, excitatory-type synapses, the latter of which were significantly more prevalent in the VTA, compared with SN. These results provide the first ultrastructural evidence indicating that NK(3) receptors are available in cytoplasmic reserve in dopaminergic neurons, but more immediately accessible at the plasmalemmal surface of non-dopaminergic dendrites in both the SN and VTA. The activation of these receptors, together with the NK(3) receptors in either the presynaptic axon terminals or glia may contribute to the diverse physiological effects of tachykinins in each region, and most prominently involving excitatory inputs to the VTA.

Glutamate synaptic inputs to ventral tegmental area neurons in the rat derive primarily from subcortical sources.

Dopamine and GABA neurons in the ventral tegmental area project to the nucleus accumbens and prefrontal cortex and modulate locomotor and reward behaviors as well as cognitive and affective processes. Both midbrain cell types receive synapses from glutamate afferents that provide an essential control of behaviorally-linked activity patterns, although the sources of glutamate inputs have not yet been completely characterized. We used antibodies against the vesicular glutamate transporter subtypes 1 and 2 (VGlut1 and VGlut2) to investigate the morphology and synaptic organization of axons containing these proteins as putative markers of glutamate afferents from cortical versus subcortical sites, respectively, in rats. We also characterized the ventral tegmental area cell populations receiving VGlut1+ or VGlut2+ synapses according to their transmitter phenotype (dopamine or GABA) and major projection target (nucleus accumbens or prefrontal cortex). By light and electron microscopic examination, VGlut2+ as opposed to VGlut1+ axon terminals were more numerous, had a larger average size, synapsed more proximally, and were more likely to form convergent synapses onto the same target. Both axon types formed predominantly asymmetric synapses, although VGlut2+ terminals more often formed synapses with symmetric morphology. No absolute selectivity was observed for VGlut1+ or VGlut2+ axons to target any particular cell population. However, the synapses onto mesoaccumbens neurons more often involved VGlut2+ terminals, whereas mesoprefrontal neurons received relatively equal synaptic inputs from VGlut1+ and VGlut2+ profiles. The distinct morphological features of VGlut1 and VGlut2 positive axons suggest that glutamate inputs from presumed cortical and subcortical sources, respectively, differ in the nature and intensity of their physiological actions on midbrain neurons. More specifically, our findings imply that subcortical glutamate inputs to the ventral tegmental area expressing VGlut2 predominate over cortical sources of excitation expressing VGlut1 and are more likely to drive the behaviorally-linked bursts in dopamine cells that signal future expectancy or attentional shifting.

Circuit specificity in the inhibitory architecture of the VTA regulates cocaine-induced behavior.

Afferent inputs to the ventral tegmental area (VTA) control reward-related behaviors through regulation of dopamine neuron activity. The nucleus accumbens (NAc) provides one of the most prominent projections to the VTA; however, recent studies have provided conflicting evidence regarding the function of these inhibitory inputs. Using optogenetics, cell-specific ablation, whole cell patch-clamp and immuno-electron microscopy, we found that NAc inputs synapsed directly onto dopamine neurons, preferentially activating GABAB receptors. GABAergic inputs from the NAc and local VTA GABA neurons were differentially modulated and activated separate receptor populations in dopamine neurons. Genetic deletion of GABAB receptors from dopamine neurons in adult mice did not affect general or morphine-induced locomotor activity, but markedly increased cocaine-induced locomotion. Collectively, our findings demonstrate notable selectivity in the inhibitory architecture of the VTA and suggest that long-range GABAergic inputs to dopamine neurons fundamentally regulate behavioral responses to cocaine.

Subcellular distribution of M2 muscarinic receptors in relation to dopaminergic neurons of the rat ventral tegmental area.

Acetylcholine can affect cognitive functions and reward, in part, through activation of muscarinic receptors in the ventral tegmental area (VTA) to evoke changes in mesocorticolimbic dopaminergic transmission. Among the known muscarinic receptor subtypes present in the VTA, the M2 receptor (M2R) is most implicated in autoregulation and also may play a heteroreceptor role in regulation of the output of the dopaminergic neurons. We sought to determine the functionally relevant sites for M2R activation in relation to VTA dopaminergic neurons by examining the electron microscopic immunolabeling of M2R and the dopamine transporter (DAT) in the VTA of rat brain. The M2R was localized to endomembranes in DAT-containing somatodendritic profiles but showed a more prominent, size-dependent plasmalemmal location in nondopaminergic dendrites. M2R also was located on the plasma membrane of morphologically heterogenous axon terminals contacting unlabeled as well as M2R- or DAT-labeled dendrites. Some of these terminals formed asymmetric synapses resembling those of cholinergic terminals in the VTA. The majority, however, formed symmetric, inhibitory-type synapses or were apposed without recognized junctions. Our results provide the first ultrastructural evidence that the M2R is expressed, but largely not available for local activation, on the plasma membrane of VTA dopaminergic neurons. Instead, the M2R in this region has a distribution suggesting more indirect regulation of mesocorticolimbic transmission through autoregulation of acetylcholine release and changes in the physiological activity or release of other, largely inhibitory transmitters. These findings could have implications for understanding the muscarinic control of cognitive and goal-directed behaviors within the VTA.

Dopamine innervation of the monkey mediodorsal thalamus: Location of projection neurons and ultrastructural characteristics of axon terminals.

Dopamine (DA) axons and receptors have recently been identified in the primate thalamus, including the mediodorsal thalamic nucleus (MD). In order to determine whether the DA innervation of the primate MD shares the anatomical features of the mesocortical or nigrostriatal DA projections, we performed tract-tracing and immunocytochemistry studies in macaque monkeys (Macaca fascicularis) to identify the location of the DA neurons that project to MD and immuno-electron microscopy to determine the distribution of the dopamine transporter (DAT) in axons within the MD. Similar to the mesocortical projection, retrogradely-labeled, tyrosine hydroxylase-containing neurons were present in dorsal tier ventral mesencephalic nuclei, such as the ventral tegmental area and the dorsal portion of the substantia nigra pars compacta. In contrast, no dual-labeled neurons were present in the ventral tier nuclei, the primary origin of the nigrostriatal DA pathway. In addition, like the DA projection to the prefrontal cortex, DAT immunoreactivity was predominantly localized to the pre-terminal portion of axons in the MD, and was infrequently found in association with synaptic vesicles, in contrast to nigrostriatal DA axons. These findings indicate that the DA projection to the MD shares anatomical features with the mesocortical DA system, suggesting that the functional properties of DA neurotransmission in the MD might be more similar to those in the cortex than in the striatum.

Anatomical and ultrastructural study of PRAF2 expression in the mouse central nervous system.

Prenylated Rab acceptor family, member 2 (PRAF2) is a four transmembrane domain protein of 19 kDa that is highly expressed in particular areas of mammalian brains. PRAF2 is mostly found in the endoplasmic reticulum (ER) of neurons where it plays the role of gatekeeper for the GB1 subunit of the GABAB receptor, preventing its progression in the biosynthetic pathway in the absence of hetero-dimerization with the GB2 subunit. However, PRAF2 can interact with several receptors and immunofluorescence studies indicate that PRAF2 distribution is larger than the ER, suggesting additional biological functions. Here, we conducted an immuno-cytochemical study of PRAF2 distribution in mouse central nervous system (CNS) at anatomical, cellular and ultra-structural levels. PRAF2 appears widely expressed in various regions of mature CNS, such as the olfactory bulbs, cerebral cortex, amygdala, hippocampus, ventral tegmental area and spinal cord. Consistent with its regulatory role of GABAB receptors, PRAF2 was particularly abundant in brain regions known to express GB1 subunits. However, other brain areas where GB1 is expressed, such as basal ganglia, thalamus and hypothalamus, contain little or no PRAF2. In these areas, GB1 subunits might reach the cell surface of neurons independently of GB2 to exert biological functions distinct from those of GABAB receptors, or be regulated by other gatekeepers. Electron microscopy studies confirmed the localization of PRAF2 in the ER, but identified previously unappreciated localizations, in mitochondria, primary cilia and sub-synaptic region. These data indicate additional modes of GABAB regulation in specific brain areas and new biological functions of PRAF2.

Impact of prenatal nicotine on the structure of midbrain dopamine regions in the rat.

In utero exposure of rats to nicotine (NIC) provides a useful animal model for studying the impact of smoking during pregnancy on human offspring. Certain sequelae of prenatal NIC exposure suggest an impact on the development of the midbrain dopamine (DA) system, which receives a robust cholinergic innervation from the mesopontine tegmentum. We therefore investigated whether prenatal NIC induced structural changes in cells and synapses within the midbrain that persisted into adulthood. Osmotic minipumps delivering either sodium bitartrate (vehicle; VEH) or NIC bitartrate at 2 mg/kg/day were implanted into nine timed-pregnant dams at E4. At birth, rat pups were culled to litters of six males each, and the litters were cross-fostered. Plasma levels of NIC and cotinine from killed pups provided evidence of NIC exposure in utero. Pups separated from dams at weaning showed a trend toward reduced locomotor activity at this time point but not when tested again in adulthood. Adult rats were killed for anatomical studies. Estimates of brain size and volume did not vary with NIC treatment. Midbrain sections stained for Nissl or by immunoperoxidase for tyrosine hydroxylase and analyzed using unbiased stereology revealed no changes in volume or cell number in the substantia nigra compacta or ventral tegmental area as a result of NIC exposure. Within the ventral tegmental area, electron microscopic physical disector analysis showed no significant differences in the number of axon terminals or the number of asymmetric (putative excitatory) or symmetric (putative inhibitory) synapses. Although too infrequent to estimate by unbiased stereology, no obvious difference in the proportion of cholinergic axons was noted in NIC- versus VEH-treated animals. These data suggest that activation of nicotinic receptors during prenatal development induces no significant modifications in the structure of cells in the ventral midbrain when assessed in adulthood.

Precise localization of alpha7 nicotinic acetylcholine receptors on glutamatergic axon terminals in the rat ventral tegmental area.

Alpha7 neuronal nicotinic acetylcholine receptors (nAChRs) constitute one of the predominant nAChR subtypes in the mammalian brain. Within the ventral tegmental area (VTA), nicotine application, paired with postsynaptic stimulation, contributes to a form of long-term potentiation, an effect attributed to presynaptic alpha7 nAChRs on glutamatergic afferents (Mansvelder and McGehee, 2000). The aim of this study was to examine the precise subcellular distribution of alpha7 nAChRs in the adult rat VTA to establish whether these receptors are indeed present on glutamatergic axon terminals and to determine their relationship with cholinergic afferents. The spatial relationship between alpha7 nAChRs, labeled using the alpha7 nAChR-specific antagonist alpha-bungarotoxin, and the local neurochemical environment was investigated by the application of multiple labeling strategies with antibodies against tyrosine hydroxylase, vesicular glutamate transporters (VGluTs), vesicular acetylcholine transporter, and glial fibrillary acidic protein. alpha7 nAChRs were localized at both somatodendritic and presynaptic loci within the VTA: on subpopulations of dopaminergic and nondopaminergic neurons and glutamatergic and nonglutamatergic terminals. There was no detectable alpha7 nAChR expression within astrocytes in the VTA. Most alpha7 nAChRs were cytoplasmic (82%), and the remainder were associated with the plasma membrane. Most presynaptic receptors (75%) were on glutamatergic axon terminals, with similar levels of alpha-bungarotoxin binding present on both VGluT1- and VGluT2-immunoreactive boutons. Both preembedding and postembedding electron microscopy revealed that presynaptic alpha7 nAChRs are often located at extrasynaptic (27%) and perisynaptic (61%) loci. alpha7 nAChRs were not associated with cholinergic synapses, consistent with their activation by a paracrine mode of acetylcholine or choline delivery.