Hui-Ling Wang

Pedunculopontine and laterodorsal tegmental nuclei contain distinct populations of cholinergic, glutamatergic and GABAergic neurons in the rat.

The pedunculopontine tegmental nucleus (PPTg) and laterodorsal tegmental nucleus (LDTg) provide cholinergic afferents to several brain areas. This cholinergic complex has been suggested to play a role in sleep, waking, motor function, learning and reward. To have a better understanding of the neurochemical organization of the PPTg/LDTg we characterized the phenotype of PPTg/LDTg neurons by determining in these cells the expression of transcripts encoding choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD) or the vesicular glutamate transporters (vGluT1, vGluT2 and vGluT3). Within the PPTg/LDTg complex we found neurons expressing ChAT, vGluT2 or GAD transcripts, these neuronal phenotypes were intermingled, but not homogeneously distributed within the PPTg or LDTg. Previous studies suggested the presence of either glutamate or γ‐aminobutyric acid (GABA) immunolabeling in a large number of PPTg/LDTg cholinergic neurons, leading to the widespread notion that PPTg/LDTg cholinergic neurons co‐release acetylcholine together with either glutamate or GABA. To assess the glutamatergic or GABAergic nature of the PPTg/LDTg cholinergic neurons, we combined in situ hybridization (to detect vGluT2 or GAD transcripts) and immunohistochemistry (to detect ChAT), and found that over 95% of all PPTg/LDTg cholinergic neurons lack transcripts encoding either vGluT2 mRNA or GAD mRNA. As the vast majority of PPTg/LDTg cholinergic neurons lack transcripts encoding essential proteins for the vesicular transport of glutamate or for the synthesis of GABA, co‐release of acetylcholine with either glutamate or GABA is unlikely to be a major factor in the interactions between acetylcholine, glutamate and GABA at the postsynaptic site.

Selective activation of cholinergic interneurons enhances accumbal phasic dopamine release: setting the tone for reward processing

Dopamine plays a critical role in motor control, addiction, and reward-seeking behaviors, and its release dynamics have traditionally been linked to changes in midbrain dopamine neuron activity. Here, we report that selective endogenous cholinergic activation achieved via in vitro optogenetic stimulation of nucleus accumbens, a terminal field of dopaminergic neurons, elicits real-time dopamine release. This mechanism occurs via direct actions on dopamine terminals, does not require changes in neuron firing within the midbrain, and is dependent on glutamatergic receptor activity. More importantly, we demonstrate that in vivo selective activation of cholinergic interneurons is sufficient to elicit dopamine release in the nucleus accumbens. Therefore, the control of accumbal extracellular dopamine levels by endogenous cholinergic activity results from a complex convergence of neurotransmitter/neuromodulator systems that may ultimately synergize to drive motivated behavior.

Mesocorticolimbic Glutamatergic Pathway

The mesocorticolimbic dopamine (DA) system plays important roles in reward, motivation, learning, memory, and movement. This system arises from the A10 region, comprising the ventral tegmental area and three adjacent midline nuclei (caudal linear nucleus, interfascicular nucleus, and rostral linear nucleus of the raphe). DAergic and GABAergic neurons are intermingled in this region with recently discovered glutamatergic neurons expressing the vesicular glutamate transporter 2 (VGluT2). Here, we show by in situ hybridization and immunohistochemistry that there are two subpopulations of neurons expressing VGluT2 mRNA in the A10 region: (1) a major subpopulation that expresses VGluT2 but lacks tyrosine hydroxylase (TH; VGluT2-only neurons), present in each nucleus of the A10 region, and (2) a smaller subpopulation that coexpresses VGluT2 and TH (VGluT2-TH neurons). By quantitative real-time PCR, we determined the mRNA copy numbers encoding VGluT2 or TH in samples of individual microdissected TH immunoreactive (IR) neurons. Data from both in situ hybridization and from mRNA quantification showed that VGluT2 mRNA is not present in every TH-IR neuron, but restricted to a subset of TH-IR neurons located in the medial portion of the A10 region. By integration of tract tracing, in situ hybridization, and immunohistochemistry, we found that VGluT2-only neurons and VGluT2-TH neurons each innervate both the prefrontal cortex and the nucleus accumbens. These findings establish that in addition to the well-recognized mesocorticolimbic DA-only and GABA-only pathways, there exist parallel mesocorticolimbic glutamate-only and glutamate-DA pathways.

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.

Role of Projections from Ventral Medial Prefrontal Cortex to Nucleus Accumbens Shell in Context-Induced Reinstatement of Heroin Seeking

In humans, exposure to contexts previously associated with heroin use can provoke relapse. In rats, exposure to heroin-paired contexts after extinction of drug-reinforced responding in different contexts reinstates heroin seeking. This effect is attenuated by inhibition of glutamate or dopamine transmission in nucleus accumbens shell, or inactivation of ventral medial prefrontal cortex (mPFC). Here, we used an anatomical asymmetrical disconnection procedure to demonstrate that an interaction between glutamatergic projections from ventral mPFC to accumbens shell and local dopamine D1 postsynaptic receptors contributes to context-induced reinstatement of heroin seeking. We also combined the marker of neuronal activity, Fos, with the retrograde tracer Fluoro-Gold to assess activation in this pathway during context-induced reinstatement. Rats were trained to self-administer heroin for 12 d; drug infusions were paired with a discrete tone–light cue. Lever pressing was subsequently extinguished in a nondrug-associated context in the presence of the discrete cue. Rats were then tested in the heroin- or extinction-associated contexts under extinction conditions. Injections of muscimol + baclofen into ventral mPFC in one hemisphere and D1-family receptor antagonist SCH 23390 into the contralateral or ipsilateral accumbens shell decreased context-induced reinstatement. Unilateral injections of muscimol + baclofen into ventral mPFC or SCH 23390 into the accumbens shell had no effect. Context-induced reinstatement was associated with increased Fos expression in ventral mPFC neurons, including those projecting to accumbens shell, with higher double-labeling in the ipsilateral projection than in the contralateral projection. Our results demonstrate that activation of glutamatergic projections from ventral mPFC to accumbens shell, previously implicated in inhibition of cocaine relapse, promotes heroin relapse.

Single rodent mesohabenular axons release glutamate and GABA

The lateral habenula (LHb) is involved in reward, aversion, addiction and depression through descending interactions with several brain structures, including the ventral tegmental area (VTA). The VTA provides reciprocal inputs to LHb, but their actions are unclear. Here we show that the majority of rat and mouse VTA neurons innervating LHb coexpress markers for both glutamate signaling (vesicular glutamate transporter 2; VGluT2) and GABA signaling (glutamic acid decarboxylase; GAD, and vesicular GABA transporter; VGaT). A single axon from these mesohabenular neurons coexpresses VGluT2 protein and VGaT protein and, surprisingly, establishes symmetric and asymmetric synapses on LHb neurons. In LHb slices, light activation of mesohabenular fibers expressing channelrhodopsin2 driven by VGluT2 (Slc17a6) or VGaT (Slc32a1) promoters elicits release of both glutamate and GABA onto single LHb neurons. In vivo light activation of mesohabenular terminals inhibits or excites LHb neurons. Our findings reveal an unanticipated type of VTA neuron that cotransmits glutamate and GABA and provides the majority of mesohabenular inputs.

Dopaminergic and glutamatergic microdomains in a subset of rodent mesoaccumbens axons

Mesoaccumbens fibers are thought to co-release dopamine and glutamate. However, the mechanism is unclear, and co-release by mesoaccumbens fibers has not been documented. Using electron microcopy, we found that some mesoaccumbens fibers have vesicular transporters for dopamine (VMAT2) in axon segments that are continuous with axon terminals that lack VMAT2, but contain vesicular glutamate transporters type 2 (VGluT2). In vivo overexpression of VMAT2 did not change the segregation of the two vesicular types, suggesting the existence of highly regulated mechanisms for maintaining this segregation. The mesoaccumbens axon terminals containing VGluT2 vesicles make asymmetric synapses, commonly associated with excitatory signaling. Using optogenetics, we found that dopamine and glutamate were released from the same mesoaccumbens fibers. These findings reveal a complex type of signaling by mesoaccumbens fibers in which dopamine and glutamate can be released from the same axons, but are not normally released at the same site or from the same synaptic vesicles.

Heterogeneous composition of dopamine neurons of the rat A10 region: molecular evidence for diverse signaling properties.

The A10 region contains different neurons: dopamine (expressing tyrosine hydroxylase; TH), GABA, glutamate-only (expressing the vesicular glutamate transporter 2; VGluT2), and TH-VGluT2 (coexpressing TH and VGluT2). We used three methods to investigate proteins necessary for the synthesis (aromatic l-amino acid decarboxylase, AADC) or transport (vesicular monoamine transporter; VMAT2 or dopamine transporter; DAT) of dopamine within TH neurons in the A10 region. By in situ hybridization–immunohistochemistry, we found that all TH neurons in the A10 region had AADC, but not all had VMAT2, DAT or D2 receptors (D2R). To determine whether TH-VGluT2 neurons account for TH neurons lacking these dopamine markers, we implemented an anatomical “mirror technique”, and found that not all TH-VGluT2 neurons lacked VMAT2, DAT or D2R. Next, by quantitative RT-PCR of individual micro-dissected TH neurons, we discovered two classes of TH-VGluT2 and three classes of TH-only neurons with different latero-medial distribution. Some of the TH-VGluT2 neurons had both VMAT2 and DAT (TH-VGluT2 Class 1); others lacked detectable levels of both transporters (TH-VGluT2 Class 2). Most of the TH-only neurons contained VMAT2 and DAT (TH-only Class 1), a few had DAT without detectable VMAT2 (TH-only Class 2), and others lacked detectable levels of both transporters (TH-only Class 3). We concluded that (a) the majority of TH neurons lacking DAT are TH-VGluT2 neurons, (b) very few TH-only neurons express DAT without VMAT2, and (c) TH-VGluT2 neurons lacking DAT also lack VMAT2. Thus, the A10 region contains dopamine neurons with differential compartmentalization and unique signaling properties.

A glutamatergic reward input from the dorsal raphe to ventral tegmental area dopamine neurons

Electrical stimulation of the dorsal raphe (DR) and ventral tegmental area (VTA) activates the fibers of the same reward pathway but the phenotype of this pathway and the direction of the reward-relevant fibers have not been determined. Here we report rewarding effects following activation of a DR-originating pathway consisting of vesicular glutamate transporter 3 (VGluT3) containing neurons that form asymmetric synapses onto VTA dopamine neurons that project to nucleus accumbens. Optogenetic VTA activation of this projection elicits AMPA-mediated synaptic excitatory currents in VTA mesoaccumbens dopaminergic neurons and causes dopamine release innucleus accumbens. Activation also reinforces instrumental behavior and establishes conditioned place preferences. These findings indicate that the DR-VGluT3 pathway to VTA utilizes glutamate as a neurotransmitter and is a substrate linking the DR—one of the most sensitive reward sites in the brain—to VTA dopaminergic neurons.

Corticotropin-releasing factor binding protein within the ventral tegmental area is expressed in a subset of dopaminergic neurons†

Corticotropin‐releasing factor (CRF) and related peptides play a role in mediating neuronal effects of stress. These peptides mediate stress responses by their interactions with the CRF receptors and the CRF‐binding protein (CRF‐BP). Because the CRF‐BP is implicated in neurotransmission within the ventral tegmental area (VTA), we investigated whether the CRF‐BP is expressed in VTA neurons. By in situ hybridization, we detected cellular expression of CRF‐BP mRNA in the VTA; no such expression was seen in neighboring substantia nigra pars compacta (SNC) or substantia nigra pars reticulata. By double in situ hybridization, we determined that VTA neurons with CRF‐BP mRNA coexpressed transcripts encoding either tyrosine hydroxylase [TH; a marker for dopamine (DA) neurons] or glutamic acid decarboxylase [GAD; synthesizing enzyme of γ‐aminobutyric acid (GABA)]. Neurons with CRF‐BP mRNA represented 25% of the total population of TH‐expressing neurons and 28% of the total population of GAD‐expressing neurons, indicating that discrete subpopulations of dopaminergic and GABAergic neurons are present in the VTA. Within the total population of neurons containing CRF‐BP mRNA, 70% coexpressed TH mRNA and only 27% coexpressed GAD mRNA. As far as we are aware, we provide the first anatomical evidence that a molecule, CRF‐BP, is encoded by DAergic neurons of the VTA but not by those of the SNC. We propose, based on the observation that the majority of VTA neurons expressing CRF‐BP mRNA are DAergic, that in the VTA interactions of CRF‐BP with CRF, or with CRF‐related peptides, are likely to be mediated predominantly by DAergic neurons. J. Comp. Neurol. 509:302–318, 2008. Published 2008 Wiley‐Liss, Inc.