Marisela Morales

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.

The Brain on Drugs: From Reward to Addiction.

Advances in neuroscience identified addiction as a chronic brain disease with strong genetic, neurodevelopmental, and sociocultural components. We here discuss the circuit- and cell-level mechanisms of this condition and its co-option of pathways regulating reward, self-control, and affect. Drugs of abuse exert their initial reinforcing effects by triggering supraphysiologic surges of dopamine in the nucleus accumbens that activate the direct striatal pathway via D1 receptors and inhibit the indirect striato-cortical pathway via D2 receptors. Repeated drug administration triggers neuroplastic changes in glutamatergic inputs to the striatum and midbrain dopamine neurons, enhancing the brains reactivity to drug cues, reducing the sensitivity to non-drug rewards, weakening self-regulation, and increasing the sensitivity to stressful stimuli and dysphoria. Drug-induced impairments are long lasting; thus, interventions designed to mitigate or even reverse them would be beneficial for the treatment of addiction.

Glutamatergic neurons are present in the rat ventral tegmental area.

The ventral tegmental area (VTA) is thought to play an important role in reward function. Two populations of neurons, containing either dopamine (DA) or γ‐amino butyric acid (GABA), have been extensively characterized in this area. However, recent electrophysiological studies are consistent with the notion that neurons that utilize neurotransmitters other than DA or GABA are likely to be present in the VTA. Given the pronounced phenotypic diversity of neurons in this region, we have proposed that additional cell types, such as those that express the neurotransmitter glutamate may also be present in this area. Thus, by using in situ hybridization histochemistry we investigated whether transcripts encoded by genes for the two vesicular glutamate transporters, VGluT1 or VGluT2, were expressed in the VTA. We found that VGluT2 mRNA but not VGluT1 mRNA is expressed in the VTA. Neurons expressing VGluT2 mRNA were differentially distributed throughout the rostro‐caudal and medio‐lateral aspects of the VTA, with the highest concentration detected in rostro‐medial areas. Phenotypic characterization with double in situ hybridization of these neurons indicated that they rarely co–expressed mRNAs for tyrosine hydroxylase (TH, marker for DAergic neurons) or glutamic acid decarboxylase (GAD, marker for GABAergic neurons). Based on the results described here, we concluded that the VTA contains glutamatergic neurons that in their vast majority are clearly non‐DAergic and non‐GABAergic.

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.

Distribution of neurons expressing immunoreactivity for the 5HT3 receptor subtype in the rat brain and spinal cord

The cellular distribution of the type 3 serotonin receptor (5HT3R) in the rat brain was established immunocytochemically by using a polyclonal antibody raised against a synthetic peptide from the deduced amino‐acid sequence of the cloned 5HT3R. The 5HT3R‐immunoreactive neurons were found in the forebrain, brainstem, and spinal cord, but within each region, the intensity of the immunoreactivity differed considerably. Within the forebrain, intensely immunoreactive cells were found in layers II–III of the neocortex, anterior olfactory nucleus, hippocampal formation, and amygdala. A few strongly immunoreactive neurons were consistently observed in the caudate putamen, and moderately or weakly labeled neurons were occasionally found in the nucleus accumbens. Within the brainstem, intensely labeled neurons were found in the trigeminal motor (V) and facial (VII) nuclei. Immunostained neurons were detected in the dorsal and the ventral horn of the spinal cord. These results reveal that the 5HT3R‐immunoreactive neurons are broadly distributed throughout the rat brain spinal cord, and suggest that this receptor can subserve significant participation in central nervous system neurotransmission. J. Comp. Neurol. 402:385–401, 1998.

Vitamin D3 attenuates cortical infarction induced by middle cerebral arterial ligation in rats

We have previously reported that intracerebral administration of glial cell line derived neurotrophic factor (GDNF) reduces the extent of middle cerebral arterial (MCA) ligation-induced cortical infarction in rats. Recent studies have shown that application of 1, 25 dihydroxyvitamin D(3) (D3) enhances GDNF mRNA expression in vitro. The purpose of the present study was to investigate if administration of D3 in vivo will protect against ischemic brain injury. Adult male Sprague-Dawley rats were injected daily with D3 or with saline for four or eight days. Animals received a 90-min right MCA ligation on the 4(th) or 8(th) day after anesthesia with chloral hydrate. Animals were sacrificed for tri-phenyl-tetrazolium chloride (TTC) staining 24 h after the onset of reperfusion. A subset of animals receiving eight days of D3 or saline treatment were used for blood gas and cerebral GDNF protein level analysis. We found that pretreatment with D3 for four days did not attenuate the ischemic injury. However, animals receiving eight days of D3 injections showed a significant reduction in the amount of infarction in the cortex. Eight day D3 treatment did not alter blood gases or blood pressure; however, it did increase calcium levels. Pretreatment with D3 significantly increased GDNF levels in the cortex. In conclusion, our data indicate that D3 reduces ischemia-induced brain damage and supports the hypothesis that this effect may be through the up-regulation of GDNF mechanisms in cortex.

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.

The type 3 serotonin receptor is expressed in a subpopulation of GABAergic neurons in the rat neocortex and hippocampus

We used in situ hybridization and immunocytochemistry to investigate the presence of GABA in neurons that express the type 3 serotonin receptor (5-HT3R). Quantitative analysis indicated that more than 90% of 5-HT3R expressing cells are GABAergic in the neocortex and hippocampus. The co-existence of 5-HT3R and GABA in cortical and hippocampal neurons indicates that serotonin, via 5-HT3R, can affect GABA release and suggests the participation of 5-HT3R in the inhibitory regulation of forebrain neurons.

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.