Identification of rat ventral tegmental area GABAergic neurons

The canonical two neuron model of opioid reward posits that mu opioid receptor (MOR) activation produces reward by disinhibiting midbrain ventral tegmental area (VTA) dopamine neurons through inhibition of local GABAergic interneurons. Although indirect evidence supports the neural circuit postulated by this model, its validity has been called into question by growing evidence for VTA neuronal heterogeneity and the recent demonstration that MOR agonists inhibit GABAergic terminals in the VTA arising from extrinsic neurons. In addition, VTA MOR reward can be dopamine-independent. To directly test the assumption that MOR activation directly inhibits local GABAergic neurons, we investigated the properties of rat VTA GABA neurons directly identified with either immunocytochemistry for GABA or GAD65/67, or in situ hybridization for GAD65/67 mRNA. Utilizing co-labeling with an antibody for the neural marker NeuN and in situ hybridization against GAD65/67, we found that 23±3% of VTA neurons are GAD65/67(+). In contrast to the assumptions of the two neuron model, VTA GABAergic neurons are heterogeneous, both physiologically and pharmacologically. Importantly, only 7/13 confirmed VTA GABA neurons were inhibited by the MOR selective agonist DAMGO. Interestingly, all confirmed VTA GABA neurons were insensitive to the GABA(B) receptor agonist baclofen (0/6 inhibited), while all confirmed dopamine neurons were inhibited (19/19). The heterogeneity of opioid responses we found in VTA GABAergic neurons, and the fact that GABA terminals arising from neurons outside the VTA are inhibited by MOR agonists, make further studies essential to determine the local circuit mechanisms underlying VTA MOR reward.     

Lateral ventral tegmental area GABAergic and glutamatergic modulation of conditioned learning

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L-谷氨酸和卡巴胆碱对中脑腹侧被盖区多巴胺能神经元簇放电的影响

中脑腹侧被盖区(ventral tegmental area,VTA)多巴胺能神经元的簇放电会导致其突触末梢多巴胺释放量瞬时大量增加,已被公认是编码奖赏效应的功能相关信号,但诱发多巴胺能神经元产生簇放电的神经调节的具体机制尚不完全清楚。为深入理解诱发VTA多巴胺能神经元产生簇放电介导奖赏信号的递质机制和不同脑区间的协同作用,本实验利用大鼠离体脑片,研究了胆碱能受体激动剂卡巴胆碱单独灌流,兴奋性谷氨酸能受体激动剂L-谷氨酸单独脉冲式给药及二者同时作用时VTA多巴胺能神经元簇放电的产生。结果显示,在离体脑片,卡巴胆碱(10μmol/L)持续灌流或L-谷氨酸(3mmol/L)脉冲式给药均能够诱发多巴胺能神经元产生簇放电。在二者单独作用不能诱发簇放电的神经元,卡巴胆碱和谷氨酸联合用药则可以诱发出簇放电。这些结果提示,卡巴胆碱和L-谷氨酸在诱发多巴胺能神经元簇放电的过程中具有协同作用。     

Comparison of the effect of morphine on locus coeruleus noradrenergic and ventral tegmental area dopaminergic neurons in vitro

Extracellular single-cell recordings were performed on rat brain slices to compare the effects of morphine on noradrenergic neurons of the locus coeruleus (LC) and on dopaminergic neurons of the ventral tegmental area (VTA). Morphine inhibited the firing of LC neurons at very low concentrations. The mean IC50 was 13.4 +/- 1nM (mean +/- SEM) (n = 7). Moreover, the inhibitory effect of morphine was identical in slices obtained from rats anesthetized with chloral hydrate or from non-anesthetized rats. On the contrary, morphine did not have any influence on the firing of most VTA neurons (N = 20) up to 100 microM, and did not modify the sensitivity of their autoreceptors (N = 8). It is concluded that morphine potently inhibits the firing of LC neurons in vitro both in slices of anesthetized and not anesthetized animals and has no direct excitatory effect on VTA dopaminergic neurons of the rat.     

Inhibition by talipexole, a thiazolo-azepine derivative, of dopaminergic neurons in the ventral tegmental area.

A microiontophoretic study using rats anesthetized with chloral hydrate and immobilized with gallamine triethiodide was carried out to compare the effect of talipexole (B-HT 920 CL2:2-amino-6-allyl-5,6,7,8-tetrahydro-4H-thiazolo [4,5-d]-azepine-dihydrochloride), a dopamine autoreceptor agonist, on dopaminergic neurons in the ventral tegmental area (VTA) to non-dopaminergic neurons in the VTA. VTA neurons were classified into two types according to the responses to antidromic stimulation of the nucleus accumbens (Acc): type I neurons with a long spike latency (8.69 +/- 0.24 msec) upon Acc stimulation and low spontaneous firing rate (6.80 +/- 1.34/sec), and type II neurons with a short latency (2.76 +/- 0.20 msec) and high spontaneous firing rate (26.77 +/- 7.05/sec), probably corresponding to dopaminergic and non-dopaminergic neurons, respectively. In type I neurons, microiontophoretic application of talipexole and dopamine inhibited antidromic spike generation elicited by Acc stimulation, and talipexole-induced inhibition was antagonized by domperidone (dopamine D-2 antagonist). In type II neurons, however, the antidromic spikes were not affected by either talipexole or dopamine. Furthermore, spontaneous firing was also inhibited by iontophoretically applied talipexole and dopamine in most type I neurons, but rarely affected by either drug. Inhibitory effects of talipexole were antagonized by domperidone. These results suggest that talipexole acts on dopamine D-2 receptors, thereby inhibiting the dopaminergic neurons in the VTA.     

The role of alpha-synuclein in the development of the dopaminergic neurons in the substantia nigra and ventral tegmental area

Alpha-synuclein is a presynaptic protein of vertebrates that belongs to the family of synucleins. Normal functions of synucleins remain unknown. Alpha-synuclein is one of the causative factors of the familial and idiopathic forms of Parkinson’s disease (PD). The progressive loss of dopaminergic (DA) neurons is characteristic of PD and the most severe damage occurs in the substantia nigra (SN). This leads to an erraticism of the synthesis and synaptic secretion of the neurotransmitters, subsequently resulting in the loss of the connections between brain areas. This work shows that alpha-synuclein is directly involved in the formation of the mature DA neurons of the midbrain at different stages of the ontogenesis and these findings are consistent with data obtained in other studies. Thus, alpha-synuclein may have a varying modulating effect on the growth dynamics and the fate of populations of DA neurons.     

Methylphenidate and amphetamine modulate differently the NMDA and AMPA glutamatergic transmission of dopaminergic neurons in the ventral tegmental area

Behavioral and neurochemical studies suggest that the induction of behavioral sensitization to psychostimulants involves transient changes at the synapses of the ventral tegmental area's dopaminergic neurons (VTA-DA). Differences in the behavioral response to amphetamine (Amph) and methylphenidate (MPD) were observed. In an attempt to understand these behavioral differences at the neuronal level, the dose-response characteristics of these two psychostimulants on electrophysiologically identified VTA-DA neurons at the glutamatergic synapse were investigated. Miniature excitatory post-synaptic currents (mEPSCs) and electrically induced EPSCs were recorded from horizontal midbrain slices of rats that had been pretreated intraperitoneally (i.p.) with saline (control), Amph (2.5, 5.0, 10.0 or 20.0 mg/kg), or MPD (2.5, 5.0, 10.0 or 20.0 mg/kg) 24 h before the recording. Perfusion of Amph through the bath (2.5, 5.0, 10.0 or 20.0 microM) increased the frequency (p<0.01) and the amplitude (p<0.05) of mEPSCs in dose-response characteristics, while MPD perfusion through the bath (2.5, 5.0, 10.0, or 20.0 microM) increased only the frequency (p<0.05) of the mEPSC. Both psychostimulants increased the prefrontal cortex's (PFC) glutamatergic EPSC in the VTA-DA neurons. However, only the higher doses of MPD induced significant effects (p<0.05) on the N-methyl-D-aspartate (NMDA) receptor-mediated EPSC but had no effects on the EPSC mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA/kainate) receptors. Contrarily, Amph increased both kinds of mediated EPSC, but mainly the EPSC mediated by AMPA/kainate receptors (p<0.01). These electrophysiological differences could represent the underlying mechanism responsible for the differences of behavioral effects, such as behavioral sensitization, elicited by MPD and Amph.     

Target-specific glutamatergic regulation of dopamine neurons in the ventral tegmental area

Dopamine (DA) neurons in the ventral tegmental area (VTA) are thought to play a critical role in affective, motivational, and cognitive functioning. There are fundamental target-specific differences in the functional characteristics of subsets of these neurons. For example, DA afferents to the prefrontal cortex (PFC) have a higher firing and transmitter turnover rate and are more responsive to some pharmacological and environmental stimuli than DA projections to the nucleus accumbens (NAc). These functional differences may be attributed in part to differences in tonic regulation by glutamate. The present study provides evidence for this mechanism: In freely moving animals, blockade of basal glutamatergic activity in the VTA by the selective alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate antagonist LY293558 produced an increase in DA release in the NAc while significantly decreasing DA release in the PFC. These data support an AMPA receptor-mediated tonic inhibitory regulation of mesoaccumbens neurons and a tonic excitatory regulation of mesoprefrontal DA neurons. This differential regulation may result in target-specific effects on the basal output of DA neurons and on the regulatory influence of voltage-gated NMDA receptors in response to phasic activation by behaviorally relevant stimuli.     

Lateral preoptic area glutamate neurons relay nociceptive information to the ventral tegmental area

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The electrophysiological actions of dopamine and dopaminergic drugs on neurons of the substantia nigra pars compacta and ventral tegmental area.

The dopaminergic neurons of the substantia nigra pars compacta and ventral tegmental area play a crucial role in regulating movement and cognition respectively. Several lines of evidence suggest that a degeneration of dopaminergic cells in the substantia nigra produces the symptoms of Parkinson's disease. On the other hand, a hyperactivity of the dopaminergic transmission in the brain induces dyskinesia, dystonia and psychosis. It is also well established that the euphoric and rewarding responses evoked by drugs of addiction, such as amphetamine and cocaine, are mediated by central dopamine systems. Electrophysiological experiments which study the activity of single dopaminergic neurons in the ventral mesencephalon have shown that dopamine and dopaminergic drugs reduce the firing frequency of these cells. This is due to the stimulation of D2-D3 autoreceptors and to a hyperpolarization of the membrane produced by an increase in potassium conductance. In addition, substances which increase the release (amphetamine), the synthesis (levodopa) or block the uptake (cocaine, nomifensine, amineptine) of dopamine in the brain inhibit the firing activity of the dopaminergic cells throughout dopamine-mediated mechanisms. In this review, we will briefly examine the literature concerning the physiological and behavioural responses caused by dopamine and dopaminergic agents on the dopaminergic neurons of the ventral mesencephalon. Our conclusion suggests that the electrophysiological actions of dopamine and dopamine-related drugs on dopaminergic cells in the ventral mesencephalon might be indicative of the pharmacological effects of these agents on the brain.     

Electrophysiological properties of mesencephalic ventral tegmental area neurons in awake rats

A statistical analysis was made of spike activity and the shape and duration of individual action potentials in cells from the mesencephalic ventral tegmental area and adjacent regions during experiments on awake rats. Four groups of cells were identified in this test population whith electrophysiological features which may relate to their distinctive neurochemical profile and to their specific afferent connections.Institute of Pharmacology, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 18, No. 6, pp. 729–737, November–December, 1986.     

Role for mTOR signaling and neuronal activity in morphine-induced adaptations in ventral tegmental area dopamine neurons

While the abuse of opiate drugs continues to rise, the neuroadaptations that occur with long-term drug exposure remain poorly understood. We describe here a series of chronic morphine-induced adaptations in ventral tegmental area (VTA) dopamine neurons, which are mediated via downregulation of AKT-mTORC2 (mammalian target of rapamycin complex-2). Chronic opiates decrease the size of VTA dopamine neurons in rodents, an effect seen in humans as well, and concomitantly increase the excitability of the cells but decrease dopamine output to target regions. Chronic morphine decreases mTORC2 activity, and overexpression of Rictor,?a component of mTORC2, prevents morphine-induced changes in cell morphology and activity. Further, local knockout of Rictor in VTA decreases DA soma size and reduces rewarding responses to morphine, consistent with the hypothesis that these adaptations represent a mechanism of reward tolerance. Together, these findings demonstrate a novel role for AKT-mTORC2 signaling in mediating neuroadaptations to opiate drugs of abuse.     

Regulatory effects of D2 receptors in the ventral tegmental area on the mesocorticolimbic dopaminergic pathway

To investigate the regulatory effects of somatodendritic D2 receptors on the terminal's extracellular dopamine (DA) concentration, a D2 antagonist (eticlopride) was infused directly into the ventral tegmental area via a microdialysis probe in chloral hydrate-anesthetized rats. Extracellular DA changes in both the nucleus accumbens (N ACC) and the medial prefrontal cortex (mPFC) were monitored. Infusion of 10.0 fM eticlopride had no effect on DA in the mPFC (110.2 +/- 10.0% of baseline) but significantly increased DA in the N ACC (150.1 +/- 11.7%). Infusion of a higher dose of eticlopride (100.0 or 1,000.0 fM) significantly augmented the DA in the mPFC (121.1 +/- 7.6 and 180.7 +/- 25.8%, respectively) but surprisingly had no effect on DA in the N ACC (111.5 +/- 7.3 and 104.1 +/- 8.7%, respectively). To further investigate whether the bluntness of DA increase in the N ACC was due to DA receptor activation in the mPFC, eticlopride or SCH23390 was infused into the mPFC prior to and during intrategmental eticlopride infusion, and the change of DA in the N ACC was simultaneously monitored. During intra-mPFC 1.0 nM eticlopride infusion but not during 10.0 nM SCH23390 administration (95.5 +/- 6.1%), intrategmental 1,000.0 fM eticlopride infusion could further elevate DA in the N ACC (130.0 +/- 4.6%). Our results indicated that (1) the mesolimbic and the mesocortical pathways were under tonic inhibition by somatodendritic D2 receptors; (2) the DA concentration in the N ACC first increased and then returned to baseline while the intrategmental infusion dose of eticlopride increased; and (3) the bluntness of DA increase in the N ACC resulted from the D2 receptor activation in the mPFC.     

Coordinated activity of ventral tegmental neurons adapts to appetitive and aversive learning

Our understanding of how value-related information is encoded in the ventral tegmental area (VTA) is based mainly on the responses of individual putative dopamine neurons. In contrast to cortical areas, the nature of coordinated interactions between groups of VTA neurons during motivated behavior is largely unknown. These interactions can strongly affect information processing, highlighting the importance of investigating network level activity. We recorded the activity of multiple single units and local field potentials (LFP) in the VTA during a task in which rats learned to associate novel stimuli with different outcomes. We found that coordinated activity of VTA units with either putative dopamine or GABA waveforms was influenced differently by rewarding versus aversive outcomes. Specifically, after learning, stimuli paired with a rewarding outcome increased the correlation in activity levels between unit pairs whereas stimuli paired with an aversive outcome decreased the correlation. Paired single unit responses also became more redundant after learning. These response patterns flexibly tracked the reversal of contingencies, suggesting that learning is associated with changing correlations and enhanced functional connectivity between VTA neurons. Analysis of LFP recorded simultaneously with unit activity showed an increase in the power of theta oscillations when stimuli predicted reward but not an aversive outcome. With learning, a higher proportion of putative GABA units were phase locked to the theta oscillations than putative dopamine units. These patterns also adapted when task contingencies were changed. Taken together, these data demonstrate that VTA neurons organize flexibly as functional networks to support appetitive and aversive learning.     

Multiple personalities in the ventral tegmental area

A small number of ventral tegmental area dopamine neurons engage in numerous and apparently contradictory functions--how can this be? A clue is provided by Lammel and colleagues in this issue of Neuron: some VTA dopamine neurons display synaptic plasticity in response to cocaine, and others in response to pain, and these populations are distinguished by their axonal projections and Ih.     

Coactivation of renal sympathetic neurons and somatic motor neurons by chemical stimulation of the midbrain ventral tegmental area

We examined whether neurons in the midbrain ventral tegmental area (VTA) play a role in generating central command responsible for autonomic control of the cardiovascular system in anesthetized rats and unanesthetized, decerebrated rats with muscle paralysis. Small volumes (60 nl) of an N-methyl-D-aspartate receptor agonist (L-homocysteic acid) and a GABAergic receptor antagonist (bicuculline) were injected into the VTA and substantia nigra (SN). In anesthetized rats, L-homocysteic acid into the VTA induced short-lasting increases in renal sympathetic nerve activity (RSNA; 66 ± 21%), mean arterial pressure (MAP; 5 ± 2 mmHg), and heart rate (HR; 7 ± 2 beats/min), whereas bicuculline into the VTA produced long-lasting increases in RSNA (130 ± 45%), MAP (26 ± 2 mmHg), and HR (66 ± 6 beats/min). Bicuculline into the VTA increased blood flow and vascular conductance of the hindlimb triceps surae muscle, suggesting skeletal muscle vasodilatation. However, neither drug injected into the SN affected all variables. Renal sympathetic nerve and cardiovascular responses to chemical stimulation of the VTA were not essentially affected by decerebration at the premammillary-precollicular level, indicating that the ascending projection to the forebrain from the VTA was not responsible for evoking the sympathetic and cardiovascular responses. Furthermore, bicuculline into the VTA in decerebrate rats produced long-lasting rhythmic bursts of RSNA and tibial motor nerve discharge, which occurred in good synchrony. It is likely that the activation of neurons in the VTA is capable of eliciting synchronized stimulation of the renal sympathetic and tibial motor nerves without any muscular feedback signal.     

The relationships between neurons containing dopamine and nitric oxide synthase in the ventral tegmental area

Ventral tegmental area (VTA) is a heterogeneous group of dopaminergic cells which contains interfascicular (IF), parabrachial (PBP) and rostral linear (RLi) nuclei. Neurons of this area are involved in the regulation of motor and motivational aspects of behavior and reveal high neuronal plasticity. Among many various neurotransmitters and neuromodulators, nitric oxide (NO) is localized in this region. In the present study, we investigated morphology and distribution of nitric oxide synthase (NOS)-positive neurons in VTA and their colocalization with dopaminergic neurons. The study was performed on six adult Wistar rats. After perfusional fixation, the brains were cut, immunostained for tyrosine hydroxylase (TH) and NOS and studied by confocal laser microscopy. In each of the three studied nuclei of VTA we investigated three different neuronal populations. Numerous TH-immunoreactive (TH-ir) and NOS-immunoreactive (NOS-ir) neurons are present in the studied region. Among them, a considerable number showed coexistence of both neurotransmitters. The populations of TH-ir and NOS-ir neurons interact with each other as manifested by the presence of NOS-ir endings on TH-ir neurons and vice versa. Taking the above into account, it may be suspected that NO is involved in the modulation of dopaminergic transmission.     

Dopaminergic ventral tegmental neurons modulated by methylphenidate

Treatment of psychostimulants leads to the development of behavioral sensitization, an augmented behavioral response to drug re-administration. The induction of behavioral sensitization to psychostimulants such as amphetamine and cocaine occurs at the ventral tegmental area's dopaminergic neurons (VTA-DA). Currently, there is limited experimental data about the physiological properties of methylphenidate (MPD) on VTA-DA neurons. Behavioral and electrophysiological experiments using male rats were performed before and after MPD treatment. The behavioral experiment included dose-response (0.6, 2.5, and 10.0 mg/kg MPD) study to select the most effective dose for the electrophysiological study. Methylphenidate increased locomotion in typical dose response characteristics. Based on this experiment, the 10.0 mg/kg MPD was used in two types of electrophysiological recordings: 1) intracellular recording of neuronal activity performed on horizontal 275-300 μm brain slices and 2) whole-cell patch clamping before and after electrical stimulation to study post-synaptic currents on neurophysiologically identified VTA-DA neurons. Methylphenidate suppressed the neuronal activity of these neurons for 210±30 sec. Stimulation of the prefrontal cortex afferent fibers to these VTA-DA neurons in the presence of TTX, saclofen, and picrotoxin led to the conclusion that this input is mediated via NMDA and kainate/AMPA receptors and may participate to induce behavioral sensitization to psychostimulants.