Self-Administration of Ethanol,Cocaine, or Nicotine Does Not Decrease the Soma Size of Ventral Tegmental Area Dopamine Neurons

Our previous observations show that chronic opiate administration, including self-administration, decrease the soma size of dopamine (DA) neurons in the ventral tegmental area (VTA) of rodents and humans, a morphological change correlated with increased firing rate and reward tolerance. Given that a general hallmark of drugs of abuse is to increase activity of the mesolimbic DA circuit, we sought to determine whether additional drug classes produced a similar morphological change. Sections containing VTA were obtained from rats that self-administered cocaine or ethanol and from mice that consumed nicotine. In contrast to opiates, we found no change in VTA DA soma size induced by any of these other drugs. These data suggest that VTA morphological changes are induced in a drug-specific manner and reinforce recent findings that some changes in mesolimbic signaling and neuroplasticity are drug-class dependent.     

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.     

GABA Neurons in the Ventral Tegmental Area Responding to Peripheral Sensory Input

Dopamine (DA) neurons in the ventral tegmental area (VTA) not only participate in reward processing, but also respond to aversive stimuli. Although GABA neurons in this area are actively involved in regulating the firing of DA neurons, few data exist concerning the responses of these neurons to aversive sensory input. In this study, by employing extracellular single-unit recording and spectral analysis techniques in paralyzed and ventilated rats, we found that the firing pattern in 44% (47 of 106) of GABA cells in the VTA was sensitive to the sensory input produced by the ventilation, showing a significant ventilation-associated oscillation in the power spectra. Detailed studies revealed that most ventilation-sensitive GABA neurons (38 of 47) were excited by the stimuli, whereas most ventilation-sensitive DA neurons (11 of 14) were inhibited. When the animals were under anesthesia or the sensory pathways were transected, the ventilation-associated oscillation failed to appear. Systemic administration of non-competitive N-methyl-D-aspartase (NMDA) receptor antagonist MK-801 completely disrupted the association between the firing of GABA neurons and the ventilation. Interestingly, local MK-801 injection into the VTA dramatically enhanced the sensitivity of GABA neurons to the ventilation. Our data demonstrate that both GABA and DA neurons in the VTA can be significantly modulated by sensory input produced by the ventilation, which may indicate potential functional roles of VTA in processing sensation-related input.     

Lesions of Cholinergic Pedunculopontine Tegmental Nucleus Neurons Fail to Affect Cocaine or Heroin Self-Administration or Conditioned Place Preference in Rats

Cholinergic input to the ventral tegmental area (VTA) is known to contribute to reward. Although it is known that the pedunculopontine tegmental nucleus (PPTg) provides an important source of excitatory input to the dopamine system, the specific role of PPTg cholinergic input to the VTA in cocaine reward has not been previously determined. We used a diphtheria toxin conjugated to urotensin-II (Dtx::UII), the endogenous ligand for urotensin-II receptors expressed by PPTg cholinergic but not glutamatergic or GABAergic cells, to lesion cholinergic PPTg neurons. Dtx::UII toxin infusion resulted in the loss of 95.78 (±0.65)% of PPTg cholinergic cells but did not significantly alter either cocaine or heroin self-administration or the development of cocaine or heroin conditioned place preferences. Thus, cholinergic cells originating in PPTg do not appear to be critical for the rewarding effects of cocaine or of heroin.     

Dopamine Modulates Persistent Synaptic Activity and Enhances the Signal-to-Noise Ratio in the Prefrontal Cortex

Background

The importance of dopamine (DA) for prefrontal cortical (PFC) cognitive functions is widely recognized, but its mechanisms of action remain controversial. DA is thought to increase signal gain in active networks according to an inverted U dose-response curve, and these effects may depend on both tonic and phasic release of DA from midbrain ventral tegmental area (VTA) neurons.

Methodology/Principal Findings

We used patch-clamp recordings in organotypic co-cultures of the PFC, hippocampus and VTA to study DA modulation of spontaneous network activity in the form of Up-states and signals in the form of synchronous EPSP trains. These cultures possessed a tonic DA level and stimulation of the VTA evoked DA transients within the PFC. The addition of high (≥1 µM) concentrations of exogenous DA to the cultures reduced Up-states and diminished excitatory synaptic inputs (EPSPs) evoked during the Down-state. Increasing endogenous DA via bath application of cocaine also reduced Up-states. Lower concentrations of exogenous DA (0.1 µM) had no effect on the up-state itself, but they selectively increased the efficiency of a train of EPSPs to evoke spikes during the Up-state. When the background DA was eliminated by depleting DA with reserpine and alpha-methyl-p-tyrosine, or by preparing corticolimbic co-cultures without the VTA slice, Up-states could be enhanced by low concentrations (0.1–1 µM) of DA that had no effect in the VTA containing cultures. Finally, in spite of the concentration-dependent effects on Up-states, exogenous DA at all but the lowest concentrations increased intracellular current-pulse evoked firing in all cultures underlining the complexity of DA''s effects in an active network.

Conclusions/Significance

Taken together, these data show concentration-dependent effects of DA on global PFC network activity and they demonstrate a mechanism through which optimal levels of DA can modulate signal gain to support cognitive functioning.     

Activation of VTA GABA neurons disrupts reward consumption

The activity of ventral tegmental area (VTA) dopamine (DA) neurons promotes behavioral responses to rewards and environmental stimuli that predict them. VTA GABA inputs synapse directly onto DA neurons and may regulate DA neuronal activity to alter reward-related behaviors; however, the functional consequences of selective activation of VTA GABA neurons remains unknown. Here, we show that in?vivo optogenetic activation of VTA GABA neurons disrupts reward consummatory behavior but not conditioned anticipatory behavior in response to reward-predictive cues. In addition, direct activation of VTA GABA projections to the nucleus accumbens (NAc) resulted in detectable GABA release but did not alter reward consumption. Furthermore, optogenetic stimulation of VTA GABA neurons directly suppressed the activity and excitability of neighboring DA neurons as well as the release of DA in the NAc, suggesting that the dynamic interplay between VTA DA and GABA neurons can control the initiation and termination of reward-related behaviors.     

Reliability in the identification of midbrain dopamine neurons

Brain regions typically contain intermixed subpopulations of neurons with different connectivity and neurotransmitters. This complicates identification of neuronal phenotypes in electrophysiological experiments without using direct detection of unique molecular markers. A prime example of this difficulty is the identification of dopamine (DA) neurons in the midbrain ventral tegmental area (VTA). Although immunocytochemistry (ICC) against tyrosine hydroxylase (TH) is widely used to identify DA neurons, a high false negative rate for TH ICC following ex vivo electrophysiology experiments was recently reported, calling into question the validity of comparing DA and non-DA VTA neurons based on post-hoc ICC. However, in whole cell recordings from randomly selected rat VTA neurons we have found that TH labeling is consistently detected in ∼55% of neurons even after long recording durations (range: 2.5–150 min). This is consistent with our prior anatomical finding that 55% of VTA neurons are TH(+). To directly estimate a false negative rate for our ICC method we recorded VTA neurons from mice in which EGFP production is driven by the TH promoter. All 12 EGFP(+) neurons recorded with a K-gluconate internal solution (as used in our rat recordings) were strongly labeled by TH ICC (recording duration 16.6±1.8 min). However, using recording electrodes with an internal solution with high Cl⁻ concentration reduced the intensity of TH co-labeling, in some cases to background (recording duration 16.7±0.9 min; n = 10). Thus TH is a highly reliable molecular marker for DA neurons in VTA patch clamp recordings provided compatible microelectrode solutions are used.     

Conjunctive processing of locomotor signals by the ventral tegmental area neuronal population

The ventral tegmental area (VTA) plays an essential role in reward and motivation. How the dopamine (DA) and non-DA neurons in the VTA engage in motivation-based locomotor behaviors is not well understood. We recorded activity of putative DA and non-DA neurons simultaneously in the VTA of awake mice engaged in motivated voluntary movements such as wheel running. Our results revealed that VTA non-DA neurons exhibited significant rhythmic activity that was correlated with the animal's running rhythms. Activity of putative DA neurons also correlated with the movement behavior, but to a lesser degree. More importantly, putative DA neurons exhibited significant burst activation at both onset and offset of voluntary movements. These findings suggest that VTA DA and non-DA neurons conjunctively process locomotor-related motivational signals that are associated with movement initiation, maintenance and termination.     

Role of Corticotropin-Releasing Factor (CRF) Receptor-1 on the Catecholaminergic Response to Morphine Withdrawal in the Nucleus Accumbens (NAc)

Stress induces the release of the peptide corticotropin-releasing factor (CRF) into the ventral tegmental area (VTA), and also increases dopamine (DA) levels in brain regions receiving dense VTA input. Since the role of stress in drug addiction is well established, the present study examined the possible involvement of CRF1 receptor in the interaction between morphine withdrawal and catecholaminergic pathways in the reward system. The effects of naloxone-precipitated morphine withdrawal on signs of withdrawal, hypothalamo-pituitary-adrenocortical (HPA) axis activity, dopamine (DA) and noradrenaline (NA) turnover in the nucleus accumbens (NAc) and activation of VTA dopaminergic neurons, were investigated in rats pretreated with vehicle or CP-154,526 (selective CRF1R antagonist). CP-154,526 attenuated the increases in body weight loss and suppressed some of withdrawal signs. Pretreatment with CRF1 receptor antagonist resulted in no significant modification of the increased NA turnover at NAc or plasma corticosterone levels that were seen during morphine withdrawal. However, blockade of CRF1 receptor significantly reduced morphine withdrawal-induced increases in plasma adrenocorticotropin (ACTH) levels, DA turnover and TH phosphorylation at Ser40 in the NAc. In addition, CP-154,526 reduced the number of TH containing neurons expressing c-Fos in the VTA after naloxone-precipitated morphine withdrawal. Altogether, these results support the idea that VTA dopaminergic neurons are activated in response to naloxone-precipitated morphine withdrawal and suggest that CRF1 receptors are involved in the activation of dopaminergic pathways which project to NAc.     

Kainic acid lesion-induced spatial memory deficits of rats

The effects of lesioning the ventral tegmental area (VTA) or substantia nigra (SN) neurons by means of bilateral stereotaxic microinjections of kainic acid (KA) (0.4 mM) were investigated to clarify the role of the VTA and the SN neurons in learning and memory processes. The present study demonstrates that KA in the SN and the VTA lesioned rats significantly decreased spontaneous alternation in Y-maze task, working memory and reference memory in radial 8 arm-maze task, suggesting effects on spatial memory performance. Our findings provide further support for the role of the VTA and the SN neurons in processing and storage of information.     

Acute pain and a motivational pathway in adult rats: influence of early life pain experience

Background

The importance of neonatal experience upon behaviour in later life is increasingly recognised. The overlap between pain and reward pathways led us to hypothesise that neonatal pain experience influences reward-related pathways and behaviours in adulthood.

Methodology/Principal Findings

Rat pups received repeat plantar skin incisions (neonatal IN) or control procedures (neonatal anesthesia only, AN) at postnatal days (P)3, 10 and 17. When adult, rats with neonatal ‘pain history’ showed greater sensory sensitivity than control rats following acute plantar skin incision. Motivational behaviour in the two groups of rats was tested in a novelty-induced hypophagia (NIH) paradigm. The sensitivity of this paradigm to pain-induced changes in motivational behaviour was shown by significant increases in the time spent in the central zone of the arena (43.7±5.9% vs. 22.5±6.7%, p<0.05), close to centrally placed food treats, and decreased number of rears (9.5±1.4 vs. 19.2±2.3, p<0.001) in rats with acute plantar skin incision compared to naive, uninjured animals. Rats with a neonatal ‘pain history’ showed the same pain-induced behaviour in the novelty-induced hypophagia paradigm as controls. However, differences were observed in reward-related neural activity between the two groups. Two hours after behavioural testing, brains were harvested and neuronal activity mapped using c-Fos expression in lateral hypothalamic orexin neurons, part of a specific reward seeking pathway. Pain-induced activity in orexin neurons of control rats (18.4±2.8%) was the same as in uninjured naive animals (15.5±2.6%), but in those rats with a ‘pain history’, orexinergic activity was significantly increased (27.2±4.1%, p<0.01). Furthermore the extent of orexin neuron activation in individual rats with a ‘pain history’ was highly correlated with their motivational behaviour (r = −0.86, p = 0.01).

Conclusions/Significance

These results show that acute pain alters motivational behaviour and that neonatal pain experience causes long-term changes in brain motivational orexinergic pathways, known to modulate mesolimbic dopaminergic reward circuitry.     

Absolute coding of stimulus novelty in the human substantia nigra/VTA

Novelty exploration can enhance hippocampal plasticity in animals through dopaminergic neuromodulation arising in the substantia nigra/ventral tegmental area (SN/VTA). This enhancement can outlast the exploration phase by several minutes. Currently, little is known about dopaminergic novelty processing and its relationship to hippocampal function in humans. In two functional magnetic resonance imaging (fMRI) studies, SN/VTA activations in humans were indeed driven by stimulus novelty rather than other forms of stimulus salience such as rareness, negative emotional valence, or targetness of familiar stimuli, whereas hippocampal responses were less selective. SN/VTA novelty responses were scaled according to absolute rather than relative novelty in a given context, unlike adaptive SN/VTA responses recently reported for reward outcome in animal studies. Finally, novelty enhanced learning and perirhinal/parahippocampal processing of familiar items presented in the same context. Thus, the human SN/VTA can code absolute stimulus novelty and might contribute to enhancing learning in the context of novelty.     

Targeted Overexpression of α-Synuclein by rAAV2/1 Vectors Induces Progressive Nigrostriatal Degeneration and Increases Vulnerability to MPTP in Mouse

Mutations, duplication and triplication of α-synuclein genes are linked to familial Parkinson’s disease (PD), and aggregation of α-synuclein (α-syn) in Lewy bodies (LB) is involved in the pathogenesis of the disease. The targeted overexpression of α-syn in the substantia nigra (SN) mediated by viral vectors may provide a better alternative to recapitulate the neurodegenerative features of PD. Therefore, we overexpressed human wild-type α-syn using rAAV2/1 vectors in the bilateral SN of mouse and examined the effects for up to 12 weeks. Delivery of rAAV-2/1-α-syn caused significant nigrostriatal degeneration including appearance of dystrophic striatal neurites, loss of nigral dopaminergic (DA) neurons and dissolving nigral neuron bodies in a time-dependent manner. In addition, the α-syn overexpressed mice also developed significant deficits in motor function at 12 weeks when the loss of DA neurons exceeded a threshold of 50%. To investigate the sensitivity to neurotoxins in mice overexpressing α-syn, we performed an MPTP treatment with the subacute regimen 8 weeks after rAAV injection. The impact of the combined genetic and environmental insults on DA neuronal loss, striatal dopamine depletion, dopamine turnover and motor dysfunction was markedly greater than that of either alone. Moreover, we observed increased phosphorylation (S129), accumulation and nuclear distribution of α-syn after the combined insults. In summary, these results reveal that the overexpressed α-syn induces progressive nigrostriatal degeneration and increases the susceptibility of DA neurons to MPTP. Therefore, the targeted overexpression of α-syn and the combination with environmental toxins may provide valuable models for understanding PD pathogenesis and developing related therapies.     

Long-term potentiation of excitatory inputs to brain reward areas by nicotine

Nicotine reinforces smoking behavior by activating nicotinic acetylcholine receptors (nAChRs) in the midbrain dopaminergic (DA) reward centers, including the ventral tegmental area (VTA). Although nicotine induces prolonged excitation of the VTA in vivo, the nAChRs on the DA neurons desensitize in seconds. Here, we show that activation of nAChRs on presynaptic terminals in the VTA enhances glutamatergic inputs to DA neurons. Under conditions where the released glutamate can activate NMDA receptors, long-term potentiation (LTP) of the excitatory inputs is induced. Both the short- and the long-term effects of nicotine required activation of presynaptic alpha7 subunit-containing nAChRs. These results can explain the long-term excitation of brain reward areas induced by a brief nicotine exposure. They also show that nicotine alters synaptic function through mechanisms that are linked to learning and memory.     

Progestins Can Have a Membrane-Mediated Action in Rat Midbrain for Facilitation of Sexual Receptivity

In rats, progesterone (P) facilitates sexual receptivity by interacting with intracellular progestin receptors in the ventromedial hypothalamus (VMH). This experiment concerns whether P can also facilitate receptivity in rats by acting extragenomically within the ventral tegmental area (VTA). Ovariectomized rats (n= 10) with bilateral guide cannulas over the VMH and VTA were primed with 2 μg subcutaneous estradiol benzoate 44 hr prior to testing. After a pretest for sexual receptivity, animals received implants to the VMH of P, P conjugated to bovine serum albumin (P:BSA), or cholesterol control (CHOL), and were retested. Two hours later, animals were again tested for receptivity, and P, P:BSA, the P metabolite 5α-pregnan-3α-ol-20-one (3α,5α-THP), or CHOL implants were applied to the VTA. Subjects were retested immediately, 30, 90, and 150 min later. Animals that received P in the VMH and had P, P:BSA, or 3α,5α-THP applied to the VTA exhibited facilitated receptivity at all time points compared with all other combination implants. That P:BSA and P were equally effective when applied to the VTA, but not the VMH, suggests that in the VTA P's membrane-mediated actions are sufficient to facilitate receptivity, whereas in the VMH they are not. Since the steroid (P) and its metabolite (3α,5α-THP) are similarly effective when applied to the VTA, given P application to the VMH earlier, P's effects in the VTA may be subsequent to metabolism and/or actions at GABA receptors. Overall, these data suggest that in rats P can act at the membrane of neurons within the VTA to modulate lordosis and that these effects may be subsequent to P's metabolism and/or actions at GABA receptors.     

Endogenous Cholinergic Inputs and Local Circuit Mechanisms Govern the Phasic Mesolimbic Dopamine Response to Nicotine

Nicotine exerts its reinforcing action by stimulating nicotinic acetylcholine receptors (nAChRs) and boosting dopamine (DA) output from the ventral tegmental area (VTA). Recent data have led to a debate about the principal pathway of nicotine action: direct stimulation of the DAergic cells through nAChR activation, or disinhibition mediated through desensitization of nAChRs on GABAergic interneurons. We use a computational model of the VTA circuitry and nAChR function to shed light on this issue. Our model illustrates that the α4β2-containing nAChRs either on DA or GABA cells can mediate the acute effects of nicotine. We account for in vitro as well as in vivo data, and predict the conditions necessary for either direct stimulation or disinhibition to be at the origin of DA activity increases. We propose key experiments to disentangle the contribution of both mechanisms. We show that the rate of endogenous acetylcholine input crucially determines the evoked DA response for both mechanisms. Together our results delineate the mechanisms by which the VTA mediates the acute rewarding properties of nicotine and suggest an acetylcholine dependence hypothesis for nicotine reinforcement.     

Functional diversity of ventral midbrain dopamine and GABAergic neurons

Recent findings indicate that VTA and SN dopaminergic (DA) and GABAergic neurons form subpopulations that are divergent in their electrophysiological features, vulnerability to neurodegeneration, and regulation by neuropeptides. This diversity can be correlated with the anatomical organization of the VTA and SN and their inputs and outputs. In this review we describe the heterogeneity in ion channels and firing patterns, especially burst firing, in subpopulations of dopamine neurons. We go on to describe variations in vulnerability to neurotoxic damage in models of Parkinson’s disease in subgroups of DA neurons and its possible relationship to developmental gene regulation, the expression of different ion channels, and the expression of different protein markers, such as the neuroprotective marker calbindin. The electrophysiological properties of subgroups of GABAergic midbrain neurons, patterns of expression of protein markers and receptors, possible involvement of GABAergic neurons in a number of processes that are usually attributed exclusively to dopaminergic neurons, and the characteristics of a subgroup of neurons that contains both dopamine and GABA are also discussed.     

Stimulation of midbrain dopaminergic structures modifies firing rates of rat lateral habenula neurons

Ventral tegmental area (VTA) and substantia nigra pars compacta (SNpc) are midbrain structures known to be involved in mediating reward in rodents. Lateral habenula (LHb) is considered as a negative reward source and it is reported that stimulation of the LHb rapidly induces inhibition of firing in midbrain dopamine neurons. Interestingly, the phasic fall in LHb neuronal activity may follow the excitation of dopamine neurons in response to reward-predicting stimuli. The VTA and SNpc give rise to dopaminergic projections that innervate the LHb, which is also known to be involved in processing painful stimuli. But it's unclear what physiological effects these inputs have on habenular function. In this study we distinguished the LHb pain-activated neurons of the Wistar rats and assessed their electrophysiological responsiveness to the stimulation of the VTA and SNpc with either single-pulse stimulation (300 μA, 0.5 Hz) or tetanic stimulation (80 μA, 25 Hz). Single-pulse stimulation that was delivered to either midbrain structure triggered transient inhibition of firing of ～90% of the LHb pain-activated neurons. However, tetanic stimulation of the VTA tended to evoke an elevation in neuronal firing rate. We conclude that LHb pain-activated neurons can receive diverse reward-related signals originating from midbrain dopaminergic structures, and thus participate in the regulation of the brain reward system via both positive and negative feedback mechanisms.