Direct bidirectional µ-opioid control of midbrain dopamine neurons.

The ventral tegmental area (VTA) is required for the rewarding and motivational actions of opioids and activation of dopamine neurons has been implicated in these effects. The canonical model posits that opioid activation of VTA dopamine neurons is indirect, through inhibition of GABAergic inputs. However, VTA dopamine neurons also express postsynaptic µ-opioid peptide (MOP) receptors. We report here that in Sprague Dawley rat, the MOP receptor-selective agonist DAMGO (0.5-3 µM) depolarized or increased the firing rate of 87 of 451 VTA neurons (including 22 of 110 dopamine neurons). This DAMGO excitation occurs in the presence of GABAA receptor blockade and its EC50 value is two orders of magnitude lower than for presynaptic inhibition of GABA release on to VTA neurons. Consistent with a postsynaptic channel opening, excitations were accompanied by a decrease in input resistance. Excitations were blocked by CdCl2 (100 µM, n = 5) and ω-agatoxin-IVA (100 nM, n = 3), nonselective and Cav2.1 Ca(2+) channel blockers, respectively. DAMGO also produced a postsynaptic inhibition in 233 of 451 VTA neurons, including 45 of 110 dopamine neurons. The mean reversal potential of the inhibitory current was -78 ± 7 mV and inhibitions were blocked by the K(+) channel blocker BaCl2 (100 µM, n = 7). Blockade of either excitation or inhibition unmasked the opposite effect, suggesting that MOP receptors activate concurrent postsynaptic excitatory and inhibitory processes in most VTA neurons. These results provide a novel direct mechanism for MOP receptor control of VTA dopamine neurons.

Opioid modulation of ventral pallidal afferents to ventral tegmental area neurons.

Activation of mu opioid receptors within the ventral tegmental area (VTA) can produce reward through the inhibition of GABAergic inputs. GABAergic neurons in the ventral pallidum (VP) provide a major input to VTA neurons. To determine the specific VTA neuronal targets of VP afferents and their sensitivity to mu opioid receptor agonists, we virally expressed channel rhodopsin (ChR2) in rat VP neurons and optogenetically activated their terminals in the VTA. Light activation of VP neuron terminals elicited GABAergic IPSCs in both dopamine (DA) and non-DA VTA neurons, and these IPSCs were inhibited by the mu opioid receptor agonist DAMGO. In addition, using a fluorescent retrograde marker to identify VTA-projecting VP neurons, we found them to be hyperpolarized by DAMGO. Both of these actions decrease GABAergic input onto VTA neurons, revealing two mechanisms by which endogenous or exogenous opioids can activate VTA neurons, including DA neurons.

Ventral tegmental area glutamate neurons: electrophysiological properties and projections.

The ventral tegmental area (VTA) has a central role in the neural processes that underlie motivation and behavioral reinforcement. Although thought to contain only dopamine and GABA neurons, the VTA also includes a recently discovered population of glutamate neurons identified through the expression of the vesicular glutamate transporter VGLUT2. A subset of VGLUT2(+) VTA neurons corelease dopamine with glutamate at terminals in the NAc, but others do not express dopaminergic markers and remain poorly characterized. Using transgenic mice that express fluorescent proteins in distinct cell populations, we now find that both dopamine and glutamate neurons in the medial VTA exhibit a smaller hyperpolarization-activated current (I(h)) than more lateral dopamine neurons and less consistent inhibition by dopamine D(2) receptor agonists. In addition, VGLUT2(+) VTA neurons project to the nucleus accumbens (NAc), lateral habenula, ventral pallidum (VP), and amygdala. Optical stimulation of VGLUT2(+) projections expressing channelrhodopsin-2 further reveals functional excitatory synapses in the VP as well as the NAc. Thus, glutamate neurons form a physiologically and anatomically distinct subpopulation of VTA projection neurons.

A novel opioid receptor-mediated enhancement of GABAA receptor function induced by stress in ventral tegmental area neurons.

Opioid receptors are G-protein-coupled receptors (GPCRs) that modulate synaptic function. Depending upon their nervous system site of action, opioid receptor agonists alter food consumption, pain perception, responses to stress, and drug reward. Opioid receptors signal primarily via G(i/o)-proteins that modulate ion channels to directly inhibit neurons or decrease neurotransmitter release from nerve terminals. Here we report that following stress, activating δ opioid receptors (DORs) on midbrain ventral tegmental area (VTA) neurons causes a novel synaptic effect: the augmentation of GABA(A) receptor (GABA(A)R)-mediated inhibitory postsynaptic currents. Most neurons showing this augmentation were identified as dopaminergic. In addition, in both stressed and unstressed animals, DOR activation decreases GABA(A)R currents in some VTA neurons. Surprisingly, both augmentation and inhibition were also observed when we bypassed the presynaptic terminal by iontophoretically applying GABA, indicating that postsynaptic mechanisms are responsible for both effects. Using a variety of blockers we determined that the augmentation is probably due to insertion of GABA(A)Rs into the synapse by a mechanism that is G-protein independent and mediated by activation of Akt via PI3K. GABA(A)Rs are inserted into the extra-synaptic plasma membrane before trafficking to the synapse, a mechanism consistent with our observation that the DOR-mediated increase in GABA(A)R signalling occurs significantly earlier in iontophoretically applied than in electrically evoked synaptic GABA. This G-protein-independent signalling pathway is not only a novel mechanism of opioid receptor-mediated inhibition, but it also represents the first reported link between activation of a GPCR and insertion of GABA(A)Rs into the plasma membrane.

Nucleus accumbens medium spiny neurons target non-dopaminergic neurons in the ventral tegmental area.

The midbrain ventral tegmental area (VTA) projection to the nucleus accumbens (NAc) is implicated in motivation and reinforcement. A significant number of NAc medium spiny neurons (MSNs) project back to the VTA, although the nature of this projection is essentially unknown. For example, do NAc MSNs directly target accumbens-projecting dopamine neurons and do they act via the GABA(A) or GABA(B) receptor? To address these issues, we expressed the light-sensitive channel rhodopsin-2 in the rat NAc and made electrophysiological recordings from VTA neurons ex vivo. We found that the NAc directly targets non-dopaminergic VTA neurons, including some that project back to the NAc. These MSN GABAergic terminals are opioid sensitive and act via GABA(A) receptors.

Substance P inhibits GABAB receptor signalling in the ventral tegmental area.

Substance P (SP) and its receptors are involved in anxiety-related behaviours and regulate the intake of drugs of abuse and alcohol. Within the midbrain ventral tegmental area (VTA), a region that is clearly involved in the control of these behaviours, SP is released by stress and has been shown to trigger relapse. SP activates neurokinin (NK) receptors, which excites midbrain dopamine (DA) neurons and leads to increased DA in target regions. In this study, we have investigated the mechanisms underlying SP actions in the VTA, specifically investigating interactions between SP and GABA(B) receptors. We show that in VTA neurons, NK receptor activation closes an inwardly rectifying potassium channel, and moreover inhibits GABA(B) receptor-mediated transmission through an interaction that depends upon phospholipase C (PLC), intracellular calcium and protein kinase C (PKC).

Delta-opioid receptor expression in the ventral tegmental area protects against elevated alcohol consumption.

Alcoholism is a complex and debilitating syndrome affecting approximately 140 million people worldwide. However, not everyone who consumes ethanol develops abuse, raising the possibility that some individuals have a protective mechanism that inhibits elevated alcohol consumption. We tested the hypothesis that the delta-opioid receptor (DOR) plays such a protective role. Here we show that DOR activity in the ventral tegmental area (VTA) robustly decreases ethanol consumption in rats and that these effects depend on baseline ethanol consumption. Intra-VTA microinjection of the DOR agonist DPDPE decreases drinking, particularly in low-drinking animals. Furthermore, VTA microinjection of the DOR selective antagonist TIPP-Psi increases drinking in low, but not high, drinkers and this increase is blocked by comicroinjection of the GABA(A) antagonist bicuculline. Using electrophysiological techniques we found that in VTA brain slices from drinking rats DPDPE presynaptically inhibits GABA(A) receptor mediated IPSCs in low drinkers, but not in high drinkers or naive animals, most likely through activation of DORs on GABA terminals. This DOR-mediated inhibition of IPSCs also correlates inversely with behavioral correlates of anxiety measured in the elevated plus maze. In contrast, presynaptic inhibition of VTA GABA(A) IPSCs by the mu-opioid receptor agonist DAMGO is significantly reduced in both high- and low-drinking rats (<30%) compared with age-matched nondrinking controls (>70%). Together, our findings demonstrate the protective nature of VTA DORs and identify an important new target for therapeutic intervention for alcoholism.

Midbrain dopamine neurons: projection target determines action potential duration and dopamine D(2) receptor inhibition.

Broad action potentials (APs) and dopamine (DA) D(2) receptor (D(2)R)-mediated inhibition are widely used to identify midbrain DA neurons. However, when these measures are taken alone they do not predict DA content in ventral tegmental area (VTA) neurons. In fact, some VTA neuronal properties correlate better with projection target than neurotransmitter content. Here we report that amygdala (AMYG)-projecting VTA DA neurons have brief APs and lack D(2)R agonist (quinpirole; 1 microM) autoinhibition. However, they are hyperpolarized by both the GABA(B) agonist baclofen (1 microM) and the kappa-opioid receptor agonist U69593 [(+)-(5alpha,7alpha,8beta)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]benzeneacetamide; 1 microM]. Furthermore, we show that accurate prediction of DA content in VTA neurons is possible when the projection target is known: in both nucleus accumbens- and AMYG-projecting neural populations, AP durations are significantly longer in DA than non-DA neurons. Among prefrontal cortex-projecting neurons, quinpirole sensitivity, but not AP duration, is a predictor of DA content. Therefore, in the VTA, AP duration and inhibition by D(2)R agonists may be valid markers of DA content in neurons of known projection target.

Acute amphetamine exposure selectively desensitizes kappa-opioid receptors in the nucleus accumbens.

In the present study, we investigated the effects of psychostimulant exposure on kappa-opioid peptide (KOP) receptor signaling in the rat mesolimbic system. A single subcutaneous (s.c.) injection of amphetamine (2.5 mg/kg) reduced the KOP receptor-mediated inhibition of glutamate release in the nucleus accumbens shell, as a consequence of KOP receptor desensitization. This effect was blocked by dopamine (DA) receptor antagonists or the nonselective opioid antagonist, naltrexone (1 mg/kg, s.c.), and mimicked by the KOP receptor agonists U69593 (0.32 mg/kg, s.c.) and dynorphin (1 microM), indicating that an amphetamine-induced release of dynorphin is producing a long-lasting desensitization of the KOP receptor. Despite the fact that amphetamine also increases dynorphin release in the ventral tegmental area (VTA), KOP receptor function in this region was not affected by amphetamine; there was no difference in the KOP receptor-mediated change in firing rate or resting membrane potential measured in VTA neurons from saline- or amphetamine-treated animals. This study demonstrates that amphetamine can produce regionally selective adaptations in KOP receptor signaling, which may, in turn, alter the effects of subsequent drug exposure.

Protein kinase inhibitors reduce GABA but not glutamate release in the nucleus accumbens.

We investigated the role of endogenous protein kinase activity on synaptic transmission in the rat nucleus accumbens slice. The isoquinolinesulfonamide H-7 (50muM), a non-selective serine/threonine protein kinase inhibitor, had no effect on pharmacologically isolated glutamatergic EPSCs. However, it reduced GABA release in a dose-dependent manner. This effect of H-7 was not mimicked by the selective cAMP-dependent protein kinase inhibitor H-89, the PKC inhibitor Bisindolylmaleimide-1, or the cGMP-dependent protein kinase inhibitor KT5823. However, bath application of the myosin light chain kinase (MLCK) inhibitor, ML-7, significantly reduced IPSC amplitudes and partially occluded the reduction in IPSCs observed following bath application of H-7. These results suggest that endogenous protein kinase activity, specifically MLCK activity, regulates GABA, but not glutamate release, onto medium spiny neurons in the nucleus accumbens.

Ventral tegmental area neurons in learned appetitive behavior and positive reinforcement.

Ventral tegmental area (VTA) neuron firing precedes behaviors elicited by reward-predictive sensory cues and scales with the magnitude and unpredictability of received rewards. These patterns are consistent with roles in the performance of learned appetitive behaviors and in positive reinforcement, respectively. The VTA includes subpopulations of neurons with different afferent connections, neurotransmitter content, and projection targets. Because the VTA and substantia nigra pars compacta are the sole sources of striatal and limbic forebrain dopamine, measurements of dopamine release and manipulations of dopamine function have provided critical evidence supporting a VTA contribution to these functions. However, the VTA also sends GABAergic and glutamatergic projections to the nucleus accumbens and prefrontal cortex. Furthermore, VTA-mediated but dopamine-independent positive reinforcement has been demonstrated. Consequently, identifying the neurotransmitter content and projection target of VTA neurons recorded in vivo will be critical for determining their contribution to learned appetitive behaviors.

The ventral tegmental area revisited: is there an electrophysiological marker for dopaminergic neurons?

The ventral tegmental area (VTA) and in particular VTA dopamine (DA) neurons are postulated to play a central role in reward, motivation and drug addiction. However, most evidence implicating VTA DA neurons in these functions is based on indirect electrophysiological characterization, rather than cytochemical identification. These physiological criteria were first established in the substantia nigra pars compacta (SNc), but their validity in the VTA is uncertain. In the current study we found that while 88 +/- 2% of SNc neurons labelled by the neuronal marker NeuN were co-labelled for the catecholamine enzyme tyrosine hydroxylase (TH), a much smaller percentage (55 +/- 2%) of VTA neurons co-expressed TH. In addition, using in vitro whole-cell recordings we found that widely accepted physiological criteria for VTA DA neurons, including the hyperpolarization-activated inwardly rectifying non-specific cation current (I(h)), spike duration, and inhibition by DA D2 receptor agonists, do not reliably predict the DA content of VTA neurons. We could not distinguish DA neurons from other VTA neurons by size, shape, input resistance, I(h) size, or spontaneous firing rate. Although the absence of an I(h) reliably predicted that a VTA neuron was non-dopaminergic, and I(h)(-) neurons differ from I(h)(+) neurons in firing rate, interspike interval (ISI) standard deviation, and ISI skew, no physiological property examined here is both sensitive and selective for DA neurons in the VTA. We conclude that reliable physiological criteria for VTA DA neuron identification have yet to be determined, and that the criteria currently being used are unreliable.

Kappa opioids selectively control dopaminergic neurons projecting to the prefrontal cortex.

Dopaminergic afferents arising from the ventral tegmental area (VTA) are crucial elements in the neural circuits that mediate arousal, motivation, and reinforcement. Two major targets of these afferents are the medial prefrontal cortex (mPFC) and the nucleus accumbens (NAc). Whereas dopamine (DA) in the mPFC has been implicated in working memory and attentional processes, DA in the NAc is required for responding to reward predictive cues. These distinct functions suggest a role for independent firing patterns of dopaminergic neurons projecting to these brain regions. In fact, DA release in mPFC and NAc can be differentially modulated. However, to date, electrophysiological studies have largely overlooked heterogeneity among VTA neurons. Here, we provide direct evidence for differential neurotransmitter control of DA neural activity and corresponding DA release based on projection target. Kappa opioid receptor agonists inhibit VTA DA neurons that project to the mPFC but not those that project to the NAc. Moreover, DA levels in the mPFC, but not the NAc, are reduced after local infusion of kappa opioid receptor agonists into the VTA. These findings demonstrate that DA release in specific brain regions can be independently regulated by opioid targeting of a subpopulation of VTA DA neurons. Selective control of VTA DA neurons projecting to the mPFC has important implications for understanding addiction, attention disorders, and schizophrenia, all of which are associated with DA dysfunction in the mPFC.

Interactions between asynchronous release and short-term plasticity in the nucleus accumbens slice.

Glutamate synapses in the nucleus accumbens (NAc) display asynchronous release in response to trains of stimulation. However, it is unclear what role this asynchronous release plays in synaptic transmission in this nucleus. This process was studied, specifically looking at the interaction between short-term depression and asynchronous release. These results indicate that synchronous and asynchronous release do not compete for a depleted readily releasable pool of vesicles.

Both kappa and mu opioid agonists inhibit glutamatergic input to ventral tegmental area neurons.

The ventral tegmental area (VTA) plays a critical role in motivation and reinforcement. Kappa and mu opioid receptor (KOP-R and MOP-R) agonists microinjected into the VTA produce powerful and largely opposing motivational actions. Glutamate transmission within the VTA contributes to these motivational effects. Therefore information about opioid control of glutamate release onto VTA neurons is important. To address this issue, we performed whole cell patch-clamp recordings in VTA slices and measured excitatory postsynaptic currents (EPSCs). There are several classes of neuron in the VTA: principal, secondary, and tertiary. The KOP-R agonist (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methane-sulfonate hydrate (U69593; 1 microM) produced a small reduction in EPSC amplitude in principal neurons (14%) and a significantly larger inhibition in secondary (47%) and tertiary (33%) neurons. The MOP-R agonist [D-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO; 3 microM) inhibited glutamate release in principal (42%), secondary (45%), and tertiary neurons (35%). Unlike principal and tertiary neurons, in secondary neurons, the magnitude of the U69593 EPSC inhibition was positively correlated with that produced by DAMGO. Finally, DAMGO did not occlude the U69593 effect in principal neurons, suggesting that some glutamatergic terminals are independently controlled by KOP and MOP receptor activation. These findings show that MOP-R and KOP-R agonists regulate excitatory input onto each VTA cell type.

Contrast enhancement: a physiological effect of striatal dopamine?

Dopamine functions as an important neuromodulator in the dorsal striatum and ventral striatum/nucleus accumbens. Evidence is accumulating for the idea that striatal neurons compete with each other for control over the animal's motor resources, and that dopamine plays an important modulatory role that allows a particular subset of neurons, encoding a specific behavior, to predominate in this competition. One means by which dopamine could facilitate selection among competing neurons is to enhance the contrast between stronger and weaker excitations (or to increase the "signal to noise ratio" among neurons, where the firing of the most excited neurons is assumed to transmit signal and the firing of the least excited to transmit noise). Here, we review the electrophysiological evidence for this hypothesis and discuss potential cellular mechanisms by which dopamine-mediated contrast enhancement could occur.

Dopamine excites nucleus accumbens neurons through the differential modulation of glutamate and GABA release.

Afferent activity into the nucleus accumbens (NAc) occurs in bursts of action potentials. However, it is unclear how synapses in this nucleus respond to such bursts, or how these responses are altered by dopamine (DA). I examined the effects of DA on excitatory and inhibitory responses to trains of stimuli in rat NAc slices. Both EPSCs and IPSCs showed use-dependent depression during trains. Although DA inhibited both glutamate and GABA release in the NAc, it differentially inhibited release during trains. The inhibition of IPSCs persisted throughout the train of stimuli, whereas the inhibition of EPSCs progressively diminished. This differential modulation may be explained by a calcium-dependent change in the recovery from depression at the GABA synapses, where DA acts by decreasing Ca2+ entry. Thus, at later stages of sustained stimulation, DA preferentially inhibits GABA release, producing a net excitatory effect during bursts suggesting a mechanism for enhancing the contrast between competing inputs into the NAc, as well as for affecting long-term plasticity in this structure.

A single cocaine exposure enhances both opioid reward and aversion through a ventral tegmental area-dependent mechanism.

Repeated exposure to drugs of abuse produces forms of experience-dependent plasticity including behavioral sensitization. Although a single exposure to many addicting substances elicits locomotor sensitization, there is little information regarding the motivational effects of such single exposures. This study demonstrates that a single cocaine exposure enhances both rewarding and aversive forms of opioid place conditioning. Rats were given a single injection of cocaine (15 mg/kg i.p.) in their home cage at different times before conditioning. This treatment enhanced conditioned place preference (CPP) to morphine (2 x 10 mg/kg s.c.) if training began 1 or 5 but not 10 days after the cocaine injection. A single cocaine exposure also enhanced conditioned place aversion (CPA) to the kappa-opioid receptor agonist U69593 (2 x 0.16 mg/kg s.c.). Compared to morphine CPP, U69593 CPA was delayed and persistent. It was not observed at 1 day but appeared if the conditioning began 5 or 10 days after the cocaine injection. Although the cocaine-induced enhancements of both morphine CPP and U69593 CPA followed different time courses, suggesting different mechanisms, both effects were blocked by injection of the N-methyl-d-aspartate receptor antagonist MK-801 (0.5 nmol bilaterally) into the ventral tegmental area, immediately before the cocaine injection. Thus, through a circuit involving the ventral tegmental area, a single cocaine exposure enhanced both micro-opioid receptor reward and kappa-opioid receptor aversion.

Kappa-opioid agonists directly inhibit midbrain dopaminergic neurons.

Dopaminergic neurons of the ventral tegmental area (VTA) play a critical role in motivation and reinforcement of goal-directed behaviors. Furthermore, excitation of these neurons has been implicated in the addictive process initiated by drugs such as morphine that act at the micro-opioid receptor (MOR). In contrast, kappa-opioid receptor (KOR) activation in the VTA produces behavioral actions opposite to those elicited by MOR activation. The mechanism underlying this functional opposition, however, is poorly understood. VTA neurons have been categorized previously as principal, secondary, or tertiary on the basis of electrophysiological and pharmacological characteristics. In the present study using whole-cell patch-clamp recordings, we demonstrate that a selective KOR agonist (U69593, 1 microm) directly inhibits a subset of principal and tertiary but not secondary neurons in the VTA. This KOR-mediated inhibition occurs via the activation of a G-protein-coupled inwardly rectifying potassium channel and is blocked by the selective KOR antagonist nor-Binaltorphimine (100 nm). Significantly, regardless of cell class, KOR-mediated inhibition was found only in tyrosine hydroxylase-immunoreactive and thus dopaminergic neurons. In addition, we found a subset of principal neurons that exhibited both disinhibition by a selective MOR agonist ([d-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin) (3 microm) and direct inhibition by KOR agonists. These results provide a cellular mechanism for the opposing behavioral effects of KOR and MOR agonists and shed light on how KORs might regulate the motivational effects of both natural rewards and addictive drugs.

Kappa opioid receptor activation in the nucleus accumbens inhibits glutamate and GABA release through different mechanisms.

Through their actions in the nucleus accumbens (NAc), kappa opioid (KOP) receptors and their endogenous ligand, dynorphin, modify behaviors associated with the administration of drugs of abuse and are regulated by exposure to such drugs. Despite their demonstrated behavioral significance, the synaptic actions of KOP receptor ligands in the NAc are not clearly understood. Using whole-cell voltage-clamp recordings of NAc medium spiny neurons, we have found that, in addition to suppressing glutamate release, the KOP receptor agonist also inhibits GABA release. Interestingly, the mechanism of inhibition of the release of glutamate differs from that controlling GABA. reduces the frequency of Ca(2+)-independent miniature excitatory postsynaptic currents, but not miniature inhibitory postsynaptic currents. Furthermore, while the inhibition of GABAergic transmission is blocked by the N-type Ca(2+) channel blocker omega-CgTx, the inhibition of excitatory glutamatergic transmission by is unaffected by N-type Ca(2+) channel blockade. These results indicate that KOP receptor activation inhibits GABA release by reducing Ca(2+) influx, but inhibits glutamate release at a step downstream of Ca(2+) entry.