Cocaine disinhibits dopamine neurons by potentiation of GABA transmission in the ventral tegmental area.

Drug-evoked synaptic plasticity in the mesolimbic system reshapes circuit function and drives drug-adaptive behavior. Much research has focused on excitatory transmission in the ventral tegmental area (VTA) and the nucleus accumbens (NAc). How drug-evoked synaptic plasticity of inhibitory transmission affects circuit adaptations remains unknown. We found that medium spiny neurons expressing dopamine (DA) receptor type 1 (D1R-MSNs) of the NAc project to the VTA, strongly preferring the GABA neurons of the VTA. Repeated in vivo exposure to cocaine evoked synaptic potentiation at this synapse, occluding homosynaptic inhibitory long-term potentiation. The activity of the VTA GABA neurons was thus reduced and DA neurons were disinhibited. Cocaine-evoked potentiation of GABA release from D1R-MSNs affected drug-adaptive behavior, which identifies these neurons as a promising target for novel addiction treatments.

GABA neurons of the VTA drive conditioned place aversion.

Salient but aversive stimuli inhibit the majority of dopamine (DA) neurons in the ventral tegmental area (VTA) and cause conditioned place aversion (CPA). The cellular mechanism underlying DA neuron inhibition has not been investigated and the causal link to behavior remains elusive. Here, we show that GABA neurons of the VTA inhibit DA neurons through neurotransmission at GABA(A) receptors. We also observe that GABA neurons increase their firing in response to a footshock and provide evidence that driving GABA neurons with optogenetic effectors is sufficient to affect behavior. Taken together, our data demonstrate that synaptic inhibition of DA neurons drives place aversion.

Bi-directional effect of increasing doses of baclofen on reinforcement learning.

In rodents as well as in humans, efficient reinforcement learning depends on dopamine (DA) released from ventral tegmental area (VTA) neurons. It has been shown that in brain slices of mice, GABA(B)-receptor agonists at low concentrations increase the firing frequency of VTA-DA neurons, while high concentrations reduce the firing frequency. It remains however elusive whether baclofen can modulate reinforcement learning in humans. Here, in a double-blind study in 34 healthy human volunteers, we tested the effects of a low and a high concentration of oral baclofen, a high affinity GABA(B)-receptor agonist, in a gambling task associated with monetary reward. A low (20 mg) dose of baclofen increased the efficiency of reward-associated learning but had no effect on the avoidance of monetary loss. A high (50 mg) dose of baclofen on the other hand did not affect the learning curve. At the end of the task, subjects who received 20 mg baclofen p.o. were more accurate in choosing the symbol linked to the highest probability of earning money compared to the control group (89.55 ± 1.39 vs. 81.07 ± 1.55%, p = 0.002). Our results support a model where baclofen, at low concentrations, causes a disinhibition of DA neurons, increases DA levels and thus facilitates reinforcement learning.

Neural bases for addictive properties of benzodiazepines.

Benzodiazepines are widely used in clinics and for recreational purposes, but will lead to addiction in vulnerable individuals. Addictive drugs increase the levels of dopamine and also trigger long-lasting synaptic adaptations in the mesolimbic reward system that ultimately may induce the pathological behaviour. The neural basis for the addictive nature of benzodiazepines, however, remains elusive. Here we show that benzodiazepines increase firing of dopamine neurons of the ventral tegmental area through the positive modulation of GABA(A) (gamma-aminobutyric acid type A) receptors in nearby interneurons. Such disinhibition, which relies on alpha1-containing GABA(A) receptors expressed in these cells, triggers drug-evoked synaptic plasticity in excitatory afferents onto dopamine neurons and underlies drug reinforcement. Taken together, our data provide evidence that benzodiazepines share defining pharmacological features of addictive drugs through cell-type-specific expression of alpha1-containing GABA(A) receptors in the ventral tegmental area. The data also indicate that subunit-selective benzodiazepines sparing alpha1 may be devoid of addiction liability.

Riluzole-induced oscillations in spinal networks.

We previously showed in dissociated cultures of fetal rat spinal cord that disinhibition-induced bursting is based on intrinsic spiking, network recruitment, and a network refractory period after the bursts. A persistent sodium current (I(NaP)) underlies intrinsic spiking, which, by recurrent excitation, generates the bursting activity. Although full blockade of I(NaP) with riluzole disrupts such bursting, the present study shows that partial blockade of I(NaP) with low doses of riluzole maintains bursting activity with unchanged burst rate and burst duration. More important, low doses of riluzole turned bursts composed of persistent activity into bursts composed of oscillatory activity at around 5 Hz. In a search for the mechanisms underlying the generation of such intraburst oscillations, we found that activity-dependent synaptic depression was not changed with low doses of riluzole. On the other hand, low doses of riluzole strongly increased spike-frequency adaptation and led to early depolarization block when bursts were simulated by injecting long current pulses into single neurons in the absence of fast synaptic transmission. Phenytoin is another I(NaP) blocker. When applied in doses that reduced intrinsic activity by 80-90%, as did low doses of riluzole, it had no effect either on spike-frequency adaptation or on depolarization block. Nor did phenytoin induce intraburst oscillations after disinhibition. A theoretical model incorporating a depolarization block mechanism could reproduce the generation of intraburst oscillations at the network level. From these findings we conclude that riluzole-induced intraburst oscillations are a network-driven phenomenon whose major accommodation mechanism is depolarization block arising from strong sodium channel inactivation.

Patterns of spontaneous activity in unstructured and minimally structured spinal networks in culture.

The rhythmic activity observed in locomotion is generated by local neuronal networks in the spinal cord. The alternating patterns are produced by reciprocal connections between these networks. Synchronous rhythmic activity, but not alternation, can be reproduced in disinhibited networks of dissociated spinal neurons of rats. This suggests that a specific network architecture is required for pattern generation but not for rhythm generation. Here we were interested in the recruitment of neurons to produce population bursts in unstructured and minimally structured cultures of rat spinal cord grown on multielectrode arrays. We tested whether two networks, connected by a small number of axons, could be functionally separated into two units and generate more complex patterns such as alternation. In the unstructured cultures, we found that the recruitment of the neurons into bursting populations is divided into two steps: the fast recruitment of a "trigger network", consisting of intrinsically firing cells connected in networks with short delays, and slow recruitment of the rest of the network. One or several trigger networks were observed in a single culture and could account for variable patterns of propagation. In the minimally structured cultures, a functional separation between loosely connected networks was achieved. Such separation led either to an independent bursting between the networks or to synchronized bursting with long and variable delays. However, no qualitatively novel pattern such as alternation could be generated. In addition, we found that the strength of reciprocal inhibitory connections was modulated by spontaneous activity.

INaP underlies intrinsic spiking and rhythm generation in networks of cultured rat spinal cord neurons.

We have shown previously that rhythm generation in disinhibited spinal networks is based on intrinsic spiking, network recruitment and a network refractory period following the bursts. This refractory period is based mainly on electrogenic Na/K pump activity. In the present work, we have investigated the role of the persistent sodium current (INaP) in the generation of bursting using patch-clamp and multielectrode array recordings. We detected INaP exclusively in the intrinsic spiking cells. The blockade of INaP by riluzole suppressed the bursting by silencing the intrinsic spiking cells and suppressing network recruitment. The blockade of the persistent sodium current produced a hyperpolarization of the membrane potential of the intrinsic spiking cells, but had no effect on non-spiking cells. We also investigated the involvement of the hyperpolarization-activated cationic current (I(h)) in the rhythmic activity. The bath application of ZD7288, a specific I(h) antagonist, slowed down the rate of the bursts by increasing the interburst intervals. I(h) was present in approximately 70% of the cells, both in the intrinsic spiking cells as well as in the non-spiking cells. We also found both kinds of cells in which I(h) was not detected. In summary, in disinhibited spinal cord cultures, a persistent sodium current underlies intrinsic spiking, which, via recurrent excitation, generates the bursting activity. The hyperpolarization-activated cationic current contributes to intrinsic spiking and modulates the burst frequency.

N-Terminal myristoylation predictions by ensembles of neural networks.

N-terminal myristoylation is a post-translational modification that causes the addition of a myristate to a glycine in the N-terminal end of the amino acid chain. This work presents neural network (NN) models that learn to discriminate myristoylated and nonmyristoylated proteins. Ensembles of 25 NNs and decision trees were trained on 390 positive sequences and 327 negative sequences. Experiments showed that NN ensembles were more accurate than decision tree ensembles. Our NN predictor evaluated by the leave-one-out procedure, obtained a false positive error rate equal to 2.1%. That was better than the PROSITE pattern for myristoylation for which the false positive error rate was 22.3%. On a recent version of Swiss-Prot (41.2), the NN ensemble predicted 876 myristoylated proteins, while 1150 proteins were predicted by the PROSITE pattern for myristoylation. Finally, compared to the well-known NMT predictor, the NN predictor gave similar results. Our tool is available under http://www.expasy.org/tools/myristoylator/myristoylator.html.

Expression of uncoupling protein-3 in subsarcolemmal and intermyofibrillar mitochondria of various mouse muscle types and its modulation by fasting.

Uncoupling protein-3 (UCP3) is a mitochondrial inner-membrane protein abundantly expressed in rodent and human skeletal muscle which may be involved in energy dissipation. Many studies have been performed on the metabolic regulation of UCP3 mRNA level, but little is known about UCP3 expression at the protein level. Two populations of mitochondria have been described in skeletal muscle, subsarcolemmal (SS) and intermyofibrillar (IMF), which differ in their intracellular localization and possibly also their metabolic role. To examine if UCP3 is differentially expressed in these two populations and in different mouse muscle types, we developed a new protocol for isolation of SS and IMF mitochondria and carefully validated a new UCP3 antibody. The data show that the density of UCP3 is higher in the mitochondria of glycolytic muscles (tibialis anterior and gastrocnemius) than in those of oxidative muscle (soleus). They also show that SS mitochondria contain more UCP3 per mg of protein than IMF mitochondria. Taken together, these results suggest that oxidative muscle and the mitochondria most closely associated with myofibrils are most efficient at producing ATP. We then determined the effect of a 24-h fast, which greatly increases UCP3 mRNA (16.4-fold) in muscle, on UCP3 protein expression in gastrocnemius mitochondria. We found that fasting moderately increases (1.5-fold) or does not change UCP3 protein in gastrocnemius SS or IMF mitochondria, respectively. These results show that modulation of UCP3 expression at the mRNA level does not necessarily result in similar changes at the protein level and indicate that UCP3 density in SS and IMF mitochondria can be differently affected by metabolic changes.