The ventral tegmental area (VTA) is the source of dopaminergic projections innervating cortical structures and ventral forebrain. Dysfunction of this mesocorticolimbic system is critically involved in psychiatric disorders such as addiction and schizophrenia. Changes in VTA dopamine (DA) neuronal activity can alter neurotransmitter release at target regions which modify information processing in the reward circuit. Here we studied the effect of α-2 noradrenergic receptor activation on the hyperpolarization-activated cation current (I h ) in DA neurons of the rat VTA. Brain slice preparations using whole-cell current and voltage-clamp techniques were employed. Clonidine and UK14304 (α-2 receptor selective agonists) were found to decrease I h amplitude and to slow its rate of activation indicating a negative shift in the current's voltage dependence. Two non-subtypeselective α-2 receptor antagonists, yohimbine and RS79948, prevented the effects of α-2 receptor activation. RX821002, a noradrenergic antagonist specific for α-2A and α-2D did not prevent I h inhibition. This result suggests that clonidine might be acting via an α-2C subtype since this receptor is the most abundant variant in the VTA. Analysis of a second messenger system associated with the α-2 receptor revealed that I h inhibition is independent of cyclic adenosine monophosphate (cAMP) and resulted from the activation of protein kinase C. It is suggested that the α-2 mediated hyperpolarizing shift in I h voltage dependence can facilitate the transition from pacemaker firing to afferent-driven burst activity. This transition may play a key role on the changes in synaptic plasticity that occurs in the mesocorticolimbic system under pathological conditions.
The ventral tegmental area (VTA) forms part of the mesocorticolimbic system and plays a pivotal role in reward and reinforcing actions of drugs of abuse. Glutamate transmission within the VTA controls important aspects of goal-directed behavior and motivation. Noradrenergic receptors also present in the VTA have important functions in the modulation of neuronal activity. Here we studied the effects of alpha-2 noradrenergic receptor activation in the alteration of glutamate neurotransmission in VTA dopaminergic neurons from male Sprague-Dawley rats. We used whole cell patch clamp recordings from putative VTA dopaminergic neurons and measured excitatory postsynaptic currents. Clonidine (40 μM) and UK 13,408 (40 μM), both alpha-2 receptor agonists, reduced (~ 40%) the amplitude of glutamate-induced excitatory postsynaptic currents. After clonidine administration, there was a dose-dependent reduction over the concentration range of 15–40 μM. Using yohimbine (20μM) and two other alpha-2 adrenergic receptor antagonists, idaxozan (40 μM) and atipemazole (20μM), we demonstrated that the inhibitory action is specifically mediated by alpha-2 receptors. Moreover, by inhibiting protein kinases with H-7 (75 μM), Rp-adenosine 3′,5′-cyclic (11 μM) and chelerythrine (1 μM) it was shown that the clonidine-induced inhibition seems to involve a selective activation of the protein kinase C intracellular pathway. An increased paired-pulse ratios and changes in spontaneous and miniature excitatory postsynaptic currents frequencies but not amplitudes indicated that the alpha-2 agonist’s effect was presynaptically mediated. It is suggested that the suppression of glutamate excitatory inputs onto VTA dopaminergic neurons might be relevant in the regulation of reward and drug seeking behaviors.
Alteration of the biological activity among neuronal components of the Mesocorticolimbic (MCL) system has been implicated in the pathophysiology of drug abuse. Changes in the electrophysiological properties of neurons involved in the reward circuit seem to be of utmost importance in addiction. The Hyperpolarization-Activated Cyclic-Nucleotide Current, Ih, is a prominent mixed cation current present in neurons. The biophysical properties of the Ih and its potential modulatory role in cell excitability depend on the expression profile of the Hyperpolarization-activated cyclic nucleotide gated channel (HCN) subunits. We investigated whether cocaine-induced behavioral sensitization, an animal model of drug addiction, elicits region-specific changes in the expression of the HCN2 channel’s subunit in the MCL system. Tissue samples from the ventral tegmental area, prefrontal cortex, nucleus accumbens and hippocampus were analyzed using Western Blot. Our findings demonstrate that cocaine treatment induced a significant increase in the expression profile of the HCN2 subunit in both, its glycosylated and non-glycosylated protein isoforms, in all areas tested. The increase in the glycosylated isoform was only observed in the ventral tegmental area. Together, these data suggest that the observed changes in MCL excitability during cocaine addiction might be associated to alterations in the subunit composition of their HCN channels.
Background: N-methyl-D-aspartate (NMDA) receptors regulate synaptic plasticity and modulate a wide variety of behaviors. Mammalian NMDA receptors are inhibited by ethanol (EtOH) even at low concentrations. In mice, the F639A mutation in transmembrane domain (TMD) 3 of the NR1 subunit reduces EtOH sensitivity of the receptor and, in some paradigms, reduces behavioral EtOH sensitivity and increases EtOH consumption. We tested the fly equivalent of the F639A and K544Q mutations for effects on EtOH sensitivity. Drosophila shows a high degree of behavioral and mechanistic conservation in its responses to EtOH.Methods: Homologous recombination and CRISPR/Cas9 genome editing were used to generate amino acid changes in the Drosophila NMDAR1 gene, yielding F654A and K558Q alleles. Animals were tested for the degree of EtOH sensitivity, the ability to acquire tolerance to EtOH, EtOH drinking preference, circadian rhythmicity, learning, and locomotor defects.Results: We observed that mutating the NMDAR1 channel also reduces EtOH sensitivity in adult flies. However, in flies, it was the K558Q mutation (orthologous to K544Q in mice) that reduces EtOH sensitivity in a recovery-from-sedation assay. The effects of the F654A mutation (orthologous to F639A in mice) were substantially different in flies than in mammals. In flies, F654A mutation produces phenotypes opposite those in mammals. In flies, the mutant allele is homozygous viable, does not seem to affect health, and increases EtOH sensitivity. Both mutations increased feeding but did not alter the animal's preference for 5% EtOH food. F654A depressed circadian rhythmicity and the capacity of males to court, but it did not depress the capacity for associative learning. K554Q, on the other hand, has little effect on circadian rhythmicity, only slightly suppresses male courtship, and is a strong learning mutant.Conclusions: Mutations in TMD 3 and in the extracellular-vestibule calcium-binding site of the NR1 NMDA subunit affect EtOH sensitivity in Drosophila.
Chronic cocaine use produces long-lasting changes in reward circuits that may underlie the transition from casual to compulsive patterns of drug use. Although strong neuroadaptations within the mesocorticolimbic system are known to occur, the specific role of these drug-induced plasticities on sensitization remains to be elucidated. Here we investigate whether PKMζ, a protein involved in maintaining long-term potentiation (LTP), plays a role in these cocaine-induced changes in synaptic strengthening. We performed whole-cell voltage clamp recordings of putative ventral tegmental area (VTA) dopamine (DA) cells 24 hours after five days of 15 mg/kg i.p. cocaine or isovolumetric saline injections. We observed that superfusion of 5µM ZIP (PKMζ inhibitory peptide) decreased AMPA currents and AMPA/NMDA ratios only in cocaine sensitized rats. In vivo ZIP microinfusions (10 nmol) into the VTA after cocaine sensitization decreased locomotor activity on a subsequent cocaine challenge only if given ZIP is given before the withdrawal period. On the other hand, ZIP microinfusions into the nucleus accumbens (NAc) core after a seven days withdrawal period disrupt the expression of locomotor sensitization. The present data provide a potentially relevant region, and time-specific PKMζ-dependent brain mechanism that enables sensitization. Our results support the vision that addiction involves a pathological learning process. They imply that if this synaptic strengthening is reversed, changes in the behavioral response may also be overturned.
The ventral tegmental area (VTA) plays an important role in the reward and motivational processes that facilitate the development of drug addiction. Presynaptic α1-AR activation modulates glutamate and Gamma-aminobutyric acid (GABA) release. This work elucidates the role of VTA presynaptic α1-ARs and their modulation on glutamatergic and GABAergic neurotransmission during cocaine sensitization. Excitatory and inhibitory currents (EPSCs and IPSCs) measured by a whole cell voltage clamp show that α1-ARs activation increases EPSCs amplitude after 1 day of cocaine treatment but not after 5 days of cocaine injections. The absence of a pharmacological response to an α1-ARs agonist highlights the desensitization of the receptor after repeated cocaine administration. The desensitization of α1-ARs persists after a 7-day withdrawal period. In contrast, the modulation of α1-ARs on GABA neurotransmission, shown by decreases in IPSCs’ amplitude, is not affected by acute or chronic cocaine injections. Taken together, these data suggest that α1-ARs may enhance DA neuronal excitability after repeated cocaine administration through the reduction of GABA inhibition onto VTA dopamine (DA) neurons even in the absence of α1-ARs’ function on glutamate release and protein kinase C (PKC) activation. α1-AR modulatory changes in cocaine sensitization increase our knowledge of the role of the noradrenergic system in cocaine addiction and may provide possible avenues for therapeutics.
Cocaine-induced psychomotor stimulation may be mediated by metabolic hypofrontality and modification of brain functional connectivity. Functional connectivity refers to the pattern of relationships among brain regions, and one way to evaluate this pattern is using interactivity correlations of the metabolic marker cytochrome oxidase among different regions. This is the first study of how repeated cocaine modifies: (1) mean cytochrome oxidase activity in neural areas using quantitative enzyme histochemistry, and (2) functional connectivity among brain regions using inter-correlations of cytochrome oxidase activity. Rats were injected with 15 mg/kg i.p. cocaine or saline for 5 days, which lead to cocaine-enhanced total locomotion. Mean cytochrome oxidase activity was significantly decreased in cocaine-treated animals in the superficial dorsal and lateral frontal cortical association areas Fr2 and Fr3 when compared to saline-treated animals. Functional connectivity showed that the cytochrome oxidase activity of the noradrenergic locus coeruleus and the infralimbic cortex were positively inter-correlated in cocaine but not in control rats. Positive cytochrome oxidase activity inter-correlations were also observed between the dopaminergic substantia nigra compacta and Fr2 and Fr3 areas and the lateral orbital cortex in cocaine-treated animals. In contrast, cytochrome oxidase activity in the interpeduncular nucleus was negatively correlated with that of Fr2, anterior insular cortex, and lateral orbital cortex in saline but not in cocaine groups. After repeated cocaine specific prefrontal areas became hypometabolic and their functional connectivity changed in networks involving noradrenergic and dopaminergic brainstem nuclei. We suggest that this pattern of hypofrontality and altered functional connectivity may contribute to cocaine-induced psychomotor stimulation.
Chronic cocaine exposure produces enduring neuroadaptations in the brain’s reward system. Persistence of early cocaine evoked neuroadaptations in the ventral tegmental area (VTA) are necessary for later synaptic alterations in the nucleus accumbens (NAc), suggesting a temporal sequence of neuroplastic changes between these two areas. However, the molecular nature of the signal that mediates this sequential event is unknown. Here we used the behavioral sensitization model and the aPKC inhibitor of late-phase LTP maintenance, ZIP, to investigate if a persistent increase in AMPA/NMDA ratio plays a role in the molecular mechanism that allows VTA neuroadaptations to induce changes in the NAc. Results showed that intra-VTA ZIP microinfusion successfully blocked cocaine evoked synaptic enhancement in the VTA and the expected AMPA/NMDA ratio decrease in the NAc following cocaine sensitization. ZIP microinfusions also blocked the expected AMPA/NMDA ratio increase in the NAc following cocaine withdrawal. These results suggest that a persistent increase in AMPA/NMDA ratio, mediated by aPKC’s, could be the molecular signal that enables the VTA to elicit synaptic alterations in the NAc following cocaine administration.
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