Catherine Le Moine

Distribution, biochemistry and function of striatal adenosine A2A receptors

It is well known that the nucleoside adenosine exerts a modulatory influence in the central nervous system by activating G protein coupled receptors. Adenosine A2A receptors, the subject of the present review, are predominantly expressed in striatum, the major area of the basal ganglia. Activation of A2A receptors interferes with effects mediated by most of the principal neurotransmitters in striatum. In particular, the inhibitory interactions between adenosine acting on A2A receptors and dopamine acting on D2 receptors have been well examined and there is much evidence that A2A receptors may be a possible target for future development of drugs for treatment of Parkinsons disease, schizophrenia and affective disorders. Our understanding of the role of striatal A2A receptors has increased dramatically over the last few years. New selective antibodies, antagonist radioligands and optimized in situ hybridization protocols have provided detailed information on the distribution of A2A receptors in rodent as well as primate striatum. Studies on the involvement of A2A receptors in the regulation of DARPP-32 and the expression of immediate early genes, such as nerve growth factor-induced clone A and c-fos, have pointed out an important role for these receptors in regulating striatopallidal neurotransmission. Moreover, by using novel selective antagonists for A2A receptors and transgenic mice lacking functional A2A receptors, crucial information on the behavioral role of striatal A2A receptors has been provided, especially concerning their involvement in the stimulatory action of caffeine and the anti-Parkinsonian properties of A2A receptor antagonists. In the present review, current knowledge on the distribution, biochemistry and function of striatal A2A receptors is summarized.

Cortical Inputs and GABA Interneurons Imbalance Projection Neurons in the Striatum of Parkinsonian Rats

The striatum receives massive cortical excitatory inputs and is densely innervated by dopamine. Striatal projection neurons form either the direct or indirect pathways. Models of Parkinsons disease propose that dopaminergic degeneration imbalances both pathways, although direct electrophysiological evidence is lacking. Here, striatal neurons were identified by electrophysiological criteria and Neurobiotin labeling combined with either immunohistochemistry or in situ hybridization. Their spontaneous discharge activity and spike response to cortical stimulation were recorded in vivo in anesthetized rats rendered hemi-parkinsonian by 6-hydroxydopamine. We showed that striatonigral neurons (direct pathway) were inhibited whereas striatopallidal neurons (indirect pathway) were activated by dopaminergic lesion. We also identified, with antidromic stimulations, corticostriatal neurons that preferentially innervate striatonigral or striatopallidal neurons and showed that dopaminergic depletion selectively decreased the spontaneous activity of the former. Therefore, dopamine degeneration induces a cascade of imbalances that spread out of the basal ganglia and affect the whole basal ganglia-thalamo-cortical circuits. Fast-spiking GABA interneurons provide potent feedforward inhibition of striatal projection neurons. We showed here that these interneurons narrowed the time window of the responses of projection neurons to cortical stimulation. In the dopamine-depleted striatum, because the intrinsic activity of these interneurons was not altered, their feedforward inhibition worsened the striatal imbalance. Indeed, the time window of the evoked responses was narrower for striatonigral neurons and wider for striatopallidal neurons. Therefore, after dopaminergic depletion, cortical inputs and GABA interneurons might imbalance striatal projection neurons and represent two novel nondopaminergic mechanisms that might secondarily contribute to the pathophysiology of Parkinsons disease.

Feedforward Inhibition of Projection Neurons by Fast-Spiking GABA Interneurons in the Rat Striatum In Vivo

Discharge activities and local field potentials were recorded in the orofacial motor cortex and in the corresponding rostrolateral striatum of urethane-anesthetized rats. Striatal projection neurons were identified by antidromic activation and fast-spiking GABAergic interneurons (FSIs) by their unique characteristics: briefer spike and burst responses. Juxtacellular injection of neurobiotin combined with parvalbumin immunohistochemistry validated this identification. Spontaneous activities and spike responses to cortical stimulation were recorded during both states of cortical activity: slow waves and desynchronization. Both FSI and projection neurons spontaneously discharged synchronously with slow waves at the maximum of cortical activity, but, on average, FSIs were much more active. Cortical desynchronization enhanced FSI activity and facilitated their spike responses to cortical stimulation, whereas opposite effects were observed regarding projection neurons. Experimental conditions favoring FSI discharge were always associated with a decrease in the firing activity of projection neurons. Spike responses to cortical stimulation occurred earlier (latency difference, 4.6 ms) and with a lower stimulation current for FSIs than for projection neurons. Moreover, blocking GABAA receptors by local picrotoxin injection enhanced the spike response of projection neurons, and this increase was larger in experimental conditions favoring FSI responses. Therefore, on average, FSIs exert in vivo a powerful feedforward inhibition on projection neurons. However, a few projection neurons were actually more sensitive to cortical stimulation than FSIs. Moreover, picrotoxin, which revealed FSI inhibition, preferentially affected projection neurons exhibiting the weakest sensitivity to cortical stimulation. Thus, feedforward inhibition by FSIs filters cortical information effectively transmitted by striatal projection neurons.

D1 and D2 Receptor Gene Expression in the Rat Frontal Cortex: Cellular Localization in Different Classes of Efferent Neurons

The dopaminergic input to the frontal cortex has an important role in motor and cognitive functions. These effects are mediated by dopamine receptors both of type D1 and of type D2, although the neural circuits involved are not completely understood. We used in situ hybridization to determine the cellular localization of D1 and D2 receptor mRNAs in the rat frontal cortex. Retrograde tracing was used in the same animals to identify the main cortical efferent populations. Fluorogold was injected into the different cortical targets of the frontal cortex and sections were hybridized with D1 and D2 35S‐labelled cRNA probes. D1 and D2 mRNA‐containing neurons were present in all the cortical areas investigated, with greater expression in the medial prefrontal, insular and cingulate cortexes and lower expression in the motor and parietal cortexes. Neurons containing D1 mRNA were most abundant in layer Vlb; they were also present in layers Vla and V of all cortical layers and in layer II of the medial prefrontal, cingulate and insular areas. Double labelling with fluorogold demonstrated that D1 mRNA was present in corticocortical, corticothalamic and corticostriatal neurons. Neurons containing D2 mRNA were essentially restricted to layer V, but only in corticostriatal and corticocortical neurons. Neither D1 nor D2 mRNA was found in corticospinal or corticopontine neurons. The present results demonstrate that D1 and D2 receptor genes are expressed in efferent cortical populations, with higher expression for D1. In spite of an overlap in some cortical layers, the expression of D1 and D2 receptor genes is specific for different categories of pyramidal neurons.

Neural correlates of the motivational and somatic components of naloxone-precipitated morphine withdrawal.

In morphine‐dependent rats, low naloxone doses have been shown to induce conditioned place aversion, which reflects the negative motivational component of opiate withdrawal. In contrast, higher naloxone doses are able to induce a ‘full’ withdrawal syndrome, including overt somatic signs. The c‐fos gene is commonly used as a marker of neuronal reactivity to map the neural substrates that are recruited by various stimuli. Using in situ hybridization, we have analysed in the brain of morphine‐dependent rats the effects of acute withdrawal syndrome precipitated by increasing naloxone doses on c‐fos mRNA expression. Morphine dependence was induced by subcutaneous implantation of slow‐release morphine pellets for 6 days and withdrawal was precipitated by increasing naloxone doses inducing the motivational (7.5 and 15 µg/kg) and somatic (30 and 120 µg/kg) components of withdrawal. Our mapping study revealed a dissociation between a set of brain structures (extended amygdala, lateral septal nucleus, basolateral amygdala and field CA1 of the hippocampus) which exhibited c‐fos mRNA dose‐dependent variations from the lowest naloxone doses, and many other structures (dopaminergic and noradrenergic nuclei, motor striatal areas, hypothalamic nuclei and periaqueductal grey) which were less sensitive and recruited only by the higher doses. In addition, we found opposite dose‐dependent variations of c‐fos gene expression within the central (increase) and the basolateral (decrease) amygdala after acute morphine withdrawal. Altogether, these results emphasize that limbic structures of the extended amygdala along with the lateral septal nucleus, the basolateral amygdala and CA1 could specifically mediate the negative motivational component of opiate withdrawal.

Chronic morphine exposure and spontaneous withdrawal are associated with modifications of dopamine receptor and neuropeptide gene expression in the rat striatum

The influence of chronic morphine and spontaneous withdrawal on the expression of dopamine receptors and neuropeptide genes in the rat striatum was investigated. Morphine dependence was induced by subcutaneous implantation of two morphine pellets for 6 days. Rats were made abstinent by removal of the pellets 1, 2 or 3 days before they were killed. The mRNA levels coding for D1‐ and D2‐dopamine receptors, dynorphin, preproenkephalin A and substance P were determined by quantitative in situ hybridization. The caudate putamen and the nucleus accumbens showed equivalent modifications in dopamine receptor and neuropeptide gene expression. After 6 days of morphine, a decrease in D2‐dopamine receptor and neuropeptide mRNA levels was observed (– 30%), but there was no change in D1‐dopamine receptor mRNA. In abstinent rats, both D1‐ and D2‐dopamine receptor mRNA levels were decreased 1 day after withdrawal (– 30% compared with chronic morphine). In contrast, neuropeptide mRNA levels were unaffected when compared with those observed after 6 days of morphine. During the second and third day of withdrawal, there was a gradual return to the levels seen in the placebo‐treated group, for both dopamine receptor and neuropeptide mRNAs. Phenotypical characterization of striatal neurons expressing μ and κ opioid receptor mRNAs showed that, in striatonigral neurons, both mRNAs were colocalized with D1‐receptor and Dyn mRNAs. Our results suggest that during morphine dependence, dopamine and morphine exert opposite effects on striatonigral neurons, and that effects occurring on striatopallidal neurons are under dopaminergic control. We also show that withdrawal is associated with a down regulation of the postsynaptic D1 and D2 receptors.

Dopamine–Adenosine Interactions in the Striatum and the Globus Pallidus: Inhibition of Striatopallidal Neurons through Either D2 or A2A Receptors Enhances D1Receptor-Mediated Effects on c-fos Expression

D1 receptors located on striatonigral neurons and D2 receptors located, together with A2Areceptors, on striatopallidal neurons are known to interact functionally. Using in situ hybridization, we examined the effects of D1 and D2 agonists and of an A2A antagonist on c-fos mRNA in identified striatal neurons and in globus pallidus. The full D1agonist, SKF 82958 (1 mg/kg), induced a homogenous increase ofc-fos mRNA in the striatum. This increase occurred to a similar extent in D1 and D2 receptor-containing striatal neurons. Conversely, the D2 agonist, quinelorane (2 mg/kg), decreased c-fos mRNA in these populations but increased it in globus pallidus. The adenosine A2A receptor antagonist, SCH 58261 (5 mg/kg), also decreased c-fosmRNA in D2 receptor-containing neurons in striatum but did not affect pallidal c-fos mRNA. Concomitant administration of either D1 plus D2 agonists or D1 agonist plus A2A antagonist caused a potentiation of c-fos mRNA in striatal neurons expressing the D1 receptor and in globus pallidus. However, only the combination of D1 and D2 agonists modified the c-fos mRNA expression to a “patchy” distribution. Our data show that (1) c-fos expression can be activated through D1 and inhibitedthrough A2A or D2 receptors in both striatal output pathways in normal rats, and (2) D2 receptor stimulation as well as A2A receptor blockade can interact with D1 receptor activation to potentiatec-fos expression in the striatum and the globus pallidus. The data also suggest that the topological alteration ofc-fos expression after coadministration of D1 and D2 agonists involves D2receptors located on interneurons or presynaptically on dopaminergic nerve terminals.

Subpopulations of cortical GABAergic interneurons differ by their expression of D1 and D2 dopamine receptor subtypes

D1 and D2 receptors have been described in different populations of efferent pyramidal neurons of the rat frontal cortex. Combined in situ hybridization and immunocytochemistry show here that these two subtypes are expressed in cortical GABAergic interneurons, with D1 and D2 mainly found in a subpopulation containing parvalbumin, whereas only 10% of the calbindin neurons express D1 receptors. These data indicate that various DA agonists may influence inhibitory circuits by distinct dopamine receptor subtypes.

A Specific Limbic Circuit Underlies Opiate Withdrawal Memories

Compulsive drug-seeking behavior and its renewal in former drug addicts is promoted by several situations, among which reactivation of drug withdrawal memories plays a crucial role. A neural hypothesis is that such memories reactivate the circuits involved in withdrawal itself and promote a motivational state leading to drug seeking or taking. To test this hypothesis, we have analyzed the neural circuits and cell populations recruited when opiate-dependent rats are reexposed to stimuli previously paired with withdrawal (memory retrieval) and compared them with those underlying acute withdrawal during conditioning (memory formation). Using in situ hybridization for c-fos expression, we report here that reexposure to a withdrawal-paired environment induced conditioned c-fos responses in a specific limbic circuit, which can be partially dissociated from the structures involved in acute withdrawal. At the amygdala level, c-fos responses were doubly dissociated between the central and basolateral (BLA) nuclei, when comparing the two situations. Detailed phenotypical analyses in the amygdala and ventral tegmental area (VTA) show that specific subpopulations in the BLA are differentially involved in the formation and retrieval of withdrawal memories, and strikingly that a population of VTA dopamine neurons is activated in both situations. Together, this indicates that withdrawal memories can drive activity changes in specific neuronal populations of interconnected limbic areas known to be involved in aversive motivational processes. This first study on the neural substrates of withdrawal memories strongly supports an incentive-motivational view of withdrawal in opiate addiction that could be crucial in compulsive drug seeking and relapse.

No Effect of Morphine on Ventral Tegmental Dopamine Neurons during Withdrawal

Substantial evidence indicates that the ventral tegmental area (VTA) of the mesocorticolimbic dopaminergic (DA) system has a key role in mechanisms of opiate dependence. Although DA neurons have been studied extensively, little is known about their activity and their response to acute morphine during morphine dependence. We recorded the activity of VTA DA neurons in five groups of anesthetized rats: drug-naive (naive) rats, morphine-dependent [(MD) implanted with pellets] rats, and three groups of withdrawn rats. Withdrawals either were precipitated by naltrexone or occurred spontaneously 24 h or 15 d after pellet removal. We confirmed that acute morphine in naive rats produced a marked increase in the firing of VTA DA neurons. We also found that the basal firing rate of VTA DA neurons was markedly higher in MD than in naive rats; however, in MD rats, acute morphine failed to increase DA activity. We confirmed inhibition of VTA DA activity in MD rats in response to precipitated withdrawal; however, this inhibition resulted only in a normalization of the firing rate to that of naive animals. In rats that had spontaneous withdrawal after 24 h or 15 d, the activity of VTA DA neurons was similar to that of naive rats, and an acute injection of morphine failed to alter their activity. Our results indicate that VTA DA neurons show long-lasting tolerance to the acute effect of morphine after withdrawal. These findings show that VTA DA neural activity is unlikely to be a factor in the altered behavioral responses that occur with acute morphine or naltrexone administration after chronic opiate exposure.