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Cocaine treatment paired with environmental cues establishes a conditioned place preference (CPP) for that environment. After expression of this preference, rats show elevated levels of immediate early genes (IEGs; e.g. c-fos) in the prelimbic cortex (PrL), basolateral amygdala complex (BLC), and nucleus accumbens core (NAcc) compared with drug-unpaired controls. These findings, together with the known connections between these regions, suggest that they function as a circuit contributing to cue-elicited craving. To investigate the function of this circuit during drug-seeking, we characterized Fos immunoreactivity of particular neuron classes in each region. To distinguish between IEG activation of GABAergic and non-GABAergic (principally, excitatory projection) neurons, we combined Fos immunohistochemistry with immunohistochemistry for glutamic acid decarboxylase 67 (GAD67) or calcium/calmodulin-dependent protein kinase II (CAMKII) proteins. Within the BLC and NAcc of drug-paired and drug-unpaired animals tested for CPP, we observed no significant differences in the percentage of Fos-immunoreactive (IR) cells that were also GAD67-IR. We also observed no group difference in the degree of Fos/CAMKII overlap in the BLC. However, in PrL, the degree of Fos/GAD67 overlap in the drug-paired group was significantly higher than in the drug-unpaired group. Also, the Fos/CAMKII overlap in the entire PrL as well as just its layer V was significantly lower in the drug-paired animals compared with controls. These findings suggest that, during CPP expression in cocaine-paired animals, the PrL GABAergic interneurons are preferentially activated while PrL output is attenuated, perhaps through greater inhibition of layer V pyramidal neurons. These results suggest a shifting prefrontal cortex cell population response during cocaine-seeking.

The globus pallidus (GP) consists of two neuron populations, distinguished according to their immunoreactivity for parvalbumin (PV). The PV-immunoreactive (PV+) neurons project preferentially to "downstream" targets such as the subthalamic and entopeduncular nuclei, whereas neurons lacking PV (PV- neurons) project preferentially to the striatum, suggesting a role for PV- cells in feedback to striatal neurons. Although dopamine D2 antagonist administration induces immediate early gene expression preferentially in PV- GP neurons, little is known about long-term regulation of PV- versus PV+ GP neurons. Nigral 6-hydroxydopamine (6-OHDA) lesions or repeated D2-class antagonist injections have been shown to increase pallidal expression of glutamate decarboxylase (GAD(67) isoform) mRNA. This increase in GAD(67) is believed to be secondary to activation of excitatory subthalamopallidal projections. The current study examined the effects of subthalamic nucleus (STN) lesion on 6-OHDA- or repeated D2 antagonist-induced changes in GP GAD(67) mRNA expression in PV+ and PV- neurons. Five or 21 d after nigral 6-OHDA injections or after 3, 7, or 21 d of D2 antagonist administration, GAD(67) mRNA increased in both the PV- and PV+ GP neurons, but the magnitude of the increase was significantly greater in PV- neurons. By contrast, STN lesion resulted in declines in GAD(67) mRNA in both cell populations, with the decreases in PV+ neurons exceeding those in PV- neurons. Furthermore, STN lesion completely blocked 6-OHDA- or D2 antagonist-induced GAD(67) mRNA increases in PV+ cells but only partly offset the GAD(67) mRNA increase in PV- pallidal neurons. Thus, the PV+ and PV- neurons are influenced in qualitatively similar ways by dopamine and the STN, but these cell types exhibit contrasting degrees of regulation by the dopaminergic and STN perturbations. This pattern of results has implications for pallidal control of striatal versus downstream basal ganglia nuclei.

The widespread biological actions of the neurotransmitter dopamine (DA) are mediated by two classes of receptor, the D(1) class (D(1) and D(5)) and the D(2) class (D(2), D(3), and D(4)), which interact synergistically in many paradigms, such as DA agonist-stimulated motor behavior and striatal c-fos expression. Understanding the mechanism(s) of this interaction has been impeded by a controversy regarding the cellular localization of D(1) and D(2) class receptors. To address this issue from a functional point of view, we elicited striatal Fos by combined administration of a D(1) class and a D(2) class agonist either in the presence or absence of the fast sodium channel blocker tetrodotoxin (TTX). Striatal Fos elicited by direct D(1)/D(2) stimulation was not reduced by TTX. By contrast, TTX greatly attenuated the Fos response evoked by cocaine or GBR 12909. In separate experiments using antagonists that distinguish among members of the D(2) class of receptors, amphetamine-stimulated Fos and motor behavior were attenuated dose-dependently by the selective D(2) antagonist L-741,626, but not by the selective D(3) antagonist U99194A or the D(4)-selective antagonist L-745,870. Because Fos expression in the paradigms that were used occurs in enkephalin-negative striatonigral neurons, which show limited coexpression of D(1) and D(2) receptors, the present findings taken together suggest the intriguing possibility that D(1)/D(2) synergism may be mediated by D(1) and D(2) receptors residing on separate striatal neurons and interacting in a manner that is not dependent on action potentials.

To examine neuronal activation associated with incentive motivation for cocaine, cocaine-seeking behavior (operant responding without cocaine reinforcement) and Fos expression were examined in rats exposed to saline and cocaine priming injections and/or a self-administration environment. Rats were first trained to self-administer cocaine or received yoked saline administration ("control"). They then received 21 daily exposures to either the self-administration environment ("extinction") or a different environment ("no extinction") without cocaine available. Extinction training, used to decrease incentive motivation for cocaine elicited by the self-administration environment, decreased cocaine-seeking behavior elicited by both the environment and the cocaine priming injection. Exposure to the self-administration environment enhanced Fos expression in the no extinction group relative to control and extinction groups in the anterior cingulate, basolateral amygdala, hippocampal CA1 region, dentate gyrus, nucleus accumbens shell and core, and central gray area, regardless of whether or not priming injections were given. The priming injections enhanced Fos expression in the ventral tegmental area, caudate putamen, substantia nigra pars reticulata, entorhinal cortex, central amygdala, lateral amygdala, arcuate nucleus, and central gray area, regardless of group. Thus, these changes likely reflect an unconditioned effect from either cocaine or injection stress. The priming injections also enhanced Fos expression in the anterior cingulate, but only in cocaine-experienced groups, suggesting that this enhancement reflects an experience-dependent motivational effect of the priming injections. The results suggest that different neural circuits may be involved in the incentive motivational effects of cocaine-paired environmental stimuli versus priming injections and that the anterior cingulate may be part of a common pathway for both.

The modulation of motor behavior by protein kinase C (PKC) signaling pathways in nigrostriatal neurons was examined by using a genetic intervention approach. Herpes simplex virus type 1 (HSV-1) vectors that encode a catalytic domain of rat PKCbetaII (PkcDelta) were developed. PkcDelta exhibited a constitutively active protein kinase activity with a substrate specificity similar to that of rat brain PKC. As demonstrated in cultured sympathetic neurons, PkcDelta caused a long-lasting, activation-dependent increase in neurotransmitter release. In the rat brain, microinjection of HSV-1 vectors that contain the tyrosine hydroxylase promoter targeted expression to dopaminergic nigrostriatal neurons. Expression of pkcDelta in a small percentage of nigrostriatal neurons (approximately 0.1-2%) was sufficient to produce a long-term (>/=1 month) change in apomorphine-induced rotational behavior. Nigrostriatal neurons were the only catecholaminergic neurons that contained PkcDelta, and the amount of rotational behavior was correlated with the number of affected nigrostriatal neurons. The change in apomorphine-induced rotational behavior was blocked by a dopamine receptor antagonist (fluphenazine). D2-like dopamine receptor density was increased in those regions of the striatum innervated by the affected nigrostriatal neurons. Therefore, this strategy enabled the demonstration that a PKC pathway or PKC pathways in nigrostriatal neurons modulate apomorphine-induced rotational behavior, and altered dopaminergic transmission from nigrostriatal neurons appears to be the affected neuronal physiology responsible for the change in rotational behavior.

Parkinson's disease (PD) and Alzheimer's disease (AD) may share certain abnormalities since a subset of PD patients suffer from dementia, and some AD individuals show extrapyramidal symptoms. In vitro quantitative autoradiography was used to examine different subtypes of excitatory amino acid (EAA) receptors (NMDA, KA, and AMPA) and dopamine transporter sites in the striatum (caudate, putamen) and nucleus accumbens (NAc) from idiopathic PD, pure AD, and mixed PD/AD patients. PD and AD groups, and to a lesser extent the PD/AD groups, showed substantially increased binding to NMDA receptors in the striatum and NAc. No statistically significant changes in binding to KA and AMPA receptors were found in any patient group. 3H-mazindol binding to dopamine transporter sites was significantly decreased in the striatum and NAc of PD and PD/AD patients, but only in the putamen and NAc of AD patients. The data indicate that (1) the majority of striatal EAA receptors are not located on dopaminergic nigrostriatal nerve terminals, and (2) elevated binding to striatal NMDA receptors correlates with binding to dopamine transporter sites in PD patients, but not in AD and PD/AD individuals. Thus, the mechanisms of NMDA receptor changes in the striatum of AD and PD patients may be different. However, it is postulated that increased binding to NMDA receptors in Parkinson and Alzheimer striatum occurs in response to an insult(s) within the striatothalamocortical circuits and that this may contribute to the clinical similarities described for subsets of PD and AD patients.

The regulation of hippocampal muscarinic M1 and M2 receptors was studied by autoradiographic methods following cholinergic denervation and reinnervation from embryonic septal transplant. In young adult male rats the density of M1 sites, labeled either with 3H-pirenzepine (PZ) or 3H-N-methylscopolamine (NMS, in the presence of excess carbachol), exceeded by 4- to 5-fold the density of M2 sites, labeled with 3H-NMS in the presence of excess PZ. Both receptors appeared to be densest in hippocampal regions lowest in acetylcholinesterase or 3H-hemicholinium-3 binding. The distribution of M1 receptors did differ from the distribution of M2 receptors within subregions of the hippocampus. Along the mediolateral axis from the subiculum to the lateral CA 1, the density of M1 receptors is uniform, but the density of M2 receptors decreases. Also apparent is the relatively small difference in density between the CA1 and dentate gyrus for M1 receptors but a significantly greater difference for M2 receptors. However, the response of M1 and M2 receptors to long-term cholinergic denervation following fimbriafornix transection of the septal cholinergic input and to cholinergic innervation by embryonic septal transplants was similar. Long-term denervation (40-60 d) resulted in a 30-60% increase in both M1 and M2 receptors within regions of the hippocampal formation. Receptor levels were reduced to normal in regions innervated by septal transplants. For both receptors, the changes in the density of sites were due to alterations in the Bmax and not the Kd for the radioligands. The specificity of this regulation is supported by the evidence that (1) the degree and topography of the normalization of muscarinic receptor density was entirely dependent on the degree and pattern of cholinergic reinnervation by the fibers of the septal transplant, (2) cholinergic fiber reinnervation by embryonic striatal grafts also down-regulated the density of M1 and M2 receptors, and (3) successfully surviving transplants (e.g., cerebellar and striatal) that did not provide innervation to the hippocampus did not induce down-regulation of muscarinic receptors. Changes in the density of sites were not related to changes in the width of the hippocampus following denervation and reinnervation. The data support the view that the majority of M1 and M2 receptors are located postsynaptically on neurons within the hippocampus and not presynaptically on cholinergic fibers.