High-affinity neurotensin receptors in the rat nucleus accumbens: subcellular targeting and relation to endogenous ligand.

Neurotensin is present in selective mesolimbic dopaminergic projections to the nucleus accumbens (NAc) shell but also is synthesized locally in this region and in the motor-associated NAc core. We examined the electron microscopic immunolabeling of the high-affinity neurotensin receptor (NTR) and neurotensin in these subdivisions of rat NAc to determine the sites for receptor activation and potential regional differences in distribution. Throughout the NAc, NTR immunoreactivity was localized discretely within both neurons and glia. NTR-labeled neuronal profiles were mainly axons and axon terminals with diverse synaptic structures, which resembled dopaminergic and glutamatergic afferents, as well as collaterals of inhibitory projection neurons. These terminals had a significantly higher numerical density in the NAc core than in the shell but were prevalent in both regions, suggesting involvement in both motor and limbic functions. In each region, neurotensin was detected in a few NTR-immunoreactive axon terminals and in terminals that formed symmetric, inhibitory type synapses with NTR-labeled somata and dendrites. The NTR labeling, however, was not seen within these synapses and, instead, was localized to segments of dendritic and glial plasma membranes often near excitatory type synapses. Neuronal NTR immunoreactivity also was associated with cytoplasmic tubulovesicles and nuclear membranes. Our results suggests that, in the NAc shell and core, NTR is targeted mainly to presynaptic sites, playing a role in the regulated secretion and/or retrograde signaling in diverse, neurotransmitter-specific neurons. The findings also support a volume mode of neurotensin actions, specifically affecting excitatory transmission through activation of not only axonal but also dendritic and glial NTR.

Ultrastructural immunocytochemical localization of the dopamine D2 receptor within GABAergic neurons of the rat striatum.

Classical antipsychotics, which block dopamine (DA) D2 receptors, showing intrastriatal variation in their effectiveness in modulating GABAergic function. To determine the cellular basis for such differences, we examined the electron microscopic immunocytochemical labeling of D2 receptors and GABA in the dorsolateral caudate-putamen (CPn) and the nucleus accumbens (Acb) shell. In both regions, peroxidase reaction product and gold-silver deposits representing D2 receptor immunoreactivity (D2-IR) and GABA immunoreactivity (GABA-IR), respectively, were detected in dendrites and perikarya having characteristics of either spiny projection neurons or aspiny interneurons. Some perikarya in both regions are dually labeled with D2-IR and GABA-IR. Neurons axon terminals in each region also contained one or both markers. However, there were notable regional differences in the immunolabeling patterns. In the CPn, D2-IR was more commonly seen in dendrites/spines than in axon terminals, and proportionally more dendrites were dually labeled than in the Acb. In the Acb shell, D2-IR was detected with similar frequency in terminals and dendrites/spines, but more terminals co-localized D2-IR and GABA-IR in this region compared with the CPn. These results provide the first ultrastructural evidence for direct D2-mediated effects of DA on striatal GABAergic neurons. They further suggest that modulation of GABAergic neurons by DA acting at D2 receptors may be relatively more postsynaptic in the CPn, but more presynaptic in the Acb shell.

Modifications of the Brandel Superfusion 600 system and other revisions in superfusion technique improve the measurement of endogenous dopamine release in vitro.

The Brandel Superfusion 600 is an automated system designed for the simultaneous measurement of neurotransmitter release from tissue slices from six chambers. In optimizing a technique for the measurement of endogenous dopamine (DA) with this system, a number of factors were identified which affected adversely both the accuracy and the precision of within and between experiments. These factors included bubbles in the perfusion chamber, an apparent loss of tissue viability during preparation of the slices, depletion of DA stores after multiple stimulations and degradation of DA in the perfusate after collection. The method was substantially improved with modifications in the Superfusion 600 system and by the addition of high Mg2+ to the preparation buffer. The addition of tyrosine to the perfusion buffer and the prevention of analyte degradation with antioxidant agents also improved accuracy and precision of measurements.

Ultrastructural immunocytochemical localization of neurotensin and the dopamine D2 receptor in the rat nucleus accumbens.

The neuroleptic-like effects of neurotensin (NT) are thought to be due to interactions with dopamine (DA) acting primarily at D2 receptors within the nucleus accumbens septi (Acb). Using electron microscopic dual labeling immunocytochemistry, we sought to demonstrate cellular substrates for functional interactions involving NT and DA D2 receptors in the adult rat Acb. Peroxidase reaction product representing D2 receptor-like immunoreactivity (D2-LI) was seen along membranes of Golgi lamellae and multivesicular bodies of perikarya containing immunogold labeling representing NT-LI. Dually labeled somata usually contained highly indented nuclei, a characteristic of aspiny neurons. Dendrites also occasionally colocalized the two immunomarkers. Other somata, dendrites, and all axon terminals were singly labeled with either NT-LI or D2-LI. In distinct sets of terminals, NT-LI was commonly associated with large, dense-cored vesicles, whereas D2-LI was found along the plasmalemma and over nearby small clear vesicles. Each type of terminal comprised approximately 20% of synaptic input to NT-immunoreactive dendrites. Similar proportions of terminals containing NT-LI or D2-LI contacted unlabeled (approximately 55%) or NT-labeled (approximately 35%) dendrites and, occasionally, were observed converging onto common dendrites. Terminals containing NT-LI or D2-LI also were often closely apposed. These findings provide the first ultrastructural evidence that: (1) NT and D2 receptors are colocalized in aspiny neurons and dendrites, (2) NT may produce a direct postsynaptic effect on neurons receiving input from terminals which are presynaptically modulated by DA via D2 receptors, and (3) NT and DA acting at D2 receptors may interact through presynaptic modulation of common axon terminals.

Protection against methamphetamine-induced neurotoxicity to neostriatal dopaminergic neurons by adenosine receptor activation.

Methamphetamine (METH)-induced neurotoxicity to nigrostriatal dopaminergic neurons in experimental animals appears to have a glutamatergic component because blockade of N-methyl-D-aspartate receptors prevents the neuropathologic consequences. Because adenosine affords neuroprotection against various forms of glutamate-mediated neuronal damage, the present studies were performed to investigate whether adenosine plays a protective role in METH-induced toxicity. METH-induced decrements in neostriatal dopamine content and tyrosine hydroxylase activity in mice were potentiated by concurrent treatment with caffeine, a nonselective adenosine antagonist that blocks both A1 and A2 adenosine receptors. In contrast, chronic treatment of mice with caffeine through their drinking water for 4 weeks, which increased the number of adenosine A1 receptors in the neostriatum and frontal cortex, followed by drug washout, prevented the neurochemical changes produced by the treatment of mice with METH treatment. In contrast, this treatment did not prevent 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine-induced dopaminergic neurotoxicity. Furthermore, concurrent administration of cyclopentyladenosine, an adenosine A1 receptor agonist, attenuated the METH-induced neurochemical changes. This protection by cyclopentyladenosine was blocked by cyclopentyltheophylline, an A1 receptor antagonist. These results indicate that activation of A1 receptors can protect against METH-induced neurotoxicity in mice.