Haloperidol but Not Clozapine Increases Neurotensin Receptor mRNA Levels in Rat Substantia Nigra

Abstract: We examined the effects of chronic (2 weeks) treatment with a typical neuroleptic, haloperidol (1 mg/kg, s.c.), and an atypical neuroleptic, clozapine (20 mg/kg, s.c.), on neurotensin receptor (NTR) mRNA levels by in situ hybridization histochemistry. Quantitative OD analysis showed haloperidol-induced NTR mRNA levels in the substantia nigra/ventral tegmental area (SN/ VTA) 110% over control levels (significant difference from the control, p < 0.05). In contrast, the same analysis applied to the sections from clozapine-treated animals showed no significant change in NTR mRNA levels compared with matched control sections ( p > 0.05). Thus, chronic treatment with haloperidol but not clozapine resulted in elevated levels of NTR mRNA within the SN/VTA. These results suggest that the high incidence of extrapyramidal side effects of typical neuroleptics could result from changes in NTR expression in the SN/VTA.     

Alterations in ionotropic glutamate receptor subunits during binge cocaine self-administration and withdrawal in rats

Chronic cocaine use in humans and animal models is known to lead to pronounced alterations in glutamatergic function in brain regions associated with reinforcement. Previous studies have examined ionotropic glutamate receptor (iGluR) subunit protein level changes following acute and chronic experimenter-administered cocaine or after withdrawal periods from experimenter-administered cocaine. To evaluate whether alterations in expression of iGluRs are associated with cocaine reinforcement, protein levels were assessed after binge (8 h/day, 15 days; 24-h access, days 16-21) cocaine self-administration and following 2 weeks of abstinence from this binge. Western blotting was used to compare levels of iGluR protein expression (NR1-3B, GluR1-7, KA2) in the ventral tegmental area (VTA), substantia nigra (SN), nucleus accumbens (NAc), striatum and prefrontal cortex (PFC) of rats. iGluR subunits were altered in a time-dependent manner in all brain regions studied; however, selective alterations in certain iGluR subtypes appeared to be associated with binge cocaine self-administration and withdrawal in a region-specific manner. In the SN and VTA, alterations in iGluR protein levels compared with controls occurred only following binge access, whereas in the striatum and PFC, iGluR alterations occurred with binge access and following withdrawal. In the NAc, GluR2/3 levels were increased following withdrawal compared with binge access, and were the only changes observed in this region. Because subunit composition determines the functional properties of iGluRs, the observed changes may indicate alterations in the excitability of dopamine transmission underlying long-term biochemical and behavioral effects of cocaine.     

Classification of cDNA array genes that have a highly significant discriminative power due to their unique distribution in four brain regions

Novel statistical methods were used to distinguish functionally distinct brain regions using their cDNA array gene expression profiles, and it was found that one of four specific factors is often associated with the most regionally discriminative genes. The gene expression profiles for the substantia nigra (SN), striatum (STR), parietal cortex (PC), and posterolateral cortical amygdaloid nucleus (PLCo) brain regions were determined from each brain region. An F-test identified 339 genes of the 1185 array genes as having a P < or = 0.01 and applied a gene ranking and selection method based on Soft Independent Modeling of Class Analogy (SIMCA) to obtain 59 of the most discriminative genes. Their discriminative power was validated in three steps. The most convincing step showed their ability to correctly predict the brain regional classifications for 18 "test" gene expression sets obtained from the four regions. A two-way Hierarchical Cluster Analysis organized the 59 genes in six clusters according to their expression differences in the brain regions. Expression patterns in the SN and STR regions greatly differed from each other and the PC and PLCo. The closer similarity in the gene expression patterns of the PC and PLCo was probably due to their functional similarity. The important factors in determining differences in the regional gene expression profiles in six clusters were (1) regional myelin/oligodendrocyte levels, (2) resident neuron types, (3) neurotransmitter innervation profiles, and (4) Ca++-dependent signaling and second messenger systems.     

Dopamine D1-D2 receptor heteromer in dual phenotype GABA/glutamate-coexpressing striatal medium spiny neurons: regulation of BDNF, GAD67 and VGLUT1/2

In basal ganglia a significant subset of GABAergic medium spiny neurons (MSNs) coexpress D1 and D2 receptors (D1R and D2R) along with the neuropeptides dynorphin (DYN) and enkephalin (ENK). These coexpressing neurons have been recently shown to have a region-specific distribution throughout the mesolimbic and basal ganglia circuits. While the functional relevance of these MSNs remains relatively unexplored, they have been shown to exhibit the unique property of expressing the dopamine D1-D2 receptor heteromer, a novel receptor complex with distinct pharmacology and cell signaling properties. Here we showed that MSNs coexpressing the D1R and D2R also exhibited a dual GABA/glutamate phenotype. Activation of the D1R-D2R heteromer in these neurons resulted in the simultaneous, but differential regulation of proteins involved in GABA and glutamate production or vesicular uptake in the nucleus accumbens (NAc), ventral tegmental area (VTA), caudate putamen and substantia nigra (SN). Additionally, activation of the D1R-D2R heteromer in NAc shell, but not NAc core, differentially altered protein expression in VTA and SN, regions rich in dopamine cell bodies. The identification of a MSN with dual inhibitory and excitatory intrinsic functions provides new insights into the neuroanatomy of the basal ganglia and demonstrates a novel source of glutamate in this circuit. Furthermore, the demonstration of a dopamine receptor complex with the potential to differentially regulate the expression of proteins directly involved in GABAergic inhibitory or glutamatergic excitatory activation in VTA and SN may potentially provide new insights into the regulation of dopamine neuron activity. This could have broad implications in understanding how dysregulation of neurotransmission within basal ganglia contributes to dopamine neuronal dysfunction.     

Stoichiometry of tyrosine hydroxylase phosphorylation in the nigrostriatal and mesolimbic systems in vivo: effects of acute haloperidol and related compounds

Electrical stimulation of the medial forebrain bundle increases (32)P incorporation into striatal tyrosine hydroxylase (TH) at Ser (19), Ser(31), and Ser(40). In the present studies, the effects of acute haloperidol and related drugs on sitespecific TH phosphorylation stoichiometry (PS) in the nigrostriatal and mesolimbic systems were determined by quantitative blot immunolabeling using phosphorylation statespecific antibodies. The striatum (Str), substantia nigra (SN), nucleus accumbens (NAc), and ventral tegmental area (VTA) from Sprague-Dawley rats were harvested 30-40 min after a single injection of either vehicle, haloperidol (2 mg/kg), raclopride (2 mg/kg), clozapine (30 mg/kg), or SCH23390 (0.5 mg/kg). In vehicle-injected control rats, Ser(19) PS was 1.5- to 2. 5-fold lower in Str and NAc than in SN and VTA, Ser(31) PS was two-to fourfold higher in Str and NAc than in SN and VTA, and Ser(40) PS was similar between the terminal field and cell body regions. After haloperidol, Ser(40) PS increased twofold in Str and NAc, whereas a smaller increase in SN and VTA was observed. The effects of haloperidol on Ser(19) PS were similar to those on Ser(40) in each region; however, haloperidol treatment increased Ser(31) PS at least 1.6-fold in all regions. The effects of raclopride on TH PS were comparable to those of haloperidol, whereas clozapine treatment increased TH PS at all sites in all regions. By contrast, the effects of SCH23390 on TH PS were relatively small and restricted to the NAc. The stoichiometries of site-specific TH phosphorylation in vivo are presented for the first time. The nigrostriatal and mesolimbic systems have common features of TH PS, distinguished by differences in TH PS between the terminal field and cell body regions and by dissimilar increases in TH PS in the terminal field and cell body regions after acute haloperidol.