Substantia Nigra Compacta Research Articles

Dopamine D2 Receptor Binding, Drd2 Expression and the Number of Dopamine Neurons in the BXD Recombinant Inbred Series: Genetic Relationships to Alcohol and Other Drug Associated Phenotypes

Background: It has not been established to what extent the natural variation in dopamine systems contribute to the variation in ethanol response. The current study addresses this issue by measuring D2 dopamine (DA) receptor binding, the expression of Drd2, the number of midbrain DA neurons in the BXD recombinant inbred (RI) series and then compares these strain means with those previously reported for a variety of ethanol and other drug‐related phenotypes.Methods: Data were collected for 21 to 23 of the BXD RI strains and the parental strains. D2 DA receptor autoradiography was performed using 125I‐epidepride as the ligand [Kanes S, Dains K, Cipp L, Gatley J, Hitzemann B, Rasmussen E, Sanderson S, Silverman S, Hitzemann R (1996) Mapping the genes for haloperidol‐induced catalepsy. J Pharmacol Exp Ther 277:1016–1025]. Drd2 expression was measured using the Affymetrix oligoarray system. Immunocytochemical techniques were used to determine the number of midbrain DA neurons [Hitzemann B, Dains K, Hitzemann R (1994) Further studies on the relationship between dopamine cell density and haloperidol response. J Pharmacol Exp Ther 271:969–976].Results and Conclusions: The range of difference in receptor binding for the RI strains was approximately 2‐fold in all regions examined, the core, the shell of the nucleus accumbens (NAc) and the dorsomedial caudate‐putamen (CPu); heritability in all regions was moderate—(h 2∼0.35). Drd2 expression in forebrain samples from the RI and parental strains ranged 1.5‐ to 2‐fold and h2 was moderate—0.47. Variation in the number of tyrosine hydroxylase (TH) positive neurons was moderate, 41% and 26% and h2 was low—0.19 and 0.15 for the ventral tegmental area (VTA) and substantia nigra compacta (SNc), respectively. Significant correlations were found between D2 DA receptor binding and the low dose (1.33 g/kg) ethanol stimulant response. (p < 0.002) and between Drd2 expression and conditioned place preference (CPP) (p < 0.0005). No significant correlations were detected between ethanol preference and either receptor binding or Drd2 expression; however, a significant correlation was found between preference and Ncam expression. Ncam is approximately 0.2 Mb from Drd2. Overall, the data suggest ethanol preference and CPP are associated with the expression of Drd2 or closely linked genetic loci.

Neonatal Ablation of the Nigrostriatal Dopamine Pathway Does Not Influence Limb Development in Rats

Hemiparkinson–hemiatrophy syndrome (HP–HA) is associated with skeletal hemiatrophy and the later development of parkinsonism. It is generally assumed that this phenotype is due to the combination of dysfunction of the basal ganglia (e.g., substantia nigra compacta and/or other related structures), causing parkinsonism, and of other areas (e.g., cerebral cortex), causing hemiatrophy. The occurrence of asymmetry of limb size in a patient with very asymmetric involvement of dopa-responsive dystonia encouraged Greene et al. (2000, Mov. Disord. 15: 537–541) to propose that lifelong deficits in nigrostriatal dopamine could account for limb asymmetry in HP–HA. The purpose of this study was to determine whether skeletal hemiatrophy could be produced in rats by unilateral, neonatal ablation of the nigrostriatal dopamine pathway. Infusion of 6-hydroxydopamine into the striatum of rat neonates resulted in loss of dopamine neurons in the ipsilateral substantia nigra, reduced striatal dopamine levels, and stimulant-induced motor asymmetry. Saline infusions neither altered the number of dopamine neurons nor produced behavioral changes. Both groups incurred discrete lesions of the ipsilateral motor cortex surrounding the infusion site and atrophy of the corresponding cerebral peduncle. Cortical, but not nigrostriatal, lesions were associated with significant atrophy of ipsilateral femora, humeri, and innominate bones, as assessed radiographically. Skeletal hemiatrophy was not observed in naive animals or in experimental animals that did not exhibit corticospinal abnormalities. The results of this study indicate that early skeletal development in rats is not affected by loss of nigrostriatal dopamine per se, but is markedly attenuated by corticospinal lesions sustained during the neonatal period.

1-Methyl-4-phenylpridinium (MPP+)-induced functional run-down of GABA(A) receptor-mediated currents in acutely dissociated dopaminergic neurons.

We have evaluated GABA(A)receptor function during treatment of 1-methyl-4-phenylpridinium (MPP+) using patch-clamp perforated whole-cell recording techniques in acutely dissociated dopaminergic (DAergic) neurons from rat substantia nigra compacta (SNc). Gamma-aminobutyric acid (GABA), glutamate or glycine induced inward currents (I(GABA), I(Glu), I(Gly)) at a holding potential (VH) of -45 mV. The I(GABA) was reversibly blocked by the GABA(A) receptor antagonist, bicuculline, suggesting that I(GABA) is mediated through the activation of GABA(A) receptors. During extracellular perfusion of MPP+ (1-10 microm), I(GABA) , but neither I(Glu) nor I(Gly), declined (termed run-down) with repetitive agonist applications, indicating that the MPP+-induced I(GABA) run-down occurred earlier than I(Gly) or I(Glu) under our experimental conditions. The MPP+-induced I(GABA) run-down can be prevented by a DA transporter inhibitor, mazindol, and can be mimicked by a metabolic inhibitor, rotenone. Using conventional whole-cell recording with different concentrations of ATP in the pipette solution, I(GABA) run-down can be induced by decreasing intracellular ATP concentrations, or prevented by supplying intracellular ATP, indicating that I(GABA) run-down is dependent on intracellular ATP concentrations. A GABA(A) receptor positive modulator, pentobarbital (PB), potentiated the declined I(GABA) and eliminated I(GABA) run-down. Corresponding to these patch-clamp data, tyrosine hydroxylase (TH) immunohistochemical staining showed that TH-positive cell loss was protected by PB during MPP+ perfusion. It is concluded that extracellular perfusion of MPP+ induces a functional run-down of GABA(A) receptors, which may cause an imbalance of excitation and inhibition of DAergic neurons.

The Neurotoxin 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine Induces Apoptosis in Mouse Nigrostriatal Glia: RELEVANCE TO NIGRAL NEURONAL DEATH AND STRIATAL NEUROCHEMICAL CHANGES

Swiss mice were given 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 25 mg/kg/day, for 5 consecutive days and killed at different days after MPTP discontinuance. Decreases in striatal tyrosine hydroxylase activity and levels of dopamine and its metabolites were observed 1 day after MPTP discontinuance. Ascorbic acid and glutamate levels had increased, dehydroascorbic acid and GSH decreased, whereas catabolites of high-energy phosphates (inosine, hypoxanthine, xanthine, and uric acid) were unchanged. In addition, gliosis was observed in both striatum and substantia nigra compacta (SNc). Sections of SNc showed some terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL)-positive cells. Neurochemical parameters of dopaminergic activity showed a trend toward recovery 3 days after MPTP discontinuance. At this time point, TUNEL-positive cells were detected in SNc; some of them showed nuclei with neuronal morphology. A late (days 6-11) increase in striatal dopamine oxidative metabolism, ascorbic acid oxidative status, and catabolites of high-energy phosphates were observed concomitant with nigral neuron and nigrostriatal glial cell apoptotic death, as revealed by TUNEL, acridine orange, and Hoechst staining, and transmission electron microscopy. These data suggest that MPTP-induced activation/apoptotic death of glial cells plays a key role in the sequential linkage of neurochemical and cellular events leading to dopaminergic nigral neuron apoptotic death.

Response of GABAergic cells in the deep mesencephalic nucleus to dopaminergic cell degeneration: an electrophysiological and in situ hybridization study

The deep mesencephalic nucleus (DMN) is a large midbrain reticular region located between the substantia nigra compacta and the superior colliculus. It contains GABAergic cells that share striatal afferents, thalamic and collicular efferents, as well as neurochemical and electrophysiological similarities, with those of the substantia nigra reticulata. In the present paper we used electrophysiological (firing rate and firing pattern) and morphological (densitometric analysis of in situ hybridization histochemical labeling for glutamic acid decarboxylase (GAD)65 and GAD67 mRNA) techniques, to study the response of DMN GABAergic cells to the degeneration of nigral dopaminergic cells. Our results showed that unilateral dopaminergic cell loss (after injection of 6-hydroxydopamine in the medial forebrain bundle) induces a bilateral and symmetrical increase in both firing rate and GAD67 mRNA levels and a decrease in GAD65 mRNA levels. These findings support the involvement of DMN GABAergic cells in the basal ganglia modifications that follow dopaminergic cell loss, also suggesting its participation in the pathophysiology of Parkinson’s disease. The symmetry of effects, together with its recently reported bilateral projections to the thalamus and superior colliculus, suggest that unlike substantia nigra reticulata, DMN is involved in the interhemispheric regulation of basal ganglia, probably keeping their functional symmetry even after asymmetric lesions.

Brain regional μ-opioid receptor function in rat lines selected for differences in alcohol preference

It has been suggested that opioid peptides play a role in the reinforcing effects of alcohol. The present study was designed to examine the function of the μ-opioid receptor system in rat lines selectively bred for alcohol preference (AA [Alko, Alcohol] rat line) and alcohol avoidance (ANA [Alko, Non-Alcohol] rat line). The functional coupling of μ-opioid receptors to G proteins was determined autoradiographically using Tyr- d-Ala-Gly-N(Me)Phe-Gly-ol-enkephalin-stimulated [ 35S]GTPγS binding in brain cryostat sections. The binding was significantly increased in the striatal patches and substantia nigra reticulata of the AA rats in comparison with that of the ANA rats. Within the AA rat line, there was a significant positive correlation between 3 mg/kg morphine-induced locomotor activity and activation of G-proteins in the substantia nigra compacta and nucleus accumbens core. These results of the selective breeding experiment suggest that brain region-specific differences in μ-opioid receptor function may correlate with innate differences in alcohol preference.

Deep brain stimulation of subthalamic neurons increases striatal dopamine metabolism and induces contralateral circling in freely moving 6-hydroxydopamine-lesioned rats

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) alleviates Parkinson's disease (PD) symptoms. Although widely used, the mechanisms of action are still unknown. In an attempt to elucidate those mechanisms, we have previously demonstrated that STN-DBS increases striatal extracellular dopamine (DA) metabolites in anaesthetized rats. PD being a movement disorder, it remains to be determined whether these findings are related to any relevant motor or behavioural changes. Thus, this study investigates concomitant behavioural changes during STN-DBS and extracellular striatal DA metabolites measured using microdialysis in freely moving 6-hydroxydopamine-lesioned rats. STN-DBS induced an increase of striatal DA metabolites in awake, freely moving animals. Furthermore, we observed concomitant contralateral circling behaviour. Taken together, these results suggest that STN-DBS could disinhibit (consequently activate) substantia nigra compacta neurons via inhibition of gamma-aminobutyric acid-ergic substantia nigra reticulata neurons.

Contradictory effects of sodium nitroprusside and S-nitroso-N-acetylpenicillamine on oxidative stress in brain dopamine neurons in vivo.

To investigate whether nitric oxide (*NO) is neurotoxic or neuroprotective in the brain, we compared the in vivo role of S-nitroso-N-acetylpenicillamine (SNAP) with that of sodium nitroprusside (SNP) on ferrous citrate-induced oxidative stress and neuronal loss in the rat nigrostriatal dopaminergic system. It is known that light irradiation releases *NO from its donor compounds; these irradiated *NO donors were used as sham controls in this study. Intranigral infusion of ferrous citrate (4.2 nmol) into the rat midbrain substantia nigra compacta area caused acute lipid peroxidation in the substantia nigra and chronic dopamine depletion in the caudate nucleus. Coinfusion of freshly prepared SNAP (0-8.4 nmol) or *NO (about 2 nmol), but not SNP, rescued iron-induced dopamine depletion in the rat brain in vivo. In fact, SNP produced prooxidative effects similar to ferrous citrate both in vivo and in vitro, since SNP is a redox iron complex. Consistently, *NO and SNAP inhibited, whereas SNP potentiated, *OH generation and lipid peroxidation evoked by ferrous citrate in vitro. We previously reported that freshly prepared, but not irradiated, S-nitroso-L-glutathione (GSNO) protected brain dopamine neurons against oxidative stress in vivo. As well as these antioxidative properties, our recent reports (see (Ref. 1)) indicate that *NO/GSNO activated guanylyl cyclase, increased cGMP and that could lead to PKG-mediated expression of MnSOD, Bcl-2, and thioredoxin for preconditioning neuroprotection against 1-methyl-4-phenylpyridinium (MPP(+)).(1) In conclusion, *NO and S-nitrosothiols (e.g., GSNO and SNAP) can scavenge reactive oxygen species and activate the heme moiety of guanylyl cyclase, resulting in protection of brain dopamine neurons through both antioxidative and antiapoptotic mechanisms.

Partial loss of dopaminergic neurons in the substantia nigra, ventrotegmental area and the retrorubral area -- model of the early beginning of Parkinson's symptomatology?

To test substances which might have protective effects on the dopaminergic system it is necessary to use models with a pathological symptomatology of the early beginning, i.e. models in which the chance exists to arrest the otherwise progressive pathological processes (see Heim et al., 2001). 6-hydroxydopamine (6-OHDA) injected unilaterally into the ventrolateral striatum of rats (6 microg dissolved in 2 microl 0.2% ascorbic acid) leads to specific stereotyped movements after subcutaneous injection of apomorphine both 3 and 13 weeks after surgery. Ten weeks after surgery decreased spontaneous motor activity could be observed. Twelve weeks after 6-hydroxydopamine injection, the animals had difficulties in performing a spatial navigation task when the submerged escape platform was moved to another position. The switching of motor programs was less pronounced. The application of tyrosine-hydroxylase-staining showed a loss of ipsilateral neurones of the substantia nigra compacta as well as of dendrites in the pars reticulata, neurones in the ventral tegmental area and in the retrorubral area ipsilaterally as well as a loss of dopaminergic fibres both ipsilaterally and contralaterally in the striatum which should belong to the contralateral acting substantia nigra afferents. The loss of the neurones and the afferents was induced by the retrograde denervation following the 6-OHDA injection within the ventrolateral striatum. The question arises whether the model used here with the partially loss of dopaminergic neurons and fibres reflects some of pathological symptoms of Parkinson's disease in the early states.

Amyloid precursor protein gene disruption attenuates degeneration of substantia nigra compacta neurons following axotomy

Our past work has shown that the C-terminal fragment of amyloid precursor protein (APP) translocated to the nucleus in neurons destined for delayed excitotoxic degeneration. To test whether nuclear APP fragments also play a role in the progressive loss of dopaminergic (DA) substantia nigra compacta (SNc) neurons, we performed unilateral medial forebrain bundle (MFB) transection on APP wild type (WT) and on mice with disruption of the APP gene (KO). In WT mice immunoreactivity for APP C-terminal, β-amyloid and Alz90 epitopes appeared in the nuclei of axotomized DA neurons at 3 days post-lesion (dpl), persisted at 7 dpl and was absent in 14 dpl mice. APP N-terminal immunoreactivity was restricted to the cytosol at all time points, precluding the possibility of full length APP in the nucleus. Nuclear localization of APP epitopes was absent in neurons of the contralateral SNc or in neurons of the ipsilateral ventral tegmental area and SN reticulata. The presence of APP C-terminal and Alz90 domains was confirmed by Western blotting performed on the nuclear fraction of the SN ipsilateral to the axotomy. Quantitative morphometric analysis revealed that WT mice demonstrated earlier and more profound loss of tyrosine hydroxylase+SNc neurons than did KO mice. These data showed that a novel nuclear C-terminal fragment appeared coincident with SNc neuron degeneration, and that APP deficiency correlated with significant neuroprotection in vivo.

APP knockout attenuates microglial activation and enhances neuron survival in substantia nigra compacta after axotomy.

Focal microglial activation and progressive dopaminergic neurodegeneration in substantia nigra compacta (SNc) have characterized Parkinson's disease (PD). We have hypothesized that the microglial response may be provoked by molecular signals from chronically stressed SNc neurons. To test whether amyloid precursor protein (APP) could serve as such a signal, we evaluated microglial activation in SN after unilateral transection of the medial forebrain bundle (MFB) in mice either wild-type (WT) or null (KO) for APP. WT and KO mice displayed comparable microglial response at the MFB transection site. In WT mice microglial activation was first apparent in the ipsilateral SN at 3 days postlesion (dpl), marked by morphological change and increased isolectin immunoreactivity. The microglial response intensified at 7 dpl and persisted in the medial nigra through 14 dpl. In contrast, in KO mice activated microglia appeared predominantly at 7 dpl, with little activation at 3 dpl and none at 14 dpl. Neuron number in affected WT SNc at 14 dpl was significantly reduced compared with loss in affected KO SNc. The delayed and limited local microglial activation and increased neuron survival in response to distal axotomy of SNc neurons in APP KO mice are consistent with the important role APP in neuronal stress responses in vivo.

Nociceptin/orphanin FQ and opioid receptor‐like receptor mRNA expression in dopamine systems

Although nociceptin/orphanin FQ (N/OFQ) influences dopamine (DA) neuronal activity, it is not known whether N/OFQ acts directly on DA neurons, indirectly by means of local circuitry, or both. We used two parallel approaches, dual in situ hybridization (ISH) and neurotoxic lesions of DA neurons by using 6-hydroxydopamine (6-OHDA), to ascertain whether N/OFQ and the N/OFQ receptor (NOP) mRNA are expressed in DA neurons in the ventral tegmental area (VTA) and substantia nigra compacta (SNc). In the VTA and SNc, small populations (approximately 6-10%) of N/OFQ-containing neurons coexpressed mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme for DA synthesis. Similarly, very few (1-2%) TH-positive neurons contained N/OFQ mRNA signal. A majority of NOP-positive neurons (approximately 75%) expressed TH mRNA and roughly half of the TH-containing neurons expressed NOP mRNA. Many N/OFQ neurons (approximately 50-60%) expressed glutamic acid decarboxylase 65 and 67 mRNAs, markers for gamma-aminobutyric acid (GABA) neurons. In the 6-OHDA lesion studies, NOP mRNA levels were nearly 80 and 85% lower in the VTA and SNc, respectively, on the lesioned side. These lesions appear to lead to compensatory changes, with N/OFQ mRNA levels approximately 60% and 300% higher in the VTA and SNc, respectively, after 6-OHDA lesions. Finally, N/OFQ-stimulated [(35)S]guanylyl-5'-O-(gamma-thio)-triphosphate levels were decreased in the VTA and SNc but not the prefrontal cortex after 6-OHDA lesions. Accordingly, it appears that N/OFQ mRNA was found largely on nondopaminergic (i.e., GABA) neurons, whereas NOP mRNA was located on DA neurons. N/OFQ is in a position to influence DA neuronal activity by means of the NOP located on DA neurons.

Neurotropism and Retrograde Axonal Transport of a Canine Adenoviral Vector: A Tool for Targeting Key Structures Undergoing Neurodegenerative Processes

Viral tropism refers to the ability of a virus to selectively infect a given subset of cells. It relies on a variety of viral and host determinants that entail virus binding and entry into target cells, in addition to the presence of genetic elements that allow or enhance viral gene expression in a specific manner. Here we report the results of neuroanatomical studies in rat brains injected in different cerebral structures with vectors derived from the canine adenovirus type 2 (CAV2), whose natural target is the respiratory epithelium. Control animals injected with vectors derived from the human adenovirus type 5 (Ad5) displayed the previously documented pattern of gene transfer into both neurons and glial cells. Injection of CAV2 vectors resulted in selective transduction of neuronal cells. Cy3-labeled CAV2 particles allowed us to establish the high affinity of this vector for neuronal processes in vitro and their rapid uptake and retrograde axonal transport in vivo. After intrahippocampal injections, labeled particles were found, within 1 hour, closely associated to the nuclei of the neurons in layer II of the entorhinal cortex. Injections into the striatum resulted in a massive transduction of dopaminergic neurons in the substantia nigra compacta. The high efficiency with which CAV2 vectors are retrogradely transported opens the possibility of targeting a transgene to neuron populations remote from the injection site and difficult to access. Our data support the possibility to target key structures undergoing a degenerative process: the enthorhinal cortex, which is affected first in Alzheimer's disease; and the substantia nigra compacta, which undergoes degeneration in Parkinson's disease.

Unilateral lesion of the nigrostriatal pathway induces an increase of neuronal activity of the pedunculopontine nucleus, which is reversed by the lesion of the subthalamic nucleus in the rat.

The role of the pedunculopontine nucleus (PPN) in the pathophysiology of Parkinson's disease is still unclear. Using microrecordings, we investigated the changes occurring in PPN neurons after lesions of the substantia nigra compacta (SNc) and the role of the subthalamic nucleus (STN) in these changes. In normal rats the firing rate of PPN neurons was 10.6 +/- 1.4 spikes/s, the majority of neurons (91%) having a regular firing pattern, 6% irregular and 3% in bursts. In rats with 6-hydroxydopamine lesions of the SNc, the firing rate increased significantly to 18.3 +/- 3.0 spikes/s compared with normal rats. In addition, the firing pattern changed significantly: 70% of the neurons discharged regularly, 27% irregularly and 3% in bursts. In rats with ibotenic acid lesions of the STN, the firing rate decreased significantly to 7.2 +/- 0.9 spikes/s and the firing pattern changed significantly: 50% of the neurons discharged regularly, 43% irregularly and 7% in bursts. The rats with combined SNc and STN lesions showed no change in the firing rate (8.5 +/- 1.0 spikes/s) compared to normal rats. The firing pattern changed significantly: 69% of the cells discharged regularly, 26% irregularly and 5% in bursts. These findings demonstrate that PPN neurons are overactive and more irregular in the 6-hydroxydopamine-lesioned rats, suggesting the implication of this nucleus in the pathophysiology of parkinsonism. Moreover, the fact that STN lesions induced a reduction in the firing rate of the PPN in normal rats and a normalization of the firing rate in rats with 6-hydroxydopamine lesions suggests that this nucleus is under major control of the STN.

Immediate and delayed effects of lead on AChE, GSH‐T and thiols in the substantia nigra, neostriatum and cortex of the rat brain

We studied the effects, at 10 and 30 min, of a single dose (10 mg kg(-1)) of lead chloride, administered by the intraperitoneal route, on the activities of acetylcholinesterase (AChE) and glutathione transferase (GSH-T) and on the concentrations of total and non-protein thiols in substantia nigra compacta (SNCO) and substantia nigra reticulata (SNRE), caudate putamen (CAU) and cerebral cortex (CC) from adult male rats in comparison with the effects of this metal at 24 and 72 h. The main immediate effects of lead consisted of decreased GSH-T activity and total and non-protein thiol concentrations in CAU and CC 10 min after administration. These effects were reversed after 30 min but with increased GSH-T activity in SNCO and AChE activity in SNRE along with diminished concentration of homogenate proteins in SNRE, CAU and CC. The GSH-T activity again was increased in SNCO but the AChE activity was decreased in CC 24 h after Pb administration; total and non-protein thiol concentrations were diminished but homogenate protein concentration was augmented in all areas. Finally, 72 h after Pb administration, AChE and GSH-T activities were decreased in CAU and CC, accompanied by an increased concentration of precipitate and supernatant proteins; supernatant protein concentration also was augmented in SNCO and SNRE; here, again, the concentrations of total and non-protein thiols were diminished and the homogenate protein concentration was augmented in all areas.

The deep mesencephalic nucleus as an output center of basal ganglia: morphological and electrophysiological similarities with the substantia nigra.

The deep mesencephalic nucleus (DMN) is a large midbrain reticular region between the superior colliculus, the substantia nigra compacta, the periaqueductal gray, and the medial geniculate body. Although some data suggest that it is involved in nociception and visceral control, its functions remain unclear. In the present study, by using morphological (combination of anterograde and retrograde tracers with immunocytochemistry and in situ hibrydization) and electrophysiological (firing activity and transynaptic response to striatal stimulation) methods, we show that a subpopulation of DMN cells shares many morphological and electrophysiological characteristics with those of the substantia nigra reticulata (SNR). These similarities include the following: 1) firing rate, firing pattern, and conduction velocity; 2) expression of GAD65, GAD67, and PV; 3) excitatory and inhibitory inputs from the striatum; and 4) projections to the ventral thalamus, superior colliculus, and pedunculopontine tegmental nucleus. Some differences were also found. In comparison with SN, DMN cells and striatal afferents are more sparsely distributed and they show conspicuous contralateral projections to the thalamus and superior colliculus. This suggests that, similarly to the SNR, the DMN acts as an output center of basal ganglia and probably facilitates the inter-hemispheric regulation of these centers.

Comparison of Cocaine‐ and Methamphetamine‐Evoked Dopamine and Glutamate Overflow in Somatodendritic and Terminal Field Regions of the Rat Brain during Acute, Chronic, and Early Withdrawal Conditions

Methamphetamine and cocaine are among the most commonly abused psychostimulants. Repeated injections of psychostimulants produce behavioral sensitization or augmented locomotion in rats. Behavioral sensitization to methamphetamine and cocaine is long lasting and persists after cessation of drug treatment. Because dopamine and glutamate are major neurotransmitters of the neostriatum, we evaluated the profile of cocaine- or methamphetamine-evoked dopamine and glutamate overflow in the caudate putamen, nucleus accumbens, ventral tegmental area, and substantia nigra compacta of the rat brain. We also compared acute exposure to these drugs with chronic treatment and early withdrawal. Acute injection of methamphetamine (1 mg/kg of body weight) or cocaine (10 mg/kg) resulted in elevated levels of extracellular dopamine in all brain regions measured, although the magnitude of increase varied between brain regions. Overall, methamphetamine caused more dopamine to accumulate in the extracellular space than did cocaine when administered to animals during early withdrawal (7 days of daily injections and challenge on day 11). For example, a challenge injection of methamphetamine produced a greater elevation of extracellular dopamine in the caudate putamen when compared to acute (naïve) exposure. By contrast, a challenge injection of cocaine resulted in dopamine levels in the caudate putamen that were lower than those observed for acute exposure. In the ventral tegmental area and the substantia nigra compacta, a challenge injection of methamphetamine or cocaine resulted in extracellular dopamine levels that were lower than those for acute exposure. Thus, it appears that behavioral sensitization to cocaine can be sustained during early withdrawal in the absence of augmented drug-evoked dopamine overflow. Acute injection of methamphetamine or cocaine did not change extracellular levels of glutamate in the neostriatum. Cocaine challenge (early withdrawal) increased glutamate overflow in the caudate putamen and the nucleus accumbens. In contrast, methamphetamine challenge increased glutamate overflow in the caudate putamen, but it decreased glutamate in the nucleus accumbens. In the ventral tegmental area and the substantia nigra compacta, acute methamphetamine exposure decreased glutamate overflow, but acute cocaine exposure increased it. Although amphetamines and cocaine induce similar behavioral responses, the results presented here demonstrate that at the neurochemical level (neurotransmitter release) they sometimes evoke opposite effects depending on the brain region studied and the duration of drug treatment. Moreover, the sensitized augmentation of locomotor activity observed by us and others in response to a challenge injection of cocaine is not dependent on elevation of the extracellular concentration of dopamine in the neostriatum. We are currently investigating the hypothesis that cocaine activates peptidergic systems of the neostriatum and that these systems modulate the synaptic release of dopamine in response to psychostimulants.

Changes in the firing pattern of globus pallidus neurons after the degeneration of nigrostriatal pathway are mediated by the subthalamic nucleus in the rat

Changes in the neuronal activity of globus pallidus (GP) have been shown in animal models of parkinsonism. In order to study the implication of the subthalamic nucleus (STN) in these changes, the effects of STN lesions alone or in combination with 6-hydroxydopamine (6-OHDA) -induced damage to the substantia nigra compacta (SNc) were examined in rats using electrophysiological recordings of GP cells. In normal rats, the firing rate was 22.1+/-1.4 spikes/s. The pattern was regular in 45%, irregular in 49% and bursty in 6% of the cases. In rats with STN lesions, the firing rate of GP units (20.15+/-1.25 spikes/s) did not differ from that of normal rats and only regular (46%) and irregular (54%) cells were found; a bursty pattern was not observed. 6-OHDA lesions of the SNc induced no change in the firing rate of GP neurons (21.5+/-1.4 spikes/s, P>0.05) but a significant decrease in the percentage of regular cells (27%, P<0.001), a significant increase in burst cells (21%, P<0.001) with no change in the percentage of irregular units (52%) were observed. In rats with combined SNc and STN lesions, the firing pattern did not change from that of normal rats. The present results show that STN lesions induced the disappearance of bursts in normal rats and normalization of firing pattern in the GP units of rats with 6-OHDA lesions suggesting that the STN plays an important role in the modulation of the pattern of activity of GP neurons which may account for the therapeutic effect of STN lesions in Parkinson's disease.

CDNA cloning and expression of a γ-aminobutyric acidAreceptor ε-subunit in rat brain

A cDNA encoding a GABAA receptor subunit was isolated from rat brain. The predicted protein is 70% identical to the human ε-subunit. It was recently reported [Sinkkonen et al. (2000), J. Neurosci., 20, 3588–3595] that the rodent ε-subunit mRNA encoded an additional sequence (≈400 residues). We provide evidence that human and rat ε-subunit are similar in size. The distribution of cells expressing the GABAAε-subunit was examined in the rat brain. In situ hybridization histochemistry revealed that ε-subunit mRNA is expressed by neurons located in septal and preoptic areas, as well as in various hypothalamic nuclei, including paraventricular, arcuate, dorsomedial and medial tuberal nuclei. The mRNA was also detected in major neuronal groups with broad-range influence, such as the cholinergic (basal nucleus), dopaminergic (substantia nigra compacta), serotonergic (raphe nuclei), and noradrenergic (locus coeruleus) systems. Immunohistochemistry using an affinity-purified antiserum directed towards the N-terminal sequence unique to the rat ε-subunit revealed the presence of ε-subunit immunoreactivity over the somatodendritic domain of neurons with a distribution closely matching that of mRNA-expressing cells. Moreover, using in situ hybridization, α3, θ and ε GABAA subunit mRNAs were all detected with an overlapping distribution in neurons of the dorsal raphe and the locus coeruleus. Our results suggest that novel GABAA receptors may regulate, neuroendocrine and modulatory systems in the brain.

CDNA cloning and expression of a gamma-aminobutyric acidA receptor epsilon-subunit in rat brain

A cDNA encoding a GABA(A) receptor subunit was isolated from rat brain. The predicted protein is 70% identical to the human epsilon-subunit. It was recently reported [Sinkkonen et al. (2000), J. Neurosci., 20, 3588-3595] that the rodent epsilon-subunit mRNA encoded an additional sequence ( approximately 400 residues). We provide evidence that human and rat epsilon-subunit are similar in size. The distribution of cells expressing the GABA(A) epsilon-subunit was examined in the rat brain. In situ hybridization histochemistry revealed that epsilon-subunit mRNA is expressed by neurons located in septal and preoptic areas, as well as in various hypothalamic nuclei, including paraventricular, arcuate, dorsomedial and medial tuberal nuclei. The mRNA was also detected in major neuronal groups with broad-range influence, such as the cholinergic (basal nucleus), dopaminergic (substantia nigra compacta), serotonergic (raphe nuclei), and noradrenergic (locus coeruleus) systems. Immunohistochemistry using an affinity-purified antiserum directed towards the N-terminal sequence unique to the rat epsilon-subunit revealed the presence of epsilon-subunit immunoreactivity over the somatodendritic domain of neurons with a distribution closely matching that of mRNA-expressing cells. Moreover, using in situ hybridization, alpha3, theta and epsilon GABA(A) subunit mRNAs were all detected with an overlapping distribution in neurons of the dorsal raphe and the locus coeruleus. Our results suggest that novel GABA(A) receptors may regulate, neuroendocrine and modulatory systems in the brain.