Jean-Jacques Soghomonian

High-Dose Methamphetamine Acutely Activates the Striatonigral Pathway to Increase Striatal Glutamate and Mediate Long-Term Dopamine Toxicity

Methamphetamine (METH) has been shown to increase the extracellular concentrations of both dopamine (DA) and glutamate (GLU) in the striatum. Dopamine, glutamate, or their combined effects have been hypothesized to mediate striatal DA nerve terminal damage. Although it is known that METH releases DA via reverse transport, it is not known how METH increases the release of GLU. We hypothesized that METH increases GLU indirectly via activation of the basal ganglia output pathways. METH increased striatonigral GABAergic transmission, as evidenced by increased striatal GAD65 mRNA expression and extracellular GABA concentrations in substantia nigra pars reticulata (SNr). The METH-induced increase in nigral extracellular GABA concentrations was D1 receptor-dependent because intranigral perfusion of the D1 DA antagonist SCH23390 (10 μm) attenuated the METH-induced increase in GABA release in the SNr. Additionally, METH decreased extracellular GABA concentrations in the ventromedial thalamus (VM). Intranigral perfusion of the GABA-A receptor antagonist, bicuculline (10 μm), blocked the METH-induced decrease in extracellular GABA in the VM and the METH-induced increase in striatal GLU. Intranigral perfusion of either a DA D1 or GABA-A receptor antagonist during the systemic administrations of METH attenuated the striatal DA depletions when measured 1 week later. These results show that METH enhances D1-mediated striatonigral GABAergic transmission (1), which in turn activates GABA-A receptors in the SNr (2), leading to a decrease in GABAergic nigrothalamic activity (3), an increase in corticostriatal GLU release (4), and a consequent long-term depletion of striatal DA content (5).

Serotonin innervation in adult rat neostriatum. II. Ultrastructural features: a radioautographic and immunocytochemical study

High-resolution radioautography after cerebroventricular administration of tritiated serotonin (5-HT) and PAP immunocytochemistry with an antiserum against 5-HT-glutaraldehyde conjugate (kindly donated by M. Geffard) were used in parallel to investigate the intrinsic and relational fine structural features of 5-HT axon varicosities (terminals) in the neostriatum of the adult rat. The uptake-labeled varicosities were examined in single thin sections from a paraventricular sector of neostriatum, whereas their immunostained counterparts were viewed in serial thin sections from the same paraventricular sector plus a dorsal neostriatal sector. The two approaches yielded complementary results in terms of varicosity dimensions, synaptic features and appositional relationships. Serotonin axon terminals were generally small and, as measured in immunostained material, even smaller in the dorsal than in the paraventricular neostriatum. Their internal features, best viewed in radioautographs, included small pleomorphic synaptic vesicles with occasional large granular vesicles and mitochondria. Junctional 5-HT terminals from both the paraventricular and the dorsal neostriatal sectors synapsed exclusively, and with equal frequency, on dendritic spines or shafts, almost always with asymmetrical membrane differentiations. The proportion of junctional varicosities, however, was very low in serial (immunocytochemical) as well as single (radioautographic) thin sections. Only 10-13% of 5-HT varicosities from either the paraventricular or the dorsal neostriatum exhibited a synaptic junction, in contrast with a junctional incidence of at least 70% for randomly selected axonal varicosities similarly sampled in the surrounding neuropil. Serotonin axon terminals, whether or not synaptic, were closely apposed to a variety of structures comprising mostly other axon terminals, dendritic spines and branches, but rarely neuronal somata. The synaptic and appositional features of immunostained 5-HT varicosities were similar for both the dorsal and the paraventricular neostriatum. In this context, it is likely that the effects of 5-HT in the neostriatum are exerted upon a multiplicity of cellular target sites in addition to the restricted number of dendritic spines and shafts synaptically contacted by this type of monoamine terminal.

Serotonin innervation in adult rat neostriatum. I. Quantified regional distribution

The distributional features of the serotonin (5-HT) innervation in adult rat neostriatum were examined and quantified using two complementary chemoanatomical methods: 5-HT-immunohistochemistry on serial histological sections and radioautography after [3H]5-HT uptake in whole cerebral hemisphere slices. As visualized and measured after peroxidase-antiperoxidase immunostaining, the neostriatal 5-HT fiber network pervading the entire neostriatum was 2-3 times denser in its ventral than dorsal parts, and showed a slight rostrocaudal increase in density. Its axonal length ranged from 1.06 to 4.18 m per mm3 of striatal tissue. Radioautographic counts of the [3H]5-HT-labeled axon varicosities within comparable sectors of the neostriatum showed good correlation with this distribution pattern. As extrapolated after appropriate corrections for incomplete detection at the chosen exposure time and from the thickness of sections examined, the number of neostriatal 5-HT varicosities (innervation density) ranged from 1.5 to 4.8 millions and averaged 2.6 millions per mm3 of tissue. These quantitative results provided new insights into the topographical organization of the dorsal raphe-neostriatal 5-HT projection system. They already allow for meaningful correlations with currently available microchemical data on intrastriatal 5-HT levels and should also be of considerable significance when as precise information becomes available on the number and localization of different 5-HT receptors and uptake carriers within rat neostriatum.

Decreased GAD65 mRNA levels in select subpopulations of neurons in the cerebellar dentate nuclei in autism: an in situ hybridization study.

The laterally positioned dentate nuclei lie in a key position in the cerebellum to receive input from Purkinje cells in the lateral cerebellar hemisphere participating in both motor and cognitive functions. Although neuropathology of the four cerebellar nuclei using Nissl staining has been qualitatively reported in children and adults with autism, surprisingly the dentate nuclei appeared less affected despite reported reductions in Purkinje cells in the posterolateral cerebellar hemisphere. To determine any underlying abnormalities in the critically important GABAergic system, the rate‐limiting GABA synthesizing enzyme, glutamic acid decarboxylase (GAD) type 65 was measured via in situ hybridization histochemistry in dentate somata. GAD65 mRNA labeling revealed two distinct subpopulations of neurons in adult control and autism postmortem brains: small‐sized cells (about 10–12 µm in diameter, presumed interneurons) and larger‐sized neurons (about 18–20 µm in diameter, likely feedback to inferior olivary neurons). A mean 51% reduction in GAD65 mRNA levels was found in the larger labeled cells in the autistic group compared with the control group (P=0.009; independent t‐test) but not in the smaller cell subpopulation. This suggests a disturbance in the intrinsic cerebellar circuitry in the autism group potentially interfering with the synchronous firing of inferior olivary neurons, and the timing of Purkinje cell firing and inputs to the dentate nuclei. Disturbances in critical neural substrates within these key circuits could disrupt afferents to motor and/or cognitive cerebral association areas in the autistic brain likely contributing to the marked behavioral consequences characteristic of autism.

Increased GAD67 mRNA Expression in Cerebellar Interneurons in Autism : Implications for Purkinje Cell Dysfunction

It has been widely reported that in autism, the number of Purkinje cells (PCs) is decreased, and recently, decreased expression of glutamic acid decarboxylase 67 (GAD67) mRNA in Purkinje cells also has been observed. However, the autism literature has not addressed key GABAergic inputs into Purkinje cells. Inhibitory basket and stellate cell interneurons in the molecular layer of the cerebellar cortex provide direct key GABAergic input into Purkinje cells and could potently influence the output of Purkinje cells to deep cerebellar nuclei. We investigated the capacity for interneuronal synthesis of gamma‐amino butyric acid (GABA) in both types of interneurons that innervate the remaining PCs in the posterolateral cerebellar hemisphere in autism. The level of GAD67 mRNA, one of the isoforms of the key synthesizing enzymes for GABA, was quantified at the single‐cell level using in situ hybridization in brains of autistic and aged‐matched controls. The National Institutes of Health imaging system showed that expression of GAD67 mRNA in basket cells was significantly up‐regulated, by 28%, in eight autistic brains compared with that in eight control brains (mean ± SEM pixels per cell, 1.03 ± 0.05 versus 0.69 ± 0.05, respectively; P < 0.0001 by independent t test). Stellate cells showed a trend toward a small increase in GAD67 mRNA levels, but this did not reach significance. The results suggest that basket cells likely provide increased GABAergic feed‐forward inhibition to PCs in autism, directly affecting PC output to target neurons in the dentate nucleus and potentially disrupting its modulatory role in key motor and/or cognitive behaviors in autistic individuals.

Striatal changes in preproenkephalin mRNA levels in parkinsonian monkeys

LEVELS of preproenkephalin (PPE) mRNA were measured in different sectors of the striatum with in situ hybridization histochemistry in both normal and parkinsonian (MPTP-treated) squirrel monkeys. In parkinsonian monkeys, a marked increase in PPE mRNA levels was noted in the dorsolateral third of the precommissural putamen and in most of the postcommissural putamen. These regions largely correspond to the sensorimotor striatal territory. The other striatal sectors, including the caudate nucleus, did not exhibit significant changes, despite the fact that the loss of the dopaminergic input was severe in most of the striatum. These results reveal that PPE mRNA expression is specifically altered in striatal regions involved in sensorimotor processing in parkinsonian monkeys.

Comparative effects of acute or chronic administration of levodopa to 6-hydroxydopamine-lesioned rats on the expression of glutamic acid decarboxylase in the neostriatum and GABAA receptors subunits in the substantia nigra, pars reticulata

Current evidence suggests that behavioral sensitization to the chronic administration of levodopa (L-DOPA) to dopamine-depleted animals involves a plasticity of GABA-mediated signaling in output regions of the basal ganglia. The purpose of this study was to compare in adult rats with a unilateral 6-hydroxydopamine (6-OHDA) lesion the effects of an acute or chronic (for 3 or 7 days) injection of L-DOPA on mRNA levels encoding for glutamic acid decarboxylase (GAD65 and GAD67) in the striatum and GABA(A) receptor alpha1, beta2 and gamma2 subunits in the substantia nigra, pars reticulata (SNr), by in situ hybridization histochemistry. In addition, immunostaining levels for the alpha1 subunit were examined in the SNr. In agreement with previous studies, we found that L-DOPA administration increased GAD mRNA levels in the striatum of 6-OHDA-lesioned rats. However, the magnitude of this effect increased with the number of injections of L-DOPA. On the other hand, we found that 6-OHDA lesions resulted in increases in alpha1, beta2 and gamma2 mRNA levels in the ipsilateral SNr, which were normalized or decreased compared with the contralateral side by the acute or chronic administration of L-DOPA. In addition, alpha1 immunostaining in the SNr was significantly decreased in rats injected for 7 days but not for 3 days or acutely with L-DOPA. Our results demonstrate that a chronic administration of L-DOPA results in a progressive increase in GAD and decrease in GABA(A) receptor expression in the striatum and SNr, respectively. They provide further evidence that behavioral sensitization and dyskinesia induced by a chronic administration of L-DOPA in an experimental model of Parkinsons disease is paralleled by a plasticity of GABA-mediated signaling in the SNr.

Metabotropic glutamate mGluR5 receptor blockade opposes abnormal involuntary movements and the increases in glutamic acid decarboxylase mRNA levels induced by l-DOPA in striatal neurons of 6-hydroxydopamine-lesioned rats

The present study examined the effect of a subchronic systemic administration of the glutamate metabotropic mGluR5 receptor antagonist MPEP on l-DOPA-induced dyskinesias and striatal gene expression in adult rats with a unilateral 6-OHDA lesion of dopamine neurons. The daily systemic administration of l-DOPA for 2 weeks induced a gradual increase in limb dyskinesia and axial dystonia. The subchronic systemic co-administration of MPEP reduced the severity of limb dyskinesia and axial dystonia over the whole duration of l-DOPA treatment. Subchronic l-DOPA administration was paralleled by a significant increase in mRNA levels of the two isoforms of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD67 and GAD65) and preprodynorphin (PPD). Single cell analysis on emulsion radioautographs indicated that l-DOPA-induced increases in GAD67 occurred predominantly in preproenkephalin-unlabeled striatonigral and, to a lesser extent, in preproenkephalin-labeled striatopallidal neurons. MPEP completely reversed the effects of l-DOPA on GAD67 and reduced the increases in GAD65 and PPD mRNA levels in striatonigral neurons. MPEP also reversed the small l-DOPA-induced increase in GAD67 mRNA levels in striatopallidal neurons. Altogether, the findings support the idea that the relative efficacy of mGluR5 receptor antagonists to oppose l-DOPA-induced abnormal involuntary movements involves an ability to oppose increases in GAD gene expression and GABA-mediated signaling in striatonigral and striatopallidal neurons. The results also confirm the potential usefulness of antagonists of mGluR5 receptors as adjuncts in the treatment of l-DOPA-induced dyskinesia in patients with Parkinsons disease.

Gene expression of the GAD67 and GAD65 isoforms of glutamate decarboxylase is differentially altered in subpopulations of striatal neurons in adult rats lesioned with 6‐OHDA as neonates

The levels of mRNAs encoding for the two isoforms of glutamate decarboxylase, GAD65 and GAD67, were measured in subpopulations of striatal neurons in adult rats depleted of dopamine as neonates with 6‐OHDA and chronically injected with vehicle or with the dopamine receptor agonists apomorphine or SKF‐38393. In adult rats depleted of dopamine as neonates, an increase of GAD65 and GAD67 mRNA levels was measured in the striatum. These changes were paralleled by an increase in preproenkephalin (PPE) and a decrease in preprodynorphin (PPD) mRNA levels. Quantitative analysis at the cellular level indicated that GAD67 mRNA levels were increased in PPE‐labeled neurons, whereas GAD65 mRNA levels were increased in PPE‐unlabeled neurons. Chronic and systemic injections of apomorphine or SKF‐38393 induced further increases in striatal GAD65 and GAD67 mRNA levels. These increases were only detected in the subpopulation of PPE‐unlabeled neurons and were paralleled by an increase in PPD mRNA levels. The increases in GAD67, GAD65, and PPD mRNA levels induced by SKF‐38393 were abolished by the administration of the D1 receptor antagonist SCH‐23390. The present results provide further evidence that GAD67 and GAD65 gene expression is differentially regulated in the two subpopulations of efferent striatal neurons. They also suggest that neonatal depletions in dopamine levels induce alterations of GABA‐mediated signaling in the two subpopulations of striatal efferent neurons. We speculate that these alterations are involved in the behavioral particularities exhibited by rats depleted of dopamine as neonates. Synapse 33:36–48, 1999.

Normalization of glutamate decarboxylase gene expression in the entopeduncular nucleus of rats with a unilateral 6-hydroxydopamine lesion correlates with increased gabaergic input following intermittent but not continuous levodopa

The expression of mRNA encoding for the 67 kilodalton isoform of glutamate decarboxylase (GAD67) was examined by in situ hybridization histochemistry in the entopeduncular nucleus (EP) of adult rats with a 6-hydroxydopamine unilaterally lesion of dopamine neurons. Our results provide original evidence that continuous or intermittent levodopa administration is equally effective at reversing the lesion-induced increase in GAD67 mRNA expression in the EP when compared with vehicle controls. To characterize the GABAergic interactions that may mediate levodopa-induced alterations in the EP, double-labeling in situ hybridization was conducted with a combination of GAD67 radioactive and preproenkephalin or preprotachykinin digoxigenin-labeled complementary RNA probes in the striatum. Levels of GAD67 mRNA labeling were significantly increased by intermittent, but not continuous levodopa. Analysis at the cellular level in a dorsal sector of the striatum revealed that GAD67 mRNA levels increased predominantly in preproenkephalin-unlabeled neuronal profiles, presumably striatal/EP neurons (+99.3%). Saturation analyses of (3)H-flunitrazepam binding to GABA(A) receptors in the EP showed that the increase in GAD67 mRNA in preproenkephalin-unlabeled neurons by intermittent levodopa paralleled a significant decrease in number of GABA(A) receptors (Bmax) in the EP ipsilateral to the lesion. Continuous levodopa failed to alter striatal GAD67 mRNA levels, or the number or affinity of GABA(A) receptors when compared with vehicle-treated controls. These results suggest the normalization of GAD gene expression in the EP by intermittent levodopa involves an increase in GABAergic inhibition by striatonigral/EP neurons of the direct pathway. Conversely, the effects of continuous levodopa on GAD mRNA levels in the EP do not appear to be mediated by GABA.