The cortico-striatal circuitry in autism-spectrum disorders: a balancing act.

The basal ganglia are major targets of cortical inputs and, in turn, modulate cortical function via their projections to the motor and prefrontal cortices. The role of the basal ganglia in motor control and reward is well documented and there is also extensive evidence that they play a key role in social and repetitive behaviors. The basal ganglia influence the activity of the cerebral cortex via two major projections from the striatum to the output nuclei, the globus pallidus internus and the substantia nigra, pars reticulata. This modulation involves a direct projection known as the direct pathway and an indirect projection via the globus pallidus externus and the subthalamic nucleus, known as the indirect pathway. This review discusses the respective contribution of the direct and indirect pathways to social and repetitive behaviors in neurotypical conditions and in autism spectrum disorders.

Increased Dopamine Type 2 Gene Expression in the Dorsal Striatum in Individuals With Autism Spectrum Disorder Suggests Alterations in Indirect Pathway Signaling and Circuitry.

Autism spectrum disorder (ASD) is behaviorally defined and diagnosed by delayed and/or impeded language, stereotyped repetitive behaviors, and difficulties with social interactions. Additionally, there are disruptions in motor processing, which includes the intent to execute movements, interrupted/inhibited action chain sequences, impaired execution of speech, and repetitive motor behaviors. Cortical loops through basal ganglia (BG) structures are known to play critical roles in the typical functioning of these actions. Specifically, corticostriate projections to the dorsal striatum (caudate and putamen) convey abundant input from motor, cognitive and limbic cortices and subsequently project to other BG structures. Excitatory dopamine (DA) type 1 receptors are predominantly expressed on GABAergic medium spiny neurons (MSNs) in the dorsal striatum as part of the "direct pathway" to GPi and SNpr whereas inhibitory DA type 2 receptors are predominantly expressed on MSNs that primarily project to GPe. This study aimed to better understand how this circuitry may be altered in ASD, especially concerning the neurochemical modulation of GABAergic MSNs within the two major BG pathways. We utilized two classical methods to analyze the postmortem BG in ASD in comparison to neurotypical cases: ligand binding autoradiography to quantify densities of GABA-A, GABA-B, 5-HT2, and DA type 1 and 2 receptors and in situ hybridization histochemistry (ISHH) to quantify mRNA for D1, D2 receptors and three key GABAergic subunits (α1, ß2, and γ2), as well as the GABA synthesizing enzymes (GAD65/67). Results demonstrated significant increases in D2 mRNA within MSNs in both the caudate and putamen, which was further verified by proenkephalin mRNA that is co-expressed with the D2 receptor in the indirect pathway MSNs. In contrast, all other GABAergic, serotonergic and dopaminergic markers in the dorsal striatum had comparable labeling densities. These results indicate alterations in the indirect pathway of the BG, with possible implications for the execution of competing motor programs and E/I imbalance in the direct/indirect motor feedback pathways through thalamic and motor cortical areas. Results also provide insights regarding the efficacy of FDA-approved drugs used to treat individuals with ASD acting on specific DA and 5-HT receptor subtypes.

Basal ganglia and autism - a translational perspective.

The basal ganglia are a collection of nuclei below the cortical surface that are involved in both motor and non-motor functions, including higher order cognition, social interactions, speech, and repetitive behaviors. Motor development milestones that are delayed in autism such as gross motor, fine motor and walking can aid in early diagnosis of autism. Neuropathology and neuroimaging findings in autism cases revealed volumetric changes and altered cell density in select basal ganglia nuclei. Interestingly, in autism, both the basal ganglia and the cerebellum are impacted both in their motor and non-motor domains and recently, found to be connected via the pons through a short disynaptic pathway. In typically developing individuals, the basal ganglia plays an important role in: eye movement, movement coordination, sensory modulation and processing, eye-hand coordination, action chaining, and inhibition control. Genetic models have proved to be useful toward understanding cellular and molecular changes at the synaptic level in the basal ganglia that may in part contribute to these autism-related behaviors. In autism, basal ganglia functions in motor skill acquisition and development are altered, thus disrupting the normal flow of feedback to the cortex. Taken together, there is an abundance of emerging evidence that the basal ganglia likely plays critical roles in maintaining an inhibitory balance between cortical and subcortical structures, critical for normal motor actions and cognitive functions. In autism, this inhibitory balance is disturbed thus impacting key pathways that affect normal cortical network activity. Autism Res 2017, 10: 1751-1775. © 2017 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: Habit learning, action selection and performance are modulated by the basal ganglia, a collection of groups of neurons located below the cerebral cortex in the brain. In autism, there is emerging evidence that parts of the basal ganglia are structurally and functionally altered disrupting normal information flow. The basal ganglia through its interconnected circuits with the cerebral cortex and the cerebellum can potentially impact various motor and cognitive functions in the autism brain.

Decreased parvalbumin mRNA levels in cerebellar Purkinje cells in autism.

Recent neuropathology studies in human brains indicate that several areas of the prefrontal cortex have decreased numbers of parvalbumin interneurons or decreased parvalbumin expression in Autism Spectrum disorders (ASD) [Hashemi, Ariza, Rogers, Noctor, & Martinez-Cerdeno, 2017; Zikopoulos & Barbas, ]. These data suggest that a deficit in parvalbumin may be a key neuropathology of ASD and contribute to altered GABAergic inhibition. However, it is unclear if a deficit in parvalbumin is a phenomenon that occurs in regions other than the cerebral cortex. The cerebellum is a major region where neuropathology was first detected in ASD over three decades ago [Bauman & Kemper, ]. In view of the documented association between parvalbumin-expressing neurons and autism, the objective of the present study was to determine if parvalbumin gene expression is also altered in Purkinje neurons of the cerebellum. Radioisotopic in situ hybridization histochemistry was used on human tissue sections from control and ASD brains in order to detect and measure parvalbumin mRNA levels at the single cell level in Purkinje cells of Crus II of the lateral cerebellar hemispheres. Results indicate that parvalbumin mRNA levels are significantly lower in Purkinje cells in ASD compared to control brains. This decrease was not influenced by post-mortem interval or age at death. This result indicates that decreased parvalbumin expression is a more widespread feature of ASD. We discuss how this decrease may be implicated in altered cerebellar output to the cerebral cortex and in key ASD symptoms. Autism Res 2017, 10: 1787-1796. © 2017 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: The cerebellum of the brain controls movement and cognition, including memory and language. This study investigated mechanisms of cerebellar function in Autism. Our hypothesis is that parvalbumin, a molecule that controls and coordinate many cellular brain functions, contributes to the excitatory/inhibitory imbalance in Autism. We report that parvalbumin expression is depressed in Purkinje cells of the cerebellum in autism. This finding contributes to elucidate the cellular and molecular underpinings of autism and should provide a direction for future therapies.

Decreased parvalbumin mRNA expression in dorsolateral prefrontal cortex in Parkinson's disease.

It has recently been shown that expression of the rate-limiting GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) is decreased in Brodmann area 9 (BA9) of the dorsolateral prefrontal cortex (DLPFC) in Parkinson's disease (PD) compared to control brains (Lanoue, A.C., Dumitriu, A., Myers, R.H., Soghomonian, JJ., 2010. Exp. Neurol. 206 (1), 207-217). A subpopulation of cortical GABAergic interneurons expresses the calcium-binding protein parvalbumin and plays a critical role in the control of pyramidal neuron excitability and the generation of cortical gamma frequency oscillations. In view of its key role in the physiology of the cerebral cortex, we sought to determine whether the expression of parvalbumin and the number of parvalbumin-expressing neurons are altered in BA9 of PD brains. First, isotopic in situ hybridization histochemistry was used to examine mRNA expression of parvalbumin on post-mortem brain sections. Second, immunohistochemistry and design-based stereology were used to determine the density of parvalbumin-positive interneurons in BA9. Quantification of mRNA labeling at the single cell level showed a significant decrease in parvalbumin expression in PD cases. In contrast, neuronal density of parvalbumin-positive neurons was not significantly different between PD and controls. Results confirm that the GABAergic system is altered in the DLPFC in PD and identify the contribution of parvalbumin-expressing neurons in these alterations. We speculate that these effects could contribute to altered cortical excitability and oscillatory activity previously documented in PD.

Decreased glutamic acid decarboxylase mRNA expression in prefrontal cortex in Parkinson's disease.

Parkinson's disease (PD) patients typically suffer from motor disorders but mild to severe cognitive deficits can also be present. Neuropathology of PD primarily involves loss of dopaminergic neurons in the substantia nigra, pars compacta, although more widespread pathology from the brainstem to the cerebral cortex occurs at different stages of the disease. Cognitive deficits in PD are thought to involve the cerebral cortex, and imaging studies have identified the dorsolateral prefrontal cortex (DLPFC) as a possible site for some of the symptoms. GABAergic neurons in the cerebral cortex play a key role in the modulation of pyramidal neurons and alterations in muscimol binding to GABA(A) receptors have been reported in Brodmann area 9 (BA9) of the prefrontal cortex in PD patients (Nishino et al., 1988). In order to further assess the likelihood that GABAergic activity is altered in the prefrontal cortex in PD, gene expression of the 67 kilodalton isoform of the GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD67 encoded by the GAD1 gene), was examined in BA9 of post-mortem brains from 19 patients and 20 controls using isotopic in situ hybridization histochemistry. GAD67 mRNA labeling was examined and quantified on X-ray films and emulsion radioautographs. We show that GAD67 mRNA labeling is significantly lower in PD compared to control cases. Analysis of emulsion radioautographs indicates that GAD67 mRNA labeling is decreased in individual neurons and is not paralleled by a decrease in the number of GAD67 mRNA-labeled neurons. Analysis of expression data from a microarray study performed in 29 control and 33 PD samples from BA9 confirms that GAD67 expression is decreased in PD. Another finding from the microarray study is a negative relationship between GAD67 mRNA expression and age at death. Altogether, the results support the possibility that GABAergic neurotransmission is impaired in the DLPFC in PD, an effect that may be involved in some of the behavioral deficits associated with the disease.

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 microm in diameter, presumed interneurons) and larger-sized neurons (about 18-20 microm 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.

Haloperidol treatment after high-dose methamphetamine administration is excitotoxic to GABA cells in the substantia nigra pars reticulata.

The therapeutic management of methamphetamine (METH)-induced psychoses often involves treatment with the typical antipsychotic drug and dopamine D2 receptor antagonist haloperidol. We report here that subchronic haloperidol administration after a high-dose regimen of METH produces a heretofore unrecognized toxicity to GABAergic cells, as reflected by GAD67 mRNA expression histochemistry, in the rat substantia nigra pars reticulata (SNr) through an acute and persistent augmentation of glutamate release, NMDA receptor activation, and DNA fragmentation. The dopaminergic cells in the substantia nigra pars compacta were unaffected by METH or haloperidol alone or the combination of METH and haloperidol. These findings suggest that the current therapeutic management of METH-induced psychoses with haloperidol may be contraindicated because of a resultant GABAergic cell death in the SNr, which may predispose some individuals to the development of hyperkinetic movement disorders and seizures.

Decreased GAD67 mRNA levels in cerebellar Purkinje cells in autism: pathophysiological implications.

The recent identification of decreased protein levels of glutamate decarboxylase (GAD) 65 and 67 isoforms in the autistic cerebellar tissue raises the possibility that abnormal regulation of GABA production in individual neurons may contribute to the clinical features of autism. Reductions in Purkinje cell number have been widely reported in autism. It is not known whether the GAD changes also occur in Purkinje cells at the level of transcription. Using a novel approach, the present study quantified GAD67 mRNA, the most abundant isoform in Purkinje cells, using in situ hybridization in adult autistic and control cases. The results indicate that GAD67 mRNA level was reduced by 40% in the autistic group (P < 0.0001; two-tailed t test), suggesting that reduced Purkinje cell GABA input to the cerebellar nuclei potentially disrupts cerebellar output to higher association cortices affecting motor and/or cognitive function. These findings may also contribute to the understanding of previous reports of alterations in the GABAergic system in limbic and cerebro-cortical areas contributing to a more widespread pathophysiology in autistic brains.

Subchronic administration of L-DOPA to adult rats with a unilateral 6-hydroxydopamine lesion of dopamine neurons results in a sensitization of enhanced GABA release in the substantia nigra, pars reticulata.

L-DOPA is the most effective pharmacological agent used for the symptomatic treatment of Parkinson's disease but long-term L-DOPA treatment induces involuntary abnormal movements such as dyskinesias. The present study, using in vivo microdialysis, investigated the effects of a single or subchronic administration of L-DOPA to adult rats with a unilateral 6-OHDA lesion of dopamine neurons on GABA release in the substantia nigra, pars reticulata (SNr). The results indicate that a challenge injection of L-DOPA (50 mg/kg, i.p.) significantly increases GABA levels in the SNr of rats treated with a daily repeated administration of L-DOPA (50 mg/kg, i.p.). Further statistical analysis between groups also showed that extracellular GABA levels were significantly higher in the subchronic L-DOPA group than in the group receiving only one injection of L-DOPA. These results show that the subchronic administration of L-DOPA results in a sensitization of enhanced extracellular GABA levels in the SNr.

L-DOPA-induced dyskinesia in adult rats with a unilateral 6-OHDA lesion of dopamine neurons is paralleled by increased c-fos gene expression in the subthalamic nucleus.

Levodopa (L-DOPA), the metabolic precursor of dopamine, is widely used as a pharmacological agent for the symptomatic treatment of Parkinson's disease. However, long-term L-DOPA use results in abnormal involuntary movements such as dyskinesias. There is evidence that abnormal cell signaling in the basal ganglia is involved in L-DOPA-induced dyskinesia. The subthalamic nucleus (STN) plays a key role in the circuitry of the basal ganglia and in the pathophysiology of Parkinson's disease. However, the contribution of the STN to L-DOPA-induced dyskinesias remains unclear. The objective of this work was to study the effects of acute or chronic systemic administration of L-DOPA to adult rats with a unilateral 6-hydroxydopamine (6-OHDA) lesion of dopamine neurons on c-fos expression in the STN and test the hypothesis that these effects correlate with L-DOPA-induced dyskinesias. c-fos mRNA expression was measured in the STN by in situ hybridization histochemistry at the single cell level. Our results confirm earlier evidence that the chronic administration of L-DOPA to rats with a unilateral 6-OHDA lesion increases c-fos expression in the STN. We also report that c-fos expression can be increased following an acute injection of L-DOPA to 6-OHDA-lesioned rats but not following a chronic injection of L-DOPA to sham-operated, unlesioned rats. Finally, we provide evidence that the occurrence and severity of dyskinesia is correlated with c-fos mRNA levels in the ipsilateral STN. These results suggest that altered cell signaling in the STN is involved in some of the behavioral effects induced by systemic L-DOPA administration.

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 microm) 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 microm), 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).

Dual effects of intermittent or continuous L-DOPA administration on gene expression in the globus pallidus and subthalamic nucleus of adult rats with a unilateral 6-OHDA lesion.

Intermittent oral doses of levodopa (L-DOPA) are routinely used to treat Parkinson's disease, but with prolonged use can result in adverse motor complications, such as dyskinesia. Continuous administration of L-DOPA achieves therapeutic efficacy without producing this effect, yet the molecular mechanisms are unclear. This study examined, by in situ hybridization histochemistry, the effects of continuous or intermittent L-DOPA administration on gene expression in the globus pallidus and subthalamic nucleus of adult rats with a unilateral 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal pathway. Results were compared to 6-OHDA-treated rats receiving vehicle. Our results provide original evidence that continuous L-DOPA normalizes the 6-OHDA-lesion-induced increase in mRNA levels encoding for the 67 kDa isoform of glutamate decarboxylase in neurons of the globus pallidus and cytochrome oxidase subunit I mRNA levels in the subthalamic nucleus. The extent of normalization did not differ between the continuous and intermittent groups. In addition, intermittent L-DOPA induced an increase in the mRNA levels encoding for the 65 kDa isoform of glutamate decarboxylase in globus pallidus neurons ipsilateral to the lesion and a bilateral increase in c-fos mRNA expression in the subthalamic nucleus. These results suggest that continuous L-DOPA tends to normalize the 6-OHDA-lesion-induced alterations in cell signaling in the pallido-subthalamic loop. On the other hand, we propose that chronic intermittent L-DOPA exerts a dual effect by normalizing cell signaling in a subpopulation of neurons in the globus pallidus and subthalamic nucleus while inducing abnormal signaling in another subpopulation.