Comparative Ultrastructural Analysis of Thalamocortical Innervation of the Primary Motor Cortex and Supplementary Motor Area in Control and MPTP-Treated Parkinsonian Monkeys.

The synaptic organization of thalamic inputs to motor cortices remains poorly understood in primates. Thus, we compared the regional and synaptic connections of vGluT2-positive thalamocortical glutamatergic terminals in the supplementary motor area (SMA) and the primary motor cortex (M1) between control and MPTP-treated parkinsonian monkeys. In controls, vGluT2-containing fibers and terminal-like profiles invaded layer II-III and Vb of M1 and SMA. A significant reduction of vGluT2 labeling was found in layer Vb, but not in layer II-III, of parkinsonian animals, suggesting a potential thalamic denervation of deep cortical layers in parkinsonism. There was a significant difference in the pattern of synaptic connectivity in layers II-III, but not in layer Vb, between M1 and SMA of control monkeys. However, this difference was abolished in parkinsonian animals. No major difference was found in the proportion of perforated versus macular post-synaptic densities at thalamocortical synapses between control and parkinsonian monkeys in both cortical regions, except for a slight increase in the prevalence of perforated axo-dendritic synapses in the SMA of parkinsonian monkeys. Our findings suggest that disruption of the thalamic innervation of M1 and SMA may underlie pathophysiological changes of the motor thalamocortical loop in the state of parkinsonism.

Glutamatergic inputs to GABAergic interneurons in the motor thalamus of control and parkinsonian monkeys.

The primate ventral motor thalamus contains a large number of GABAergic interneurons of poorly understood function and anatomical connectivity. Glutamatergic inputs to these cells arise predominantly from corticothalamic (in both basal ganglia- and cerebellar-receiving ventral motor thalamic territories; BGMT and CBMT, respectively) and cerebellothalamic terminals (in CBMT). In Parkinson's disease patients and animal models, neuronal activity is abnormal within both BGMT and CBMT. Historically, such motor thalamic dysregulation has been largely attributed to changes in inhibitory tone from the basal ganglia output nuclei, ignoring the potential role of other thalamic inputs in such processes, particularly within the CBMT, which is largely devoid of direct basal ganglia afferents. We have recently reported changes in the abundance and structural morphology of corticothalamic terminals in BGMT of parkinsonian monkeys. In this study, we assessed potential changes in the prevalence of cortical (vesicular glutamate transporter 1-positive, vGluT1-positive) and subcortical (vGluT2-positive) glutamatergic inputs in contact with GABAergic interneurons in BGMT and CBMT of MPTP-treated parkinsonian monkeys. Our findings revealed that interneurons represent a major target of both sets of glutamatergic terminals. In both BGMT and CBMT of control and parkinsonian monkeys, 29%-38% of total asymmetric axodendritic synapses (putative glutamatergic) were formed by vGluT1-positive terminals and 11%-17% of total vGluT1-positive terminals targeted dendrites of GABAergic interneurons. In CBMT, 16%-18% of asymmetric synaptic inputs on interneurons involved vGluT2-containing terminals. No major differences in the extent of glutamatergic innervation of thalamic GABAergic interneurons were found between control and parkinsonian monkeys.

Striatal glutamate delta-1 receptor regulates behavioral flexibility and thalamostriatal connectivity.

Impaired behavioral flexibility and repetitive behavior is a common phenotype in autism and other neuropsychiatric disorders, but the underlying synaptic mechanisms are poorly understood. The trans-synaptic glutamate delta (GluD)-Cerebellin 1-Neurexin complex, critical for synapse formation/maintenance, represents a vulnerable axis for neuropsychiatric diseases. We have previously found that GluD1 deletion results in reversal learning deficit and repetitive behavior. In this study, we show that selective ablation of GluD1 from the dorsal striatum impairs behavioral flexibility in a water T-maze task. We further found that striatal GluD1 is preferentially found in dendritic shafts, and more frequently associated with thalamic than cortical glutamatergic terminals suggesting localization to projections from the thalamic parafascicular nucleus (Pf). Conditional deletion of GluD1 from the striatum led to a selective loss of thalamic, but not cortical, terminals, and reduced glutamatergic neurotransmission. Optogenetic studies demonstrated functional changes at thalamostriatal synapses from the Pf, but no effect on the corticostriatal system, upon ablation of GluD1 in the dorsal striatum. These studies suggest a novel molecular mechanism by which genetic variations associated with neuropsychiatric disorders may impair behavioral flexibility, and reveal a unique principle by which GluD1 subunit regulates forebrain circuits.

Group I metabotropic glutamate receptors in the primate motor thalamus: subsynaptic association with cortical and sub-cortical glutamatergic afferents.

Preclinical evidence indicates that mGluR5 is a potential therapeutic target for Parkinson's disease and L-DOPA-induced dyskinesia. However, the mechanisms through which these therapeutic benefits are mediated remain poorly understood. Although the regulatory role of mGluR5 on glutamatergic transmission has been examined in various basal ganglia nuclei, very little is known about the localization and function of mGluR5 in the ventral motor and intralaminar thalamic nuclei, the main targets of basal ganglia output in mammals. Thus, we used immuno-electron microscopy to map the cellular and subcellular localization of group I mGluRs (mGluR1a and mGluR5) in the ventral motor and caudal intralaminar thalamic nuclei in rhesus monkeys. Furthermore, using double immuno-electron microscopy, we examined the subsynaptic localization of mGluR5 in relation to cortical and sub-cortical glutamatergic afferents. Four major conclusions can be drawn from these data. First, mGluR1a and mGluR5 are expressed postsynaptically on the plasma membrane of dendrites of projection neurons and GABAergic interneurons in the basal ganglia- and cerebellar-receiving regions of the ventral motor thalamus and in CM. Second, the plasma membrane-bound mGluR5 immunoreactivity is preferentially expressed perisynaptically at the edges of cortical and sub-cortical glutamatergic afferents. Third, the mGluR5 immunoreactivity is more strongly expressed in the lateral than the medial tiers of CM, suggesting a preferential association with thalamocortical over thalamostriatal neurons in the primate CM. Overall, mGluR5 is located to subserve powerful modulatory role of cortical and subcortical glutamatergic transmission in the primate ventral motor thalamus and CM.

Sub-synaptic localization of Cav3.1 T-type calcium channels in the thalamus of normal and parkinsonian monkeys.

T-type calcium channels (Cav3) are key mediators of thalamic bursting activity, but also regulate single cells excitability, dendritic integration, synaptic strength and transmitter release. These functions are strongly influenced by the subcellular and subsynaptic localization of Cav3 channels along the somatodendritic domain of thalamic cells. In Parkinson's disease, T-type calcium channels dysfunction in the basal ganglia-receiving thalamic nuclei likely contributes to pathological thalamic bursting activity. In this study, we analyzed the cellular, subcellular, and subsynaptic localization of the Cav3.1 channel in the ventral anterior (VA) and centromedian/parafascicular (CM/Pf) thalamic nuclei, the main thalamic targets of basal ganglia output, in normal and parkinsonian monkeys. All thalamic nuclei displayed strong Cav3.1 neuropil immunoreactivity, although the intensity of immunolabeling in CM/Pf was significantly lower than in VA. Ultrastructurally, 70-80 % of the Cav3.1-immunoreactive structures were dendritic shafts. Using immunogold labeling, Cav3.1 was commonly found perisynaptic to asymmetric and symmetric axo-dendritic synapses, suggesting a role of Cav3.1 in regulating excitatory and inhibitory neurotransmission. Significant labeling was also found at non-synaptic sites along the plasma membrane of thalamic neurons. There was no difference in the overall pattern and intensity of immunostaining between normal and parkinsonian monkeys, suggesting that the increased rebound bursting in the parkinsonian state is not driven by changes in Cav3.1 expression. Thus, T-type calcium channels are located to subserve neuronal bursting, but also regulate glutamatergic and non-glutamatergic transmission along the whole somatodendritic domain of basal ganglia-receiving neurons of the primate thalamus.

Effects of Optogenetic Activation of Corticothalamic Terminals in the Motor Thalamus of Awake Monkeys.

The role of the corticothalamic projection in the ventral motor thalamus remains poorly understood. Therefore, we studied the electrophysiological responses of neurons in the basal ganglia and cerebellar receiving-territories of the motor thalamus (BGMT and CbMT, respectively) using optogenetic activation of corticothalamic projections in awake rhesus macaques. After injections of viral vectors carrying the excitatory opsins ChR2 or C1V1 into the primary motor and premotor cortices of two monkeys, we used optrodes to light activate opsin-expressing neurons in cortex or their terminals in the thalamus while simultaneously recording the extracellular activity of neurons in the vicinity of the stimulation sites. As expected, light activation of opsins in the cerebral cortex evoked robust, short-latency increases in firing of cortical neurons. In contrast, light stimulation of corticothalamic terminals induced small-amplitude, long-latency increases and/or decreases of activity in thalamic neurons. In postmortem material, opsins were found to be expressed in cell bodies and dendrites of cortical neurons and along their corticothalamic projections. At the electron microscopic level, opsin labeling was confined to unmyelinated preterminal axons and small terminals that formed asymmetric synapses with dendrites of projection neurons or GABAergic interneurons in BGMT and CbMT and with neurons in the reticular thalamic nucleus. The morphological features of the transfected terminals, along with the long latency and complex physiological responses of thalamic neurons to their activation, suggest a modulatory role of corticothalamic afferents upon the primate ventral motor thalamus. SIGNIFICANCE STATEMENT: This study provides the first analysis of the physiological effects of cortical inputs on the activity of neurons in the primate ventral motor thalamus using light activation of opsin-containing corticothalamic terminals in awake monkeys. We found that selective light activation of corticothalamic terminals in contact with distal dendrites of thalamocortical neurons and GABAergic interneurons elicits complex patterns of slowly developing excitatory and inhibitory effects in thalamic neurons of the basal ganglia- and cerebellar-receiving regions of the motor thalamus. Our observations suggest a modulatory (instead of a "driver") role of the corticothalamic system in the primate ventral motor thalamus.

Anatomical localization of Cav3.1 calcium channels and electrophysiological effects of T-type calcium channel blockade in the motor thalamus of MPTP-treated monkeys.

Conventional anti-Parkinsonian dopamine replacement therapy is often complicated by side effects that limit the use of these medications. There is a continuing need to develop nondopaminergic approaches to treat Parkinsonism. One such approach is to use medications that normalize dopamine depletion-related firing abnormalities in the basal ganglia-thalamocortical circuitry. In this study, we assessed the potential of a specific T-type calcium channel blocker (ML218) to eliminate pathologic burst patterns of firing in the basal ganglia-receiving territory of the motor thalamus in Parkinsonian monkeys. We also carried out an anatomical study, demonstrating that the immunoreactivity for T-type calcium channels is strongly expressed in the motor thalamus in normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys. At the electron microscopic level, dendrites accounted for >90% of all tissue elements that were immunoreactive for voltage-gated calcium channel, type 3.2-containing T-type calcium channels in normal and Parkinsonian monkeys. Subsequent in vivo electrophysiologic studies in awake MPTP-treated Parkinsonian monkeys demonstrated that intrathalamic microinjections of ML218 (0.5 µl of a 2.5-mM solution, injected at 0.1-0.2 µl/min) partially normalized the thalamic activity by reducing the proportion of rebound bursts and increasing the proportion of spikes in non-rebound bursts. The drug also attenuated oscillatory activity in the 3-13-Hz frequency range and increased gamma frequency oscillations. However, ML218 did not normalize Parkinsonism-related changes in firing rates and oscillatory activity in the beta frequency range. Whereas the described changes are promising, a more complete assessment of the cellular and behavioral effects of ML218 (or similar drugs) is needed for a full appraisal of their anti-Parkinsonian potential.

In vivo optogenetic control of striatal and thalamic neurons in non-human primates.

Electrical and pharmacological stimulation methods are commonly used to study neuronal brain circuits in vivo, but are problematic, because electrical stimulation has limited specificity, while pharmacological activation has low temporal resolution. A recently developed alternative to these methods is the use of optogenetic techniques, based on the expression of light sensitive channel proteins in neurons. While optogenetics have been applied in in vitro preparations and in in vivo studies in rodents, their use to study brain function in nonhuman primates has been limited to the cerebral cortex. Here, we characterize the effects of channelrhodopsin-2 (ChR2) transfection in subcortical areas, i.e., the putamen, the external globus pallidus (GPe) and the ventrolateral thalamus (VL) of rhesus monkeys. Lentiviral vectors containing the ChR2 sequence under control of the elongation factor 1α promoter (pLenti-EF1α -hChR2(H134R)-eYFP-WPRE, titer 109 particles/ml) were deposited in GPe, putamen and VL. Four weeks later, a probe combining a conventional electrode and an optic fiber was introduced in the previously injected brain areas. We found light-evoked responses in 31.5% and 32.7% of all recorded neurons in the striatum and thalamus, respectively, but only in 2.5% of recorded GPe neurons. As expected, most responses were time-locked increases in firing, but decreases or mixed responses were also seen, presumably via ChR2-mediated activation of local inhibitory connections. Light and electron microscopic analyses revealed robust expression of ChR2 on the plasma membrane of cell somas, dendrites, spines and terminals in the striatum and VL. This study demonstrates that optogenetic experiments targeting the striatum and basal ganglia-related thalamic nuclei can be successfully achieved in monkeys. Our results indicate important differences of the type and magnitude of responses in each structure. Experimental conditions such as the vector used, the number and rate of injections, or the light stimulation conditions have to be optimized for each structure studied.

Thalamic contributions to Basal Ganglia-related behavioral switching and reinforcement.

Although the existence of prominent connections between the intralaminar thalamic nuclei and the basal ganglia has long been established, the limited knowledge of the functional relevance of this network has considerably hampered progress in our understanding of the neural mechanisms by which the thalamostriatal system integrates and regulates the basal ganglia circuitry. In this brief commentary, we will address this gap of knowledge through a discussion of the key points of a symposium entitled "Thalamic Contributions to Basal Ganglia-Related Behavioral Switching and Reinforcement" that will be presented at the 2011 Society for Neuroscience meeting. Recent anatomical and physiological data that support the role of the thalamostriatal system in action selection, attentional shifting, and reinforcement will be discussed. We will also address the possibility that degeneration of the thalamostriatal system could underlie some of the deficits in redirection of attention in response to salient stimuli seen in Parkinson's disease.

Differential structural plasticity of corticostriatal and thalamostriatal axo-spinous synapses in MPTP-treated Parkinsonian monkeys.

Striatal spine loss is a key pathological feature of Parkinson's disease (PD). Knowing that striatal glutamatergic afferents target dendritic spines, these data appear difficult to reconcile with evidence for an increased expression of the vesicular glutamate transporter 1 (vGluT1) in the striatum of PD patients and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys, as well as in some electrophysiological studies showing overactivity of the corticostriatal glutamatergic system in models of parkinsonism. To address the possibility that structural changes in glutamatergic afferents may underlie these discrepancies, we undertook an ultrastructural analysis of vGluT1-positive (i.e., corticostriatal) and vGluT2-positive (i.e., mostly thalamostriatal) axo-spinous glutamatergic synapses using a 3D electron microscopic approach in normal and MPTP-treated monkeys. Three main conclusions can be drawn: 1) spines contacted by vGluT1-containing terminals have larger volume and harbor significantly larger postsynaptic densities (PSDs) than those contacted by vGluT2-immunoreactive boutons; 2) a subset of vGluT2-, but not vGluT1-immunoreactive, terminals display a pattern of multisynaptic connectivity in normal and MPTP-treated monkeys; and 3) VGluT1- and vGluT2-positive axo-spinous synapses undergo ultrastructural changes (larger spine volume, larger PSDs, increased PSD perforations, larger presynaptic terminal) indicative of increased synaptic activity in parkinsonian animals. Furthermore, spines contacted by cortical terminals display an increased volume of their spine apparatus in MPTP-treated monkeys, suggesting an increased protein synthesis at corticostriatal synapses. These findings demonstrate that corticostriatal and thalamostriatal glutamatergic axo-spinous synapses display significantly different ultrastructural features, and that both systems undergo complex morphological changes that could underlie the pathophysiology of corticostriatal and thalamostriatal systems in PD.

The thalamostriatal systems: anatomical and functional organization in normal and parkinsonian states.

Although we have gained significant knowledge in the anatomy and microcircuitry of the thalamostriatal system over the last decades, the exact function(s) of these complex networks remain(s) poorly understood. It is now clear that the thalamostriatal system is not a unique entity, but consists of multiple neural systems that originate from a wide variety of thalamic nuclei and terminate in functionally segregated striatal territories. The primary source of thalamostriatal projections is the caudal intralaminar nuclear group which, in primates, comprises the centromedian and parafascicular nuclei (CM/Pf). These two nuclei provide massive, functionally organized glutamatergic inputs to the whole striatal complex. There are several anatomical and physiological features that distinguish this system from other thalamostriatal projections. Although all glutamatergic thalamostriatal neurons express vGluT2 and release glutamate as neurotransmitter, CM/Pf neurons target preferentially the dendritic shafts of striatal projection neurons, whereas all other thalamic inputs are almost exclusively confined to the head of dendritic spines. This anatomic arrangement suggests that transmission of input from sources other than CM/Pf to the striatal neurons is likely regulated by dopaminergic afferents in the same manner as cortical inputs, while the CM/Pf axo-dendritic synapses do not display any particular relationships with dopaminergic terminals. A better understanding of the role of these systems in the functional circuitry of the basal ganglia relies on future research of the physiology and pathophysiology of these networks in normal and pathological basal ganglia conditions. Although much remains to be known about the role of these systems, recent electrophysiological studies from awake monkeys have provided convincing evidence that the CM/Pf-striatal system is the entrance for attention-related stimuli to the basal ganglia circuits. However, the processing and transmission of this information likely involves intrinsic GABAergic and cholinergic striatal networks, thereby setting the stage for complex physiological responses of striatal output neurons to CM/Pf activation. Finally, another exciting development that will surely generate significant interest towards the thalamostriatal systems in years to come is the possibility that CM/Pf may be a potential surgical target for movement disorders, most particularly Tourette syndrome and Parkinson's disease. Although the available clinical evidence is encouraging, these procedures remain empirical at this stage because of the limited understanding of the thalamostriatal systems.

Differential synaptic plasticity of the corticostriatal and thalamostriatal systems in an MPTP-treated monkey model of parkinsonism.

Two cardinal features of Parkinson's disease (PD) pathophysiology are a loss of glutamatergic synapses paradoxically accompanied by an increased glutamatergic transmission to the striatum. The exact substrate of this increased glutamatergic drive remains unclear. The striatum receives glutamatergic inputs from the thalamus and the cerebral cortex. Using vesicular glutamate transporters (vGluTs) 1 and 2 as markers of the corticostriatal and thalamostriatal afferents, respectively, we examined changes in the synaptology and relative prevalence of striatal glutamatergic inputs in methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys using electron microscopic immunoperoxidase and confocal immunofluorescence methods. Our findings demonstrate that the prevalence of vGluT1-containing terminals is significantly increased in the striatum of MPTP-treated monkeys (51.9 +/- 3.5% to 66.5 +/- 3.4% total glutamatergic boutons), without any significant change in the pattern of synaptic connectivity; more than 95% of vGluT1-immunolabeled terminals formed axo-spinous synapses in both conditions. In contrast, the prevalence of vGluT2-immunoreactive terminals did not change after MPTP treatment (21.7 +/- 1.3% vs. 21.6 +/- 1.2% total glutamatergic boutons). However, a substantial increase in the ratio of axo-spinous to axo-dendritic synapses formed by vGluT2-immunoreactive terminals was found in the pre-caudate and post-putamen striatal regions of MPTP-treated monkeys, suggesting a certain degree of synaptic reorganization of the thalamostriatal system in parkinsonism. About 20% of putative glutamatergic terminals did not show immunoreactivity in striatal tissue immunostained for both vGluT1 and vGluT2, suggesting the expression of another vGluT in these boutons. These findings provide striking evidence that suggests a differential degree of plasticity of the corticostriatal and thalamostriatal system in PD.

Differential synaptology of vGluT2-containing thalamostriatal afferents between the patch and matrix compartments in rats.

The striatum is divided into two compartments named the patch (or striosome) and the matrix. Although these two compartments can be differentiated by their neurochemical content or afferent and efferent projections, the synaptology of inputs to these striatal regions remains poorly characterized. By using the vesicular glutamate transporters vGluT1 and vGluT2, as markers of corticostriatal and thalamostriatal projections, respectively, we demonstrate a differential pattern of synaptic connections of these two pathways between the patch and the matrix compartments. We also demonstrate that the majority of vGluT2-immunolabeled axon terminals form axospinous synapses, suggesting that thalamic afferents, like corticostriatal inputs, terminate preferentially onto spines in the striatum. Within both compartments, more than 90% of vGluT1-containing terminals formed axospinous synapses, whereas 87% of vGluT2-positive terminals within the patch innervated dendritic spines, but only 55% did so in the matrix. To characterize further the source of thalamic inputs that could account for the increase in axodendritic synapses in the matrix, we undertook an electron microscopic analysis of the synaptology of thalamostriatal afferents to the matrix compartments from specific intralaminar, midline, relay, and associative thalamic nuclei in rats. Approximately 95% of PHA-L-labeled terminals from the central lateral, midline, mediodorsal, lateral dorsal, anteroventral, and ventral anterior/ventral lateral nuclei formed axospinous synapses, a pattern reminiscent of corticostriatal afferents but strikingly different from thalamostriatal projections arising from the parafascicular nucleus (PF), which terminated onto dendritic shafts. These findings provide the first evidence for a differential pattern of synaptic organization of thalamostriatal glutamatergic inputs to the patch and matrix compartments. Furthermore, they demonstrate that the PF is the sole source of significant axodendritic thalamic inputs to striatal projection neurons. These observations pave the way for understanding differential regulatory mechanisms of striatal outflow from the patch and matrix compartments by thalamostriatal afferents.

The thalamostriatal system: a highly specific network of the basal ganglia circuitry.

Although the existence of thalamostriatal projections has long been known, the role(s) of this system in the basal ganglia circuitry remains poorly characterized. The intralaminar and ventral motor nuclei are the main sources of thalamic inputs to the striatum. This review emphasizes the high degree of anatomical and functional specificity of basal ganglia-thalamostriatal projections and discusses various aspects of the synaptic connectivity and neurochemical features that differentiate this glutamate system from the corticostriatal network. It also discusses the importance of thalamostriatal projections from the caudal intralaminar nuclei in the process of attentional orientation. A major task of future studies is to characterize the role(s) of corticostriatal and thalamostriatal pathways in regulating basal ganglia activity in normal and pathological conditions.

Group I metabotropic glutamate receptors in the monkey striatum: subsynaptic association with glutamatergic and dopaminergic afferents.

Group I metabotropic glutamate receptors (mGluRs) are involved in long-term synaptic plasticity and neuroprotection in the striatum, but the specific role(s) of mGluR1 and mGluR5 remain poorly understood. In this study, we used electron-microscopic immunocytochemistry to compare the pattern of subsynaptic and subcellular distribution of mGluR1a and mGluR5 in relation to putative glutamatergic and dopaminergic inputs to the monkey striatum. At the light-microscopic level, both group I mGluRs are expressed in the striatal neuropil. In addition, numerous perikarya of striatal output neurons are immunostained for mGluR5, but much less frequently for mGluR1a. At the electron-microscopic level, immunoreactivity for both receptor subtypes is primarily expressed postsynaptically in dendrites and spines, although presynaptic mGluR1a labeling of glutamatergic thalamostriatal boutons and, less frequently, dopaminergic and corticostriatal terminals is also seen. In contrast to mGluR1a, mGluR5 immunoreactivity is rarely encountered presynaptically. In postsynaptic elements, 40-70% of immunoreactivity for both receptor subtypes is expressed intracellularly, whereas 30-60% is apposed to the plasma membrane. More than 80% of the labeling apposed to the plasma membrane is extrasynaptic. The remaining 20% is located at the edges of putative glutamatergic synapses or in the active zone of symmetric synapses. In mGluR5-, but not mGluR1a-immunostained sections, approximately 70% of dopaminergic symmetric synapses are labeled perisynaptically. These data emphasize the differential pattern of subsynaptic localization of the two group I mGluRs and provide various presynaptic and postsynaptic sites whereby mGluR1 and mGluR5 could mediate different, but complementary, effects on glutamatergic and dopaminergic transmission in the primate striatum.