Adriana Galvan

Pathophysiology of Parkinsonism

The motor signs of Parkinsons disease are thought to result in large part from a reduction of the level of dopamine in the basal ganglia. Over the last few years, many of the functional and anatomical consequences of dopamine loss in these structures have been identified, both in the basal ganglia and in related areas in thalamus and cortex. This knowledge has contributed significantly to our understanding of the link between the degeneration of dopamine neurons in the midbrain and the development of parkinsonism. This review discusses the evidence that implicates electrophysiologic changes (including altered discharge rates, increased incidence of burst firing, interneuronal synchrony, oscillatory activity, and altered sensorimotor processing) in basal ganglia, thalamus, and cortex, in parkinsonism. From these studies, parkinsonism emerges as a complex network disorder, in which abnormal activity in groups of neurons in the basal ganglia strongly affects the excitability, oscillatory activity, synchrony and sensory responses of areas of the cerebral cortex that are involved in the planning and execution of movement, as well as in executive, limbic or sensory functions. Detailed knowledge of these changes will help us to develop more effective and specific symptomatic treatments for patients with Parkinsons disease.

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 Parkinsons disease. Although the available clinical evidence is encouraging, these procedures remain empirical at this stage because of the limited understanding of the thalamostriatal systems.

The thalamostriatal system in normal and diseased states

Because of our limited knowledge of the functional role of the thalamostriatal system, this massive network is often ignored in models of the pathophysiology of brain disorders of basal ganglia origin, such as Parkinson’s disease (PD). However, over the past decade, significant advances have led to a deeper understanding of the anatomical, electrophysiological, behavioral and pathological aspects of the thalamostriatal system. The cloning of the vesicular glutamate transporters 1 and 2 (vGluT1 and vGluT2) has provided powerful tools to differentiate thalamostriatal from corticostriatal glutamatergic terminals, allowing us to carry out comparative studies of the synaptology and plasticity of these two systems in normal and pathological conditions. Findings from these studies have led to the recognition of two thalamostriatal systems, based on their differential origin from the caudal intralaminar nuclear group, the center median/parafascicular (CM/Pf) complex, or other thalamic nuclei. The recent use of optogenetic methods supports this model of the organization of the thalamostriatal systems, showing differences in functionality and glutamate receptor localization at thalamostriatal synapses from Pf and other thalamic nuclei. At the functional level, evidence largely gathered from thalamic recordings in awake monkeys strongly suggests that the thalamostriatal system from the CM/Pf is involved in regulating alertness and switching behaviors. Importantly, there is evidence that the caudal intralaminar nuclei and their axonal projections to the striatum partly degenerate in PD and that CM/Pf deep brain stimulation (DBS) may be therapeutically useful in several movement disorders.

Glutamate and GABA receptors and transporters in the basal ganglia: What does their subsynaptic localization reveal about their function?

GABA and glutamate, the main transmitters in the basal ganglia, exert their effects through ionotropic and metabotropic receptors. The dynamic activation of these receptors in response to released neurotransmitter depends, among other factors, on their precise localization in relation to corresponding synapses. The use of high resolution quantitative electron microscope immunocytochemical techniques has provided in-depth description of the subcellular and subsynaptic localization of these receptors in the CNS. In this article, we review recent findings on the ultrastructural localization of GABA and glutamate receptors and transporters in monkey and rat basal ganglia, at synaptic, extrasynaptic and presynaptic sites. The anatomical evidence supports numerous potential locations for receptor-neurotransmitter interactions, and raises important questions regarding mechanisms of activation and function of synaptic versus extrasynaptic receptors in the basal ganglia.

Alterations in neuronal activity in basal ganglia-thalamocortical circuits in the parkinsonian state

In patients with Parkinson’s disease and in animal models of this disorder, neurons in the basal ganglia and related regions in thalamus and cortex show changes that can be recorded by using electrophysiologic single-cell recording techniques, including altered firing rates and patterns, pathologic oscillatory activity and increased inter-neuronal synchronization. In addition, changes in synaptic potentials or in the joint spiking activities of populations of neurons can be monitored as alterations in local field potentials (LFPs), electroencephalograms (EEGs) or electrocorticograms (ECoGs). Most of the mentioned electrophysiologic changes are probably related to the degeneration of diencephalic dopaminergic neurons, leading to dopamine loss in the striatum and other basal ganglia nuclei, although degeneration of non-dopaminergic cell groups may also have a role. The altered electrical activity of the basal ganglia and associated nuclei may contribute to some of the motor signs of the disease. We here review the current knowledge of the electrophysiologic changes at the single cell level, the level of local populations of neural elements, and the level of the entire basal ganglia-thalamocortical network in parkinsonism, and discuss the possible use of this information to optimize treatment approaches to Parkinson’s disease, such as deep brain stimulation (DBS) therapy.

GABAergic circuits in the basal ganglia and movement disorders

GABA is the major inhibitory neurotransmitter in the basal ganglia, and GABAergic pathways dominate information processing in most areas of these structures. It is therefore not surprising that abnormalities of GABAergic transmission are key elements in pathophysiologic models of movement disorders involving the basal ganglia. These include hypokinetic diseases such as Parkinsons disease, and hyperkinetic diseases, such as Huntingtons disease or hemiballism. In this chapter, we will briefly review the major anatomic features of the GABAergic pathways in the basal ganglia, and then describe in greater detail the changes of GABAergic transmission, which are known to occur in movement disorders.

Effects of stimulation of the centromedian nucleus of the thalamus on the activity of striatal cells in awake rhesus monkeys

Although the existence of a massive projection from the caudal intralaminar nuclei of the thalamus [i.e. the centromedian (CM) and parafascicular nuclei] to the striatum is well documented, the effects of CM activation upon striatal cells remain poorly understood. Therefore, we studied the effects of electrical stimulation of CM on the electrophysiological activity of striatal neurons, and on striatal levels of γ‐aminobutyric acid (GABA) and acetylcholine in rhesus monkeys. Striatal cells did not respond to single‐pulse stimulation (bipolar biphasic stimulation, 175–500 μA), but the large majority of recorded neurons responded to burst stimulation (100 Hz, 1 s, 150–175 μA) of CM, often with a delay of tens of milliseconds. Striatal phasically active neurons, which likely correspond to projection neurons, responded mainly with increases in firing (13/28 cells), while tonically active neurons (likely cholinergic interneurons) often showed combinations of increases and decreases in firing (24/46 cells). In microdialysis studies, CM stimulation led to a reduction of striatal acetylcholine levels. This effect was prevented by addition of the GABA‐A receptor antagonist gabazine to the microdialysis fluid. We conclude that CM stimulation frequently results in striatal response patterns with excitatory and inhibitory components. Under the conditions chosen here, the specific patterns of striatal responses to CM stimulation are likely the result of striatal processing of thalamic inputs. Through these indirect effects, local CM stimulation may engage large portions of the striatum. These effects may be relevant in the interpretation of the therapeutic effects of CM stimulation for the treatment of neurological disorders.

Differential subcellular and subsynaptic distribution of GABAA and GABAB receptors in the monkey subthalamic nucleus

The activation of GABA receptor subtype A (GABA(A)) and GABA receptor subtype B (GABA(B)) receptors mediates differential effects on GABAergic and non-GABAergic transmission in the basal ganglia. To further characterize the anatomical substrate that underlies these functions, we used immunogold labeling to compare the subcellular and subsynaptic localization of GABA(A) and GABA(B) receptors in the subthalamic nucleus (STN). Our findings demonstrate major differences and some similarities in the distribution of GABA(A) and GABA(B) receptors in the monkey STN. The immunoreactivity for GABA(A) receptor alpha1 subunits is mostly bound to the plasma membrane, whereas GABA(B) R1 subunit alpha1 immunoreactivity is largely expressed intracellularly. Plasma membrane-bound GABA(A) alpha1 subunit aggregate in the main body of putative GABAergic synapses, while GABA(B) R1 receptors are found at the edges of putative glutamatergic or GABAergic synapses. A large pool of plasma membrane-bound GABA(A) and GABA(B) receptors is extrasynaptic. In conclusion, these findings demonstrate a significant degree of heterogeneity between the distributions of the two major GABA receptor subtypes in the monkey STN. Their pattern of synaptic localization puts forward interesting questions regarding their mechanisms of activation and functions at GABAergic and non-GABAergic synapses.

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.

Localization and function of GABA transporters in the globus pallidus of parkinsonian monkeys

The GABA transporters GAT-1 and GAT-3 are abundant in the external and internal segments of the globus pallidus (GPe and GPi, respectively). We have shown that pharmacological blockade of either of these transporters results in decreased neuronal firing, and in elevated levels of extracellular GABA in normal monkeys. We now studied whether the electrophysiologic and biochemical effects of local intra-pallidal injections of GAT-1 and GAT-3 blockers, or the subcellular localization of these transporters, are altered in monkeys rendered parkinsonian by the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The subcellular localization of the transporters in GPe and GPi, studied with electron microscopy immunoperoxidase, was similar to that found in normal animals: i.e., GAT-3 immunoreactivity was mostly confined to glial processes, while GAT-1 labeling was expressed in unmyelinated axons and glial processes. A combined injection/recording device was used to record the extracellular activity of single neurons in GPe and GPi, before, during and after administration of small volumes (1microl) of either the GAT-1 inhibitor, SKF-89976A hydrochloride (720ng), or the GAT-3 inhibitor, (S)-SNAP-5114 (500ng). In GPe, the effects of GAT-1 or GAT-3 blockade were similar to those seen in normal monkeys. However, unlike the findings in the normal state, the firing of most neurons was not affected by blockade of either transporter in GPi. These results suggest that, after dopaminergic depletion, the functions of GABA transporters are altered in GPi; without major changes in their subcellular localization.