The motor symptoms of Parkinson’s disease (PD) are commonly attributed to striatal dopamine loss, but reduced dopamine innervation of basal ganglia output nuclei, the internal globus pallidus (GPi) and the substantia nigra pars reticulata (SNr) may also contribute to symptoms and signs of PD. Both structures express dopamine D1 and D5 receptors under normal conditions, and we have recently demonstrated that their local activation reduces neuronal discharge rates and enhances bursts and oscillatory activity in both nuclei of normal monkeys [M.A. Kliem et al. (2007)
J. Neurophysiol., 89, 1489–1500]. Here, we determined the ultrastructural localization and function of D1-like receptors in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated parkinsonian monkeys. In both normal and MPTP-treated monkeys, most of the D1 and D5 receptor immunoreactivity was associated with unmyelinated axons, but we also found significant postsynaptic D5 receptor immunostaining in dendrites of GPi and SNr neurons. A significant proportion of axonal D1 immunostaining was bound to the plasma membrane in both normal and MPTP-treated monkeys. Local microinjections of the D1 / D5 receptor agonist SKF82958 significantly reduced discharge rates in GPi and SNr neurons, while they increased burst firing and oscillatory activity in the 3–15-Hz band in SNr, but not in GPi, of parkinsonian monkeys. Together with our recent findings from normal monkeys, these data provide evidence that functional D1 / D5 receptors are expressed in GPi and SNr in both normal and parkinsonian states, and that their activation by endogenous dopamine (under normal conditions) or dopamine receptor agonists (in parkinsonism) may regulate basal ganglia outflow.
D1; D5; globus pallidus; MPTP; Parkinson’s disease; substantia nigra
Regulator of G Protein Signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates G protein and MAPK signaling pathways. In the adult mouse brain, RGS14 mRNA and protein are found almost exclusively in hippocampal CA2 neurons. We have shown that RGS14 is a natural suppressor of CA2 synaptic plasticity and hippocampal-dependent learning and memory. However, the protein distribution and spatiotemporal expression patterns of RGS14 in mouse brain during postnatal development are unknown. Here, using a newly characterized monoclonal anti-RGS14 antibody, we demonstrate that RGS14 protein immunoreactivity is undetectable at birth (P0) with very low mRNA expression in the brain. However, RGS14 protein and mRNA are upregulated during early postnatal development, with protein first detected at P7, and both increasing over time until reaching highest sustained levels throughout adulthood. Our immunoperoxidase data demonstrate that RGS14 protein is expressed in regions outside of hippocampal CA2 during development including the primary olfactory areas, the anterior olfactory nucleus and piriform cortex, and the olfactory associated orbital and entorhinal cortices. RGS14 is also transiently expressed in neocortical layers II/III and V during postnatal development. Finally, we show that RGS14 protein is first detected in the hippocampus at P7 with strongest immunoreactivity in CA2 and fasciola cinerea and sporadic immunoreactivity in CA1; labeling intensity in hippocampus increases until adulthood. These results show that RGS14 mRNA and protein are upregulated throughout postnatal mouse development, and RGS14 protein exhibits a dynamic localization pattern that is enriched in hippocampus and primary olfactory cortex in the adult mouse brain.
RGS14; hippocampus; hippocampal CA2; synaptic plasticity; RGS proteins
High frequency deep brain stimulation (DBS) of the subthalamic nucleus (STN) is a successful treatment for patients with advanced Parkinson’s disease (PD). While its exact mechanism of action is unknown, it is currently believed that the beneficial effects of the stimulation are mediated either by alleviating pathological basal ganglia output patterns of activity, or by activation of axons of passage which arise from the cerebral cortex and other sources.
In this study, we show that the anatomical composition of the primate STN provides a substrate through which DBS may elicit widespread changes in brain activity via stimulation of fibers of passage. Using quantitative high-resolution electron microscopy, we found that the primate STN is traversed by numerous myelinated axons which occupy as much as 45% of its sensorimotor territory, and 36% of its associative region. In comparison, myelinated axons occupy only 27% of the surface areas of the sensorimotor and associative regions of the internal segment of the globus pallidus (GPi), another target for therapeutic DBS in PD. We also noted that myelinated axons in the STN have, on average, a larger diameter than those in GPi, which may render them more susceptible to electrical stimulation.
Because axons are more excitable than other neuronal elements, our findings support the hypothesis that STN DBS, even when carried out entirely within the confines of the nucleus, mediates some of its effects by activating myelinated axons of passage.
deep brain stimulation; Parkinson’s disease; fibers of passage; globus pallidus; antidromic
In the striatum, the dendritic tree of the two main populations of projection neurons, called “Medium Spiny Neurons (MSNs)”, are covered with spines that receive glutamatergic inputs from the cerebral cortex and thalamus. In Parkinson’s disease (PD), striatal MSNs undergo an important loss of dendritic spines, whereas aberrant overgrowth of striatal spines occurs following chronic cocaine exposure. This review examines the possibility that opposite dopamine dysregulation is one of the key factors that underlies these structural changes. In PD, nigrostriatal dopamine degeneration results in a significant loss of dendritic spines in the dorsal striatum, while rodents chronically exposed to cocaine and other psychostimulants, display an increase in the density of “thin and immature” spines in the nucleus accumbens (NAc). In rodent models of PD, there is evidence that D2 dopamine receptor-containing MSNs are preferentially affected, while D1-positive cells are the main targets of increased spine density in models of addiction. However, such specificity remains to be established in primates. Although the link between the extent of striatal spine changes and the behavioral deficits associated with these disorders remains controversial, there is unequivocal evidence that glutamatergic synaptic transmission is significantly altered in both diseased conditions. Recent studies have suggested that opposite calcium-mediated regulation of the transcription factor myocyte enhancer factor 2 (MEF2) function induces these structural defects. In conclusion, there is strong evidence that dopamine is a major, but not the sole, regulator of striatal spine pathology in PD and addiction to psychostimulants. Further studies of the role of glutamate and other genes associated with spine plasticity in mediating these effects are warranted.
striatum; cocaine; accumbens; corticostriatal; thalamostriatal; glutamate
Striatal cholinergic interneurons (ChIs) are involved in reward-dependent learning and the regulation of attention. The activity of these neurons is modulated by intrinsic and extrinsic γ-aminobutyric acid (GABA)ergic and glutamatergic afferents, but the source and relative prevalence of these diverse regulatory inputs remain to be characterized. To address this issue, we performed a quantitative ultrastructural analysis of the GABAergic and glutamatergic innervation of ChIs in the postcommissural putamen of rhesus monkeys. Postembedding immunogold localization of GABA combined with peroxidase immunostaining for choline acetyltransferase showed that 60% of all synaptic inputs to ChIs originate from GABAergic terminals, whereas 21% are from putatively glutamatergic terminals that establish asymmetric synapses, and 19% from other (non-GABAergic) sources of symmetric synapses. Double pre-embedding immunoelectron microscopy using substance P and Met-/Leu-enkephalin antibodies to label GABAergic terminals from collaterals of “direct” and “indirect” striatal projection neurons, respectively, revealed that 47% of the indirect pathway terminals and 36% of the direct pathway terminals target ChIs. Together, substance P- and enkephalin-positive terminals represent 24% of all synapses onto ChIs in the monkey putamen. These findings show that ChIs receive prominent GABAergic inputs from multiple origins, including a significant contingent from axon collaterals of direct and indirect pathway projection neurons.
medium spiny neurons; substance P; enkephalin; GABA; striatum
The perirhinal cortex plays a critical role in recognition and associative memory. However, the network properties that support perirhinal contributions to memory are unclear. To shed light on this question, we compared the synaptic articulation of short- and long-range inputs from the perirhinal cortex or temporal neocortex with perirhinal neurons in rats. Iontophoretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHAL) were performed at different rostrocaudal levels of the ventral temporal neocortex or perirhinal cortex, and electron microscopic observations of anterogradely labeled (PHAL+) axon terminals found at perirhinal sites adjacent to or rostrocaudally distant from the injection sites were performed. After neocortical injections, the density of PHAL+ axons in the perirhinal cortex decreased steeply with rostrocaudal distance from the injection sites, much more so than following perirhinal injections. Otherwise, similar results were obtained with neocortical and perirhinal injections. In both cases, most (76–86%) PHAL+ axon terminals formed asymmetric synapses, typically with spines (type A, 83–89%) and less frequently with dendritic profiles (type B, 11–17%). The remaining terminals formed symmetric synapses with dendritic profiles (type C, 14– 23%). Type B and C synapses were 2.4–2.6 times more frequent in short- than long-range connections. The postsynaptic elements in type A–C synapses were identified with immunocytochemistry for CAMKIIa, a marker of glutamatergic cortical neurons. Type A and C terminals contacted CAMKII α- positive principal cells, whereas type B synapses contacted presumed inhibitory neurons. Overall, these results suggest that principal perirhinal neurons are subjected to significantly more inhibition from short- than from long-range cortical inputs, an organization that likely impacts perirhinal contributions to memory.
perirhinal; GABA; interneuron; electron microscopy; tract-tracing
Astrocytes are relatively resistant to injury compared to neurons and oligodendrocytes. Here, we report transient region-specific loss of astrocytes in mice early after pilocarpine-induced status epilepticus (SE). In the dentate hilus, immunoreactivity for glial acidic fibrillary protein (GFAP) was decreased, and the number of healthy appearing GFAP- or S100β-positive cells was significantly reduced (≥ 65%) 1 and 3 days after pilocarpine-induced SE. Many remaining GFAP-positive cells were shrunken, and 1 day after SE electron microscopy revealed numerous electron-dense degenerating astrocyte processes and degenerating glial somata in the hilus. Degeneration of GFAP-expressing cells may be linked to hilar neuronal death, because we did not observe loss of astrocytes after kainate-induced SE, after which hilar neurons remained intact. Ten days after SE, hilar GFAP immunoreactivity had returned, partially from GFAP-positive cells in the hilus. Unlike control mice, many GFAP-positive hilar processes originated from cell bodies located in the subgranular zone (SGZ). To investigate whether proliferation contributes to hilar repopulation, we injected 5-bromo-2′-deoxyuridine (BrdU) 3 days after SE. Five hours later and up to 31 days after SE, many BrdU/GFAP colabeled cells were found in the hilus and the SGZ, some with hilar processes, indicating that proliferation in both areas contributes to generation of hilar astrocytes and astrocyte processes. In contrast to pilocarpine-induced SE in mice, astrocyte degeneration was not found after pilocarpine-induced SE in rats. These findings demonstrate astrocyte degeneration in the mouse dentate hilus specifically in the mouse pilocarpine epilepsy model, followed by astrogenesis leading to hilar repopulation.
Stem cell; Seizure; Hilus; Kainate; Glial fibrillary acidic protein; Adult progenitor cell; Mouse strain
The dystonias are comprised of a group of disorders that share common neurological abnormalities of involuntary twisting or repetitive movements and postures. The most common inherited primary dystonia is DYT1 dystonia, which is due to loss of a GAG codon in the TOR1A gene that encodes torsinA. Autopsy studies of brains from patients with DYT1 dystonia have revealed few abnormalities, although recent neuroimaging studies have implied the existence of microstructural defects that might not be detectable with traditional histopathological methods. The current studies took advantage of a knock-in mouse model for DYT1 dystonia to search for subtle anatomical abnormalities in the striatum, a region often implicated in studies of dystonia. Multiple abnormalities were identified using a combination of quantitative stereological measures of immunohistochemical stains for specific neuronal populations, morphometric studies of Golgi-stained neurons, and immuno-electron microscopy of synaptic connectivity. In keeping with other studies, there was no obvious loss of striatal neurons in the DYT1 mutant mice. However, interneurons immunoreactive for choline acetyltransferase or parvalbumin were larger in the mutants than in control mice. In contrast, interneurons immunoreactive for neuronal nitric oxide synthase were smaller in the mutants than in controls. Golgi histochemical studies of medium spiny projection neurons in the mutant mice revealed slightly fewer and thinner dendrites, and a corresponding loss of dendritic spines. Electron microscopic studies showed a reduction in the ratio of axo-spinous to axo-dendritic synaptic inputs from glutamatergic and dopaminergic sources in mutant mice compared with controls. These results suggest specific anatomical substrates for altered signaling in the striatum and potential correlates of the abnormalities implied by human imaging studies of DYT1 dystonia.
Mouse mutant; anatomy; Golgi histochemistry; stereology; striatum; electron microscopy
Group III metabotropic glutamate receptors (mGluR4,7,8) are widely distributed in the basal ganglia. Injection of group III mGluR agonists into the striatopallidal complex alleviates parkinsonian symptoms in 6-hydroxydopamine-treated rats. In vitro rodent studies have suggested that this may be partly due to modulation of synaptic transmission at striatopallidal and corticostriatal synapses through mGluR4 activation. However, the in vivo electrophysiological effects of group III mGluRs activation upon basal ganglia neurons activity in nonhuman primates remain unknown. Thus, in order to examine the anatomical substrates and physiological effects of group III mGluRs activation upon striatal and pallidal neurons in monkeys, we used electron microscopy immunohistochemistry to localize mGluR4, combined with local administration of the group III mGluR agonist L-AP4, or the mGluR4 positive allosteric modulator VU0155041, to assess the effects of group III mGluR activation on the firing rate and pattern of striatal and pallidal neurons in 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP)-treated parkinsonian monkeys.
At the ultrastructural level, striatal mGluR4 immunoreactivity was localized in pre- (60%) and post-synaptic (30%) elements, while in the GPe, mGluR4 was mainly expressed presynaptically (90%). In the putamen, terminals expressing mGluR4 were evenly split between putative excitatory and inhibitory terminals, while in the GPe, most labeled terminals displayed the ultrastructural features of striatal-like inhibitory terminals, though putative excitatory boutons were also labeled. No significant difference was found between normal and parkinsonian monkeys. Extracellular recordings in awake MPTP-treated monkeys revealed that local microinjections of small volumes of L-AP4 resulted in increased firing rates in one half of striatal cells and one third of pallidal cells, while a significant number of neurons in both structures showed either opposite effects, or did not display any significant rate changes following L-AP4 application. VU0155041 administration had little effect on firing rates. Both compounds also had subtle effects on bursting and oscillatory properties, acting to increase the irregularity of firing. The occurrence of pauses in firing was reduced in the majority (80%) of GPe neurons after L-AP4 injection. Our findings indicate that glutamate can mediate multifarious physiological effects upon striatal and pallidal neurons through activation of pre-synaptic group III mGluRs at inhibitory and excitatory synapses in parkinsonian monkeys.
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.
thalamus; Parkinson’s disease; intralaminar nuclei; glutamate; vesicular glutamate transporter; attention; striatum; Tourette’s syndrome
The sodium/bicarbonate transporter NBCn1 plays an essential role in intracellular pH regulation and transepithelial HCO3− movement in the body. NBCn1 also has sodium channel-like activity uncoupled to Na/HCO3 cotransport. We previously reported that NBCn1 interacts with the postsynaptic density protein PSD-95 in the brain. Here, we elucidated the structural determinant and functional consequence of NBCn1/PSD-95 interaction.
In rat hippocampal CA3 neurons, NBCn1 was localized to the postsynaptic membranes of both dendritic shafts and spines and occasionally to the presynaptic membranes. A GST/NBCn1 fusion protein containing the C-terminal 131 amino acids of NBCn1 pulled down PSD-95 from rat brain lysates, whereas GST/NBCn1-ΔETSL (deletion of the last four amino acids) and GST/NBCn2 (NCBE) lacking the same ETSL did not. NBCn1 and PSD-95 were coimmunoprecipitated in HEK 293 cells, and their interaction did not affect the efficacy of PSD-95 to bind to the NMDA receptor NR2A. PSD-95 has negligible effects on intracellular pH changes mediated by NBCn1 in HEK 293 cells and Xenopus oocytes. However, PSD-95 increased an ionic conductance produced by NBCn1 channel-like activity. This increase was abolished by NBCn1-ΔETSL or by the peptide containing the last 15 amino acids of NBCn1.
Our data suggest that PSD-95 interacts with NBCn1 and increases its channel-like activity while negligibly affecting Na/HCO3 cotransport. The possibility that the channel-like activity occurs via an intermolecular cavity of multimeric NBCn1 proteins is discussed.
Bicarbonate transport; Acid-base; pH; Electrophysiology; Neuron; Xenopus
Brainstem noradrenergic neurons innervate the mesocorticolimbic reward pathway both directly and indirectly, with norepinephrine facilitating dopamine (DA) neurotransmission via α1-adrenergic receptors (α1ARs). Although α1AR signaling in the prefrontal cortex (PFC) promotes mesolimbic transmission and drug-induced behaviors, the potential contribution of α1ARs in other parts of the pathway, such as the ventral tegmental area (VTA) and nucleus accumbens (NAc), has not been investigated before. We found that local blockade of α1ARs in the medial NAc shell, but not the VTA, attenuates cocaine- and morphine-induced locomotion. To determine the neuronal substrates that could mediate these effects, we analyzed the cellular, subcellular, and subsynaptic localization of α1ARs and characterized the chemical phenotypes of α1AR-containing elements within the mesocorticolimbic system using single and double immunocytochemical methods at the electron microscopic (EM) level. We found that α1ARs are found mainly extra-synaptically in axons and axon terminals in the NAc and are enriched in glutamatergic and dopaminergic elements. α1ARs are also abundant in glutamatergic terminals in the PFC, and in GABA-positive terminals in the VTA. In line with these observations, microdialysis experiments revealed that local blockade of α1ARs attenuated the increase in extracellular DA in the medial NAc shell following administration of cocaine. These data indicate that local α1ARs control DA transmission in the medial NAc shell and behavioral responses to drugs of abuse.
norepinephrine; dopamine; alpha-1 adrenergic; nucleus accumbens; ventral tegmental area; cocaine; addiction & substance abuse, psychostimulants, catecholamines, dopamine, norepinephrine, rat, morphine, adrenergic receptor, microdialysis
Intrapallidal application of GAT-1 or GAT-3 transporter blockers (SKF 89976A or SNAP 5114) reduces the activity of pallidal neurons in monkey. This effect could be mediated through activation of presynaptic GABAB heteroreceptors in glutamatergic terminals by GABA spillover following GABA transporters (GATs) blockade. To test this hypothesis, we applied the whole-cell recording technique to study the effects of SKF 89976A and SNAP 5114 on evoked excitatory post synaptic currents (eEPSCs) in presence of gabazine, a GABAA receptor antagonist, in rat GP slice preparations. Under the condition of postsynaptic GABAB receptor blockade by intracellular application of OX314, bath application of SKF 89976A (10 μM) or SNAP 5114 (10 μM) decreased the amplitude of eEPSCs, without significant effect on its holding current and whole cell input resistance. The inhibitory effect of GATs blockade on eEPSCs was blocked by CGP 58845, a GABAB receptor antagonist. The paired-pulse ratio (PPR) of evoked EPSCs was increased, while the frequency, but not the amplitude, of miniature excitatory postsynaptic currents (mEPSCs) was reduced in presence of either GAT blockers, demonstrating a presynaptic effect. These results suggest that synaptically released GABA can inhibit glutamatergic transmission through activation of presynaptic GABAB heteroreceptors following GAT-1 or GAT-3 blockade.
In conclusion, our findings demonstrate that pre-synaptic GABAB heteroreceptors in putative glutamatergic subthalamic afferents to GP are sensitive to increases in extracellular GABA induced by GATs inactivation, thereby suggesting that GATs blockade represents a potential mechanism by which overactive subthalamopallidal activity may be reduced in parkinsonism.
EPSC; patch-clamp; striatum; GATs; GABA receptor
Clinical, epidemiological, and genetic evidence suggest overlapping pathogenic mechanisms between autism spectrum disorder (ASD) and schizophrenia. We tested this hypothesis by asking if mutations in the ASD gene MECP2 which cause Rett syndrome affect the expression of genes encoding the schizophrenia risk factor dysbindin, a subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), and associated interacting proteins. We measured mRNA and protein levels of key components of a dysbindin interaction network by, quantitative real time PCR and quantitative immunohistochemistry in hippocampal samples of wild-type and Mecp2 mutant mice. In addition, we confirmed results by performing immunohistochemistry of normal human hippocampus and quantitative qRT-PCR of human inducible pluripotent stem cells (iPSCs)-derived human neurons from Rett syndrome patients. We defined the distribution of the BLOC-1 subunit pallidin in human and mouse hippocampus and contrasted this distribution with that of symptomatic Mecp2 mutant mice. Neurons from mutant mice and Rett syndrome patients displayed selectively reduced levels of pallidin transcript. Pallidin immunoreactivity decreased in the hippocampus of symptomatic Mecp2 mutant mice, a feature most prominent at asymmetric synapses as determined by immunoelectron microcopy. Pallidin immunoreactivity decreased concomitantly with reduced BDNF content in the hippocampus of Mecp2 mice. Similarly, BDNF content was reduced in the hippocampus of BLOC-1 deficient mice suggesting that genetic defects in BLOC-1 are upstream of the BDNF phenotype in Mecp2 deficient mice. Our results demonstrate that the ASD-related gene Mecp2 regulates the expression of components belonging to the dysbindin interactome and these molecular differences may contribute to synaptic phenotypes that characterize Mecp2 deficiencies and ASD.
The adenosine A2A receptor (A2AR) is a potential drug target for the treatment of Parkinson’s disease and other neurological disorders. In rodents, the therapeutic efficacy of A2AR modulation is improved by concomitant modulation of the metabotropic glutamate receptor 5 (mGluR5). To elucidate the anatomical substrate(s) through which these therapeutic benefits could be mediated, pre-embedding electron microscopy immunohistochemistry was used to conduct a detailed, quantitative ultrastructural analysis of A2AR localization in the primate basal ganglia and to assess the degree of A2AR/mGluR5 colocalization in the striatum. A2AR immunoreactivity was found at the highest levels in the striatum and external globus pallidus (GPe). However, the monkey, but not the rat, substantia nigra pars reticulata (SNr) also harbored a significant level of neuropil A2AR immunoreactivity. At the electron microscopic level, striatal A2AR labeling was most commonly localized in postsynaptic elements (58% ± 3% of labeled elements), whereas, in the GPe and SNr, the labeling was mainly presynaptic (71% ± 5%) or glial (27% ± 6%). In both striatal and pallidal structures, putative inhibitory and excitatory terminals displayed A2AR immunoreactivity. Striatal A2AR/mGluR5 colocalization was commonly found; 60–70% of A2AR-immunoreactive dendrites or spines in the monkey striatum coexpress mGluR5. These findings provide the first detailed account of the ultrastructural localization of A2AR in the primate basal ganglia and demonstrate that A2AR and mGluR5 are located to interact functionally in dendrites and spines of striatal neurons. Together, these data foster a deeper understanding of the substrates through which A2AR could regulate primate basal ganglia function and potentially mediate its therapeutic effects in parkinsonism.
mGluR5; Parkinson’s disease; primate; immunogold; globus pallidus; substantia nigra; putamen
An extensive literature shows that astrocytes exhibit metabotropic glutamate receptor 5 (mGluR5)–dependent increases in cytosolic calcium ions (Ca2+) in response to glutamatergic transmission and, in turn, modulate neuronal activity by their Ca2+-dependent release of gliotransmitters. These findings, based on studies of young rodents, have led to the concept of the tripartite synapse, in which astrocytes actively participate in neurotransmission. Using genomic analysis, immunoelectron microscopy, and two-photon microscopy of astrocytic Ca2+ signaling in vivo, we found that astrocytic expression of mGluR5 is developmentally regulated and is undetectable after postnatal week 3. In contrast, mGluR3, whose activation inhibits adenylate cyclase but not calcium signaling, was expressed in astrocytes at all developmental stages. Neuroglial signaling in the adult brain may therefore occur in a manner fundamentally distinct from that exhibited during development.
The demonstration that dopamine loss is the key pathological feature of Parkinson's disease (PD), and the subsequent introduction of levodopa have revolutionalized the field of PD therapeutics. This review will discuss the significant progress that has been made in the development of new pharmacological and surgical tools to treat PD motor symptoms since this major breakthrough in the 1960s. However, we will also highlight some of the challenges the field of PD therapeutics has been struggling with during the past decades. The lack of neuroprotective therapies and the limited treatment strategies for the nonmotor symptoms of the disease (ie, cognitive impairments, autonomic dysfunctions, psychiatric disorders, etc.) are among the most pressing issues to be addressed in the years to come. It appears that the combination of early PD nonmotor symptoms with imaging of the nigrostriatal dopaminergic system offers a promising path toward the identification of PD biomarkers, which, once characterized, will set the stage for efficient use of neuroprotective agents that could slow down and alter the course of the disease.
dopamine; nonmotor symptoms; striatum; substantia nigra; deep brain stimulation; transplantation; dopamine; movement disorders; neurodegeneration/neuroprotection; neurology; basal ganglia; substantia nigra; deep brain stimulation; nigrostriatal; adenosine
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.
There is an increasing amount of literature data showing the positive effects on preclinical anti-Parkinsonian rodent models with selective positive allosteric modulators of metabotropic glutamate receptor 4 (mGlu4).1 However, most of the data generated utilize compounds that have not been optimized for drug-like properties and, as a consequence, they exhibit poor pharmacokinetic properties and thus do not cross the blood-brain barrier. Herein, we report on a series of N-4-(2,5-dioxopyrrolidin-1-yl)-phenylpicolinamides with improved PK properties with excellent potency and selectivity as well as improved brain exposure in rodents. Finally, ML182 was shown to be orally active in the haloperidol induced catalepsy model, a well-established anti-Parkinsonian model.
metabotropic glutamate receptors; mGlu4; positive allosteric modulators; Parkinson’s disease; haloperidol-induced catalepsy; structure-activity relationship (SAR); oral efficacy; brain penetration
The striatum receives glutamatergic inputs from two main thalamostriatal systems that originate either from the centre median/parafascicular complex (CM/PF-striatal system) or the rostral intralaminar, midline, associative and relay thalamic nuclei (non-CM/PF-striatal system). These dual thalamostriatal systems display striking differences in their anatomical and, most likely, functional organization. The CM/PF-striatal system is topographically organized, and integrated within functionally segregated basal ganglia-thalamostriatal circuits that process sensorimotor, associative and limbic information. CM/PF neurons are highly responsive to attention-related sensory stimuli, suggesting that the CM/PF-striatal system, through its strong connections with cholinergic interneurons, may play a role in basal ganglia-mediated learning, behavioral switching and reinforcement. In light of evidence for prominent CM/PF neuronal loss in Parkinson’s disease, we propose that the significant CM-striatal system degeneration, combined with the severe nigrostriatal dopamine loss in sensorimotor striatal regions, may alter normal automatic actions, and shift the processing of basal ganglia-thalamocortical motor programs towards goal-directed behaviors.
Centromedian; Parafascicular; set shifting; striatum; learning; cholinergic interneuron
The Biogenesis of Lysosome-Related Organelles Complex 1 (BLOC-1) is a protein complex containing the schizophrenia susceptibility factor dysbindin, which is encoded by the gene DTNBP1. However, mechanisms engaged by dysbindin defining schizophrenia susceptibility pathways have not been quantitatively elucidated. Here, we discovered prevalent and novel cellular roles of the BLOC-1 complex in neuronal cells by performing large-scale Stable Isotopic Labeling of Cells in Culture quantitative proteomics (SILAC) combined with genetic analyses in dysbindin-null mice (Mus musculus) and the genome of schizophrenia patients. We identified 24 proteins that associate with the BLOC-1 complex many of which were altered in content/distribution in cells or tissues deficient in BLOC-1. New findings include BLOC-1 interactions with the COG complex, a Golgi apparatus tether, and antioxidant enzymes peroxiredoxins 1-2. Importantly, loci encoding eight of the 24 proteins are affected by genomic copy number variation in schizophrenia patients. Thus, our quantitative proteomic studies expand the functional repertoire of the BLOC-1 complex and provide insight into putative molecular pathways of schizophrenia susceptibility.
The positron emission tomography (PET) tracer 2β-carbomethoxy-3β-(4-chlorophenyl)-8-(2-[18F]-fluoroethyl)-nortropane (18F-FECNT) is a highly specific ligand for dopamine transporter (DAT) that yields higher peak striatum-to-cerebellum ratios and offers more favorable kinetics than most 18F-radiolabeled DAT ligands currently available. The goal of this study is to validate the use of 18F-FECNT as a PET radiotracer to assess the degree of striatal dopamine terminals denervation and midbrain dopaminergic cell loss in MPTP-treated parkinsonian monkeys. Three rhesus monkeys received weekly injections of MPTP (0.2-0.5 mg/kg) for 21 weeks, which resulted in the progressive development of a moderate level of parkinsonism. We carried out 18F-FECNT PET at baseline (twice; ten weeks apart) and at week 21 post-MPTP injections. Postmortem stereological cell counts of dopaminergic neurons in the ventral midbrain, and intensity measurements of DAT and tyrosine hydroxylase (TH) immunoreactivity in the striatum were performed and correlated with striatal and ventral midbrain PET data. Three additional monkeys were used as controls for midbrain dopaminergic cell counts, and striatal DAT or TH immunoreactivity measurements. The correlation and coefficient of variance between 18F-FECNT test-retest specific uptake ratios were 0.99 (R2) and 2.65%, respectively. The 18F-FECNT binding potential of the ventral midbrain and striatal regions was tightly correlated with postmortem stereological cell counts of nigral dopaminergic neurons (R2 = 0.91), and striatal DAT (R2 = 0.83) or TH (R2 = 0.88) immunoreactivity intensity measurements. These findings demonstrate that 18F-FECNT is a highly sensitive PET imaging ligand to quantify both striatal dopamine denervation and midbrain dopaminergic cell loss associated with parkinsonism.
Parkinson’s disease; brain imaging; transporter; dopamine; striatum; MPTP; monkey; primate; substantia nigra; midbrain; animal model
Degeneration of the dopaminergic nigrostriatal system and of noradrenergic neurons in the locus coeruleus are important pathological features of Parkinson’s disease. There is an urgent need to develop therapies that slow down the progression of neurodegeneration in Parkinson’s disease. In the present study, we tested whether the highly specific metabotropic glutamate receptor 5 antagonist, 3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine, reduces dopaminergic and noradrenergic neuronal loss in monkeys rendered parkinsonian by chronic treatment with low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Weekly intramuscular 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine injections (0.2–0.5 mg/kg body weight), in combination with daily administration of 3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine or vehicle, were performed until the development of parkinsonian motor symptoms in either of the two experimental groups (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine versus 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/vehicle). After 21 weeks of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment, all 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/vehicle-treated animals displayed parkinsonian symptoms, whereas none of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine-treated monkeys were significantly affected. These behavioural observations were consistent with in vivo positron emission tomography dopamine transporter imaging data, and with post-mortem stereological counts of midbrain dopaminergic neurons, as well as striatal intensity measurements of dopamine transporter and tyrosine hydroxylase immunoreactivity, which were all significantly higher in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine-treated animals than in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/vehicle-treated monkeys. The 3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine treatment also had a significant effect on the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced loss of norepinephrine neurons in the locus coeruleus and adjoining A5 and A7 noradrenaline cell groups. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/vehicle-treated animals, almost 40% loss of tyrosine hydroxylase-positive norepinephrine neurons was found in locus coeruleus/A5/A7 noradrenaline cell groups, whereas the extent of neuronal loss was lower than 15% of control values in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine-treated monkeys. Our data demonstrate that chronic treatment with the metabotropic glutamate receptor 5 antagonist, 3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine, significantly reduces 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity towards dopaminergic and noradrenergic cell groups in non-human primates. This suggests that the use of metabotropic glutamate receptor 5 antagonists may be a useful strategy to reduce degeneration of catecholaminergic neurons in Parkinson’s disease.
substantia nigra; locus coeruleus; striatum; neuroprotection; noradrenaline
Changes in GABAergic transmission in the external and internal segments of the globus pallidus (GPe and GPi) contribute to the pathophysiology of the basal ganglia network in Parkinson’s disease. Because GABA-B receptors are involved in the modulation of GABAergic transmission in GPe and GPi, it is possible that changes in the functions or localization of these receptors contribute to the changes in GABAergic transmission. To further examine this question, we investigated the anatomical localization of GABA-B receptors and the electrophysiologic effects of microinjections of GABA-B receptor ligands in GPe and GPi of MPTP-treated (parkinsonian) monkeys. We found that the pattern of cellular and ultrastructural localization of the GABA-BR1 subunit of the GABA-B receptor in GPe and GPi was not significantly altered in parkinsonian monkeys. However, the magnitude of reduction in firing rate of GPe and GPi neurons produced by microinjections of the GABA-B receptor agonist baclofen was larger in MPTP-treated animals than in normal monkeys. Injections of the GABA-B receptor antagonist CGP55845A were more effective in reducing the firing rate of GPi neurons in parkinsonian monkeys than in normal animals. In addition, the injections of baclofen in GPe and GPi, or of CGP55845A in GPi lead to a significant increase in the proportion of spikes in rebound bursts in parkinsonian animals, but not in normal monkeys. Thus, despite the lack of changes in the localization of GABA-BR1 subunits in the pallidum, GABA-B receptor-mediated effects are altered in the GPe and GPi of parkinsonian monkeys. These changes in GABA-B receptors function may contribute to bursting activities in the parkinsonian state.
External segment of the globus pallidus; internal segment of the globus pallidus; Parkinson’s disease; GABAergic transmission; ultrastructural localization; nonhuman primate; in vivo electrophysiology