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In both Parkinson’s disease (PD) patients and MPTP-treated non-human primates, there is a profound neuronal degeneration of the intralaminar centromedian/parafascicular (CM/Pf) thalamic complex. Although this thalamic pathology has long been established in PD (and other neurodegenerative disorders), the impact of CM/Pf cell loss on the integrity of the thalamo-striatal glutamatergic system and its regulatory functions upon striatal neurons remain unknown. In the striatum, cholinergic interneurons (ChIs) are important constituents of the striatal microcircuitry and represent one of the main targets of CM/Pf-striatal projections. Using light and electron microscopy approaches, we have analyzed the potential impact of CM/Pf neuronal loss on the anatomy of the synaptic connections between thalamic terminals (vGluT2-positive) and ChIs neurons in the striatum of parkinsonian monkeys treated chronically with MPTP. The following conclusions can be drawn from our observations: (1) As reported in PD patients, and in our previous monkey study, CM/Pf neurons undergo profound degeneration in monkeys chronically treated with low doses of MPTP. (2) In the caudate (head and body) nucleus of parkinsonian monkeys, there is an increased density of ChIs. (3) Despite the robust loss of CM/Pf neurons, no significant change was found in the density of thalamostriatal (vGluT2-positive) terminals, and in the prevalence of vGluT2-positive terminals in contact with ChIs in parkinsonian monkeys. These findings provide new information about the state of thalamic innervation of the striatum in parkinsonian monkeys with CM/Pf degeneration, and bring up an additional level of intricacy to the consequences of thalamic pathology upon the functional microcircuitry of the thalamostriatal system in parkinsonism. Future studies are needed to assess the importance of CM/Pf neuronal loss, and its potential consequences on the neuroplastic changes induced in the synaptic organization of the thalamostriatal system, in the development of early cognitive impairments in PD. development of early cognitive impairments in PD.

The glutamate receptor delta 1 (GluD1) is strongly expressed in the striatum. Knockout of GluD1 expression in striatal neurons elicits cognitive deficits and disrupts the thalamostriatal system in mice. To understand the potential role of GluD1 in the primate striatum, we compared the cellular and subcellular localization of striatal GluD1 immunoreactivity (GluD1-IR) in mice and monkeys. In both species, striatal GluD1-IR displayed a patchy pattern of distribution in register with the striosome/matrix compartmentation, but in an opposite fashion. While GluD1 was more heavily expressed in the striosomes than the matrix in the monkey caudate nucleus, the opposite was found in the mouse striatum. At the electron microscopic level, GluD1-IR was preferentially expressed in dendritic shafts (47.9 ± 1.2%), followed by glia (37.7 ± 2.5%), and dendritic spines (14.3 ± 2.6%) in the matrix of the mouse striatum. This pattern was not statistically different from the labeling in the striosome and matrix compartments of the monkey caudate nucleus, with the exception of a small amount of GluD1-positive unmyelinated axons and axon terminals in the primate striatum. Immunogold staining revealed synaptic and perisynaptic GluD1 labeling at putative axo-dendritic and axo-spinous glutamatergic synapses, and intracellular labeling on the surface of mitochondria. Confocal microscopy showed that GluD1 is preferentially colocalized with thalamic over cortical terminals in both the striosome and matrix compartments. These data provide the anatomical substrate for a deeper understanding of GluD1 regulation of striatal glutamatergic synapses, but also suggest possible extrasynaptic, glial, and mitochondrial GluD1 functions.

In Parkinson's disease (PD) patients and animal models of PD, the progressive degeneration of the nigrostriatal dopamine (DA) projection leads to two major changes in the morphology of striatal projection neurons (SPNs), i.e., a profound loss of dendritic spines and the remodeling of axospinous glutamatergic synapses. Striatal spine loss is an early event tightly associated with the extent of striatal DA denervation, but not the severity of parkinsonian motor symptoms, suggesting that striatal spine pruning might be a form of homeostatic plasticity that compensates for the loss of striatal DA innervation and the resulting dysregulation of corticostriatal glutamatergic transmission. On the other hand, the remodeling of axospinous corticostriatal and thalamostriatal glutamatergic synapses might represent a form of late maladaptive plasticity that underlies changes in the strength and plastic properties of these afferents and the resulting increased firing and bursting activity of striatal SPNs in the parkinsonian state. There is also evidence that these abnormal synaptic connections might contribute to the pathophysiology of L-DOPA-induced dyskinesia. Despite the significant advances made in this field over the last thirty years, many controversial issues remain about the striatal SPN subtypes affected, the role of spine changes in the altered activity of SPNs in the parkinsonisn state, and the importance of striatal spine plasticity in the pathophysiology of L-DOPA-induced dyskinesia. In this review, we will examine the current state of knowledge of these issues, discuss the limitations of the animal models used to address some of these questions, and assess the relevance of data from animal models to the human-diseased condition.

In light of postmortem human studies showing extensive degeneration of the center median (CM) and parafascicular (Pf) thalamic nuclei in Parkinson's disease patients, the present study assessed the extent of neuronal loss in CM/Pf of non-human primates that were rendered parkinsonian by repeated injections of low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In order to determine the course of CM/Pf degeneration during the MPTP intoxication, motor-asymptomatic animals with partial striatal dopamine denervation were also used. The Cavalieri's principle for volume estimation and the unbiased stereological cell count method with the optical dissector technique were used to estimate the total number of neurons in the CM/Pf. We found substantial neurons loss in the CM/Pf in both, motor-symptomatic MPTP-treated monkeys in which the striatal dopamine innervation was reduced by more than 80 %, and in motor-asymptomatic MPTP-treated animals with 40–50 % striatal dopamine loss. In MPTP-treated parkinsonian monkeys, 60 and 62 % neurons loss was found in CM and Pf, respectively, while partially dopamine-depleted asymptomatic animals displayed 59 and 52 % neurons loss in the CM and Pf, respectively. Thus, our study demonstrates that the CM/Pf neurons loss is an early phenomenon that occurs prior to the development of parkinsonian motor symptoms in these animals. In contrast, the neighboring mediodorsal nucleus of the thalamus was only mildly affected (18 % neurons loss) in the parkinsonian monkeys. Together with recent findings about the possible role of the CM/Pf-striatal system in cognition, our findings suggest that the pathology of the thalamostriatal system may precede the development of motor symptoms in PD, and may account for some of the cognitive deficits in attentional set-shifting often seen in these patients. Future studies in this animal model, and in monkeys with selective lesion of CM or Pf, are needed to further elucidate the role of the CM/Pf-striatal system in normal and parkinsonian conditions.

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 positron emission tomography (PET) tracer 2β-carbomethoxy-3β-(4-chlorophenyl)-8-(2-[18F]-fluoroethyl)-nortropane ((18)F-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 (18)F-radiolabeled DAT ligands currently available. The goal of this study is to validate the use of (18)F-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 (18)F-FECNT PET at baseline (twice; 10 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 (18)F-FECNT test-retest specific uptake ratios were 0.99 (R²) and 2.65%, respectively. The (18)F-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 (18)F-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 (PD) is characterized by a dramatic loss of dopamine that underlies complex structural and functional changes in striatal projection neurons. A key alteration that has been reported in various rodent models and PD patients is a significant reduction in striatal dendritic spine density. Our recent findings indicate that striatal spine loss is also a prominent feature of parkinsonism in MPTP-treated monkeys. In these animals, striatal spine plasticity is tightly linked with the degree of striatal dopamine denervation. It affects predominantly the sensorimotor striatal territory (i.e. the post-commissural putamen) and targets both direct and indirect striatofugal neurons. However, electron microscopic 3D reconstruction studies demonstrate that the remaining spines in the dopamine-denervated striatum of parkinsonian monkeys undergo major morphological and ultrastructural changes characteristic of increased synaptic efficacy. Although both corticostriatal and thalamostriatal glutamatergic afferents display such plastic changes, the ultrastructural features of pre- and post-synaptic elements at these synapses are consistent with a higher strength of corticostriatal synapses over thalamic inputs in both normal and pathological conditions. Thus, striatal projection neurons and their glutamatergic afferents are endowed with a high degree of structural and functional plasticity. In parkinsonism, the striatal dopamine denervation induces major spine loss on medium spiny neurons and generates a significant remodeling of corticostriatal and thalamostriatal glutamatergic synapses, consistent with increased synaptic transmission. Future studies are needed to further characterize the mechanisms underlying striatal spine plasticity, and determine if it represents a pathological feature or compensatory process of PD.

Degeneration of the nigrostriatal dopaminergic system is the characteristic neuropathological feature of Parkinson's disease and therapy is primarily based on a dopamine replacement strategy. Dopamine has long been recognized to be a key neuromodulator of basal ganglia function, essential for normal motor activity. The recent years have witnessed significant advances in our knowledge of dopamine function in the basal ganglia. Although the striatum remains the main functional target of dopamine, it is now appreciated that there is dopaminergic innervation of the pallidum, subthalamic nucleus, and substantia nigra. A new dopaminergic- thalamic system has also been uncovered, setting the stage for a direct dopamine action on thalamocortical activity. The differential distribution of D1 and D2 receptors on neurons in the direct and indirect striato-pallidal pathways has been re-emphasized, and cholinergic interneurons are recognized as an intermediary mediator of dopamine-mediated communication between the two pathways. The importance and specificity of dopamine in regulating morphological changes in striatal projection neurons provides further evidence for the complex and multifarious mechanisms through which dopamine mediates its functional effects in the basal ganglia. In this review, the role of basal ganglia dopamine and its functional relevance in normal and pathological conditions will be discussed.

The existence of the thalamostriatal projection has long been known, but the functional role of this system remains enigmatic. The thalamostriatal system originates from two main groups of thalamic nuclei: the centromedian/parafascicular (CM/Pf) complex and the non-CM/Pf thalamic nuclei. In addition to their differences in thalamic origin, these two thalamostriatal systems display striking differences in their pattern of termination, synaptic properties, and glutamate receptors expression. The responses of striatal neurons to CM/Pf activation are complex and likely involve intrastriatal GABAergic networks. Behaviorally, the CM/Pf-striatal system is involved in attention-related cognitive processes. Through regulation of striatal cholinergic interneuron responses to salient stimuli, the CM/Pf-striatal system contributes to behavioral switching and response biases for reward-oriented actions and learning. Because the CM/Pf complex degenerates in Parkinson's disease and Huntington's chorea, this nondopaminergic pathology may underlie attention-related cognitive deficits in these disorders. The CM/Pf complex is a promising neurosurgical target for Tourette's syndrome and possibly Parkinson's disease.

The basal ganglia play a critical role in a wide range of cognitive functions. Consequently, neurodegenerative diseases that affect the basal ganglia, such as Parkinson’s disease (PD) and Huntington’s disease (HD), are commonly associated with cognitive impairments. Although dysfunction of the relationships between the prefrontal cortex and the caudate nucleus is likely to be involved in these deficits, data collected over the past decade strongly suggest that the thalamostriatal system from the centromedian (CM) and parafascicular (PF) nuclei is another important regulator of cognitive functions in the basal ganglia. The fact that the CM/PF undergoes severe degeneration in HD and PD further supports the possible contribution of the thalamostriatal system pathology to cognitive dysfunctions in these disorders.

The striatum and the subthalamic nucleus are the main entry doors for extrinsic inputs to reach the basal ganglia circuitry. The cerebral cortex, thalamus and brainstem are the key sources of glutamatergic inputs to these nuclei. There is functional and neurochemical evidence that glutamatergic neurotransmission is altered in the striatum and subthalamic nucleus of animal models of Parkinson’s disease, and that these changes may contribute to aberrant network neuronal activity in the basal ganglia-thalamocortical circuitry. Postmortem studies of animal models and PD patients have revealed significant pathology of glutamatergic synapses, dendritic spines and microcircuits in the striatum of parkinsonians. More recent findings have also demonstrated a significant breakdown of the glutamatergic corticosubthalamic system in parkinsonian monkeys. In this review, we will discuss evidence for synaptic glutamatergic dysfunction and pathology of cortical and thalamic inputs to the striatum and subthalamic nucleus in models of Parkinson’s disease. The potential functional implication of these alterations on synaptic integration, processing and transmission of extrinsic information through the basal ganglia circuits will be considered. Finally, the significance of these pathological changes in the pathophysiology of motor and non-motor symptoms in Parkinson’s disease will be examined.

Strong -aminobutyric acid type B (GABAB) receptor binding has been shown throughout the thalamus, but the distribution of the two GABAB receptor subunits, GABAB receptor subunit 1 (GABABR1) and GABAB receptor subunit 2 (GABABR2), remains poorly characterized. In primates, the caudal intralaminar nuclei, centromedian and parafascicular (CM/ PF), are an integral part of basal ganglia circuits and a main source of inputs to the striatum. In this study, we analyzed the subcellular and subsynaptic distribution of GABAB receptor subunits by using light and electron microscopic immunocytochemical techniques. Quantitative immunoperoxidase and immunogold analysis showed that both subunits display a similar pattern of distribution in CM/PF, being expressed largely at extrasynaptic and perisynaptic sites in neuronal cell bodies, dendrites, and axon-like processes and less abundantly in axon terminals. Postsynaptic GABABR1 labeling was found mostly on the plasma membrane (70 – 80%), whereas GABABR2 was more evenly distributed between the plasma membrane and intracellular compartments of CM/PF neurons. A few axon terminals forming symmetric and asymmetric synapses were also labeled for GABABR1 and GABABR2, but the bulk of presynaptic labeling was expressed in small axon-like processes. About 20% of presynaptic vesicle-containing dendrites of local circuit neurons displayed GABABR1/R2 immunoreactivity. Vesicular glutamate transporters (vGluT1)-containing terminals forming asymmetric synapses expressed GABABR1 and/or displayed postsynaptic GABABR1 at the edges of their asymmetric specialization. Overall, these findings provide evidence for multiple sites where GABAB receptors could modulate GABAergic and glutamatergic transmission in the primate CM/PF complex. J. Comp. Neurol. 496:269 –287, 2006. © 2006 Wiley-Liss, Inc. Indexing terms: immunocytochemistry; GABABR1; GABABR2; vGluT1; intralaminar thalamic nuclei; Parkinson’s disease

Neurons in the external and internal segment of the globus pallidus (GPe and GPi, respectively) receive substantial GABAergic inputs from the striatum and through axon collaterals of neighboring pallidal neurons. The effects of GABA on pallidal activity depend on the synaptic localization of GABA receptors and the distribution and activity of GABA transporters (GATs). To explore the contribution of GABA receptors and transporters to pallidal function, we recorded the activity of single neurons in GPe or GPi before, during, and after local microinjections of GABAergic compounds in awake rhesus monkeys. Activation of GABAA or GABAB receptors with muscimol or baclofen, respectively, inhibited pallidal activity. These effects were reversed by concomitant infusion of the respective GABA receptor antagonists, gabazine and CGP-55845. Given alone, the antagonists were without consistent effect. Application of the selective GAT-1 inhibitor, SKF-89976A, and the semiselective GAT-3 blocker, SNAP-5114, decreased pallidal activity. Both GAT inhibitors increased GABA levels in the pallidum, as measured by microdialysis. Electron microscopic observations revealed that these transporters are located on glial processes and unmyelinated axonal segments, but rarely on terminals. Our results indicate that activation of GABAA and GABAB receptors inhibits neuronal activity in both segments of the pallidum. GAT-1 and GAT-3 are involved in the modulation of endogenous GABA levels and may be important in regulating the extrasynaptic levels of GABA. Together with previous evidence that a considerable proportion of pallidal GABA receptors are located outside the synaptic cleft, our experiments strongly support the importance of extrasynaptic GABAergic transmission in the primate pallidum.

GABA is one of the key neurotransmitters that regulate the firing activity of neurones in the supraoptic (SON) and paraventricular (PVN) nuclei. In the present study, we used immunohistochemical techniques to study the distribution and subcellular localisation of metabotropic GABA(B) receptors in magnocellular neurones in the SON and PVN. Robust GABA(B) receptor immunoreactivity (GABA(B)R; both subunit 1 and subunit 2 of the heterodimer), was observed in the SON and PVN. At the light microcope level, GABA(B)R immonoreactivity displayed a clustered pattern localised both intracytoplasmically and at the plasma membrane. Densitometry analysis indicated that GABA(B)R immunoreactivity was significantly more intense in vasopressin cells than in oxytocin cells, both in male, virgin female and lactating rats, and was denser in males than in virgin females. Light and electron microscope studies indicated that cytoplasmic GABA(B)R was localised in various organelles, including the Golgi, early endosomes and lysosomes, suggesting the cycling of the receptor within the endocytic and trafficking pathways. Some smaller clusters at the level of the cell plasma membrane were apposed to glutamic acid decarboxylase 67 immunoreactive boutons, and appeared to be colocalised with gephyrin, a constituent protein of the postsynaptic density at inhibitory synapses. The presence of GABA(B)R immunoreactivity at synaptic and extrasynaptic sites was supported by electron microscopy. These results provide anatomical evidence for the expression of postsynaptic GABA(B) receptors in magnocellular neurosecretory cells.

The striatum and the subthalamic nucleus are the main entry points for cortical information to the basal ganglia. Parkinson’s disease affects not only the function, but also the morphological integrity of some of these inputs and their synaptic targets in the basal ganglia. Significant morphological changes in the cortico-striatal system have already been recognized in patients with Parkinson’s disease and in animal models of the disease. To find out whether the primate cortico-subthalamic system is also subject to functionally relevant morphological alterations in parkinsonism, we used a combination of light and electron microscopy anatomical approaches and in vivo electrophysiological methods in monkeys rendered parkinsonian following chronic exposure to low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). At the light microscopic level, the density of vesicular glutamate transporter 1-positive (i.e. cortico-subthalamic) profiles in the dorsolateral part of the subthalamic nucleus (i.e. its sensorimotor territory) was 26.1% lower in MPTP-treated parkinsonian monkeys than in controls. These results were confirmed by electron microscopy studies showing that the number of vesicular glutamate transporter 1-positive terminals and of axon terminals forming asymmetric synapses in the dorsolateral subthalamic nucleus was reduced by 55.1% and 27.9%, respectively, compared with controls. These anatomical findings were in line with in vivo electrophysiology data showing a 60% reduction in the proportion of pallidal neurons that responded to electrical stimulation of the cortico-subthalamic system in parkinsonian monkeys. These findings provide strong evidence for a partial loss of the hyperdirect cortico-subthalamic projection in MPTP-treated parkinsonian monkeys. * Abbreviations : GPe : external globus pallidus GPi : internal globus pallidus MPTP : 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine STN : subthalamic nucleus vGluT1 : vesicular glutamate transporter 1

One of the roadblocks to developing effective therapeutics for Huntington disease (HD) is the lack of animal models that develop progressive clinical traits comparable to those seen in patients. Here we report a longitudinal study that encompasses cognitive and motor assessment, and neuroimaging of a group of transgenic HD and control monkeys from infancy to adulthood. Along with progressive cognitive and motor impairment, neuroimaging revealed a progressive reduction in striatal volume. Magnetic resonance spectroscopy at 48 months of age revealed a decrease of N-acetylaspartate (NAA), further suggesting neuronal damage/loss in the striatum. Postmortem neuropathological analyses revealed significant neuronal loss in the striatum. Our results indicate that HD monkeys share similar disease patterns with HD patients, making them potentially suitable as a preclinical HD animal model.

Metabotropic glutamate receptors (mGluRs) modulate somatosensory, autonomic, and motor functions at spinal levels. mGluR postsynaptic actions over spinal neurons display the pharmacologic characteristics of type I mGluRs; however, the spinal distribution of type I mGluR isoforms remains poorly defined. In this study, the authors describe a differential distribution of immunoreactivity to various type I mGluR isoforms (mGluR1a, mGluR5a,b, and mGluR1b) that suggests a correlation between specific isoforms and particular aspects of spinal cord function. Two different antisera raised against mGluR5a,b detected intense immunoreactivity within nociceptive afferent terminal fields (laminae I and II) and also in autonomic regions (parasympathetic and sympathetic). In contrast, two of three anti-mGluR1a antibodies did not immunostain lamina I or II. Laminae I and II immunostaining by a third anti-mGluR1a antibody was competed by a peptide sequence obtained from a homologous region in mGluR5, suggesting possible cross reactivity in fixed tissue. Autonomic neurons did not express mGluR1a immunoreactivity. All anti-mGluR1a antibodies strongly and specifically immunolabeled dendritic and somatic membranes of neurons in the deep dorsal horn (lamina III-V) and the ventral horn (lamina VI-IX). Somatic motoneurons expressed mGluR1a immunoreactivity but little or no mGluR5 immunoreactivity. Phrenic and pudendal motoneurons expressed the highest level of mGluR1a immunoreactivity in the spinal cord. Intense mGluR1b immunoreactivity was restricted to a few scattered neurons and a prominent group of neurons in lamina X. Lamina II neurons expressed low levels of mGluR1b immunoreactivity. Ultrastructurally, type I mGluR immunoreactivity was found mostly at extrasynaptic sites on the plasma membrane, but it was also found perisynaptically, in the body of the postsynaptic regions or in relation to intracytoplasmic structures.

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. 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 centre 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 Parkinson’s disease and that CM/Pf deep brain stimulation may be therapeutically useful in several movement disorders.

Synaptic plasticity is the ability of synapses to modulate the strength of neuronal connections; however, the molecular factors that regulate this feature are incompletely understood. Here, we demonstrated that mice lacking brain-specific angiogenesis inhibitor 1 (BAI1) have severe deficits in hippocampus-dependent spatial learning and memory that are accompanied by enhanced long-term potentiation (LTP), impaired long-term depression (LTD), and a thinning of the postsynaptic density (PSD) at hippocampal synapses. We showed that compared with WT animals, mice lacking Bai1 exhibit reduced protein levels of the canonical PSD component PSD-95 in the brain, which stems from protein destabilization. We determined that BAI1 prevents PSD-95 polyubiquitination and degradation through an interaction with murine double minute 2 (MDM2), the E3 ubiquitin ligase that regulates PSD-95 stability. Restoration of PSD-95 expression in hippocampal neurons in BAI1-deficient mice by viral gene therapy was sufficient to compensate for Bai1 loss and rescued deficits in synaptic plasticity. Together, our results reveal that interaction of BAI1 with MDM2 in the brain modulates PSD-95 levels and thereby regulates synaptic plasticity. Moreover, these results suggest that targeting this pathway has therapeutic potential for a variety of neurological disorders.

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.

The present study provides light- and electron-microscopic immunocytochemical data on the presence of neurons that are immunoreactive to the C-terminal flanking peptide of neuropeptide Y, C-PON, in the neostriatum of the hedgehog (Erinaceus europaeus). Positive neurons have mostly fusiform or round perikarya from which two to four poorly branched processes arise. Immunostained fibers and puncta are also evenly distributed throughout the neostriatum. Ultrastructurally, each neuron exhibits a deeply invaginated nucleus surrounded by abundant cytoplasm with a well-developed rough endoplasmic reticulum and Golgi apparatus. Positive neurons receive symmetric and asymmetric synapses from unlabeled terminals. The results of this study can be correlated with previous findings, as the C-PON-positive neurons of the hedgehog resemble medium-sized neostriatal neurons that are known to be local circuit neurons exhibiting C-PON in the rat. Thus, a high degree of C-PON neuronal system phylogenetic conservation and function can be postulated for the neostriatum of mammals.

Striatal dopamine denervation is the pathological hallmark of Parkinson's disease (PD). Another major pathological change described in animal models and PD patients is a significant reduction in the density of dendritic spines on medium spiny striatal projection neurons. Simultaneously, the ultrastructural features of the neuronal synaptic elements at the remaining corticostriatal and thalamostriatal glutamatergic axo-spinous synapses undergo complex ultrastructural remodeling consistent with increased synaptic activity (Villalba and Smith, 2011). The concept of tripartite synapses (TS) was introduced a decade ago, according to which astrocytes process and exchange information with neuronal synaptic elements at glutamatergic synapses (Araque et al., 1999a). Although there has been compelling evidence that astrocytes are integral functional elements of tripartite glutamatergic synaptic complexes in the cerebral cortex and hippocampus, their exact functional role, degree of plasticity and preponderance in other CNS regions remain poorly understood. In this review, we discuss our recent findings showing that neuronal elements at cortical and thalamic glutamatergic synapses undergo significant plastic changes in the striatum of MPTP-treated parkinsonian monkeys. We also present new ultrastructural data that demonstrate a significant expansion of the astrocytic coverage of striatal TS synapses in the parkinsonian state, providing further evidence for ultrastructural compensatory changes that affect both neuronal and glial elements at TS. Together with our limited understanding of the mechanisms by which astrocytes respond to changes in neuronal activity and extracellular transmitter homeostasis, the role of both neuronal and glial components of excitatory synapses must be considered, if one hopes to take advantage of glia–neuronal communication knowledge to better understand the pathophysiology of striatal processing in parkinsonism, and develop new PD therapeutics.

The effect of a recent hyperammonemic model, consisting of a high ammonia diet for 3, 7, 15, 45, and 90 days, on glial fibrillary acidic protein (GFAP) in the rat spinal cord and on blood ammonia levels has been studied. The high ammonia diet was prepared by mixing a standard diet with ammonium acetate (20% wt/wt); in addition, 5 mM of ammonium acetate was added to the water supply. GFAP contents were determined by means of immunoblotting analysis. The results demonstrated that this high ammonia diet model neither induces significant changes in GFAP immunoreactivity, nor modifies total protein concentration, and only induces significant blood hyperammonemic levels in the first days of treatment. An adaptative response to the diet is suggested and discussed to explain these results. A relation between ammonia and GFAP expression is suggested because transient hyperammonemia induces transient, although no significant, changes on GFAP expression.

Although the presence of the neuropeptide C-terminal flanking peptide of neuropeptide Y, C-PON, has been described in the central nervous system (CNS) of mammals, to date there is no information related with its involvement in brain functions. An analysis of the location of C-PON in specific neuronal circuits of known anatomy and physiological action should provide light on its physiological role. The presence, distribution and morphology of C-PON-containing neurons in the olfactory cortex of the hedgehog was studied by immunocytochemistry. Immunoreactive neurons to C-PON were widely distributed in the three layers of the olfactory cortex of this primitive mammal. These neurons were medium sized and showed two or three immunostained, poorly branched, dendrites. In some positive neurons, a fine, beaded axon-like process was also immunostained. Although direct evidence of a physiological function of C-PON in the olfactory cortex of the hedgehog cannot be accurately stated from our findings, the morphology of C-PON neurons and their distribution in the deep cortical layers, where the majority of pyramidal neurons are located, suggest that this neuropeptide may play a role in the intrinsic neuronal circuitry of the relatively well-developed hedgehog paleocortex. A regulatory vascular role of some peptide-immunoreactive neurons can be inferred since occasional C-PON-positive neurons have been located near blood vessels.

The present study was performed in order to follow the response of rat cerebellum astroglial cells (Bergmann glial cells and astrocytes) to long-term portacaval shunt (PCS), by means of glial fibrillary acidic protein (GFAP) and vimentin immunoreactivities. Bergmann glia accumulated GFAP in response to PCS, whereas astrocytes decreased GFAP immunoreactivity when compared to control rats. The increase of GFAP occurs in cells located in the cerebellar layer where glutamate is mainly released. Since the vimentin content remained unaltered in response to PCS, when compared to control rats, it can be concluded that only the GFAP filaments are affected by PCS. Nevertheless, GFAP immunoreactivity presents regional differences in the cerebellar astroglial population, and the factors responsible for these variations are still unknown. © 1992 Wiley-Liss, Inc.

Dopamine (DA) plays a critical role in regulating spine density on medium-sized spiny neurons (MSNs) in the striatum. Quantitative analysis of Golgi-impregnated MSNs in striatal sections at the level of the anterior commissure (commissural striatum) from MPTP-treated monkeys with complete DA denervation showed a significant reduction in spine density in both the caudate nucleus and putamen compared with that in controls. Similar analysis in partially DA-denervated striata revealed that the loss of spines was tightly correlated with the relative degree of dopamine depletion. Quantitative electron microscopy (EM) analyses showed a significant decrease in the density of both D1-immunolabeled and immunonegative spines in the commissural putamen of MPTP-treated monkeys, suggesting that DA depletion causes a reduction of spines on both direct and indirect pathway MSNs in the striatum of parkinsonian monkeys. Both the degree of striatal dopamine loss and extent of spine degeneration are correlated with low level of calbindin expression in striatal subregions, suggesting an important role of calcium in striatal pathogenesis of Parkinson’s disease. This is further supported by electron microscopic data showing that calbindin-containing spines are less sensitive to degeneration in the caudate nucleus of partially depleted MPTP-treated monkeys. These findings provide strong evidence that striatal spine loss is an early sign of parkinsonism pathogenesis that is tightly correlated with the degree of striatal dopamine denervation and affects both direct and indirect striatofugal pathway neurons predominantly in calbindin-poor striatal subregions of MPTP-treated monkeys.

Striatal spine loss is a key pathological feature of human Parkinson's disease (PD) that can be induced after complete degeneration of the nigrostriatal dopaminergic system in rodent models of parkinsonism. In line with these observations, our findings reveal a significant (30-50%) reduction in spine density in both the caudate nucleus and putamen of severely DA-depleted striata of MPTP-treated monkeys; the sensorimotor post-commissural putamen being the most severely affected region for both dopamine depletion and spine loss. Using MPTP-treated monkeys with complete or partial striatal dopamine (DA) denervation, we also demonstrate that striatal spine loss is an early pathological feature of parkinsonism, which progresses along a positive rostrocaudal and mediolateral gradient in parallel with the extent of striatal dopamine denervation. Quantitative electron microscopy immunocytochemistry for D1 dopamine receptor (D1) in the striatum of control and severely DA-depleted animals revealed that both D1-immunoreactive and immunonegative spines are lost in the putamen of MPTP-treated monkeys. These data demonstrate that striatal spine loss in MPTP-treated monkeys is an early pathological event of parkinsonism, tightly correlated with the degree of nigrostriatal dopamine denervation that likely affects both direct and indirect striatofugal pathways.

Since the original description of dendritic spines using the Golgi impregnation method by Ramon y Cajal in the late 1800s, our knowledge of the importance of these neuronal structures in normal physiological processes and brain dysfunctions underwent tremendous advances. The development of sophisticated high resolution approaches to visualize dendritic spines, and assess their complex plastic properties and capabilities in rapid structural remodeling in response to physiological or pathophysiological changes in brain activity has led to a clear understanding that these neuronal elements are key substrates of synaptic transmission in the normal and diseased brain. Because of their abundance on dendrites of specific neuronal populations (i.e. cortical pyramidal neurons, cerebellar Purkinje cells, striatal medium spiny neurons, etc…), and the fact that they represent the main synaptic target of glutamatergic excitatory synapses in the central nervous system, dysregulation of their homeostatic properties can have dramatic influences upon normal synaptic communication, and lead to complex behavioral changes seen in neurological and neuropsychiatric disorders. At the cellular level, pruning, growth and morphological remodeling of dendritic spines are closely related to synaptic function and plasticity, thereby allowing synaptic phenomena such as long-term potentiation and long-term depression of excitatory transmission to occur and underlie complex cognitive processes associated with learning and memory. Understanding the molecular mechanisms that regulate dendritic spine plasticity and pathology is another area of rapid growth and tremendous relevance for our knowledge of the etiologies and the development of new therapeutics for brain disorders. Taking into consideration the knowledge gained in the past decades about the importance of dendritic spine pathology in brain disorders, the main goal of this Special Issue is to provide a current overview of recent advances that have been made in our understanding of the morphological, functional and molecular underpinnings of dendritic spine alterations in various neurological and neuropsychiatric disorders (Fragile X syndrome, Schizophrenia, Depression, Huntington’s disease, Aging, Drug addiction, Parkinson’s disease, Alzheimer’s disease, and Epilepsy). Because such advances are highly dependent on the continued development and refinement of methods that allow localizing, reconstructing, molecularly dissecting and functionally monitoring individual spines, manuscripts of this Special Issue are also devoted to technical breakthroughs in studies of dendritic spine architecture and dynamic regulation (3D EM reconstruction methods, Optical imaging techniques). In many reviews, authors provide a comprehensive assessment of the literature related to spine pathology in specific disorders, and discuss their current understanding of spine pathology as being the cause or consequences of brain diseases. As is the case for most studies of the central nervous system, advances of our knowledge of the role of dendritic spines in normal and abnormal brain activity rely on the development of collaborative and multidisciplinary research programs that cut across a broad spectrum of genetic, molecular, anatomical and physiological approaches. The combination of these methods will allow dissecting out the complex substrates that mediate the unique properties of dendritic spine plasticity, and examine their relevance in the context of changes in neuronal circuits and complex brain networks that underlie neurological and neuropsychiatric disorders. Two other areas of research that warrant significant development in this field is the urgent need for more in-depth studies of spine pathologies in diseased human brains, and the development of animal models that more closely mimic the human diseased condition. These two areas are critical if one hopes to translate more efficiently knowledge gained about dendritic spine pathology in the laboratory to human brain disorders and therapeutics.

This experimental study was made to know the effect of hyperammonemia on glial fibrillary acidic protein (GFAP) in the glial cells of posterior rhombencephalon in the goldfish (Carassius auratus L.). Hyperammonemia was induced by elevating the ammonia concentration in the tank water to 0.88 mM with ammonium chloride; the ammonia level in the control tank water was < 0.1 mM. The GFAP levels were measured at 8, 16, 30, 60, 90 and 120 days. GFAP was quantified with a digital analysis system and a transient heterogeneous decrease of GFAP was observed. Hyperammonemia mostly affected GFAP in the astrocyte processes associated with cholinergic pathways. An explanation for the adaptive response to hyperammonemia by fish astrocytes is suggested.

In this study we have demonstrated that high ammonia concentration in tank water induces changes in the glial fibrillary acidic protein (GFAP) of ependymal cells and radial astrocytes in the goldfish spinal cord. Hyperammonemia was induced by elevating the ammonia concentration in the tank water to 0.88 mM using ammonium chloride; ammonia in control water was less than 0.1 mM. Immunohistochemical methods were used for GFAP and vimentin, and levels were measured at 4, 8, 16, 30, 60, 90 and 120 days. GFAP quantification was made by means of a digital analysis system. The GFAP immunoreactivity was significantly lower at 30 and 60 days of treatment and at 90 days it had returned to control levels. However, no changes in vimentin immunoreactivity were appreciated in any case. GFAP loss was general and was not selective in any specific spinal cord region. To explain this transient generalized loss of GFAP and its posterior recuperation, a possible relation between glutamine synthetase distribution and GFAP changes is discussed.

The topographical distribution, histochemical characteristics, and anatomical relationships of the cellular elements containing choline acetyltransferase (ChAT) immunoreactivity, demonstrated with specific monoclonal antibodies to ChAT following the unlabelled antibody peroxidase-antiperoxidase (PAP) procedure at the optical and electron microscopic levels, were investigated in the rat substantia nigra (SN). Scarce, large (20–30 μm in maximum soma extent) cholinergic cell bodies and processes were found within the boundaries of the SN, in the borders of the pars compacta and pars reticulata, principally at caudal levels. Occasionally, cholinergic neurons were also found at intermediate levels of the SN, in the borders of the pars reticulata and pars lateralis. Cytologically, these large cells resembled ChAT-positive neurons localized in other areas of the central nervous system (CNS) of the rat—for example, the pontomesencephalotegmental (PMT) cholinergic complex (Ch5-Ch6) and the nucleus basalis of Meynert (nbM) (Ch4). Histochemically, ChAT-positive cells in the SN were characterized by their ability to utilize the reduced cofactor nicotinamide adenine dinucleotide phosphate (NADPH). Identified ChAT-positive neurons in the light microscope were subsequently studied in the electron microscope. All cholinergic neurons in the SN share essentially the same ultrastructural characteristics. The copious cytoplasm was rich in organelles with large lipofuscin granules. The synaptic input onto cell bodies and their dendrites was studied in serial sections. Synaptic contacts onto the perikarya and proximal dendrites were sparse and of asymmetric type. Both symmetric and asymmetric synaptic specializations onto ChAT-positive distal dendrites were detected. Asymmetric synaptic contacts onto cell bodies and dendrites were often defined by the presence of subjunctional dense bodies associated with the postsynaptic membrane. The pattern of the synaptic input to these cells differs strikingly from that onto unlabelled neighboring neurons. The perikarya and dendrites of the latter were characteristically covered with synaptic boutons. Scarce immunoreactive terminals in asymmetric synaptic contact with unlabelled dendritic profiles were also detected in portions of SN compacta with no ChAT-positive cells. Extranigrally located ChAT-positive cells of the PMT cholinergic complex were also examined in the electron microscope for comparison purposes. These cells exhibited, on the basis of their morphology and synaptic input pattern, very similar characteristics to those shown by SN cholinergic neurons. On the basis of histochemical and morphological comparison between nigral cholinergic neurons and extranigrally located ChAT-positive cells of the PMT complex, it is concluded that cholinergic neurons in the SN of the rat may be projection neurons of the PMT cholinergic complex ectopically located and that they might play an important role in the regulation of forebrain activation and in locomotion. Our electron microscopic results suggest that substance P and GABA may influence the function of these cells.

The presence of the neuropeptide C-terminal flanking peptide of neuropeptide-Y, C-PON, has been investigated in the main olfactory bulb of the rat using conventional fluorescence and peroxidase-antiperoxidase immunocytochemical techniques. The distribution of immunoreactive structures to C-PON was examined in both horizontal and coronal sections. Endogenous C-PON was localized within two types of short-axon cells including (1) superficial short-axon cells in the glomerular layer and (2) deep short-axon cells lying in the deepest portion of the granule cell layer and in the adjacent white matter. In addition, varicose immunoreactive processes were detected in all layers, although they were more numerous in the deepest portion of the granule cell layer. Immunoreactive cell bodies and processes were also observed in the nucleus olfactorius anterior and in the intrabulbar portion of the anterior commissure. Nevertheless, immunoreactive structures were not localized in the lateral olfactory tract. The indirect immunofluorescence technique to detect endogenous C-PON in combination with the enzyme histochemical demonstration of NADPH-diaphorase activity, in single sections, showed that the NADPH-diaphorase procedure is a reliable marker for these C-PON positive cells. Also, indirectly, that, in the rat main olfactory bulb, C-PON and neuropeptide-Y are contained in the same cell types. Many glomeruli were stained following the NADPH-diaphorase procedure, but they were not C-PON immunoreactives. Results of this study provide evidence suggesting that C-PON may influence polysynaptically the function of mitral cells and, therefore, the olfactory bulb output.

This investigation was carried out on the distribution of enkephalin-containing nerve fibres and terminals in the region of the nucleus basalis magnocellularis (NBM) of the rat. At the light microscope (LM) level, enkephalin-immunoreactive sites and endogenous choline acetyltransferase (ChAT) were demonstrated by employing the two-colour immunoperoxidase staining technique, using highly specific monoclonal antibodies against enkephalin and ChAT. A pharmacohistochemical procedure to reveal acetylcholinesterase (AChE)-synthesizing neurons combined with the peroxidase-antiperoxidase (PAP) immunocytochemical technique to detect endogenous enkephalins, provided ultrastructural data on the relationships of neuronal elements containing AChE and enkephalins in the region of the NBM. At the LM level, cholinergic neurons of the NBM were surrounded by a dense network of enkephalin-immunoreactive nerve fibres. Electron microscopic (EM) observations of histochemically characterized structures, that were first identified in the LM, revealed that intensely AChE-stained structures in the region of the NBM received sparse synaptic inputs from enkephalin immunoreactive terminals. Synaptic inputs of immunoreactive terminals onto the intensely AChE-stained neuron cell bodies were not detected. Synaptic contacts onto proximal AChE-positive dendrites were sparse, but the density increased on more distal regions of the dendrites. All immunoreactive boutons studied established symmetrical synaptic contacts with AChE-positive post-synaptic structures. The pattern of the synaptic input to these cells differs strikingly from that onto typical globus pallidus neurons. The perikarya and dendrites of the latter neurons were characteristically ensheathed in immunoreactive synaptic boutons. Results are consistent with the view that enkephalin-like substances in the rat might be synaptic transmitters or neuromodulators in the area of the NBM and that cholinergic neurons of the NBM (Ch4) are integrated into the circuitry of the basal ganglia. Enkephalins may play an important role regulating the extrinsic cholinergic innervation of the neocortex.

The presence of cholinergic terminals in the substantia nigra (SN) of the rat was investigated under the electron microscope using a monoclonal antibody against choline actyltransferase (ChAT), the acetylcholine (ACh)-synthesizing enzyme, following the unlabelled antibody peroxidase-antiperoxidase (PAP) procedure. ChAT-immunoreactive terminals were found making synaptic contacts with unlabelled dendrites in the SN pars compacta (SNC). Synaptic contacts established between cholinergic boutons and immunonegative dendrites were observed in serial sections to be of asymmetric type. The unlabelled postsynaptic dendrites to immunoreactive terminals displayed similar morphological aspects to typical dopamine-containing dendrites of the SN. Results of this study provide fine ultrastructural neurochemical support for the existence of a cholinergic innervation of the rat SNC and are consistent with the reported excitatory action of ACh on SNC dopaminergic neurons.

The indirect immunofluorescence technique to detect endogenous C-flanking peptide of neuropeptide Y (NPY), C-PON, incombination with NADPH-diaphorase (NADPH-d) histochemistry, were applied to the same sections to establish whether C-PON containing cell bodies in the rat striatum can be labelled by their content of NADPH-diaphorase activity. NADPH-diaphorase activity proved to be a reliable marker for these positive C-PON neurons. Our results suggest that, in the rat striatum, C-PON, SOM and NPY co-exist in the same cells.