Rosa M. Villalba

Vesicular Glutamate Transporters in the Spinal Cord, With Special Reference to Sensory Primary Afferent Synapses

Spinal cord sensory synapses are glutamatergic, but previous studies have found a great diversity in synaptic vesicle structure and have suggested additional neurotransmitters. The identification of several vesicular glutamate transporters (VGLUTs) similarly revealed an unexpected molecular diversity among glutamate‐containing terminals. Therefore, we quantitatively investigated VGLUT1 and VGLUT2 content in the central synapses of spinal sensory afferents by using confocal and electron microscopy immunocytochemistry. VGLUT1 localization (most abundant in LIII/LIV and medial LV) is consistent with an origin from cutaneous and muscle mechanoreceptors. Accordingly, most VGLUT1 immunoreactivity disappeared after rhizotomy and colocalized with markers of cutaneous (SSEA4) and muscle (parvalbumin) mechanoreceptors. With postembedding colloidal gold, intense VGLUT1 immunoreactivity was found in 88–95% (depending on the antibody used) of CII dorsal horn glomerular terminals and in large ventral horn synapses receiving axoaxonic contacts. VGLUT1 partially colocalized with CGRP in some large dense‐core vesicles (LDCVs). However, immunostaining in neuropeptidergic afferents was inconsistent between VGLUT1 antibodies and rather weak with light microscopy. VGLUT2 immunoreactivity was widespread in all spinal cord laminae, with higher intensities in LII and lateral LV, complementing VGLUT1 distribution. VGLUT2 immunoreactivity did not change after rhizotomy, suggesting a preferential intrinsic origin. However, weak VGLUT2 immunoreactivity was detectable in primary sensory nociceptors expressing lectin (GSA‐IB4) binding and in 83–90% of CI glomerular terminals in LII. Additional weak VGLUT2 immunoreactivity was found over the small clear vesicles of LDCV‐containing afferents and in 50–60% of CII terminals in LIII. These results indicate a diversity of VGLUT isoform combinations expressed in different spinal primary afferents. J. Comp. Neurol. 472:257–280, 2004.

Striatal and extrastriatal dopamine in the basal ganglia: An overview of its anatomical organization in normal and Parkinsonian brains†

Degeneration of the nigrostriatal dopaminergic system is the characteristic neuropathological feature of Parkinsons 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.

Dopaminergic denervation and spine loss in the striatum of MPTP-treated monkeys

Striatal spine loss is a key pathological feature of human Parkinsons 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.

The thalamostriatal system in normal and diseased states

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

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

Striatal spine loss is a key pathological feature of Parkinsons 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. J. Comp. Neurol. 519:989–1005, 2011.

Striatal spine plasticity in Parkinson's disease.

Striatal dopamine (DA) denervation results in a significant loss of dendritic spines on medium spiny projection neurons in Parkinsons disease. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated parkinsonian monkeys, spines contacted either by cortical or thalamic glutamatergic terminals are severely affected on both direct and indirect striatofugal neurons. In rodents, indirect pathway neurons appear to be more sensitive, at least in early stages of acute dopamine denervation. The remaining corticostriatal and thalamostriatal axo-spinous synapses undergo complex ultrastructural remodeling consistent with increased synaptic activity in the DA-denervated primate striatum, which may explain the pathophysiological overactivity of the corticostriatal system reported in various animal models of parkinsonism. The calcium-mediated regulation of the transcription factor myocyte enhancer factor 2 was recognized as a possible underlying mechanism for striatal spine plasticity. Future studies to determine how alterations in striatal spine plasticity contribute to the symptomatology of parkinsonism are warranted.

Astroglial pattern in the spinal cord of the adult barbel (Barbus comiza)

The distribution and the structural, ultrastructural and immunohistochemical characteristics of the astroglial cells in the spinal cord of the adult barbel (Barbus comiza) have been studied by means of metallic impregnations (Golgi and gold-sublimate), immunohistochemical (GFAP and vimentin) and electron microscopic techniques. GFAP-positive cells were mainly distributed in the ependyma and in the periependymal region, but they have also been observed at subpial level in the anterior column. The ependymocytes were heterogeneous cells because they showed different immunohistochemical characteristics: GFAP-positive, vimentin-positive or non-immunoreactive cells. The radial astrocytes showed only GFAP immunoreactivity, and their processes ended at the subpial zone forming a continuous subpial glia limitans. Desmosomes and gap junctions between soniata and processes of radial astrocytes were numerous, and a relationship between radial astroglial processes and the nodes of Ranvier was also described. The perivascular glia limitans was poorly developed and it was not complete in the blood vessels of the periependymal zone; in this case, the basal lamina was highly developed. An important characteristic in the barbel spinal cord was the existence of a zone with an abundant extracellular space near the ependyma. The presence of radial astroglial somata at subpial level, the existence of vimentin-positive ependymocytes and the abundant extracellular space in the periependymal zone is discussed in relation to the regeneration capacity and the continuous growth showed by fish. Moreover, the abundance of gliofilaments and desmosomes leads us to suggest that mechanical support might be an important function for the astroglial cells in the barbel spinal cord.

Electron microscopic localization of cholinergic terminals in the rat substantia nigra: an immunocytochemical study.

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 acetyltransferase (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.

Striatal spine plasticity in Parkinson’s disease: pathological or not?

Parkinsons 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.

Immunocytochemical localization of cholinergic terminals in the region of the nucleus basalis magnocellularis of the rat: A correlated light and electron microscopic study

The cholinergic circuitry in the nucleus basalis magnocellularis of the rat was investigated in a correlated light and electron microscopic study by using monoclonal antibodies against the acetylcholine-synthesizing enzyme, choline acetyltransferase, following the unlabelled antibody peroxidase-antiperoxidase immunocytochemical procedure. After the immunocytochemical approach, large cholinergic cells and a few immunoreactive fibres exhibiting a varicose appearance, were detected by light microscopy in portions of the nucleus basalis magnocellularis located within the anatomical limits of the globus pallidus, mostly in its ventromedial part. Cholinergic neurons and fibre-like structures were also found within the substantia innominata on the edge of globus pallidus. The same material studied by light microscopy was analysed with the electron microscope. At the ultrastructural level, the immunopositive neurons showed the same cytological characteristics and pattern of synaptic input as cholinergic basal forebrain cells. Additionally, scarce immunoreactive preterminal axons and terminal boutons were detected in the region. The immunoreactive terminals were scattered or formed occasional clusters and appeared as heavily immunostained vesicle-filled boutons making exclusively axodendritic synaptic contacts principally with immunonegative distal dendrites. Both symmetric and asymmetric synaptic contacts established between these structures were detected, although the symmetric contacts were the more numerous. The surface of postsynaptic immunonegative dendrites in asymmetric synaptic contact with immunoreactive terminals was generally covered by terminals that lacked detectable immunoreactivity. In contrast, those in symmetric synaptic contact with labelled terminals showed much sparser input from immunonegative terminals, suggesting that they may belong to interneurons. Very rarely, cholinergic terminals were detected in asymmetric synaptic contact with dendrites which also contained positive immunoreaction product. Asymmetric contacts were frequently characterized by the presence of subjunctional dense bodies. The detection of cholinergic terminals in the region of the nucleus basalis magnocellularis of the rat indicates that this region not only contains cholinergic projecting neurons, but receives a cholinergic input itself. Results of this study provide evidence of the existence of a cholinergic transmission in the basal forebrain of the rat, and also that acetylcholine might play a role in the regulation of the extrinsic cortical cholinergic innervation. The possible sources of this innervation are discussed.