Structural Plasticity of GABAergic Pallidothalamic Terminals in MPTP-Treated Parkinsonian Monkeys: A 3D Electron Microscopic Analysis.

The internal globus pallidus (GPi) is a major source of tonic GABAergic inhibition to the motor thalamus. In parkinsonism, the firing rate of GPi neurons is increased, and their pattern switches from a tonic to a burst mode, two pathophysiological changes associated with increased GABAergic pallidothalamic activity. In this study, we used high-resolution 3D electron microscopy to demonstrate that GPi terminals in the parvocellular ventral anterior nucleus (VApc) and the centromedian nucleus (CM), the two main GPi-recipient motor thalamic nuclei in monkeys, undergo significant morphometric changes in parkinsonian monkeys including (1) increased terminal volume in both nuclei; (2) increased surface area of synapses in both nuclei; (3) increased number of synapses/GPi terminals in the CM, but not VApc; and (4) increased total volume, but not number, of mitochondria/terminals in both nuclei. In contrast to GPi terminals, the ultrastructure of putative GABAergic nonpallidal terminals was not affected. Our results also revealed striking morphological differences in terminal volume, number/area of synapses, and volume/number of mitochondria between GPi terminals in VApc and CM of control monkeys. In conclusion, GABAergic pallidothalamic terminals are endowed with a high level of structural plasticity that may contribute to the development and maintenance of the abnormal increase in pallidal GABAergic outflow to the thalamus in the parkinsonian state. Furthermore, the evidence for ultrastructural differences between GPi terminals in VApc and CM suggests that morphologically distinct pallidothalamic terminals from single pallidal neurons may underlie specific physiological properties of pallidal inputs to VApc and CM in normal and diseased states.

Neuronal loss in the caudal intralaminar thalamic nuclei in a primate model of Parkinson's disease.

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.

Differential striatal spine pathology in Parkinson's disease and cocaine addiction: a key role of dopamine?

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.

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

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.

Expression of GABAB receptors in magnocellular neurosecretory cells of male, virgin female and lactating rats.

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.

Differential distribution of metabotropic glutamate receptors 1a, 1b, and 5 in the rat spinal cord.

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.

C-PON immunoreactive neurons in the neostriatum of the hedgehog (Erinaceus europaeus): a correlated light- and electron-microscopic study.

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.

High ammonia diet: its effect on the glial fibrillary acidic protein (GFAP).

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 adaptive 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.

Presence of C-flanking peptide of neuropeptide Y(C-PON)-immunoreactive neurons in the olfactory cortex of the hedgehog (Erinaceus europaeus).

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.

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 somata 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.

Effects of experimentally induced hyperammonemia on glial fibrillary acidic protein (GFAP) in the rhombencephalon of goldfish (Carassius auratus L.).

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.

Hyperammonemia induces transient GFAP immunoreactivity changes in goldfish spinal cord (Carassius auratus L.).

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.

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.

Light and electron microscopic study of galanin-immunoreactive nerve fibers in the rat posterior thalamus.

Light and electron microscopic immunocytochemistry was used to study certain cell groups in the posteromedial thalamus which contain galanin-immunoreactive (GAL-IR) fibers. The nuclei subparafascicularis pars parvicellularis (SPFpc) and parafascicularis (PF) contain a dense network of GAL-IR fibers which form basketlike structures around unstained cells. The periventricular area also contains numerous GAL-IR fibers and these also occasionally form basketlike structures. The GAL-IR terminal fields continue caudally in the mesodiencephalic junction and merge with other GAL-IR fibers in the dorsal aspects of the substantia nigra and around the dorsolateral tip of the medial lemniscus. Ultrastructural analysis of the GAL-IR basketlike structures revealed that GAL-IR terminals make numerous synapses with the cell bodies and proximal dendrites of SPFpc neurones. These results suggest that the activity of cells in the SPFpc and PF nuclei may be strongly influenced by galanin-containing nerve fibers probably originating in the spinal cord.

Vasoactive intestinal polypeptide-immunoreactive neurons in the main olfactory bulb of the hedgehog (Erinaceus europaeus).

Vasoactive intestinal polypeptide (VIP) immunoreactivity was localized by the indirect antibody enzyme method (PAP technique) in the main olfactory bulb of the hedgehog. Most VIP-immunoreactive cells were located in the glomerular layer and throughout the external plexiform layer. Fewer cells were observed in the granule cell layer. At the morphological level they exhibit the characteristics of periglomerular, external tufted, superficial short axon, horizontal and Van Gehuchten cells. It should be mentioned that another specific neuronal type was found in the inner third of the external plexiform layer, which is not described in other animals. These results revealed that a high number of intrinsic neuronal types of the olfactory bulb of the hedgehog display a strong VIP immunoreactivity.

Localization of C-PON immunoreactivity in the rat main olfactory bulb. Demonstration that the population of neurons containing endogenous C-PON display NADPH-diaphorase activity.

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.

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.

C-PON containing neurons in the rat striatum are also positive for NADPH-diaphorase activity. A light microscopic study.

The indirect immunofluorescence technique to detect endogenous C-flanking peptide of neuropeptide Y (NPY), C-PON, in combination 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.