Astrocytes display ultrastructural alterations and heterogeneity in the hippocampus of aged APP-PS1 mice and human post-mortem brain samples.

The past decade has witnessed increasing evidence for a crucial role played by glial cells, notably astrocytes, in Alzheimer's disease (AD). To provide novel insights into the roles of astrocytes in the pathophysiology of AD, we performed a quantitative ultrastructural characterization of their intracellular contents and parenchymal interactions in an aged mouse model of AD pathology, as aging is considered the main risk factor for developing AD. We compared 20-month-old APP-PS1 and age-matched C57BL/6J male mice, among the ventral hippocampus CA1 strata lacunosum-moleculare and radiatum, two hippocampal layers severely affected by AD pathology. Astrocytes in both layers interacted more with synaptic elements and displayed more ultrastructural markers of increased phagolysosomal activity in APP-PS1 versus C57BL6/J mice. In addition, we investigated the ultrastructural heterogeneity of astrocytes, describing in the two examined layers a dark astrocytic state that we characterized in terms of distribution, interactions with AD hallmarks, and intracellular contents. This electron-dense astrocytic state, termed dark astrocytes, was observed throughout the hippocampal parenchyma, closely associated with the vasculature, and possessed several ultrastructural markers of cellular stress. A case study exploring the hippocampal head of an aged human post-mortem brain sample also revealed the presence of a similar electron-dense, dark astrocytic state. Overall, our study provides the first ultrastructural quantitative analysis of astrocytes among the hippocampus in aged AD pathology, as well as a thorough characterization of a dark astrocytic state conserved from mouse to human.

Ultrastructural characterization of dark microglia during aging in a mouse model of Alzheimer's disease pathology and in human post-mortem brain samples.

A diverse heterogeneity of microglial cells was previously described in Alzheimer's disease (AD) pathology, including dark microglia, a state characterized by ultrastructural markers of cellular stress. To provide novel insights into the roles of dark microglia during aging in the context of AD pathology, we performed a quantitative density and ultrastructural analysis of these cells using high-throughput scanning electron microscopy in the ventral hippocampus CA1 stratum lacunosum-moleculare of 20-month-old APP-PS1 vs C57BL/6J male mice. The density of dark microglia was significantly higher in APP-PS1 vs C57BL/6J mice, with these cells accounting for nearly half of all microglia observed near amyloid-beta (Aß) plaques. This dark microglial state interacted more with dystrophic neurites compared to other APP-PS1 microglia and possessed glycogen granules, associated with a metabolic shift toward glycolysis, which provides the first ultrastructural evidence of their presence in microglia. Dark microglia were further observed in aging human post-mortem brain samples showing similar ultrastructural features as in mouse. Overall, our results provide a quantitative ultrastructural characterization of a microglial state associated with cellular stress (i.e., dark microglia) that is primarily restricted near Aß plaques and dystrophic neurites. The presence of this microglial state in the aging human post-mortem brain is further revealed.

The highly selective mGlu2 receptor positive allosteric modulator LY-487,379 alleviates l-DOPA-induced dyskinesia in the 6-OHDA-lesioned rat model of Parkinson's disease.

l-3,4-Dihydroxyphenylalanine (l-DOPA) is the most effective treatment for Parkinson's disease (PD), but its use over a long period is marred by motors complications such as dyskinesia. We previously demonstrated that selective metabotropic glutamate 2/3 (mGlu2/3 ) receptor activation with LY-354,740 alleviates dyskinesia in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned marmoset and the 6-hydroxydopamine (6-OHDA)-lesioned rat. Here, we sought to determine the role played by selective mGlu2 activation in the anti-dyskinetic effect of mGlu2/3 stimulation and have investigated the effect of the highly selective mGlu2 positive allosteric modulator LY-487,379 at alleviating established, and preventing the development of, l-DOPA-induced dyskinesia in the 6-OHDA-lesioned rat. First, dyskinetic 6-OHDA-lesioned rats were administered l-DOPA in combination with LY-487,379 (0.1, 1 and 10 mg/kg) or vehicle, and the severity of dyskinesia was determined. Second, 6-OHDA-lesioned rats were administered LY-487,379 (0.1 or 1 mg/kg), started concurrently with l-DOPA, once daily for 22 days, and dyskinesia severity was evaluated weekly for four consecutive weeks. We also assessed the effect of LY-487,379 on l-DOPA anti-parkinsonian effect. We found that acute challenges of LY-487,379 0.1 mg/kg in combination with l-DOPA, significantly diminished dyskinesia severity, by ≈54% (p < .01), when compared to vehicle. Moreover, animals treated with l-DOPA/LY-487,379 0.1 and 1 mg/kg during the dyskinesia induction phase exhibited milder dyskinesia, by ≈74% and ≈61%, respectively (both p < .01), when compared to l-DOPA/vehicle. LY-487,379 did not impair l-DOPA anti-parkinsonian activity. These results suggest that mGlu2 activation may be an effective and promising therapeutic strategy to alleviate the severity and prevent the development of dyskinesia.

Somatotopic organization of ferret thalamus.

The stereotaxic reference marks of ferret skull have large variability and the reference point for stereotaxic experiments in ferret brain is difficult to define. Here, using extracellular single-unit recordings, we studied the somatotopic organization of cutaneous receptive fields in the ventroposterior medial (VPM) and the ventral posterolateral (VPL) nuclei of the ferret thalamus. The mechanical stimulation of the skin was done through air puffs. The skull was positioned according to Horsley-Clarke coordinate system. Most of the neurons responding to face skin stimulation were located +7-+9 mm anterior, 2-3.9 mm lateral and 7-9.6 mm from cortical surface, whereas those responding to body skin stimulation were located +7-+10 mm anterior, 3.3-5.5 mm lateral and 6.7-10 mm from cortical surface. Out of 90 thalamic neurons recorded in this study, 58 responded to the body and the other neurons to the face stimulation. All neurons responded with spikes to stimulus onset, 37% of neurons responded only to stimuli onset and offset and 22% neurons fired tonically throughout stimulating epoch. The whiskers representation was located in the middle of the VPM nucleus, whereas those of the tongue, nose, bridge of the nose, supraorbital areas, upper and lower lips, and lower jaw were surrounding the whiskers representation. Within the VPL nucleus there was a clear topological correspondence from forelimb to hindlimb in the medial-to-lateral direction. Our findings indicate the whiskers representation in VPM or the forelimb-hindlimb representation in the VPL nucleus can be considered as a reliable reference in the ferret thalamus.

Serotonin and dopamine striatal innervation in Parkinson's disease and Huntington's chorea.

In contrast to our vast knowledge of the dopamine (DA) system, much less is known about the involvement of serotonin (5-HT) in neurodegenerative diseases affecting the basal ganglia. Therefore, we designed a study that aimed at characterizing the status of the striatal DA and 5-HT systems in patients who suffered from either Parkinson's (PD) or Huntington's disease (HD), compared to age-matched controls. Antibodies against tyrosine hydroxylase (TH) and 5-HT transporter (SERT) were used as markers of DA and 5-HT axonal profiles, respectively. The density and pattern of TH+ and SERT + innervation were determined by optical density measurements as well as by direct stereological estimates of labeled axon varicosities. The results reveal a significant decrease in TH immunoreactivity and TH + axon terminals throughout the striatum in both PD and HD, whereas the intensity of SERT immunostaining and the density of SERT + axon varicosities were found to be slightly increased in the striatum of PD and HD patients compared to controls. These findings reveal that the nigrostriatal DA system is significantly impaired in both PD and HD compared to the striatal 5-HT innervation, which is slightly increased in these two conditions. The striatal 5-HT augmentation observed in PD might be the result of a neural mechanism designed to compensate for DA denervation, whereas the marked atrophy of the striatum might explain the increase in the 5-HT innervation noted in HD. These findings underline the importance of the complex interplay between DA and 5-HT striatal afferents in the elaboration of appropriate motor behaviour.

Serotonin innervation of basal ganglia in monkeys and humans.

This review paper summarizes our previous contributions to the study of serotonin (5-hydroxytryptamine; 5-HT) innervation of basal ganglia in human and nonhuman primates under normal conditions. We have visualized the 5-HT neuronal system in squirrel monkey (Saimiri sciureus) and human postmortem materials with antibodies directed against either 5-HT, 5-HT transporter (SERT) or 5-HT synthesizing enzyme tryptophan hydroxylase (TPH). Confocal microscopy was used to compare the distribution of 5-HT and dopamine (DA; tyrosine hydroxylase-immunolabeled) axons in human, while the ultrastructural features of 5-HT axon terminals in monkey subthalamic nucleus were characterized at electron microscopic level. In monkeys and humans, midbrain raphe neurons emit axons that traverse the brainstem via the transtegmental system, ascend within the medial forebrain bundle and reach their targets by coursing along the major output pathways of the basal ganglia. These 5-HT axons arborize in virtually all basal ganglia components with the substantia nigra receiving the densest innervation and the striatum the most heterogeneous one. Although the striatum - the major basal ganglia input structure - appears to be a common termination site for many of 5-HT ascending axons, our results reveal that the widely distributed 5-HT neuronal system can also act directly upon neurons located within the two major output structures of the basal ganglia, namely the internal pallidum and the substantia nigra pars reticulata in monkeys and humans. This system also has a direct access to neurons of the DA nigrostriatal pathway, a finding that underlines the importance of the 5-HT/DA interactions in the physiopathology of basal ganglia.

Serotonin innervation of human basal ganglia.

This study aimed to provide a first detailed description of the serotonin (5-hydroxytryptamine, 5-HT) innervation of the human basal ganglia under nonpathological conditions. We applied an immunohistochemical approach to postmortem human brain material with antibodies directed against the 5-HT transporter and the 5-HT-synthesizing enzyme (tryptophane hydroxylase) to visualize 5-HT axons and cell bodies, respectively. Adjacent sections were immunostained for tyrosine hydroxylase to compare the distribution of 5-HT axons with that of dopamine axons. Human basal ganglia are innervated by 5-HT axons that emerge chiefly from the dorsal and, less abundantly, from the median raphe nuclei. These axons form thick ascending fascicles that fragment themselves as they penetrate the decussation of the superior cerebellar peduncle. They regroup within the ventral tegmental area and ascend along the medial forebrain bundle, immediately beneath the dopamine ascending fibers. At regular intervals along their course, 5-HT axons detach themselves from the medial forebrain bundle and sweep laterally to arborize within all basal ganglia components, where they display highly variable densities and patterns of innervation. The substantia nigra is the most densely innervated component of the basal ganglia, whereas the caudate nucleus is more heterogeneously innervated than the putamen and pallidum. The subthalamic nucleus harbors 5-HT-immunoreactive fibers that display a mediolateral-decreasing gradient. The fact that all components of human basal ganglia receive a dense 5-HT input indicates that, in concert with dopamine, 5-HT plays a crucial role in the functional organization of these motor-related structures, which are often targeted in neurodegenerative diseases.

Intense dopamine innervation of the subventricular zone in Huntington's disease.

Dopamine exerts a robust promoting effect on adult neurogenesis. Here, we report the presence of an intense dopamine (tyrosine hydroxylase immunoreactive) zone along the ventricular border of the caudate nucleus in patients with Huntington's disease, but not in age-matched controls. This thin (150-400 microm) paraventricular zone was composed of numerous small and densely packed dopamine axon varicosities and overlapped the deep layers of the subventricular zone. Immunoreactivity in the paraventricular zone was 50% higher than in adjacent striatal areas. This intense dopamine zone concurs with the striking increase of neurogenesis noted in the subventricular zone of Huntington's disease patients and indicates that dopamine might play a crucial role in intrinsic mechanisms designed to compensate for the massive striatal neuronal losses that occur in Huntington's disease.

Distribution and ultrastructural features of the serotonin innervation in rat and squirrel monkey subthalamic nucleus.

The main purpose of this light and electron microscopic immunocytochemical study was to characterize and compare the serotonin (5-HT) innervation of the subthalamic nucleus (STN) in rats and squirrel monkeys (Saimiri sciureus) following labeling with an antibody against the 5-HT transporter (SERT). Unbiased counts of SERT+ axon varicosities revealed an average density of 5-HT innervation higher in monkeys (1.52 x 10(6) varicosities/mm3) than rats (1.17 x 10(6)), particularly in the anterior half of the nucleus (1.70 x 10(6)). As measured by electron microscopy, SERT+ axon varicosity profiles in the STN of both species were smaller than unlabeled profiles. The number of SERT+ profiles displaying a synaptic junction indicated that, in both rat and monkey STN, approximately half of 5-HT axon varicosities were asynaptic. In monkeys, all synaptic junctions made by SERT+ varicosities were asymmetrical, as opposed to only 77% in rats. Despite the higher density of 5-HT innervation in the anterior half of monkey STN, the ultrastructural features of its SERT+ varicosities, including synaptic incidence, did not significantly differ from those in its posterior half. These findings suggest that, throughout the rat and monkey STN, 5-HT afferents may exert their influence via both synaptic delivery and diffusion of 5-HT, and that an ambient level of 5-HT maintained in STN by these two modes of transmission might also modulate neuronal activity and influence motor behavior. A better understanding of the factors governing the complex interplay between these signaling processes would greatly improve our knowledge of the physiopathology of the STN.

The fate of the large striatal interneurons expressing calretinin in Huntington's disease.

Huntington's disease (HD) is characterized by the atrophy of the striatum due to losses of projection neurons, while interneurons are relatively spared. However, little is known about the fate of the large interneurons that express calretinin (Cr) in HD. We addressed this issue by applying a double immunofluorescent labeling technique to postmortem striatum from HD patients and controls. We compared the distribution and density of Cr-positive (+) interneurons and their degree of choline acetyltransferase (ChAT) coexpression in normal and HD cases. Large interneurons containing only Cr, ChAT, or both occurred in the normal human striatum and a twofold decrease in the density of Cr+/ChAT+ and Cr-/ChAT+ neurons was recorded in HD striatum compared to controls. However, studies undertaken with neurokinin-1 receptor as a marker of large Cr+ and ChAT+ neurons revealed that these neurons are selectively spared in HD. Hence, the apparent decrease in the number of Cr+/ChAT+ and Cr-/ChAT+ neurons in HD is better explained by a diminution in the expression of Cr and ChAT than by the degeneration of these cells. Altogether, our data suggest that neurodegenerative processes at play in HD affect the expression of Cr and ChAT in the large striatal interneurons without causing their death.

Neurokinin-1 and neurokinin-3 receptors in primate substantia nigra.

Striatonigral axons co-release GABA and substance P (SP) at their target sites, but little is known about the action of SP at nigral level. Therefore, we studied immunohistochemically the cellular and subcellular localization of SP and its high affinity receptors neurokinin-1 (NK-1R) and neurokinin-3 (NK-3R) at nigral level in squirrel monkeys. Immunofluorescent studies revealed that, although SP+ fibers arborised more densely in the pars reticulata (SNr) than in the pars compacta (SNc), the two nigral divisions harbored numerous neurons expressing NK-1R and NK-3R. Confocal microscopic analyses showed that numerous SNr neurons and virtually all SNc dopaminergic neurons contained both NK-1R and NK-3R. At the electron microscope level, NK-1R and NK-3R were mainly associated with intracellular sites or located at extrasynaptic position on plasma membrane. A small proportion of SP+ boutons also showed NK-3R immunoreactivity. The distribution of NK-1R and NK-3R in SNr and SNc suggests that SP exerts its effect through postsynaptic receptors, as well as via presynaptic autoreceptors and heteroreceptors. These findings indicate that the excitatory peptide SP can modulate the inhibitory action of GABA at nigral level and suggest that the co-release of these two neuroactive substances should be taken into account when considering the functional organization of the basal ganglia.

Striosomes are enriched in glutamic acid decarboxylase in primates.

The compartmental distribution of glutamic acid decarboxylase (GAD) in the striatum was investigated in squirrel monkeys and rats with antibodies raised against the two isoforms of this enzyme (GAD65 and GAD67) and with calbindin D-28k (CB) and/or micro-opiate receptor (MOR) as striosomal markers. In primates, immunostaining for both GAD65 and GAD67 was much more intense in striosomes than in the surrounding matrix. A thin immunoreactive strip of GAD labeling was also present in the dorsolateral part of both caudate nucleus and putamen. This narrow band appears to correspond to the so-called subcallosal streak (SS) found in rodent striatum. Although the immunostaining intensity for the two enzymes was similar at pallidal level, that for GAD65 was more intense than that for GAD67 at the striatal level. The GAD immunostaining was more uniformly distributed in the rat striatum, which did not display GAD-rich patches that corresponded to MOR-positive striosomes. Moreover, in contrast to the findings obtained in monkeys, the subcallosal streak in rats was less intensely stained for GAD than for the remaining regions of the striatum. These results reveal that GAD65 and GAD67 are faithful markers of striosomes in primates but not in rodents. They suggest the existence of a significant species difference between rodents and primates in respect to the chemical organization of the striatum, a difference that should be taken into account when using rodents as animal models to study the functional organization of the basal ganglia and the pathogenesis of neurodegenerative diseases that affect the striatum.