The dynamic interplay between ATP/ADP levels and autophagy sustain neuronal migration in vivo.

Cell migration is a dynamic process that entails extensive protein synthesis and recycling, structural remodeling, and considerable bioenergetic demand. Autophagy is one of the pathways that maintain cellular homeostasis. Time-lapse imaging of autophagosomes and ATP/ADP levels in migrating cells in the rostral migratory stream of mouse revealed that decreases in ATP levels force cells into the stationary phase and induce autophagy. Pharmacological or genetic impairments of autophagy in neuroblasts using either bafilomycin, inducible conditional mice, or CRISPR/Cas9 gene editing decreased cell migration due to the longer duration of the stationary phase. Autophagy is modulated in response to migration-promoting and inhibiting molecular cues and is required for the recycling of focal adhesions. Our results show that autophagy and energy consumption act in concert in migrating cells to dynamically regulate the pace and periodicity of the migratory and stationary phases to sustain neuronal migration.

Levodopa partially rescues microglial numerical, morphological, and phagolysosomal alterations in a monkey model of Parkinson's disease.

Parkinson's disease (PD) is the most common neurodegenerative motor disorder. The mechanisms underlying the onset and progression of Levodopa (L-Dopa)-induced dyskinesia (LID) during PD treatment remain elusive. Emerging evidence implicates functional modification of microglia in the development of LID. Thus, understanding the link between microglia and the development of LID may provide the knowledge required to preserve or promote beneficial microglial functions, even during a prolonged L-Dopa treatment. To provide novel insights into microglial functional alterations in PD pathophysiology, we characterized their density, morphology, ultrastructure, and degradation activity in the sensorimotor functional territory of the putamen, using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) cynomolgus monkeys. A subset of MPTP monkeys was treated orally with L-Dopa and developed LID similar to PD patients. Using a combination of light, confocal and transmission electron microscopy, our quantitative analyses revealed alterations of microglial density, morphology and phagolysosomal activity following MPTP intoxication that were partially normalized with L-Dopa treatment. In particular, microglial density, cell body and arborization areas were increased in the MPTP monkeys, whereas L-Dopa-treated MPTP animals presented a microglial phenotype similar to the control animals. At the ultrastructural level, microglia did not differ between groups in their markers of cellular stress or aging. Nevertheless, microglia from the MPTP monkeys displayed reduced numbers of endosomes, compared with control animals, that remained lower after L-Dopa treatment. Microglia from MPTP monkeys treated with L-Dopa also had increased numbers of primary lysosomes compared with non-treated MPTP animals, while secondary and tertiary lysosomes remained unchanged. Moreover, a decrease microglial immunoreactivity for CD68, considered a marker of phagocytosis and lysosomal activity, was measured in the MPTP monkeys treated with L-Dopa, compared with non-treated MPTP animals. Taken together, these findings revealed significant changes in microglia during PD pathophysiology that were partially rescued by L-Dopa treatment. Albeit, this L-Dopa treatment conferred phagolysosomal insufficiency on microglia in the dyskinetic Parkinsonian monkeys.

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.

Intraoperative fiber optic guidance during chronic electrode implantation in deep brain stimulation neurosurgery: proof of concept in primates.

OBJECTIVE: The clinical outcome of deep brain stimulation (DBS) surgery relies heavily on the implantation accuracy of a chronic stimulating electrode into a small target brain region. Most techniques that have been proposed to precisely target these deep brain regions were designed to map intracerebral electrode trajectory prior to chronic electrode placement, sometimes leading to positioning error of the final electrode. This study was designed to create a new intraoperative guidance tool for DBS neurosurgery that can improve target detection during the final implantation of the chronic electrode. METHODS: Taking advantage of diffuse reflectance spectroscopy, the authors developed a new surgical tool that senses proximal brain tissue through the tip of the chronic electrode by means of a novel stylet, which provides rigidity to DBS leads and houses fiber optics. RESULTS: As a proof of concept, the authors demonstrated the ability of their noninvasive optical guidance technique to precisely locate the border of the subthalamic nucleus during the implantation of commercially available DBS electrodes in anesthetized parkinsonian monkeys. Innovative optical recordings combined to standard microelectrode mapping and detailed postmortem brain examination allowed the authors to confirm the precision of optical target detection. They also show the optical technique's ability to detect, in real time, upcoming blood vessels, reducing the risk of hemorrhage during the chronic lead implantation. CONCLUSIONS: The authors present a new optical guidance technique that can detect target brain regions during DBS surgery from within the implanted electrode using a proof of concept in nonhuman primates. The technique discriminates tissue in real time, contributes no additional invasiveness to the procedure by being housed within the electrode, and can provide complementary information to microelectrode mapping during the implantation of the chronic electrode. The technique may also be a powerful tool for providing direct anatomical information in the case of direct implantations wherein microelectrode mapping is not performed.

Evidence for Sprouting of Dopamine and Serotonin Axons in the Pallidum of Parkinsonian Monkeys.

This light and electron microscopie immunohistochemical quantitative study aimed at determining the state of the dopamine (DA) and serotonin (5-HT) innervations of the internal (GPi) and external (GPe) segments of the pallidum in cynomolgus monkeys (Macaca fascicularis) rendered parkinsonian by systemic injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In contrast to the prominent DA denervation of striatum, the GPi in MPTP monkeys was found to be markedly enriched in DA (TH+) axon varicosities. The posterior sensorimotor region of this major output structure of the basal ganglia was about 8 times more intensely innervated in MPTP monkeys (0.71 ± 0.08 × 106 TH+ axon varicosities/mm3) than in controls (0.09 ± 0.01 × 106). MPTP intoxication also induced a two-fold increase in the density of 5-HT (SERT+) axon varicosities in both GPe and GPi. This augmentation was particularly pronounced anteriorly in the so-called associative and limbic pallidal territories. The total length of the labeled pallidal axons was also significantly increased in MPTP monkeys compared to controls, but the number of DA and 5-HT axon varicosities per axon length unit remained the same in the two groups, indicating that the DA and 5-HT pallidal hyperinnervations seen in MPTP monkeys result from axon sprouting rather than from the appearance of newly formed axon varicosities on non-growing axons. At the ultrastructural level, pallidal TH+ and SERT+ axons were morphologically similar in MPTP and controls, and their synaptic incidence was very low suggesting a volumic mode of transmission. Altogether, our data reveal a significant sprouting of DA and 5-HT pallidal afferents in parkinsonian monkeys, the functional significance of which remains to be determined. We suggest that the marked DA hyperinnervation of the GPi represents a neuroadaptive change designed to normalize pallidal firing patterns associated with the delayed appearance of motor symptoms, whereas the 5-HT hyperinnervation might be involved in the early expression of non-motor symptoms in Parkinson's disease.

A dense cluster of D1 + cells in the mouse nucleus accumbens.

The striatum is known to be largely composed of intermingled medium-sized projection neurons expressing either the D1 or the D2 dopamine receptors. In the present study, we took advantage of the double BAC Drd1a-TdTomato/Drd2-GFP (D1 /D2 ) transgenic mice to reveal the presence of a peculiar cluster of densely-packed D1 + cells located in the shell compartment of the nucleus accumbens. This spherical cluster has a diameter of 110 µm and is exclusively composed by D1 + cells, which are all immunoreactive for the neuronal nuclear marker (NeuN). However, in contrast to other D1 + or D2 + striatal cells, those that form the accumbens cluster are devoid of calbindin (CB) and DARPP-32, two faithful markers for striatal projection neurons. Using GAD-GFP transgenic mice, we confirm the GABAergic nature of the D1 + clustered neurons. Intracellular injections from fixed brain slices indicate that these neurons are endowed with distinctive morphological features, including a small (5-6 µm), round cell body giving rise to a single primary dendrite that branches into two secondary processes. Single-neuronal injections combined to electron microscopy reveal the existence of GAP junctions linking these D1 + cells. Based on their location, morphological characteristics and neurochemical phenotype, we conclude that the D1 + accumbens cluster form a highly compact group of small neurons distinct from the larger and more diffusely distributed D1 + or D2 + striatal projection neurons that surround it. This remarkable nucleus might play a crucial role in the limbic function of the murine striatum.

Distribution of VGLUT3 in highly collateralized axons from the rat dorsal raphe nucleus as revealed by single-neuron reconstructions.

This study aimed at providing the first detailed morphological description, at the single-cell level, of the rat dorsal raphe nucleus neurons, including the distribution of the VGLUT3 protein within their axons. Electrophysiological guidance procedures were used to label dorsal raphe nucleus neurons with biotinylated dextran amine. The somatodendritic and axonal arborization domains of labeled neurons were reconstructed entirely from serial sagittal sections using a computerized image analysis system. Under anaesthesia, dorsal raphe nucleus neurons display highly regular (1.72 ± 0.50 Hz) spontaneous firing patterns. They have a medium size cell body (9.8 ± 1.7 µm) with 2-4 primary dendrites mainly oriented anteroposteriorly. The ascending axons of dorsal raphe nucleus are all highly collateralized and widely distributed (total axonal length up to 18.7 cm), so that they can contact, in various combinations, forebrain structures as diverse as the striatum, the prefrontal cortex and the amygdala. Their morphological features and VGLUT3 content vary significantly according to their target sites. For example, high-resolution confocal analysis of the distribution of VGLUT3 within individually labeled-axons reveals that serotonin axon varicosities displaying VGLUT3 are larger (0.74 ± 0.03 µm) than those devoid of this protein (0.55 ± 0.03 µm). Furthermore, the percentage of axon varicosities that contain VGLUT3 is higher in the striatum (93%) than in the motor cortex (75%), suggesting that a complex trafficking mechanism of the VGLUT3 protein is at play within highly collateralized axons of the dorsal raphe nucleus neurons. Our results provide the first direct evidence that the dorsal raphe nucleus ascending projections are composed of widely distributed neuronal systems, whose capacity to co-release serotonin and glutamate varies from one forebrain locus to the other.

Striatal allografts in patients with Huntington's disease: impact of diminished astrocytes and vascularization on graft viability.

Neuronal transplantation has been proposed as a potential therapy to replace lost neurons in Huntington's disease. Transplant vascularization and trophic support are important for graft survival. However, very few studies have specifically addressed graft vascularization in patients with neurological disorders. In the present study, we analysed the vasculature of the host putamen and solid grafts of foetal striatal tissue transplanted into patients with Huntington's disease 9 and 12 years previously. Grafts were characterized by a significantly reduced number of large calibre blood vessels in comparison with the host brain. There were also significantly fewer astrocytes and gap junctions, suggesting a lack of functional blood-brain barrier components within the grafted tissue. Additionally, grafts demonstrated a nearly complete absence of pericytes (compared with the striatum) that are considered important for vascular stabilization and angiogenesis. Finally, the host striatum had a marked increase in atrophic astrocytes in comparison with controls and grafts. The extent to which the lower number of large calibre vessels and astrocytes within the transplants contributed to suboptimal graft survival is unknown. The marked increase in atrophic astrocytes in the host brain surrounding the grafts suggests that reduced host trophic support may also contribute to poor graft survival in Huntington's disease. A better understanding of the way in which these components support allografted tissue is critical to the future development of cell-based therapies for the treatment of Huntington's disease.

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.

Association of common polymorphisms in the fractalkine receptor (CX3CR1) with obesity.

The inflammatory component in obesity is now well established. The CX3CR1 gene encodes the fractalkine (CX3CL1) receptor and has two coding single-nucleotide polymorphisms, V249I and T280M, linked to a lower risk of other inflammatory diseases such as coronary artery disease (CAD) and asthma. To determine whether CX3CR1 is associated with obesity, we genotyped the V249I and T280M polymorphisms of the CX3CR1 gene in subjects with a BMI ≥30 kg/m² and nonobese controls with a BMI <30 kg/m². Binary logistic regression analyses revealed that the 280MM genotype was associated with obesity (P = 0.022). A gender-specific one-way ANOVA was also conducted to investigate mean BMI and waist circumference differences between genotypes of each polymorphism. For both polymorphisms independently, women carrying two copies of the minor allele had significant higher mean waist circumference than those carrying only one copy of the minor allele (MM > TM, P = 0.031; II > VI, P = 0.013) or those who were homozygous for the major allele (MM > TT, P = 0.005; II > VV, P = 0.006). We also observed significant higher mean waist circumference in men carrying one copy of the minor allele when compared to those who were homozygous for the major allele for the T280M polymorphism (TM > TT, P = 0.029). This study suggests that CX3CR1, a biomarker of obesity in this sample, constitutes a potential target for further investigation of the role of inflammation in the expression of obesity-related phenotypes.