Carole Carcenac

Pallidal Origin of GABA Release within the Substantia Nigra Pars Reticulata during High-Frequency Stimulation of the Subthalamic Nucleus

High-frequency stimulation of the subthalamic nucleus (HFS-STN) is an effective treatment for alleviating the motor symptoms of parkinsonian patients. However, the neurochemical basis of its effects remains unknown. We showed previously that 1 h of HFS-STN in normal rats increases extracellular glutamate (Glu) level in the output nuclei of the STN, the globus pallidus (GP), and the substantia nigra pars reticulata (SNr), consistent with an increase in the activity of STN neurons. HFS-STN also increases GABA levels in the SNr, but the origin of this increase is unclear. We investigated the effectiveness of HFS-STN for improving Parkinsons disease symptoms, using intracerebral microdialysis to determine the extracellular Glu and GABA levels of the GP and SNr in response to HFS-STN in anesthetized hemiparkinsonian rats [6-hydroxydopamine lesion of the substantia nigra pars compacta (SNc)]. Basal levels of Glu and GABA in the GP and SNr were significantly higher in hemiparkinsonian than in intact rats. HFS-STN did not affect extracellular Glu level in the SNr of hemiparkinsonian rats but doubled the level of GABA. Ibotenic acid lesion of the GP abolished the increase in GABA levels in the SNr induced by HFS-STN in SNc-lesioned rats. These results provide neurochemical confirmation of the hyperactivity of the STN after dopaminergic denervation and suggest that the therapeutic effects of HFS-STN may result partly from the stimulation of pallidonigral fibers, thereby revealing a potential role for pallidal GABA in the inhibition of basal ganglial output structures during HFS-STN.

Subthalamic Stimulation-Induced Forelimb Dyskinesias Are Linked to an Increase in Glutamate Levels in the Substantia Nigra Pars Reticulata

The neurobiological mechanisms by which high-frequency stimulation of the subthalamic nucleus (STN–HFS) alleviates the motor symptoms of Parkinsons disease (PD) remain unclear. In this study, we analyzed the effects of STN–HFS on motor behavior in intact or hemiparkinsonian rats (6-hydroxydopamine lesion of the substantia nigra pars compacta) and investigated the correlation between these effects and extracellular glutamate (Glu) and GABA levels, assessed by intracerebral microdialysis in the substantia nigra pars reticulata (SNr). STN–HFS at an intensity corresponding to the threshold inducing contralateral forelimb dyskinesia, increased Glu levels in the SNr of both intact and hemiparkinsonian rats. In contrast, STN–HFS at half this intensity did not affect Glu levels in the SNr in intact or hemiparkinsonian rats but increased GABA levels in hemiparkinsonian rats only. STN–HFS-induced forelimb dyskinesia was blocked by microinjection of the Glu receptor antagonist kynurenate into the SNr and facilitated by microinjection of a mixture of the Glu receptor agonists AMPA and NMDA into the SNr. These new neurochemical data suggest that STN–HFS-induced forelimb dyskinesia is mediated by glutamate, probably via the direct activation of STN axons, shedding light on the mechanisms of STN–HFS in PD.

Loss of dopaminergic nigrostriatal neurons accounts for the motivational and affective deficits in Parkinson's disease.

Parkinson’s disease (PD) involves the degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) that is thought to cause the classical motor symptoms of this disease. However, motivational and affective impairments are also often observed in PD patients. These are usually attributed to a psychological reaction to the general motor impairment and to a loss of some of the neurons within the ventral tegmental area (VTA). We induced selective lesions of the VTA and SNc DA neurons that did not provoke motor deficits, and showed that bilateral dopamine loss within the SNc, but not within the VTA, induces motivational deficits and affective impairments that mimicked the symptoms of PD patients. Thus, motivational and affective deficits are a core impairment of PD, as they stem from the loss of the major group of neurons that degenerates in this disease (DA SNc neurons) and are independent of motor deficits.

High-frequency stimulation of the subthalamic nucleus prolongs the increase in striatal dopamine induced by acute l-3,4-dihydroxyphenylalanine in dopaminergic denervated rats

High‐frequency stimulation of the subthalamic nucleus (STN‐HFS) is a powerful approach for treating the motor symptoms of Parkinsons disease (PD). It results in clinical improvement in patients with PD, further reducing the l‐3,4‐dihydroxyphenylalanine (L‐DOPA) requirement and thus L‐DOPA‐induced dyskinesia. However, it remains unclear how STN‐HFS modifies the response to L‐DOPA. We investigated the effect of STN‐HFS on striatal extracellular concentrations of dopamine and its metabolites following acute L‐DOPA administration in intact or partially dopaminergic denervated (DA‐PL) rats. L‐DOPA treatment significantly increased striatal dopamine levels in intact and DA‐PL animals, with the maximal effect observed 1 h after L‐DOPA injection. This increase was more pronounced in DA‐PL rats (ipsilateral to the lesion) than in intact animals. It remained fairly stable 1 h after the maximal effect of L‐DOPA and then decreased towards basal values. STN‐HFS in intact rats had no effect on the maximal L‐DOPA‐induced increase in striatal extracellular dopamine concentration or the return to basal values, the profiles observed being similar to those for non‐stimulated intact animals. Conversely, STN‐HFS amplified the L‐DOPA‐induced increase in striatal dopamine levels during the stimulation period (1 h) in DA‐PL rats and this increase was sustained throughout the post‐stimulation period (2.5 h), without the return to basal levels observed in stimulated intact and non‐stimulated rats. These new neurochemical data suggest that STN‐HFS interferes with L‐DOPA effects, probably synergically, by stabilizing dopamine levels in the striatum and shed light on the mechanisms of STN‐HFS in PD.

Discovering New Bioactive Neuropeptides in the Striatum Secretome Using in Vivo Microdialysis and Versatile Proteomics

The striatum, a major component of the brain basal nuclei, is central for planning and executing voluntary movements and undergoes lesions in neurodegenerative disorders such as Huntington disease. To perform highly integrated tasks, the striatum relies on a complex network of communication within and between brain regions with a key role devoted to secreted molecules. To characterize the rat striatum secretome, we combined in vivo microdialysis together with proteomics analysis of trypsin digests and peptidomics studies of native fragments. This versatile approach, carried out using different microdialysis probes and mass spectrometer devices, allowed evidencing with high confidence the expression of 88 proteins and 100 processed peptides. Their secretory pathways were predicted by in silico analysis. Whereas high molecular weight proteins were mainly secreted by the classical mode (94%), low molecular weight proteins equally used classical and non-classical modes (53 and 47%, respectively). In addition, our results suggested alternative secretion mechanisms not predicted by bioinformatics tools. Based on spectrum counting, we performed a relative quantification of secreted proteins and peptides in both basal and neuronal depolarization conditions. This allowed detecting a series of neuropeptide precursors and a 6-fold increase for neurosecretory protein VGF and proenkephalin (PENK) levels. A focused investigation and a long peptide experiment led to the identification of new secreted non-opioid PENK peptides, referred to as PENK 114–133, PENK 239–260, and PENK 143–185. Moreover we showed that injecting synthetic PENK 114–133 and PENK 239–260 into the striatum robustly increased glutamate release in this region. Thus, the combination of microdialysis and versatile proteomics methods shed new light on the secreted protein repertoire and evidenced novel neuropeptide transmitters.

Pramipexole reverses Parkinson's disease‐related motivational deficits in rats

Recent evidence suggests that Parkinsons disease affects not only movement, but also cognitive and psychiatric functions. Among these nonmotor complications, apathy, which is defined as a lack of motivation and operationalized as a quantitative reduction in goal‐directed behavior, may even precede motor impairments, disappearing with the introduction of dopaminergic (DA) therapies and possibly reappearing with its discontinuation, suggesting a causal role of DA. We recently developed a lesion‐based model, with stereotaxic infusion of 6‐hydroxydopamine (6‐OHDA) into precise areas of the rat SNc or ventral tegmental area and showed, in several operant tasks, that a partial denervation of the nigrostriatal, but not of the mesocorticolimbic, DA system induced profound motivational deficits during instrumental action. We investigated the time course of the effects of nigrostriatal DA denervation on motivation in rats, by assessing the negative effect of SNc bilateral 6‐OHDA infusion on preacquired operant behavior, and determining whether the induced deficits were sensitive to the introduction and withdrawal of a clinically relevant PD treatment, the DA D2/D3 receptor agonist, pramipexole (PRA). Partial nigrostriatal DA denervation was accompanied by a significant reduction in operant behavior. This deficit, indicative of a decrease in motivation, was fully reversed by PRA and reappeared after treatment withdrawal. This longitudinal preclinical study provides evidence for the implication of the DA nigrostriatal system in PD‐associated apathy. Moreover, by showing a good isomorphy and predictive value, our model highlights the relevance of D2/D3 receptors as potential targets for alleviating apathy in PD.

Chronic Intermittent Hypoxia Induces Chronic Low-Grade Neuroinflammation in the Dorsal Hippocampus of Mice.

STUDY OBJECTIVES Obstructive sleep apnea (OSA) induces cognitive impairment that involves intermittent hypoxia (IH). Because OSA is recognized as a low-grade systemic inflammatory disease and only some patients develop cognitive deficits, we investigated whether IH-related brain consequences shared similar pathophysiology and required additional factors such as systemic inflammation to develop. DESIGN Nine-week-old male C57BL/6J mice were exposed to 1 day, 6 or 24 w of IH (alternating 21-5% FiO2 every 30 sec, 8 h/day) or normoxia. Microglial changes were assessed in the functionally distinct dorsal (dH) and ventral (vH) regions of the hippocampus using Iba1 immunolabeling. Then the study concerned dH, as vH only tended to be lately affected. Seven proinflammatory and anti-inflammatory cytokine messenger RNA (mRNA) were assessed at all time points using semiquantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Similar mRNA analysis was performed after 6 w IH or normoxia associated for the past 3 w with repeated intraperitoneal low-dose lipopolysaccharide or saline. MEASUREMENTS AND RESULTS Chronic (6, 24 w) but not acute IH induced significant microglial changes in dH only, including increased density and morphological features of microglia priming. In dH, acute but not chronic IH increased IL-1β and RANTES/CCL5 mRNA, whereas the other cytokines remained unchanged. In contrast, chronic IH plus lipopolysaccharide increased interleukin (IL)-6 and IL10 mRNA whereas lipopolysaccharide alone did not affect these cytokines. CONCLUSION The obstructive sleep apnea component intermittent hypoxia (IH) causes low-grade neuroinflammation in the dorsal hippocampus of mice, including early but transient cytokine elevations, delayed but long-term microglial changes, and cytokine response alterations to lipopolysaccharide inflammatory challenge. These changes may contribute to IH-induced cognitive impairment and pathological brain aging.

Mitochondrial permeability transition pore inhibitors prevent ethanol-induced neuronal death in mice.

Ethanol induces brain injury by a mechanism that remains partly unknown. Mitochondria play a key role in cell death processes, notably through the opening of the permeability transition pore (PTP). Here, we tested the effect of ethanol and PTP inhibitors on mitochondrial physiology and cell viability both in vitro and in vivo. Direct addition of ethanol up to 100 mM on isolated mouse brain mitochondria slightly decreased oxygen consumption but did not affect PTP regulation. In comparison, when isolated from ethanol-treated (two doses of 2 g/kg, 2 h apart) 7-day-old mouse pups, brain mitochondria displayed a transient decrease in oxygen consumption but no change in PTP regulation or H2O2 production. Conversely, exposure of primary cultured astrocytes and neurons to 20 mM ethanol for 3 days led to a transient PTP opening in astrocytes without affecting cell viability and to a permanent PTP opening in 10 to 20% neurons with the same percentage of cell death. Ethanol-treated mouse pups displayed a widespread caspase-3 activation in neurons but not in astrocytes and dramatic behavioral alterations. Interestingly, two different PTP inhibitors (namely, cyclosporin A and nortriptyline) prevented both ethanol-induced neuronal death in vivo and ethanol-induced behavioral modifications. We conclude that PTP opening is involved in ethanol-induced neurotoxicity in the mouse.

Implication of dopamine D3 receptor activation in the reversion of Parkinson's disease-related motivational deficits.

In addition to the classical motor symptoms, motivational and affective deficits are core impairments of Parkinsons disease (PD). We recently demonstrated, by lesional approaches in rats, that degeneration of the substantia nigra pars compacta (SNc) dopaminergic (DA) neurons is likely to have a crucial role in the development of these neuropsychiatry symptoms. We have also shown that, as in clinical investigations, chronic treatment with levodopa or the DA D2/D3 receptor (D2/D3R) agonist ropinirole specifically reverses these PD-related motivational deficits. The roles of specific DA receptor subtypes in such reversal effects remain, however, unknown. We therefore investigated here the precise involvement of D1, D2 and D3R in the reversal of the motivational and affective deficits related to SNc DA neuronal loss. Three weeks after bilateral and partial 6-hydroxydopamine (6-OHDA) SNc lesions, rats received 14 daily intraperitoneal administrations of the selective D1R agonist SKF-38393 (2.5 or 3.5 mg kg−1), the selective D2R agonist sumanirole (0.1 or 0.15 mg kg−1), or the preferring D3R gonist PD-128907 (0.1 or 0.15 mg kg−1). Anxiety-, depressive-like and motivated behaviors were assessed in an elevated-plus maze, a forced-swim test, and an operant sucrose self-administration procedure, respectively. All DA agonists attenuated anxiety- and depressive-like behaviors. However, only PD-128907 reversed the motivational deficits induced by 6-OHDA SNc lesions. This effect was blocked by a selective D3R (SB-277011A, 10 mg kg−1), but not D2R (L-741,626, 1.5 mg kg−1), antagonist. These data provide strong evidence for the role of D3R in motivational processes and identify this receptor as a potentially valuable target for the treatment of PD-related neuropsychiatric symptoms.

High-frequency stimulation of the subthalamic nucleus modifies the expression of vesicular glutamate transporters in basal ganglia in a rat model of Parkinson’s disease

BackgroundIt has been suggested that glutamatergic system hyperactivity may be related to the pathogenesis of Parkinson’s disease (PD). Vesicular glutamate transporters (VGLUT1-3) import glutamate into synaptic vesicles and are key anatomical and functional markers of glutamatergic excitatory transmission. Both VGLUT1 and VGLUT2 have been identified as definitive markers of glutamatergic neurons, but VGLUT 3 is also expressed by non glutamatergic neurons. VGLUT1 and VGLUT2 are thought to be expressed in a complementary manner in the cortex and the thalamus (VL/VM), in glutamatergic neurons involved in different physiological functions. Chronic high-frequency stimulation (HFS) of the subthalamic nucleus (STN) is the neurosurgical therapy of choice for the management of motor deficits in patients with advanced PD. STN-HFS is highly effective, but its mechanisms of action remain unclear. This study examines the effect of STN-HFS on VGLUT1-3 expression in different brain nuclei involved in motor circuits, namely the basal ganglia (BG) network, in normal and 6-hydroxydopamine (6-OHDA) lesioned rats.ResultsHere we report that: 1) Dopamine(DA)-depletion did not affect VGLUT1 and VGLUT3 expression but significantly decreased that of VGLUT2 in almost all BG structures studied; 2) STN-HFS did not change VGLUT1-3 expression in the different brain areas of normal rats while, on the contrary, it systematically induced a significant increase of their expression in DA-depleted rats and 3) STN-HFS reversed the decrease in VGLUT2 expression induced by the DA-depletion.ConclusionsThese results show for the first time a comparative analysis of changes of expression for the three VGLUTs induced by STN-HFS in the BG network of normal and hemiparkinsonian rats. They provide evidence for the involvement of VGLUT2 in the modulation of BG cicuits and in particular that of thalamostriatal and thalamocortical pathways suggesting their key role in its therapeutic effects for alleviating PD motor symptoms.