Ontogenetic changes in the tyrosine hydroxylase immunoreactive preoptic area in the small-spotted catshark Scyliorhinus canicula (L., 1758) females: catecholaminergic involvement in sexual maturation.

Introduction: The catecholaminergic component of the brain-pituitary-gonadal axis, which mediates the influence of external and internal stimuli on the central nervous system and gonad development in vertebrates, is largely unexplored in Chondrichthyes. We considered Scyliorhinus canicula (L., 1758) females as a model for this vertebrate's class, to assess the involvement of the catecholaminergic system of the brain in its reproduction. Along the S. canicula reproductive cycle, we characterized and evaluated differences in somata morphometry and the number of putative catecholaminergic neurons in two brain nuclei: the periventricular preoptic nucleus, hypothesized to be a positive control for ovarian development, and the suprachiasmatic nucleus, examined as a negative control. Materials and methods: 16 S. canicula wild females were sampled and grouped in maturity stages (immature, maturing, mature, and mature egg-laying). The ovary was histologically processed for the qualitative description of maturity stages. Anti-tyrosine hydroxylase immunofluorescence was performed on the diencephalic brain sections. The immunoreactive somata were investigated for morphometry and counted using the optical fractionator method, throughout the confocal microscopy. Results and discussions: Qualitative and quantitative research confirmed two separate populations of immunoreactive neurons. The modifications detected in the preoptic nucleus revealed that somata were more numerous, significantly smaller in size, and more excitable during the maturing phase but decreased, becoming slightly bigger and less excitable in the egg-laying stage. This may indicate that the catecholaminergic preoptic nucleus is involved in the control of reproduction, regulating both the onset of puberty and the imminent spawning. In contrast, somata in the suprachiasmatic nucleus grew in size and underwent turnover in morphometry, increasing the total number from the immature-virgin to maturing stage, with similar values in the more advanced maturity stages. These changes were not linked to a reproductive role. These findings provide new valuable information on Chondrichthyes, suggesting the existence of an additional brain system implicated in the integration of internal and environmental cues for reproduction.

The Intranigral Infusion of Human-Alpha Synuclein Oligomers Induces a Cognitive Impairment in Rats Associated with Changes in Neuronal Firing and Neuroinflammation in the Anterior Cingulate Cortex.

Parkinson's disease (PD) is a complex pathology causing a plethora of non-motor symptoms besides classical motor impairments, including cognitive disturbances. Recent studies in the PD human brain have reported microgliosis in limbic and neocortical structures, suggesting a role for neuroinflammation in the development of cognitive decline. Yet, the mechanism underlying the cognitive pathology is under investigated, mainly for the lack of a valid preclinical neuropathological model reproducing the disease's motor and non-motor aspects. Here, we show that the bilateral intracerebral infusion of pre-formed human alpha synuclein oligomers (H-αSynOs) within the substantia nigra pars compacta (SNpc) offers a valid model for studying the cognitive symptoms of PD, which adds to the classical motor aspects previously described in the same model. Indeed, H-αSynOs-infused rats displayed memory deficits in the two-trial recognition task in a Y maze and the novel object recognition (NOR) test performed three months after the oligomer infusion. In the anterior cingulate cortex (ACC) of H-αSynOs-infused rats the in vivo electrophysiological activity was altered and the expression of the neuron-specific immediate early gene (IEG) Npas4 (Neuronal PAS domain protein 4) and the AMPA receptor subunit GluR1 were decreased. The histological analysis of the brain of cognitively impaired rats showed a neuroinflammatory response in cognition-related regions such as the ACC and discrete subareas of the hippocampus, in the absence of any evident neuronal loss, supporting a role of neuroinflammation in cognitive decline. We found an increased GFAP reactivity and the acquisition of a proinflammatory phenotype by microglia, as indicated by the increased levels of microglial Tumor Necrosis Factor alpha (TNF-α) as compared to vehicle-infused rats. Moreover, diffused deposits of phospho-alpha synuclein (p-αSyn) and Lewy neurite-like aggregates were found in the SNpc and striatum, suggesting the spreading of toxic protein within anatomically interconnected areas. Altogether, we present a neuropathological rat model of PD that is relevant for the study of cognitive dysfunction featuring the disease. The intranigral infusion of toxic oligomeric species of alpha-synuclein (α-Syn) induced spreading and neuroinflammation in distant cognition-relevant regions, which may drive the altered neuronal activity underlying cognitive deficits.

Changes in Dendritic Spine Morphology and Density of Granule Cells in the Olfactory Bulb of Anguilla anguilla (L., 1758): A Possible Way to Understand Orientation and Migratory Behavior.

Olfaction could represent a pivotal process involved in fish orientation and migration. The olfactory bulb can manage olfactive signals at the granular cell (GC) and dendritic spine levels for their synaptic plasticity properties and changing their morphology and structural stability after environmental odour cues. The GCs' dendritic spine density and morphology were analysed across the life stages of the catadromous Anguilla anguilla. According to the head and neck morphology, spines were classified as mushroom (M), long thin (LT), stubby (S), and filopodia (F). Total spines' density decreased from juvenile migrants to no-migrant stages, to increase again in the adult migrant stage. Mean spines' density was comparable between glass and silver eels as an adaptation to migration. At non-migrating phases, spines' density decreased for M and LT, while M, LT, and S density increased in silver eels. A great dendritic spine development was found in the two migratory phases, regressing in trophic phases, but that could be recreated in adults, tracing the migratory memory of the routes travelled in juvenile phases. For its phylogenetic Elopomorph attribution and its complex life cycle, A. anguilla could be recommended as a model species to study the development of dendritic spines in GCs of the olfactory bulb as an index of synaptic plasticity involved in the modulation of olfactory stimuli. If olfaction is involved in the orientation and migration of A. anguilla and if eels possess a memory, these processes could be influenced by the modification of environmental stimuli (ocean alterations and rapid climate change) contributing to threatening this critically endangered species.

Modeling Parkinson's Disease Neuropathology and Symptoms by Intranigral Inoculation of Preformed Human α-Synuclein Oligomers.

The accumulation of aggregated α-synuclein (αSyn) is a hallmark of Parkinson's disease (PD). Current evidence indicates that small soluble αSyn oligomers (αSynOs) are the most toxic species among the forms of αSyn aggregates, and that size and topological structural properties are crucial factors for αSynOs-mediated toxicity, involving the interaction with either neurons or glial cells. We previously characterized a human αSynO (H-αSynO) with specific structural properties promoting toxicity against neuronal membranes. Here, we tested the neurotoxic potential of these H-αSynOs in vivo, in relation to the neuropathological and symptomatic features of PD. The H-αSynOs were unilaterally infused into the rat substantia nigra pars compacta (SNpc). Phosphorylated αSyn (p129-αSyn), reactive microglia, and cytokine levels were measured at progressive time points. Additionally, a phagocytosis assay in vitro was performed after microglia pre-exposure to αsynOs. Dopaminergic loss, motor, and cognitive performances were assessed. H-αSynOs triggered p129-αSyn deposition in SNpc neurons and microglia and spread to the striatum. Early and persistent neuroinflammatory responses were induced in the SNpc. In vitro, H-αSynOs inhibited the phagocytic function of microglia. H-αsynOs-infused rats displayed early mitochondrial loss and abnormalities in SNpc neurons, followed by a gradual nigrostriatal dopaminergic loss, associated with motor and cognitive impairment. The intracerebral inoculation of structurally characterized H-αSynOs provides a model of progressive PD neuropathology in rats, which will be helpful for testing neuroprotective therapies.

Neuroprotection by the Immunomodulatory Drug Pomalidomide in the Drosophila LRRK2WD40 Genetic Model of Parkinson's Disease.

The search for new disease-modifying drugs for Parkinson's disease (PD) is a slow and highly expensive process, and the repurposing of drugs already approved for different medical indications is becoming a compelling alternative option for researchers. Genetic variables represent a predisposing factor to the disease and mutations in leucine-rich repeat kinase 2 (LRRK2) locus have been correlated to late-onset autosomal-dominant PD. The common fruit fly Drosophila melanogaster carrying the mutation LRRK2 loss-of-function in the WD40 domain (LRRK2WD40), is a simple in vivo model of PD and is a valid tool to first evaluate novel therapeutic approaches to the disease. Recent studies have suggested a neuroprotective activity of immunomodulatory agents in PD models. Here the immunomodulatory drug Pomalidomide (POM), a Thalidomide derivative, was examined in the Drosophila LRRK2WD40 genetic model of PD. Mutant and wild type flies received increasing POM doses (1, 0.5, 0.25 mM) through their diet from day 1 post eclosion, until postnatal day (PN) 7 or 14, when POM's actions were evaluated by quantifying changes in climbing behavior as a measure of motor performance, the number of brain dopaminergic neurons and T-bars, mitochondria integrity. LRRK2WD40 flies displayed a spontaneous age-related impairment of climbing activity, and POM significantly and dose-dependently improved climbing performance both at PN 7 and PN 14. LRRK2WD40 fly motor disability was underpinned by a progressive loss of dopaminergic neurons in posterior clusters of the protocerebrum, which are involved in the control of locomotion, by a low number of T-bars density in the presynaptic bouton active zones. POM treatment fully rescued the cell loss in all posterior clusters at PN 7 and PN 14 and significantly increased the T-bars density. Moreover, several damaged mitochondria with dilated cristae were observed in LRRK2WD40 flies treated with vehicle but not following POM. This study demonstrates the neuroprotective activity of the immunomodulatory agent POM in a genetic model of PD. POM is an FDA-approved clinically available and well-tolerated drug used for the treatment of multiple myeloma. If further validated in mammalian models of PD, POM could rapidly be clinically tested in humans.

Beneficial effects of curtailing immune susceptibility in an Alzheimer's disease model.

BACKGROUND: Currently, there are no effective therapeutic options for Alzheimer's disease, the most common, multifactorial form of dementia, characterized by anomalous amyloid accumulation in the brain. Growing evidence points to neuroinflammation as a major promoter of AD. We have previously shown that the proinflammatory cytokine TNFSF10 fuels AD neuroinflammation, and that its immunoneutralization results in improved cognition in the 3xTg-AD mouse. METHODS: Here, we hypothesize that inflammatory hallmarks of AD might parallel with central and peripheral immune response dysfunction. To verify such hypothesis, we used a triple transgenic mouse model of AD. 3xTg-AD mice were treated for 12 months with an anti-TNFSF10 antibody, and thereafter immune/inflammatory markers including COX2, iNOS, IL-1ß and TNF-α, CD3, GITR, and FoxP3 (markers of regulatory T cells) were measured in the spleen as well as in the hippocampus. RESULTS: Spleens displayed accumulation of amyloid-ß1-42 (Aß1-42), as well as high expression of Treg cell markers FoxP3 and GITR, in parallel with the increased levels of inflammatory markers COX2, iNOS, IL-1ß and TNF-α, and blunted IL-10 expression. Moreover, CD3 expression was increased in the hippocampus, consistently with FoxP3 and GITR. After chronic treatment of 3xTg-AD mice with an anti-TNFSF10 antibody, splenic FoxP3, GITR, and the above-mentioned inflammatory markers expression was restored to basal levels, while expression of IL-10 was increased. A similar picture was observed in the hippocampus. Such improvement of peripheral and CNS inflammatory/immune response was associated with decreased microglial activity in terms of TNFα production, as well as decreased expression of both amyloid and phosphorylated tau protein in the hippocampus of treated 3xTg-AD mice. Interestingly, we also reported an increased expression of both CD3 and FoxP3, in sections from human AD brain. CONCLUSIONS: We suggest that neuroinflammation in the brain of 3xTg-AD mice triggered by TNFSF10 might result in a more general overshooting of the immune response. Treatment with an anti-TNFSF10 antibody blunted inflammatory processes both in the spleen and hippocampus. These data confirm the detrimental role of TNFSF10 in neurodegeneration, and corroborate the hypothesis of the anti-TNFSF10 strategy as a potential treatment to improve outcomes in AD.

Immunomodulatory drugs alleviate l-dopa-induced dyskinesia in a rat model of Parkinson's disease.

BACKGROUND: Thalidomide and closely related analogues are used clinically for their immunomodulatory and antiangiogenic properties mediated by the inhibition of the proinflammatory cytokine tumor necrosis factor α. Neuroinflammation and angiogenesis contribute to classical neuronal mechanisms underpinning the pathophysiology of l-dopa-induced dyskinesia, a motor complication associated with l-dopa therapy in Parkinson's disease. The efficacy of thalidomide and the more potent derivative 3,6'-dithiothalidomide on dyskinesia was tested in the 6-hydroxydopamine Parkinson's disease model. METHODS: Three weeks after 6-hydroxydopamine infusion, rats received 10 days of treatment with l-dopa plus benserazide (6 mg/kg each) and thalidomide (70 mg/kg) or 3,6'-dithiothalidomide (56 mg/kg), and dyskinesia and contralateral turning were recorded daily. Rats were euthanized 1 hour after the last l-dopa injection, and levels of tumor necrosis factor-α, interleukin-10, OX-42, vimentin, and vascular endothelial growth factor immunoreactivity were measured in their striatum and substantia nigra reticulata to evaluate neuroinflammation and angiogenesis. Striatal levels of GLUR1 were measured as a l-dopa-induced postsynaptic change that is under tumor necrosis factor-α control. RESULTS: Thalidomide and 3,6'-dithiothalidomide significantly attenuated the severity of l-dopa-induced dyskinesia while not affecting contralateral turning. Moreover, both compounds inhibited the l-dopa-induced microgliosis and excessive tumor necrosis factor-α in the striatum and substantia nigra reticulata, while restoring physiological levels of the anti-inflammatory cytokine interleukin-10. l-Dopa-induced angiogenesis was inhibited in both basal ganglia nuclei, and l-dopa-induced GLUR1 overexpression in the dorsolateral striatum was restored to normal levels. CONCLUSIONS: These data suggest that decreasing tumor necrosis factor-α levels may be useful to reduce the appearance of dyskinesia, and thalidomide, and more potent derivatives may provide an effective therapeutic approach to dyskinesia. © 2019 International Parkinson and Movement Disorder Society.

Noradrenergic terminals are the primary source of α2-adrenoceptor mediated dopamine release in the medial prefrontal cortex.

In various psychiatric disorders, deficits in dopaminergic activity in the prefrontal cortex (PFC) are implicated. Treatments involving selective augmentation of dopaminergic activity in the PFC primarily depend on the inhibition of α2-adrenoreceptors singly or in combination with the inhibition of the norepinephrine transporter (NET). We aimed to clarify the relative contribution of dopamine (DA) release from noradrenergic and dopaminergic terminals to DA output induced by blockade of α2-adrenoreceptors and NET. To this end, we assessed whether central noradrenergic denervation modified catecholamine output in the medial PFC (mPFC) of rats elicited by atipamezole (an α2-adrenoreceptor antagonist), nisoxetine (an NET inhibitor), or their combination. Intraventricular administration of anti-dopamine-beta-hydroxylase-saporin (aDBH) caused a loss of DBH-positive fibers in the mPFC and almost total depletion of tissue and extracellular NE level; however, it did not reduce tissue DA level but increased extracellular DA level by 70% in the mPFC. Because noradrenergic denervation should have caused a loss of NET and reduced NE level at α2-adrenoceptors, the actual effect of an aDBH-induced lesion on DA output elicited by blockade of α2-adrenoceptors and NET was evaluated by comparing denervated and control rats following blockade of α2-adrenoceptors and NET with atipamezole and nisoxetine, respectively. In the control rats, extracellular NE and DA levels increased by approximately 150% each with 3 mg/kg atipamezole; 450% and 230%, respectively, with 3 mg/kg nisoxetine; and 2100% and 600%, respectively, with combined atipamezole and nisoxetine. In the denervated rats, consistent with the loss of NET, nisoxetine failed to modify extracellular DA level, whereas atipamezole, despite the lack of NE-induced stimulation of α2-adrenoceptors, increased extracellular DA level by approximately 30%. Overall, these results suggest that atipamezole-induced DA release mainly originated from noradrenergic terminals, possibly through the inhibition of α2-autoreceptors. Furthermore, while systemic and local administration of the α2-adrenoceptor agonist clonidine into the mPFC of the controls rats reduced extracellular NE level by 80% and 60%, respectively, and extracellular DA level by 50% and 60%, respectively, it failed to reduce DA output in the denervated rats, consistent with the loss of α2-autoreceptors. To eliminate the possibility that denervation reduced DA release potential via the effects at dopaminergic terminals in the mPFC, the effect of systemic administration of the D2-DA antagonist raclopride (0.5 mg/kg IP) on DA output was analyzed. In the control rats, raclopride was found to be ineffective when administered alone, but it increased extracellular DA level by 380% following NET inhibition with nisoxetine. In the denervated rats, as expected due to the loss of NET, raclopride-alone or with nisoxetine-increased DA release to approximately the same level as that observed in the control rats after NET inhibition. Overall, these results suggest that noradrenergic terminals in the mPFC are the primary source of DA released by blockade of α2-adrenoreceptors and NET and that α2-autoreceptors, and not α2-heteroreceptors, mediate DA output induced by α2-adrenoceptor blockade.

Dopamine Restores Limbic Memory Loss, Dendritic Spine Structure, and NMDAR-Dependent LTD in the Nucleus Accumbens of Alcohol-Withdrawn Rats.

Alcohol abuse leads to aberrant forms of emotionally salient memory, i.e., limbic memory, that promote escalated alcohol consumption and relapse. Accordingly, activity-dependent structural abnormalities are likely to contribute to synaptic dysfunctions that occur from suddenly ceasing chronic alcohol consumption. Here we show that alcohol-dependent male rats fail to perform an emotional-learning task during abstinence but recover their functioning by l-3,4-dihydroxyphenylalanin (l-DOPA) administration during early withdrawal. l-DOPA also reverses the selective loss of dendritic "long thin" spines observed in medium spiny neurons of the nucleus accumbens (NAc) shell of alcohol-dependent rats during abstinence, as well as the reduction in tyrosine hydroxylase immunostaining and postsynaptic density-95-positive elements. Patch-clamp experiments in NAc slices reveal that both in vivo systemic l-DOPA administration and in vitro exposure to dopamine can restore the loss of long-term depression (LTD) formation, counteract the reduction in NMDAR-mediated synaptic currents and rectify the altered NMDAR/AMPAR ratio observed in alcohol-withdrawn rats. Further, in vivo microdialysis experiments show that blunted dopaminergic signaling is revived after l-DOPA treatment during early withdrawal. These results suggest a key role of an efficient dopamine signaling for maintaining, and restore, neural trophism, NMDA-dependent LTD, and ultimately optimal learning.SIGNIFICANCE STATEMENT Blunted dopamine signaling and altered glutamate connectivity in the nucleus accumbens represent the neuroanatomical basis for the impairment in aversive limbic memory observed during withdrawal in alcohol dependence. Supplying l-DOPA during withdrawal re-establishes synaptic morphology and functional neuroadaptations, suggesting a complete recovery of nucleus accumbens glutamatergic synaptic plasticity when dopamine is revived. Importantly, restoring dopamine transmission allows those synapses to encode emotionally relevant information and rescue flexibility in the neuronal circuits that process limbic memory formation. Under these conditions, drugs capable of selectively boosting the dopaminergic function during the "fluid" and still responsive state of the early withdrawn maladaptive synapses may help in the treatment of alcohol addiction.

Neuroinflammation in L-DOPA-induced dyskinesia: beyond the immune function.

Neuroinflammation is a main component of Parkinson's disease (PD) neuropathology, where unremitting reactive microglia and microglia-secreted soluble molecules such as cytokines, contribute to the neurodegenerative process as part of an aberrant immune reaction. Besides, pro-inflammatory cytokines, predominantly TNF-α, play an important neuromodulatory role in the healthy and diseased brain, being involved in neurotransmitter metabolism, synaptic scaling and brain plasticity. Recent preclinical studies have evidenced an exacerbated neuroinflammatory reaction in the striatum of parkinsonian rats that developed dyskinetic responses following L-DOPA administration. These findings prompted investigation of non-neuronal mechanisms of L-DOPA-induced dyskinesia (LID) involving glial cells and glial-secreted soluble molecules. Hence, besides the classical mechanisms of LID that include abnormal corticostriatal neurotransmission and maladaptive changes in striatal medium spiny neurons (MSNs), here we review studies supporting a role of striatal neuroinflammation in the development of LID, with a focus on microglia and the pro-inflammatory cytokine TNF-α. Moreover, we discuss several mechanisms that have been involved in the development of LID, which are directly or indirectly under the control of TNF-α, and might be abnormally affected by its chronic overproduction and release by microglia in PD. It is proposed that TNF-α may contribute to the altered neuronal responses occurring in LID by targeting receptor trafficking and function in MSNs, but also dopamine synthesis in preserved dopaminergic terminals and serotonin metabolism in serotonergic neurons. Therapeutic approaches specifically targeting glial-secreted cytokines may represent a novel target for preventing or treating LID.

Boosting phagocytosis and anti-inflammatory phenotype in microglia mediates neuroprotection by PPARγ agonist MDG548 in Parkinson's disease models.

BACKGROUND AND PURPOSE: Microglial phenotype and phagocytic activity are deregulated in Parkinson's disease (PD). PPARγ agonists are neuroprotective in experimental PD, but their role in regulating microglial phenotype and phagocytosis has been poorly investigated. We addressed it by using the PPARγ agonist MDG548. EXPERIMENTAL APPROACH: Murine microglial cell line MMGT12 was stimulated with LPS and/or MDG548, and their effect on phagocytosis of fluorescent microspheres or necrotic neurons was investigated by flow cytometry. Cytokines and markers of microglia phenotype, such as mannose receptor C type 1; MRC1), Ym1 and CD68 were measured by elisa and fluorescent immunohistochemistry. Levels of Beclin-1, which plays a role in microglial phagocytosis, were measured by Western blotting. In the in vivo MPTP-probenecid (MPTPp) model of PD in mice, MDG548 was tested on motor impairment, nigral neurodegeneration, microglial activation and phenotype. KEY RESULTS: In LPS-stimulated microglia, MDG548 increased phagocytosis of both latex beads and necrotic cells, up-regulated the expression of MRC1, CD68 and to a lesser extent IL-10, while blocking the LPS-induced increase of TNF-α and iNOS. MDG548 also induced Beclin-1. Chronic MPTPp treatment in mice down-regulated MRC1 and TGF-ß and up-regulated TNF-α and IL-1ß immunoreactivity in activated CD11b-positive microglia, causing the death of nigral dopaminergic neurons. MDG548 arrested MPTPp-induced cell death, enhanced MRC1 and restored cytokine levels. CONCLUSIONS AND IMPLICATIONS: This study adds a novel mechanism for PPARγ-mediated neuroprotection in PD and suggests that increasing phagocytic activity and anti-inflammatory markers may represent an effective disease-modifying approach.

Microglial phenotypes in Parkinson's disease and animal models of the disease.

Over the last decade the important concept has emerged that microglia, similar to other tissue macrophages, assume different phenotypes and serve several effector functions, generating the theory that activated microglia can be organized by their pro-inflammatory or anti-inflammatory and repairing functions. Importantly, microglia exist in a heterogenous population and their phenotypes are not permanently polarized into two categories; they exist along a continuum where they acquire different profiles based on their local environment. In Parkinson's disease (PD), neuroinflammation and microglia activation are considered neuropathological hallmarks, however their precise role in relation to disease progression is not clear, yet represent a critical challenge in the search of disease-modifying strategies. This review will critically address current knowledge on the activation states of microglia as well as microglial phenotypes found in PD and in animal models of PD, focusing on the expression of surface molecules as well as pro-inflammatory and anti-inflammatory cytokine production during the disease process. While human studies have reported an elevation of both pro- or anti-inflammatory markers in the serum and CSF of PD patients, animal models have provided insights on dynamic changes of microglia phenotypes in relation to disease progression especially prior to the development of motor deficits. We also review recent evidence of malfunction at multiple steps of NFκB signaling that may have a causal interrelationship with pathological microglia activation in animal models of PD. Finally, we discuss the immune-modifying strategies that have been explored regarding mechanisms of chronic microglial activation.

l-DOPA-induced dyskinesia and neuroinflammation: do microglia and astrocytes play a role?

In Parkinson's disease (PD), l-DOPA therapy leads to the emergence of motor complications including l-DOPA-induced dyskinesia (LID). LID relies on a sequence of pre- and postsynaptic neuronal events, leading to abnormal corticostriatal neurotransmission and maladaptive changes in striatal projection neurons. In recent years, additional non-neuronal mechanisms have been proposed to contribute to LID. Among these mechanisms, considerable attention has been focused on l-DOPA-induced inflammatory responses. Microglia and astrocytes are the main actors in neuroinflammatory responses, and their double role at the interface between immune and neurophysiological responses is starting to be elucidated. Both microglia and astrocytes express a multitude of neurotransmitter receptors and via the release of several soluble molecules modulate synaptic function in neuronal networks. Here we review preclinical and clinical evidence of glial overactivation by l-DOPA, supporting a role of microglia and astrocytes in the development of LID. We propose that in PD, chronically and abnormally activated microglia and astrocytes lead to an aberrant neuron-glia communication, which affect synaptic activity and neuroplasticity contributing to the development of LID.

Differential induction of dyskinesia and neuroinflammation by pulsatile versus continuous l-DOPA delivery in the 6-OHDA model of Parkinson's disease.

Neuroinflammation is associated with l-DOPA treatment in Parkinson's disease (PD), suggesting a role in l-DOPA-induced dyskinesia (LID), however it is unclear whether increased inflammation is specifically related to the dyskinetic outcome of l-DOPA treatment. Diversely from oral l-DOPA, continuous intrajejunal l-DOPA infusion is associated with very low dyskinetic outcome in PD patients. We reproduced these regimens of administration in 6-OHDA-lesioned hemiparkinsonian rats, where dyskinetic responses and striatal neuroinflammation induced by chronic pulsatile (DOPAp) or continuous (DOPAc) l-DOPA were compared. Moreover, we investigated the contribution of a peripheral inflammatory challenge with lipopolysaccharide (LPS), to DOPAp-induced dyskinetic and neuroinflammatory responses. Rats 6-OHDA-infused in the medial forebrain bundle received two weeks treatment with DOPAp, DOPAc via subcutaneous osmotic minipumps, or DOPAp followed by DOPAc. l-DOPA plasma levels were measured in all experimental groups. An independent group of rats received one peripheral dose of LPS 24h before DOPAp treatment. Abnormal involuntary movements (AIMs) were evaluated as a rat model of LID. Immunoreactivity (IR) for OX-42, microglial and neuronal TNF-α, iNOS and GFAP was quantified in denervated and contralateral striatum. In addition, serum TNF-α was measured. The 6-OHDA denervation induced a mild microgliosis in the striatum two weeks after neurotoxin infusion, and increased TNF-α IR in microglia. Rats receiving the DOPAp treatment developed AIMs and displayed increased striatal OX-42, microglial TNF-α, iNOS and GFAP. Moreover, TNF-α IR was also increased in a subpopulation of striatal neurons. Conversely, DOPAc did not induce AIMs or inflammatory responses in either drug-naïve animals or rats that were previously dyskinetic when exposed to DOPAp. Serum TNF-α was not altered by any l-DOPA treatment. LPS pre-treatment increased the degree of DOPAp-induced AIMs and striatal IR for OX-42, TNF-α, iNOS and GFAP. Altogether the present findings indicate that in the 6-OHDA model, chronic l-DOPA induces striatal inflammatory responses, which however depend upon the administration regimen and the dyskinetic outcome of drug treatment. The potentiation of dyskinetic responses by LPS suggests a reciprocal causal link between neuroinflammation and LID.

α2A adrenergic receptors highly expressed in mesoprefrontal dopamine neurons.

α2 adrenoreceptors (α2-ARs) play a key role in the control of noradrenaline and dopamine release in the medial prefrontal cortex (mPFC). Here, using UV-laser microdissection-based quantitative mRNA expression in individual neurons we show that in hTH-GFP rats, a transgenic line exhibiting intense and specific fluorescence in dopaminergic (DA) neurons, α2A adrenoreceptor (α2A-AR) mRNA is expressed at high and low levels in DA cells in the ventral tegmental area (VTA) and substantia nigra compacta (SNc), respectively. Confocal microscopy fluorescence immunohistochemistry revealed that α2A-AR immunoreactivity colocalized with tyrosine hydroxylase (TH) in nearly all DA cells in the VTA and SNc, both in hTH-GFP rats and their wild-type Sprague-Dawley (SD) counterparts. α2A-AR immunoreactivity was also found in DA axonal projections to the mPFC and dorsal caudate in the hTH-GFP and in the anterogradely labeled DA axonal projections from VTA to mPFC in SD rats. Importantly, the α2A-AR immunoreactivity localized in the DA cells of VTA and in their fibers in the mPFC was much higher than that in DA cells of SNc and their fibers in dorsal caudate, respectively. The finding that α2A-ARs are highly expressed in the cell bodies and axons of mesoprefrontal dopaminergic neurons provides a morphological basis to the vast functional evidence that somatodendritic and nerve-terminal α2A-AR receptors control dopaminergic activity and dopamine release in the prefrontal cortex. This finding raises the question whether α2A-ARs might function as autoreceptors in the mesoprefrontal dopaminergic neurons, replacing the lack of D2 autoreceptors.

The "addicted" spine.

Units of dendritic branches called dendritic spines represent more than simply decorative appendages of the neuron and actively participate in integrative functions of "spinous" nerve cells thereby contributing to the general phenomenon of synaptic plasticity. In animal models of drug addiction, spines are profoundly affected by treatments with drugs of abuse and represent important sub cellular markers which interfere deeply into the physiology of the neuron thereby providing an example of the burgeoning and rapidly increasing interest in "structural plasticity". Medium Spiny Neurons (MSNs) of the Nucleus Accumbens (Nacc) show a reduced number of dendritic spines and a decrease in TH-positive terminals upon withdrawal from opiates, cannabinoids and alcohol. The reduction is localized "strictly" to second order dendritic branches where dopamine (DA)-containing terminals, impinging upon spines, make synaptic contacts. In addition, long-thin spines seems preferentially affected raising the possibility that cellular learning of these neurons may be selectively hampered. These findings suggest that dendritic spines are affected by drugs widely abused by humans and provide yet another example of drug-induced aberrant neural plasticity with marked reflections on the physiology of synapses, system structural organization, and neuronal circuitry remodeling.

Dynamic changes in pro- and anti-inflammatory cytokines in microglia after PPAR-γ agonist neuroprotective treatment in the MPTPp mouse model of progressive Parkinson's disease.

Neuroinflammatory changes play a pivotal role in the progression of Parkinson's disease (PD) pathogenesis. Recent findings have suggested that activated microglia may polarize similarly to peripheral macrophages in the central nervous system (CNS), assuming a pro-inflammatory M1 phenotype or the alternative anti-inflammatory M2 phenotype via cytokine production. A skewed M1 activation over M2 has been related to disease progression in Alzheimer disease, and modulation of microglia polarization may be a therapeutic target for neuroprotection. By using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-probenecid (MPTPp) mouse model of progressive PD, we investigated dynamic changes in the production of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1ß, and anti-inflammatory cytokines, such as transforming growth factor (TGF)-ß and IL-10, within Iba-1-positive cells in the substantia nigra compacta (SNc). In addition, to further characterize changes in the M2 phenotype, we measured CD206 in microglia. Moreover, in order to target microglia polarization, we evaluated the effect of the peroxisome-proliferator-activated receptor (PPAR)-γ agonist rosiglitazone, which has been shown to exert neuroprotective effects on nigral dopaminergic neurons in PD models, and acts as a modulator of cytokine production and phenotype in peripheral macrophages. Chronic treatment with MPTPp induced a progressive degeneration of SNc neurons. The neurotoxin treatment was associated with a gradual increase in both TNF-α and IL-1ß colocalization with Iba-1-positive cells, suggesting an increase in pro-inflammatory microglia. In contrast, TGF-ß colocalization was reduced by the neurotoxin treatment, while IL-10 was mostly unchanged. Administration of rosiglitazone during the full duration of MPTPp treatment reverted both TNF-α and IL-1ß colocalization with Iba-1 to control levels. Moreover, rosiglitazone induced an increase in TGF-ß and IL-10 colocalization compared with the MPTPp treatment. CD206 was gradually reduced by the chronic MPTPp treatment, while rosiglitazone restored control levels, suggesting that M2 anti-inflammatory microglia were stimulated and inflammatory microglia were inhibited by the neuroprotective treatment. The results show that the dopaminergic degeneration was associated with a gradual microglia polarization to the inflammatory over the anti-inflammatory phenotype in a chronic mouse model of PD. Neuroprotective treatment with rosiglitazone modulated microglia polarization, boosting the M2 over the pro-inflammatory phenotype. PPAR-γ agonists may offer a novel approach to neuroprotection, acting as disease-modifying drugs through an immunomodulatory action in the CNS.

Hampered long-term depression and thin spine loss in the nucleus accumbens of ethanol-dependent rats.

Alcoholism involves long-term cognitive deficits, including memory impairment, resulting in substantial cost to society. Neuronal refinement and stabilization are hypothesized to confer resilience to poor decision making and addictive-like behaviors, such as excessive ethanol drinking and dependence. Accordingly, structural abnormalities are likely to contribute to synaptic dysfunctions that occur from suddenly ceasing the use of alcohol after chronic ingestion. Here we show that ethanol-dependent rats display a loss of dendritic spines in medium spiny neurons of the nucleus accumbens (Nacc) shell, accompanied by a reduction of tyrosine hydroxylase immunostaining and postsynaptic density 95-positive elements. Further analysis indicates that "long thin" but not "mushroom" spines are selectively affected. In addition, patch-clamp experiments from Nacc slices reveal that long-term depression (LTD) formation is hampered, with parallel changes in field potential recordings and reductions in NMDA-mediated synaptic currents. These changes are restricted to the withdrawal phase of ethanol dependence, suggesting their relevance in the genesis of signs and/or symptoms affecting ethanol withdrawal and thus the whole addictive cycle. Overall, these results highlight the key role of dynamic alterations in dendritic spines and their presynaptic afferents in the evolution of alcohol dependence. Furthermore, they suggest that the selective loss of long thin spines together with a reduced NMDA receptor function may affect learning. Disruption of this LTD could contribute to the rigid emotional and motivational state observed in alcohol dependence.

Co-transplantation of endothelial progenitor cells and pancreatic islets to induce long-lasting normoglycemia in streptozotocin-treated diabetic rats.

Graft vascularization is a crucial step to obtain stable normoglycemia in pancreatic islet transplantation. Endothelial progenitor cells (EPCs) contribute to neoangiogenesis and to the revascularization process during ischaemic events and play a key role in the response to pancreatic islet injury. In this work we co-transplanted EPCs and islets in the portal vein of chemically-induced diabetic rats to restore islet vascularization and to improve graft survival. Syngenic islets were transplanted, either alone or with EPCs derived from green fluorescent protein (GFP) transgenic rats, into the portal vein of streptozotocin-induced diabetic rats. Blood glucose levels were monitored and intraperitoneal glucose tolerance tests were performed. Real time-PCR was carried out to evaluate the gene expression of angiogenic factors. Diabetic-induced rats showed long-lasting (6 months) normoglycemia upon co-transplantation of syngenic islets and EPCs. After 3-5 days from transplantation, hyperglycaemic levels dropped to normal values and lasted unmodified as long as they were checked. Further, glucose tolerance tests revealed the animals' ability to produce insulin on-demand as indexed by a prompt response in blood glucose clearance. Graft neovascularization was evaluated by immunohistochemistry: for the first time the measure of endothelial thickness revealed a donor-EPC-related neovascularization supporting viable islets up to six months after transplant. Our results highlight the importance of a newly formed viable vascular network together with pancreatic islets to provide de novo adequate supply in order to obtain enduring normoglycemia and prevent diabetes-related long-term health hazards.

Simultaneous Golgi-Cox and immunofluorescence using confocal microscopy.

Visualization of neuronal elements is of fundamental importance in modern neuroscience. Golgi-Cox impregnation is a widely employed method that provides detailed information about morphological characteristics of neurons, but none regarding their neurochemical features. Immunocytochemical procedures, on the other hand, can provide a high degree of biochemical specificity but poorer morphological details, in particular if compared to Golgi-Cox impregnation. Hence, the combined use of these two approaches is highly desirable, especially for confocal microscopy that can exploit the advantages of both methods simultaneously. Here we show an innovative procedure of perfusion and fixation of brain tissue, that allows, by applying Golgi-Cox impregnation and immunofluorescence in the same histological section, to obtain high-quality histological material, with a very simple and inexpensive method. This procedure is based on three simple fixation steps: (1) a paraformaldehyde perfusion followed by a standard post-fixation to stabilize the subsequent immunofluorescence reaction; (2) the classical Golgi-Cox impregnation and (3) an immunofluorescence reaction in previously impregnated material. This combination allows simultaneous visualization of (a) the structural details (Golgi-Cox impregnated neurons), (b) the antigens' characterization, (c) the anatomical interactions between discrete neuronal elements and (d) the 3D reconstruction and modeling. The method is easy to perform and can be reproducibly applied by small laboratories and expanded through the use of different antibodies. Overall, the method presented in this study offers an innovative and powerful approach to study the nervous system, especially by using confocal microscopy.