Données nouvelles sur l’innervation à dopamine du striatum et son co-phénotype glutamatergique

Une sous-population des neurones à dopamine (DA) du mésencéphale ventral du rat et de la souris étant connue pour exprimer l'ARN messager du transporteur vésiculaire 2 du glutamate (VGLUT2), nous avons eu recours à l'immunocytochimie en microscopie électronique, après simple ou double marquage de l'enzyme de synthèse tyrosine hydroxylase (TH) et de VGLUT2, pour déterminer la présence de l'une et/ou l'autre protéine dans les terminaisons (varicosités) axonales de ces neurones et caractériser leur morphologie ultrastructurale dans diverses conditions expérimentales. Dans un premier temps, des rats jeunes (P15) ou adultes (P90), ainsi que des rats des deux âges soumis à l'administration intraventriculaire cérébrale de la cytotoxine 6-hydroxydopamine (6-OHDA) dans les jours suivant la naissance, ont été examinés, afin d'étayer l'hypothèse d'un rôle de VGLUT2 au sein des neurones DA, au cours du développement normal ou pathologique de ces neurones. Chez le jeune rat, ces études ont montré: i) la présence de VGLUT2 dans une fraction importante des varicosités axonales TH immunoréactives du coeur du noyau accumbens ainsi que du néostriatum; ii) une augmentation de la proportion de ces terminaisons doublement marquées dans le noyau accumbens par suite de la lésion 6-OHDA néonatale; iii) le double marquage fréquent des varicosités axonales appartenant à l'innervation DA aberrante (néoinnervation), qui se développe dans la substance noire, par suite de la lésion 6-OHDA néonatale. Des différences significatives ont aussi été notées quant à la dimension des terminaisons axonales marquées pour la TH seulement, VGLUT2 seulement ou TH et VGLUT2. Enfin, à cet âge (P15), toutes les terminaisons doublement marquées sont apparues dotées d'une spécialisation membranaire synaptique, contrairement aux terminaisons marquées pour la TH ou pour VGLUT2 seulement. Dans un deuxième temps, nous avons voulu déterminer le devenir du double phénotype chez le rat adulte (P90) soumis ou non à la lésion 6-OHDA néonatale. Contrairement aux observations recueillies chez le jeune rat, nous avons alors constaté: i) l'absence complète de terminaisons doublement marquées dans le coeur du noyau accumbens et le néostriatum d'animaux intacts, de même que dans les restes de la substance noire des animaux 6-OHDA lésés; ii) une très forte baisse de leurnombre dans le coeur du noyau accumbens des animaux 6-OHDA lésés. Ces observations, suggérant une régression du double phénotype TH/VGLUT2 avec l'âge, sont venues renforcer l'hypothèse d'un rôle particulier d'une co-libération de glutamate par les neurones mésencéphaliques DA au cours du développement. Dans ces conditions, il est apparu des plus intéressants d'examiner l'innervation DA méso-striatale chez deux lignées de souris dont le gène Vglut2 avait été sélectivement invalidé dans les neurones DA du cerveau, ainsi que leurs témoins et des souris sauvages. D'autant que malgré l'utilisation croissante de la souris en neurobiologie, cette innervation DA n'avait jamais fait l'objet d’une caractérisation systématique en microscopie électronique. En raison de possibles différences entre le coeur et la coque du noyau accumbens, l'étude a donc porté sur les deux parties de ce noyau ainsi que le néostriatum et des souris jeunes (P15) et adultes (P70-90) de chaque lignée, préparées pour l'immunocytochimie de la TH, mais aussi pour le double marquage TH et VGLUT2, selon le protocole précédemment utilisé chez le rat. Les résultats ont surpris. Aux deux âges et quel que soit le génotype, les terminaisons axonales TH immunoréactives des trois régions sont apparues comparables quant à leur taille, leur contenu vésiculaire, le pourcentage contenant une mitochondrie et une très faible incidence synaptique (5% des varicosités, en moyenne). Ainsi, chez la souris, la régression du double phénotype pourrait être encore plus précoce que chez le rat, à moins que les deux protéines ne soient très tôt ségréguées dans des varicosités axonales distinctes des mêmes neurones DA. Ces données renforcent aussi l’hypothèse d’une transmission diffuse (volumique) et d’un niveau ambiant de DA comme élément déterminant du fonctionnement du système mésostriatal DA chez la souris comme chez le rat.

Differential regulation of FGF-2 in neurons and reactive astrocytes of axotomized rat hypoglossal nucleus. A possible therapeutic target for neuroprotection in peripheral nerve pathology

Despite the favorable treatment of cranial nerve neuropathology in adulthood, some cases are resistant to therapy leading to permanent functional impairments In many cases, suitable treatment is problematic as the therapeutic target remains unknown Basic fibroblast growth factor (bFGF, FGF 2) is involved in neuronal maintenance and wound repair following nervous system lesions It is one of few neurotrophic molecules acting in autocrine, paracrine and intracrine fashions depending upon specific circumstances Peripheral cranial somatic motor neurons, i e hypoglossal (XII) neurons, may offer a unique opportunity to study cellular FGF 2 mechanisms as the molecule is present in the cytoplasm of neurons and in the nuclei of astrocytes of the central nervous system FGF-2 may trigger differential actions during development, maintenance and lesion of XII neurons because axotomy of those cells leads to cell death during neonatal ages, but not in adult life Moreover, the modulatory effects of astroglial FGF 2 and the Ca+2 binding protein S100 beta have been postulated in paracrine mechanisms after neuronal lesions In our study, adult Wistar rats received a unilateral crush or transection (with amputation of stumps) of XII nerve, and were sacrificed after 72 h or 11 days Brains were processed for immunohistochemical localization of neurofilaments (NF), with or without counterstaining for Nissl substance, ghat fibrillary acidic protein (GFAP, as a marker of astrocytes), S100 beta and FGF-2 The number of Nissl positive neurons of axotomized XII nucleus did not differ from controls The NF immunoreactivity increased in the perikarya and decreased in the neuropil of axotomized XII neurons 11 days after nerve crush or transection An astrocytic reaction was seen in the ipsilateral XII nucleus of the crushed or transected animals 72 h and 11 days after the surgery The nerve lesions did not change the number of FGF-2 neurons in the ipsilateral XII nucleus, however, the nerve transection increased the number of FGF-2 ghat profiles by 72 h and 11 days Microdensitometric image analysis revealed a short lasting decrease in the intensity of FGF 2 immunoreactivity in axotomized XII neurons by 72 h after nerve crush or transection and also an elevation of FGF-2 in the ipsilateral of ghat nuclei by 72h and 11 days after the two lesions S100 beta decreased in astrocytes of 11-day transected XII nucleus The two-color immunoperoxidase for the simultaneous detection of the GFAP/FGF-2 indicated FGF-2 upregulation in the nuclei of reactive astrocytes of the lesioned XII nucleus Astroglial FGF-2 may exert paracrine trophic actions in mature axotomized XII neurons and might represent a therapeutic target for neuroprotection in peripheral nerve pathology (C) 2009 Elsevier GmbH All rights reserved

Hemiparkinsonian rats rotate toward the side with the weaker dopaminergic neurotransmission

Rats with unilateral lesion of the substantia nigra pars compacta (SNpc) have been used as a model of Parkinson`s disease. Depending on the lesion protocol and on the drug challenge, these rats rotate in opposite directions. The aim of the present study was to propose a model to explain how critical factors determine the direction of these turns. Unilateral lesion of the SNpc was induced with 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Separate analysis showed that neither the type of neurotoxin nor the site of lesion along the nigrostriatal. pathway was able to predict the direction of the turns these rats made after they were challenged with apomorphine. However, the combination of these two factors determined the magnitude of the lesion estimated by tyrosine-hydroxylase immunohistochemistry and HPLC-ED measurement of striatal dopamine. Very small lesions did Dot cause turns, medium-size lesions caused ipsiversive turns, and large lesions caused contraversive turns. Large-size SNpc lesions resulted in an increased binding of [H-3] raclopride to D2 receptors, while medium-size lesions reduced the binding of [H-3]SCH-23390 D1 receptors in the ipsilateral striatum. These results are coherent with the model proposing that after challenged with a dopamine receptor agonist, unilaterally SNpc-lesioned rats rotate toward the side with the weaker activation of dopamine receptors. This activation is weaker on the lesioned side in animals with small SNpc lesions due to the loss of dopamine, but stronger in animals with large lesions due to dopamine receptor supersensitivity. (C) 2008 Elsevier B.V. All rights reserved.

Role of Locus coeruleus noradrenergic neurons in cardiorespiratory and thermal control during hypoxia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudo dos efeitos comportamentais do neuropeptídeos em camundongos submetidos a modelos animais de Parkinson

Neuropeptide S (NPS) is the endogenous ligand of a G-protein coupled receptor. Preclinical studies have shown that NPSR receptor activation can promote arousal, anxiolytic-like behavioral, decrease in food intake, besides hyperlocomotion, which is a robust but not well understood phenomenon. Previous findings suggest that dopamine transmission plays a crucial role in NPS hyperactivity. Considering the close relationship between dopamine and Parkinson Disease (PD), and also that NPSR receptors are expressed on dopaminergic nuclei in the brain, the current study attempted to investigate the effects of NPS in motor deficits induced by intracerebroventricular (icv) administration of 6-OHDA and systemic administration of haloperidol. Motor deficits induced by 6-OHDA and haloperidol were evaluated on Swiss mice in the rota-rod and catalepsy test. Time on the rotating rod and time spent immobile in the elevated bar were measured respectively in each test. L-Dopa, a classic antiparkinsonian drug, and NPS were administrated in mice submitted to one of the animal models of PD related above. 6-OHDA injection evoked severe motor impairments in rota-rod test, while the cataleptic behavior of 6-OHDA injected mice was largely variable. The administration of L-Dopa (25 mg/kg) and NPS (0,1 and 1 nmol) reversed motor impairments induced by 6-OHDA in the rota-rod. Haloperidolinduced motor deficits on rota-rod and catalepsy tests which were reversed by L-Dopa (100 e 400 mg/kg), but not by NPS (0,1 and 1 nmol) administration. The association of L-Dopa 10 mg/kg and NPS 1 nmol was also unable to counteract haloperidol-induced motor deficits. To summarize, 6-OHDA-, but not haloperidol-, induced motor deficits were reversed by the central administration of NPS. These data suggest that NPS possibly facilitates dopamine release in basal ganglia, what would explain the overcome of motor performance promoted by NPS administration in animals pretreated with 6-OHDA, but not haloperidol. Finally, the presented findings point, for the first time, to the potential of NPSR agonist as an innovative treatment for PD.

Locus coeruleus noradrenergic neurons and CO2 drive to breathing

The Locus coeruleus (LC) has been suggested as a CO2 chemoreceptor site in mammals. In the present study, we assessed the role of LC noradrenergic neurons in the cardiorespiratory and thermal responses to hypercapnia. To selectively destroy LC noradrenergic neurons, we administered 6-hydroxydopamine (6-OHDA) bilaterally into the LC of male Wistar rats. Control animals had vehicle (ascorbic acid) injected (sham group) into the LC. Pulmonary ventilation (plethysmograph), mean arterial pressure (MAP), heart rate (HR), and body core temperature (T-c, data loggers) were measured followed by 60 min of hypercapnic exposure (7% CO2 in air). To verify the correct placement and effectiveness of the chemical lesions, tyrosine hydroxylase immunoreactivity was performed. Hypercapnia caused an increase in pulmonary ventilation in all groups, which resulted from increases in respiratory frequency and tidal volume (V-T) in sham-operated and 6-OHDA-lesioned groups. The hypercapnic ventilatory response was significantly decreased in 6-OHDA-lesioned rats compared with sham group. This difference was due to a decreased V-T in 6-OHDA rats. LC chemical lesion or hypercapnia did not affect MAP, HR, and T-c. Thus, we conclude that LC noradrenergic neurons modulate hypercapnic ventilatory response but play no role in cardiovascular and thermal regulation under resting conditions.

Effects of lithium and myoinositol on the rat bisected vas deferens

1. The effects of lithium (Li+) on the concentration-response curves (CRC) to norepinephrine (NE) and acetylcholine (Ach) on the bisected rat vas deferens (RVD) were investigated, as well as its action on the neuronal uptake of [H-3] NE.2. Li+ did not affect the 50% effective concentration (EC(50)) of NE and Ach in the epididymal (EP) portion of the RVD.3. Li+ caused a significant increase of the EC(50) to NE and Ach in the prostatic (PP) portion of the RVD. This shift to the right of the CRC to NE was prevented by the presence of myoinositol.4. Incubation of the PP of the RVD with Li+, increased the neuronal uptake of NE. The simultaneous incubation with myoinositol prevented this increase.5. After the pre-treatment of the rats with 6-hydroxydopamine (6-OHDA), or in the presence of cocaine, Li+ failed to desensitize the PP of the RVD to NE.6. These results suggest that the effect of Li+ on the PP of the RVD occurs mainly at the pre-synaptic level and may be related to the increase of neuronal uptake and to the interference of Li+ on phosphatidylinositol hydrolysis.

Análise comportamental e histológica de um modelo animal da doença de Parkinson em camundongos suíços

A Doença de Parkinson (DP) é uma das doenças neurodegenerativas mais comuns relacionadas com a idade, e apresenta sintomatologia com alterações motoras clássicas que estão relacionadas com a degeneração dos neurônios dopaminérgicos da SNpc e a diminuição de dopamina no estriado. Modelos animais da DP são instrumentos importantes utilizados por pesquisadores para uma maior compreensão de mecanismos patológicos envolvidos na doença e para a avaliação de possíveis intervenções terapêuticas. Tais modelos devem mimetizar algum aspecto da doença, como a degeneração dos neurônios dopaminérgicos nigrais. Neste contexto, o modelo da DP induzido pela injeção da neurotoxina 6- hidroxidopamina (6-OHDA) já se encontra bem estabelecido em ratos, mas necessita ainda de melhor caracterização das alterações comportamentais e lesões no sistema nigro-estriatal em camundongos de diferentes linhagens a fim de que haja interpretações confiáveis quando o modelo for usado em testes terapêuticos. O presente estudo teve como objetivo melhorar a caracterização do modelo unilateral da DP com 6-OHDA em camundongos suíços, avaliando alterações comportamentais e o efeito sobre os neurônios dopaminérgicos da SNpc. Nesta investigação utilizou-se uma única injeção intraestriatal unilateral de 6-OHDA, em duas diferentes concentrações da toxina: 5µg/µl e 10µg/µl. Os nossos resultados mostraram que ambas as concentrações utilizadas causaram perda severa de neurônios dopaminérgicos na SNpc, com uma média de 74,5% e 89,5% de per da, respectivamente. Esta perda apresentou uma correlação alta com o comportamento rotatório induzido por apomorfina e uma correlação baixa com a ambulação no teste do campo aberto. Desta forma, injeções intraestriatais de 5µg/µl ou 10µg/µl de 6-OHDA, em camundongos suíços, reproduzem de forma efetiva o modelo animal unilateral da DP com 6-OHDA, podendo ser utilizadas de forma confiável em experimentos que visem a investigação de terapias farmacológicas, celulares e/ou de neuroproteção para a DP.

Caracterização dos efeitos do extrato de folhas de Swietenia macrophylla em modelo experimental de doença de Parkinson

Estudos prévios indicam que o extrato de folhas de mogno Swietenia macrophylla possui composição química rica em substâncias antioxidantes com efeito neuroprotetor em cultura. Um dos principais mecanismos envolvidos na neurodegeração da Doença de Parkinson (DP) é o estresse oxidativo. Portanto, substâncias antioxidantes são candidatas potenciais para terapias que retardem o processo neurodegenerativo da doença. Este estudo tem por objetivo caracterizar os efeitos do extrato de folhas de mogno frente à degeneração nigroestriatal e alterações comportamentais de camundongos expostos a uma única injeção intraestriatal de 6-OHDA unilateralmente. Foram utilizados camundongos machos, os quais foram submetidos à cirurgia estereotáxica para a injeção de 20 μg de 6-OHDA no estriado esquerdo. Os animais foram subdivididos em 4 grupos, de acordo com a dose de extrato de mogno administrada. O extrato foi aplicado por via intraperitoneal nos 7 primeiros dias após a injeção de 6-OHDA nas doses de 0,0 (controle), 0,5 (G1), 1,0 (G2) e 5,0 mg/kg (G3). O grupo controle (GC) recebeu injeções de salina a 0,9% (veículo). Foi feita análise da ambulação no campo aberto antes, no 7º e no 21º dias e do número de rotações induzidas por apomorfina no 7º e no 21º dias após a cirurgia. Avaliação da neurodegeneração foi realizada através da contagem de neurônios dopaminérgicos TH+ na substância negra por estereologia. Como resultado, encontrou-se diferença estatisticamente significativa no 21º dia, onde os grupos G2 e G3 apresentaram redução no comportamento ambulatório em relação aos grupos G1 e GC; este dois últimos tiveram comportamento ambulatório equivalente entre si. Em relação às rotações induzidas por apomorfina, no 21º dia, o G1 apresentou média de rotações significativamente menor do que os grupos GC, G2 e G3. Na contagem de células, G1 apresentou diminuição na perda dos neurônios dopaminérgicos estatisticamente significativa em relação ao controle. Assim, concluímos que o extrato de mogno na concentração de 0,5 mg/kg promoveu neuroproteção na neurodegeneração do sistema nigroestriatal induzida por 6-OHDA.

Neuroprotective strategies against neurodegeneration in cellular model systems

In the present study we analyzed new neuroprotective therapeutical strategies in PD (Parkinson’s disease) and AD (Alzheimer’s disease). Current therapeutic strategies for treating PD and AD offer mainly transient symptomatic relief but it is still impossible to block the loss of neuron and then the progression of PD and AD. There is considerable consensus that the increased production and/or aggregation of α- synuclein (α-syn) and β-amyloid peptide (Aβ), plays a central role in the pathogenesis of PD, related synucleinopathies and AD. Therefore, we identified antiamyloidogenic compounds and we tested their effect as neuroprotective drug-like molecules against α-syn and β-amyloid cytotoxicity in PC12. Herein, we show that two nitro-catechol compounds (entacapone and tolcapone) and 5 cathecol-containing compounds (dopamine, pyrogallol, gallic acid, caffeic acid and quercetin) with antioxidant and anti-inflammatory properties, are potent inhibitors of α-syn and β-amyloid oligomerization and fibrillization. Subsequently, we show that the inhibition of α-syn and β-amyloid oligomerization and fibrillization is correlated with the neuroprotection of these compounds against the α-syn and β-amyloid-induced cytotoxicity in PC12. Finally, we focused on the study of the neuroprotective role of microglia and on the possibility that the neuroprotection properties of these cells could be use as therapeutical strategy in PD and AD. Here, we have used an in vitro model to demonstrate neuroprotection of a 48 h-microglial conditioned medium (MCM) towards cerebellar granule neurons (CGNs) challenged with the neurotoxin 6-hydroxydopamine (6-OHDA), which induces a Parkinson-like neurodegeneration, with Aβ42, which induces a Alzheimer-like neurodegeneration, and glutamate, involved in the major neurodegenerative diseases. We show that MCM nearly completely protects CGNs from 6-OHDA neurotoxicity, partially from glutamate excitotoxicity but not from Aβ42 toxin.

Molecular approach to Neurodegenerative Disorders: Role of Nociceptin/Orphanin FQ - NOP System in Parkinson and Epigenetic Mechanism in Alzheimer's Disease

With life expectancies increasing around the world, populations are getting age and neurodegenerative diseases have become a global issue. For this reason we have focused our attention on the two most important neurodegenerative diseases: Parkinson’s and Alzheimer’s. Parkinson’s disease is a chronic progressive neurodegenerative movement disorder of multi-factorial origin. Environmental toxins as well as agricultural chemicals have been associated with PD. Has been observed that N/OFQ contributes to both neurotoxicity and symptoms associated with PD and that pronociceptin gene expression is up-regulated in rat SN of 6-OHDA and MPP induced experimental parkinsonism. First, we investigated the role of N/OFQ-NOP system in the pathogenesis of PD in an animal model developed using PQ and/or MB. Then we studied Alzheimer's disease. This disorder is defined as a progressive neurologic disease of the brain leading to the irreversible loss of neurons and the loss of intellectual abilities, including memory and reasoning, which become severe enough to impede social or occupational functioning. Effective biomarker tests could prevent such devastating damage occurring. We utilized the peripheral blood cells of AD discordant monozygotic twin in the search of peripheral markers which could reflect the pathology within the brain, and also support the hypothesis that PBMC might be a useful model of epigenetic gene regulation in the brain. We investigated the mRNA levels in several genes involve in AD pathogenesis, as well DNA methylation by MSP Real-Time PCR. Finally by Western Blotting we assess the immunoreactivity levels for histone modifications. Our results support the idea that epigenetic changes assessed in PBMCs can also be useful in neurodegenerative disorders, like AD and PD, enabling identification of new biomarkers in order to develop early diagnostic programs.

Microglial involvement in brain physiopathology: in vitro studies using rat primary cultures

Microglial involvement in neurological disorders is well-established, being microglial activation not only associated with neurotoxic consequences, but also with neuroprotective effects. The studies presented here, based on microglia rat primary cell cultures and mainly on microglial conditioned medium (MCM), show insights into the mechanism of Superoxide dismutase 1 (SOD1) and Apolipoprotein E (ApoE) secretion by microglia as well as their neuroprotective effect towards primary cerebellar granule neurons (CGNs) exposed to the dopaminergic toxin 6-hydroxydopamine (6-OHDA). SOD1 and ApoE are released respectively through non-classical lysosomal or the classical ER/Golgi-mediated secretion pathway. Microglial conditioned medium, in which SOD1 and ApoE accumulated, protected CGNs from degeneration and these effects were replicated when exogenous SOD1 or ApoE was added to a non-conditioned medium. SOD1 neuroprotective action was mediated by increased cell calcium from an external source. ApoE release is negatively affected by microglia activation, both with lipopolysaccharide (LPS) and Benzoylbenzoyl-ATP (Bz-ATP) but is stimulated by neuronal-conditioned medium as well as in microglia-neurons co-culture conditions. This neuronal-stimulated microglial ApoE release is differently regulated by activation states (i.e. LPS vs ATP) and by 6-hydroxydopamine-induced neurodegeneration. In co-culture conditions, microglial ApoE release is essential for neuroprotection, since microglial ApoE silencing through siRNA abrogated protection of cerebellar granule neurons against 6-OHDA toxicity. Therefore, these molecules could represent a target for manipulation aimed at promoting neuroprotection in brain diseases. Considering a pathological context, and the microglial ability to adopt a neuroprotective or neurotoxic profile, we characterize the microglial M1/M2 phenotype in transgenic rats (McGill-R-Thy1-APP) which reproduce extensively the Alzheimer’s-like amyloid pathology. Here, for the first time, cortical, hippocampal and cerebellar microglia of wild type and transgenic adult rats were compared, at both early and advanced stages of the pathology. In view of possible therapeutic translations, these findings are relevant to test microglial neuroprotection, in animal models of neurodegenerative diseases.

Neurodegenerative diseases: molecular mechanisms and new strategies for neuroprotection

The term neurodegeneration defines numerous conditions that modify neuron’s normal functions in the human brain where is possible to observe a progressive and consistent neuronal loss. The mechanisms involved in neurodegenerative chronic and acute diseases evolution are not completely understood yet, however they share common characteristics such as misfolded proteins, oxidative stress, inflammation, excitotoxicity, and neuronal loss. Many studies have shown the frequency to develop neurodegenerative chronic diseases several years after an acute brain injury. In addition, many patients show, after a traumatic brain injury, motor and cognitive manifestations that are close to which are observed in neurodegenerative chronic patients. For this reason it is evident how is fundamental the concept of neuroprotection as a way to modulate the neurodegenerative processes evolution. Neuroinflammation, oxidative stress and the apoptotic process may be functional targets where operate to this end. Taking into account these considerations, the aim of the present study is to identify potential common pathogenetic pathways in neurodegenerative diseases using an integrated approach of preclinical studies. The goal is to delineate therapeutic strategies for the prevention of neuroinflammation, neurodegeneration and dysfunctions associated to Parkinson’s disease (PD) and cerebral ischemia. In the present study we used a murine model of PD treated with an isothiocyanate, 6-MSITC, able to quench ROS formation, restore the antioxidant GSH system, slow down the apoptotic neuronal death and counteract motor dysfunction induced by 6-OHDA. In the second study we utilized a transgenic mouse model knockout for CD36 receptor to investigate the inflammation involvement in a long term study of MCAo, which shows a better outcome after the damage induced. In conclusion, results in this study allow underlying the connection among these pathologies, and the importance of a neuroprotective strategy able to restore neurons activity where current drugs therapies have shown palliative but not healing abilities.

ROLE OF DOPAMINE OF NUCLEUS ACCUMBENS IN BEHAVIORAL SENSITIZATION TO METHYLPHENIDATE

Behavioral sensitization is defined as the subsequent augmentation of the locomotor response to a drug following repeated administrations of the drug. It is believed to occur due to alterations in the motive circuit in the brain by stressors, central nervous system stimulants, and similar stimuli. The motive circuit (or mesocorticolimbic system) consists of several interconnected nuclei that determine the behavioral response to significant biological stimuli. A final target of the mesocorticolimbic system is the nucleus accumbens (NAc), which is a key structure linking motivation and action. In particular, the dopaminergic innervations of the Nac are considered to be essential in regulating motivated states of behavior such as goal-directed actions, stimulus-reward associations and reinforcement by addictive substances. Therefore, the objective of this study was to investigate the role of dopaminergic afferents of the NAc in the behavioral sensitization elicited by chronic treatment with methylphenidate (MPD), a psychostimulant that is widely used to treat attention deficit hyperactivity disorder. The dopaminergic afferents can be selectively destroyed using catecholamine neurotoxin 6-hydroxydopamine (6-OHDA). In order to determine whether destruction of dopaminergic afferents of the NAc prevents sensitization, I compared locomotor activity in rats that had received infusions of 6-hydroxydopamine (6-OHDA) into the NAc with that of control and sham-operated animals. All groups of rats received six days of single daily MPD injections after measuring their pre and post surgery locomotor baseline. Following the consecutive MPD injections, there was a washout period of 4 days, where no injections were given. Then, a rechallenge injection of MPD was given. Behavioral responses after repeated MPD were compared to those after acute MPD to assess behavioral sensitization. Expression of sensitization to MPD was not prevented by 6-OHDA infusion into the NAc. Moreover, two distinct responses were seen to the acute injection of MPD: one group of rats had essentially no response to acute MPD, while the other had an augmented (‘sensitized’-like) acute response. Among rats with 6-OHDA infusions, the animals with diminished acute response to MPD had intact behavioral sensitization to repeated MPD, while the animals with increased acute response to MPD did not exhibit further sensitization to it. This suggests that the acute and chronic effects of MPD have distinct underlying neural circuitries.

A pharmacological investigation of noradrenergic receptor mechanisms in neophobia

The dorsal noradrenergic bundle (DB) is a major ascending pathway which originates in the locus coeruleus of the brainstem and projects to the forebrain. The behavioral role of the DB remains unclear, despite a great deal of effort. Selective attention and anxiety are two areas which have been the focus of recent research. Some studies of the DB utilize the neurotoxin 6-hydroxydopamine (6-OHDA), since 6-OHDA injection into this pathway results in greater than 90 percent depletion of cortical and hippocampal norepinephrine (NE). Neophobia, the fear of novelty, has been reported to be either increased or decreased by 6-OHDA lesions of the DB, depending on conditions. The selective attention hypothesis would be supported by increased neophobia after 6-OHDA lesions, while the anxiety hypothesis would be supported by decreased neophobia. We have examined the effects of 6-OHDA DB lesions on neophobia under conditions in which the test environment and/or the test food were novel. We found that the lesion attenuates neophobia, defined as an increased preference for novel food, when both the environment and food were novel. The lesion had no effect on neophobia when only the environment or food was novel.^ We examined the effects of chronic intraventricular NE infusions on behavior in our neophobia test, in sham and 6-OHDA DB lesioned animals. We found that chronic NE infusions into lesioned animals significantly reversed the lesion-induced attenuation of neophobia. Sham/NE infused animals demonstrated a 40 percent greater preference for familiar food compared to sham/saline infused animals. These data suggest that infusions of NE have an effect opposite to lesion-induced attenuation of neophobia. Chronic infusions of the alpha adrenoceptor agonists had no consistent effects on neophobia. The beta adrenoceptor agonist, isoproterenol reversed the lesion-induced attenuation of neophobia but not to a statistically significant degree. Isoproterenol increased neophobia in sham animals. Forskolin, an adenylate cyclase activator, mimicked the effects of NE infusion by significantly reversing the lesion-induced attenuation of neophobia, while increasing neophobia in sham animals. These results suggest that increased release of NE during stress increases neophobia in part by stimulating beta adrenoceptors which activate adenylate cyclase. ^