Chronic pain induced by nerve damage due to trauma or invasion of cancer to the bone elicits severe ongoing pain as well as hyperalgesia and allodynia likely reflecting adaptive changes within central circuits that amplify nociceptive signals. The present study explored the possible contribution of the mesolimbic dopaminergic circuit in promoting allodynia related to neuropathic and cancer pain. Mice with ligation of the sciatic nerve or treated with intrafemoral osteosarcoma cells showed allodynia to a thermal stimulus applied to the paw on the injured side. Patch clamp electrophysiology revealed that the intrinsic neuronal excitability of ventral tegmental area (VTA) dopamine neurons projecting to the nucleus accumbens (N.Acc.) was significantly reduced in those mice. We used tyrosine hydroxylase (TH)-cre mice that were microinjected with adeno-associated virus (AAV) to express channelrhodopsin-2 (ChR2) to allow optogenetic stimulation of VTA dopaminergic neurons in the VTA or in their N.Acc. terminals. Optogenetic activation of these cells produced a significant but transient anti-allodynic effect in nerve injured or tumor-bearing mice without increasing response thresholds to thermal stimulation in sham-operated animals. Suppressed activity of mesolimbic dopaminergic neurons is likely to contribute to decreased inhibition of N.Acc. output neurons and to neuropathic or cancer pain-induced allodynia suggesting strategies for modulation of pathological pain states.

The present experiments investigated the effects of different antidepressant treatments on dopamine (DA) transmission by employing in vivo microdialysis in the nucleus accumbens (NAC) and the striatum of freely moving rats. The treatments were: a) the tricyclic antidepressant desipramine (DMI), b) the novel antidepressant drug bupropion, and c) electrically induced seizures (ECS). The following results were obtained: 1) Neither acute (5 mg/kg), nor chronic (5 mg/kg, b.i.d. X 21) DMI influenced basal interstitial concentrations of DA in the NAC or the striatum. Chronic DMI did not influence apomorphine (25 μg/kg, s.c.)-induced decreases in extracellular DA in the NAC. In contrast, d-amphetamine (1.5 mg/kg, s.c.)-induced increases in extracellular DA were significantly enhanced in the NAC (not in striatum) of the chronic DMI group. d-Amphetamine-induced hypermotility was also enhanced in the chronic DMI group. 2) Bupropion (10, 25 and 100 mg/kg, i.p.) increased extracellular striatal DA concentrations in a dose-, time-, and action potential-dependent manner. Bupropion produced similar responses in the NAC. The in vivo neurochemical effects of bupropion were compared with the effects of other DA uptake inhibitors such as d-amphetamine, GBR 12909, cocaine, nomifensine, methylphenidate, and benztropine by direct administration of the drugs to the striatum via the perfusion fluid in increasing concentrations (1 to 1000 μM). The rank order of potency of these drugs as determined by the increases in extracellular DA produced by 10 or 100 μM (following correction for dialysis efficiency of the test compounds in vitro) was: GBR 12909> benztropine> amphetamine= nomifensine= methylphenidate> cocaine> bupropion. Simultaneous in vivo microdialysis in the NAC and striatum was employed to investigate the effects of chronic (10 mg/kg, b.i.d. X 21) bupropion treatment on bupropion (25 mg/kg, i.p.)-induced increases in extracellular DA concentrations. The effect of the challenge bupropion injection was significantly enhanced in the NAC (not in striatum) of the chronic bupropion group. Bupropion-induced hyperlocomotion was also enhanced in the chronic bupropion group. 3) Following a single ECS (150 V, 0.75 sec) interstitial concentrations of DA in the NAC and striatum increased sharply to 130% and 300%, respectively. The ECS-induced DA increase in the striatum was Ca⁺⁺-sensitive, partially TTX-independent, and was not influenced by barbiturate-induced anaesthesia. Seizure activity induced by flurothyl did not influence dialysate DA concentrations from the striatum, but increased dialysate DA from the NAC to 150%. These results suggest that the ECS-induced DA release in the striatum (not in the NAC) is related to the passage of current and not to the seizure activity. A course of ECS (8 treatments, one every second day) did not influence basal extracellular DA concentrations in the striatum or the NAC, while it significantly increased the DA metabolites in the striatum. Chronic ECS did not influence apomorphine (25 μg/kg, s.c.)-induced decreases in extracellular DA in the NAC. d-Amphetamine (1.5 mg/kg s.c.)-induced increases in extracellular DA were significantly enhanced in the NAC of the chronic ECS group. d-Amphetamine-induced hypermotility was also enhanced in the chronic ECS group. These results provide in vivo neurochemical confirmation that chronically administered DMI or ECS do not produce DA autoreceptor subsensitivity. They also demonstrate that chronic DMI- or chronic ECS-induced increases in the locomotor stimulant effects of d-amphetamine are accompanied by a potentiation of its effects on interstitial DA concentrations in the NAC. Moreover, these results demonstrate that chronic bupropion-induced behavioral sensitization is accompanied by a selective potentiation of its effects on interstitial DA concentrations in the NAC. Taken together, the present data provide direct neurochemical evidence that these antidepressant treatments can increase the functional output of the meso-accumbens dopaminergic system. / Medicine, Faculty of / Graduate

Chemical-induced models have revealed the crucial role of oxidative stress and mito-chondrial dysfunction in the development of Parkinson’s Disease. In this project, firstly, we in-vestigated the mechanism of action of rotenone, a commercialized pesticide that was previously described to reproduce the bradykinetic dopaminergic neurodegeneration symptoms of Parkin-son’s Disease in zebrafish by inhibition of the mitochondrial complex I. We found out that rote-none caused change in the morphology of the zebrafish dopaminergic mitochondrial network. We also observed the altered expression of various genes involves in mitochondrial fusion and fission in response to rotenone exposure. Secondly, to develop the use of adult zebrafish as a toxin-based model for Parkinson’s Disease, we sought to minimize any off-target effects by exposure of rotenone specifically to the brain. We demonstrated that microinjection of rotenone into the forebrain ventricular zone of adult zebrafish decreases the number of dopaminergic neurons. However, behavioural tests suggested that did not translate into locomotor impairment in these fish. Taken together, these results gave us more information about the potential use of zebrafish to study the physiological mechanism leading to dopaminergic degeneration and allow for the development of therapeutic strategies for Parkinson’s Disease.

Lactate has been long thought of as a dead-end waste product of glycolysis. In the brain, recent evidence has revealed a key role of L-lactate creating a paradigm-shift in our understanding of the neuronal energy metabolism. The Astrocyte neuron lactate shuttle (ANLS) model, has shown L-Lactate as the main energy substrate delivered by astrocytes to neurons to sustain neuronal oxidative metabolism. This metabolic coupling is an essential mechanism for long-term memory formation. Experimental evidence indicates that the role of lactate in cognitive function is not limited to being a neuronal metabolic substrate, but rather it is also an important signaling molecule for synaptic plasticity. One of the new emerging roles of lactate is its effect on gene expression levels; however, our current understanding of the mechanism of lactate effect on gene expression is rudimentary. Here, I investigate the role of lactate as an epigenetic modulator in neuronal cultures. First, I explored the effect of lactate on the transcriptome and methylome of the neuronal cells using primary neuronal cell culture models. Our results reveal a significant role for lactate in inducing neuronal cell differentiation. Following, I characterized a neuroblastoma cell line as our neuronal differentiation cell model and assessed its metabolic features relative to other immortal cell lines. Further, using the cell line in vitro model, I looked into the metabolic reprograming that occurs in parallel with the first indications of differentiation, focusing on lactate production rates. Subsequently, I investigated the role of lactate in differentiation through transcriptomic analysis. We show that lactate induced histone acetylation and promoted expression of dopaminergic markers, with a stronger effect of D-lactate over L-lactate. Further studies to establish potential linkages between those two pathways will enhance our understanding of the effect of lactate.

The present study ln male Wistar rats was designed to rate and analyze six specific cocaine-induced behaviors. These behavioral parameters have been defined by others as either dopaminergic (sniffing, grooming, and locomotor activity) or serotonergic (repetitive head movements, rearing, and Straub tail) in origin. Results were analyzed by analysis of variance in two ways : (i) as grouped dopaminergic or serotonergic scores, and (ii) as the net behavioral index (dopaminergic scores minus the serotonergic scores). The purpose of approaching the data in this way was to attempt to define the behavioral interactions of the two neurotransmitters. One conclusion that developed from this study was the indication that dopaminergic behaviors peak at lower doses of cocaine than do serotonergic behaviors. This relationship held true for all the individual parameters in addition to the dopaminergic and serotonergic totals. A dopaminergic blocker, haloperidol, significantly attenuated all responses elicited by cocaine. When the net behavioral index was analyzed, it was found that the response of the median dose of cocaine was significantly altered from a net dopaminergic score towards a net serotonergic score. In this sense, haloperidol was shown to have the capacity to attenuate dopaminergic-associated parameters to a greater extent than the serotonergic-associated parameters. Cyproheptadine, an antiserotonergic agent, did not significantly affect the net behavioral index; however, this compound did significantly increase the dopaminergic parameter of grooming at the high doses of cocaine and cyproheptadine. Also at this dose combination , gnawing was elicited -- a dopaminergic response seen under no other experimental conditions. Due to the antiserotonergic agent causing an increase in the dopaminergic parameters of grooming and gnawing, it is proposed that the serotonergic influence on these two dopaminergic behaviors is of an inhibitory type.

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease, featuring motor signs such as tremors, bradykinesia, and impaired gait that are often preceded by nonmotor symptoms such as anxiety/depression and olfactory dysfunction. Interestingly, significant olfactory loss was found to be manifested in the majority of PD patients and may precede motor symptoms by years, and thus can be used for the risk assessment of developing PD in asymptomatic individuals. The main pathological feature of PD is the progressive and irreversible loss of dopaminergic (DA) neurons in the substantia nigra pars compacta of the midbrain. Although the detailed etiology of PD remains unclear, most PD cases were found to be sporadic and can be associated with environmental factors. Only 5–10% of patients result from familial PD. With considerable effort in the past two decades, a number of genes associated with familial PD have been identified and interestingly, many of these genes are involved in regulating and maintaining mitochondrial function. The presenilin-associated rhomboid-like (PARL) gene was found to contribute to mitochondrial morphology and function and was linked to familial Parkinson’s disease (PD). The PARL gene product is a mitochondrial intramembrane cleaving protease that acts on a number of mitochondrial proteins involved in mitochondrial morphology, apoptosis, and mitophagy. To date, functional and genetic studies of PARL have been mainly performed in mammals. However, little is known about PARL function and its role in dopaminergic (DA) neuron development in vertebrates. The zebrafish genome comprises two PARL paralogs: parla and parlb. Here, we show novel information concerning the role of PARL in zebrafish by establishing a loss-of-function mutation in parla and parlb via CRISPR/Cas9- mediated mutagenesis. We examined DA neuron numbers in the adult brain and expression of genes associated with DA neuron function in larvae and adults. We show that loss of parla function, as well as loss of both parla and parlb function result in loss of DA neurons in the olfactory bulb and telencephalon of adult zebrafish brain. Changes in the levels of tyrosine hydroxylase transcripts supported this neuronal loss. Expression of fis1, a gene involved in mitochondrial fission, was increased in parla mutants and in fish with loss of parla and parlb function. Furthermore, we showed that loss of parla and/or parlb function translates into altered locomotion parameters and that loss of parla but not parlb function results in impaired olfaction. Finally, increased susceptibility to neurotoxin exposure was identified in mutants with loss of both parla and parlb function but not with loss of parla or parlb function. These results suggest an evident role for parla in the development and/or maintenance of DA neuron function in zebrafish and confirm the existence of redundant and non-redundant functions for the two paralogs, parla and parlb.

The hypothalamic tuberoinfundibular dopaminergic (TIDA) neurones secrete dopamine, which inhibits prolactin secretion. TIDA neurone numbers are deficient in Ames (df/df) and Snell (dw/dw) dwarf mice, which lack prolactin, growth hormone and thyroid-stimulating hormone. Prolactin therapy initiated before 21 days maintains normal-sized TIDA neurone numbers in df/df mice and, when initiated as early as 7 days, maintains the maximum TIDA neurone numbers observed in dw/dw development, which are decreased compared to those in normal mice. The present study investigated the effect of prolactin dose and species on TIDA neurone development. Snell dwarf and normal mice were treated with saline, 5 μg of ovine prolactin (oPRL), 50 μg of oPRL, or 50 μg of recombinant mouse prolactin (rmPRL) beginning at 3days of age. Brains were analysed at 45 days using catecholamine histofluorescence, and immunohistochemistry for tyrosine hydroxylase or bromodeoxyuridine. Normal mice had greater (P ≤ 0.01) TIDA neurones than dw/dw, regardless of treatment. TIDA neurones in 50 μg oPRL-treated dw/dw mice were greater (P ≤ 0.05) than those in 5 μg oPRL- and rmPRL-treated dw/dw mice, which were greater (P ≤ 0.01) than those in saline-treated dw/dw mice. Fifty microgram oPRL-treated dw/dw mice also had greater (P < 0.01) TIDA neurone numbers than the maximum numbers observed in untreated dw/dw mice development. Among saline, 5 μg oPRL and 50 μg oPRL treatments, but not rmPRL, A14 neurone numbers were higher (P ≤ 0.01) in normal compared to in dw/dw mice. The mechanism of TIDA neurone recruitment was investigated using bromodeoxyuridine (BrdU) treatment at intervals after 21 days. Mice treated with rmPRL, but not oPRL, had increased BrdU incorporation in the periventricular area surrounding the third ventricle and median eminence and in the arcuate nucleus. The data obtained in the present study indicate that oPRL, but not rmPRL, when given at a high enough dose, induces TIDA neurone differentiation in dw/dw mice. This supports neurotrophic effects of prolactin on TIDA neurones in early postnatal development that extends beyond maintenance of the cell population.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第20284号 / 医科博第75号 / 新制||医科||5(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 宮本 享, 教授 林 康紀, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Nitric oxide (NO) has been implicated in large number of pathologies and in normal physiological function of the brain. The aim of this study was to recognize the effect of Nitro-L-Arginine Methyl Ester ·HCl (NAME) and L-Arginine Ethyl Ester.HCl (ARGININE) on reactivity of the central DA D3 receptor to agonist (Quinpirole) in rats. For this reason we have been used specific behavioural procedure such yawning behaviour which is mediated via central DA D3 receptors. Experiments were perform in adult male Wistar rats treated daily with quinpirole (0.05 mg/kg IP) or vehicle (0.9% NaCl) for the first 11 days from birth to obtain of the central D3 receptor supersensitivity. NAME and ARGININE in different way modified response of the central DA receptor to quinpirole estimated by means yawning behavioural procedure.