1. Institute of Marine Biology, National Sun Yat-sen University 2. Department of Biology, National Changhua University of Education Abstract Brain serotonin (5-hydroxytryptamine; 5-HT) generally serves an inhibitory role in aggressive behavior. But little is known about how 5-HT works during agonistic interaction and where the related works take place in the brain. Paradise fish has regular and ritual process of agonistic interaction which can be separated into three phases, namely, initial phase, threatening phase, and fighting phase. In initial phase, two fish encounter and swim close to each other. In threatening phase, two fish display shaking, head-tail swimming to threat their opponent. In fighting phase, two fish bite each other. With its stereotyped pattern of agonistic behaviors and amenability for pharmacological manipulation, paradise fish represents an excellent model for studies on neurochemical basis of aggressive behaviors. The results suggested that proper visual stimulus stemming from the interacting opponents elicits a socially stressful state that activates the telencephalic serotonergic system of the receipting paradise fish. The elevated serotonergic activity appears to inhibit the interacting individuals from entering fighting phase by constraining them to threatening phase. Presumably, diminishing activity of the telencephalic serotonergic system ushers in physical fighting behaviors.

Mitofusin 1 and 2 are outer-mitochondrial membrane proteins that have been shown to be involved in fusion. Mitofusin 2 has also been associated with apoptosis and development. When Mfn1 and Mfn2 were each conditionally knocked out from the cerebellum, Purkinje cells in Mfn2 deficient cerebellum during development had undergone neurodegeneration. Mutations in Mfn2 have also been associated with the Charcot Marie Tooth Type 2A (CMT2A). We want to asses the effect Mfn2 and Mfn1 might have on the development of other regions of the brain such as the telencephalon. We generated Mfn1 and Mfn2 conditional knockouts in the telencephalon by crossing them with Foxg1 Cre - a cre expressed in the telencephalon. We found that Mfn1 deficient mice have lost their corpus callosum at the midline, but survive over 6 months with a decrease in progenitor cells postnatally. Mfn2 deficient mice die between P9 and P12 with a decrease in progenitor cells postnatally and a decrease in number of neurons in the cortex. Therefore, our results suggest that Mfn1 and Mfn2 play a significant role in the development of the telencephalon.

Mitofusin 1 and 2 are outer-mitochondrial membrane proteins that have been shown to be involved in fusion. Mitofusin 2 has also been associated with apoptosis and development. When Mfn1 and Mfn2 were each conditionally knocked out from the cerebellum, Purkinje cells in Mfn2 deficient cerebellum during development had undergone neurodegeneration. Mutations in Mfn2 have also been associated with the Charcot Marie Tooth Type 2A (CMT2A). We want to asses the effect Mfn2 and Mfn1 might have on the development of other regions of the brain such as the telencephalon. We generated Mfn1 and Mfn2 conditional knockouts in the telencephalon by crossing them with Foxg1 Cre - a cre expressed in the telencephalon. We found that Mfn1 deficient mice have lost their corpus callosum at the midline, but survive over 6 months with a decrease in progenitor cells postnatally. Mfn2 deficient mice die between P9 and P12 with a decrease in progenitor cells postnatally and a decrease in number of neurons in the cortex. Therefore, our results suggest that Mfn1 and Mfn2 play a significant role in the development of the telencephalon.

Psychostimulants are known to alter visual function acutely and on withdrawal, and can cause attention deficit following prenatal exposure. However, psychostimulants can also improve visual attention in patients with attention deficit. The mechanisms involved in these contrasting effects remain largely unknown. To determine the role of specific brain regions and the dopamine system in the impact of cocaine exposure on visual processing, we employed two-photon microscopy and a transgenic larval zebrafish expressing the calcium indicator GCaMP-HS. We documented neuronal responses to contrasting visual stimuli, red light (LF) and dark (DF) flashes. We found that in the optic tectum neuropil (TOn), both stimuli elicited similar responses, though after repeated stimulus presentation, habituation developed to dark flash only. The dorsal telencephalon (dTe) responded and habituated to LF only. Acute cocaine (0.5 μM) reduced neuronal responses to LF in both brain regions and prevented habituation of dTe neurons to LF, but did not modify responses or habituation to dark flash. Prenatal cocaine exposure (PCE) did not modify baseline responses, but it prevented the acute effects of cocaine on LF responses in both regions and habituation in dTe, with no impact on dark flash responses. PCE also significantly reduced D1 dopamine receptor expression in TOn and cerebellum, but not dTe or the eye. Fish lacking the dopamine transporter (DATKO) retained normal D1 expression throughout the brain, baseline responses to LF in both TOn and dTe, and response reduction following cocaine in TOn. However, they demonstrated abnormal swimming behavior, and neither their swimming behavior nor dTe responses to LF were modified by cocaine. We discovered that in zebrafish, a diurnal vertebrate, responses to light not only require the primary visual processing center TO (superior colliculus in mammals), but also higher level processing by dTe. Responses to light but not darkness are modified by cocaine, unless the fish lack DAT or were exposed to PCE. Together, our results demonstrate specific effects of cocaine on visual processing mediated by the dopamine system, and provide a novel animal model for further investigation of these phenomena and development of new therapeutic approaches.

Early visual processing in the avian brain has been studied extensively, the retina and midbrain in particular. However, a clear understanding of the higher visual centers in the forebrain (the telencephalon and thalamus) remains poor. Two structures located within the avian visual telencephalon, the entopallium (E) and the lateral portion of the intermediate nidopallium (NIL), merit extensive investigation based on their critical role in visual processing. The goal of the current study was to further clarify the anatomical characteristics of E and NIL. Visual information that reaches these telencephalic structures is mostly from the contralateral retina. Thus, blocking visual input on one side affects the opposite hemisphere, but leaves the hemisphere on the same side largely unaffected. This unique property of the avian visual system was used in order to emphasize neurochemical expression in the higher visual structures. After blocking visual input to one hemisphere either by monocular occlusion or unilateral lesion of the nucleus rotundus we examined the expression of specific neuroanatomical markers; namely cytochrome oxidase (CO) and the calcium binding protein, parvalbumin (PV) in E and ZENK protein expression in NIL. . Our results showed there were significantly more PV-ir cell bodies in the inner region of E compared to the outer region. In terms of PV-ir neuropil, regional differences within E were significant. In particular, the ventrolateral E tended to have a higher density of PV-ir neuropil than other regions except the most ventromedial area which was also high in PV-ir. Differential CO staining patterns were observed as well. However, unlike PV-ir neuropil the ventro-intermedial area showed a low level of CO staining compared to the other areas. Finally, there was a significant reduction of ZENK-ir cell bodies on NIL in the experimental hemisphere compared to control side. Based on this differential expression of ZENK, it was possible for the first time to visualize the location of NIL. The findings presented here are discussed in terms of their relevance to the identification of the size and extent of NIL and the heterogeneous nature of E.

No description available.

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 3 uppsatser.

A atribuição da percepção da dor pelos peixes é um assunto controverso no meio científico. Alguns autores associam a percepção da dor a estruturas neocorticais que estão ausentes em peixes. Entretanto, estudos recentes têm demonstrado que os peixes são capazes de perceber e responder a estímulos nocivos de maneira semelhante ao que é observado em mamíferos, sendo estas respostas sensíveis à administração de morfina. Além disso, estudos pioneiros de nosso laboratório demonstraram a existência de um sistema analgésico endógeno em peixes. O objetivo deste estudo foi avaliar se este sistema analgésico endógeno pode ser ativado pelo estresse. A natureza neuroquímica deste sistema e a participação de uma região telencefálica, o telencéfalo dorsomedial (Dm), na modulação da antinocicepção também foram investigados. Nossos dados demonstram que o estresse de restrição de 3 e 5 minutos de duração inibe a resposta comportamental à injeção subcutânea de formalina a 3% na região da nadadeira adiposa no peixe Leporinus macrocephalus, sugerindo que este procedimento é capaz de ativar um sistema antinociceptivo endógeno. Além disso, a antinocicepção induzida pelo estresse de restrição de 3 e 5 min é de curta duração, sendo observada apenas por 5 min após o término da restrição. A análise da natureza neuroquímica da antinocicepção induzida pelo estresse de restrição revelou participação do sistema opióde e canabinoide na modulação desta resposta. O tratamento prévio com injeção intraperitoneal de naloxona (30 mg.kg-1), um antagonista opioide não seletivo, bloqueou a antinocicepção induzida pela restrição de 3 min de duração, mas não foi capaz de inibir a antinocicepção induzida pela restrição de 5 min de duração. Já o tratamento prévio com injeção intraperitoneal de AM251 (3 mg.kg-1), um antagonista de receptores canabinoides tipo 1, bloqueou a antinocicepção induzida pelo estresse de restrição de 3 e 5 min de duração, sugerindo que o sistema canabinoide desempenha um papel fundamental na antinocicepção induzida por esta modalidade de estresse na espécie estudada. Nosso estudo também demonstrou que a região do telencéfalo dorsomedial está envolvida na modulação da antinocicepção induzida pelo estresse de restrição no peixe L. macrocephalus. A microinjeção de midazolan (40 e 80 nmol), um agonista de receptores benzodiazepínicos, no telencéfalo Dm bloqueou a antinocicepção induzida pela restrição de 3 e 5 min de duração. Além disso, o tratamento prévio com flumazenil (80 e 160 nmol), um antagonista específico de receptores benzodiazepínicos, inibiu os efeitos do tratamento com midazolan, demonstrando que o bloqueio da antinocicepção promovido pelo midazolan ocorre pela ativação específica dos receptores benzodiazepínicos. Juntos estes resultados trazem novas perspectivas acerca do entendimento sobre a percepção nociceptiva em peixes. Este é o primeiro trabalho que traz evidências acerca da existência de um sistema de modulação da dor ativado pelo estresse e demonstra a participação de uma região encefálica específica na modulação desta antinocicepção. Estes resultados indicam que as vias analgésicas endógenas em peixes são ativadas de maneira semelhante aos mamíferos, sugerindo que estes animais possuem um processamento complexo da informação nociceptiva. / The assignment of pain perception by fish is controversial among scientists. Some authors associate the pain perception to neocortical structures that are absent in fish. However, recent studies have shown that fish are able to perceive and respond to noxious stimuli, similar to observed in mammals, and this responses are sensitive to morphine administration. Furthermore, pioneering studies from our laboratory have demonstrated the existence of an endogenous analgesic system in fish. This study aimed to evaluate if this endogenous analgesic system can be activated by stress, the neurochemical nature of this system and involvement of a telencephalic region, the dorsomedial (Dm) telencephalon, in the antinociception modulation. Our data demonstrate that 3 and 5 min of restraint stress inhibits the behavioral response to subcutaneous injection of formalin 3 % in the adipose fin in the fish Leporinus macrocephalus, suggesting that this procedure can activate an endogenous antinociceptive system. Furthermore, stress-induced antinociception induced by 3 and 5 min of restraint is short, with the antinociceptive effects being observed only for 5 min after the restriction. The analysis of the neurocheamical nature of antinociception induced by restraint stress revealed the involvement of opioid and cannabinoid systems in the modulation of this response. The pre-treatment with intraperitoneal injection of naloxone (30 mg.kg-1), a non-selective opioid receptors antagonist, blocked the antinociception induced by 3 min of restraint, but was not able to inhibit the antinociception induced by 5 min of restraint. The pre-treatment with intraperitoneal injection of AM251 ( 3 mg.kg-1), a type 1 cannabinoid receptors antagonist, blocked the stress-induced antinociception promoted by 3 and 5 min of restraint, suggesting that the cannabinoid system plays a critical role in this type of stress-induced antinociception in the studied species. Our study also showed that the dorsomedial telencephalon is involved in the modulation of stress-induced antinociception in fish L. macrocephalus. The microinjection of midazolan (40 and 80 nmol), a benzodiazepine receptors agonist, in the Dm blocked the stress-induced antinociception promoted by 3 and 5 min of restraint. Furthermore, pre-treatment with flumazenil (80 and 160 nmol), a benzodiazepine receptors selective antagonist, inhibited the effects of the midazolan treatment, demonstrating that the antinociception blockade by midazolan is promoted by specific activation of benzodiazepine receptors. Together these results provide new insights on the understanding of nociceptive perception in fish. This is the first study that demonstrates evidence for the existence of a pain modulation system activated by stress in fish and demonstrates the involvement of a specific brain region in the modulation of this antinociception. These results indicate that the endogenous analgesic pathways in fish are activated in a similar manner to mammals, suggesting that these animals have a complex processing of nociceptive information.

The vertebrate nervous system is a highly complex and intriguing structure with diverse functions. To understand the functional nervous system, we first have to be aware of how it is assembled during development. In this thesis the mechanism of early diversification and regionalisation necessary for subsequent formation of part of the nervous system, namely the telencephalon and the placodes, will be addressed. We have identified signalling molecules involved in the dorsoventral patterning of the telencephalon and we propose a mechanism for the induction and differential specification of the olfactory and lens placodes. The telencephalon is regionalised along the dorsoventral axis during development. The cells situated dorsally will give rise to the cerebral cortex while the ventral and intermediate cells are mainly progenitors for the basal ganglia. The cerebral cortex is associated with higher cognitive functions whereas the basal ganglia control movements. We provide evidence that dorsal and intermediate telencephalic cells are re-specified from cells with an intrinsic ventral character. Dorsal telencephalic cells are specified at stage 10 in chick, while the intermediate cells are specified a few hours later, at stage 14. The expression of Wnt and Fibroblast growth factors (Fgfs) coincides with the time point when the dorsal cells are specified, and we provide evidence that Wnt and FGF signals act in a sequential way to specify dorsal telencephalic cells. The retinoic acid (RA) synthesising enzyme Raldh3 is expressed in proximity to the telencephalon, and our result suggests that RA is both required and sufficient to induce intermediate telencephalic cell types. Additionally, Fgf8 is expressed in the anterior neural ridge and the ventral telencephalic cells require FGF signals that oppose RA to maintain their character. The olfactory and lens placodes contribute to the special sense organs associated with olfaction and vision, respectively. Olfactory and lens placodes are specified at gastrula stage in chick, and become spatially separated at the neural fold stage. We provide evidence that Bone morphogenetic protein (BMP) signalling is required for the induction of a pool of placodal progenitor cells. Furthermore, time of exposure to BMP signals plays a key role in the differential specification of the olfactory and lens placodes, where continued exposure to BMP signals promotes lens character at the expense of olfactory placodal cells.

The brain has been one of the central foci in studies of vertebrate evolution. Work in East African cichlids and other emerging fish models like the Mexican cavefish (Astyanax mexicanus) offer new insight on the role of patterning on brain evolution. These fish can be grouped into two major categories according to habitat; for cichlids it is rock-dwelling (known locally as mbuna) and sand-dwelling (non-mbuna) lineage. The brain development of mbuna versus non-mbuna is defined by changes in gene deployment working along the dorsal/ventral (DV) and anterior/posterior (AP) neuraxes, respectively. Comparison of disparate fish ecotypes offer a new perspective of the role of patterning on brain evolution; through the slight and early modification of signal pathways working across 3-D axes, and a subsequent magnifying effect across ontogeny, evolution can generate widespread changes in the brain. To illustrate this patterning model of brain evolution, two comparative studies were done between mbuna and non-mbuna, examining the action of gene pathways that work to pattern the cichlid forebrain. The first study found that non-mbuna cichlids have a more rapid AP expansion of a gene pathway (Wingless) into the presumptive midbrain and diencephalon versus mbuna. These forebrain structures are involved in sight processing and could be of ecological benefit to vision-focused non-mbuna. The second study described a difference within the developing telencephalon. The embryonic telencephalon is split into the pallium, which processes visual signals, and the subpallium, which develops into the olfactory bulbs. Mbuna possess a larger subpallium relative to non-mbuna, which have a larger pallium. This was correlated to a more rapid expansion of another gene pathway (Hedgehog) along the DV axis. The difference in size of the pallial vs. subpallialial comparments between cichlids can be correlated to expanded olfaction in mbuna and vision in non-mbuna adult brains. Overall, East African cichlids are an excellent system to investigate the role of patterning on brain evolution because they allow for the comparison of the earliest patterning events in brain ontogeny between distinct ecotypes. These fish systems link study in brain development to the brain morphology comparisons employed in classic studies of brain evolution.