Thesis (Ph. D.)--University of Chicago, 1917. / "Author's abstract of this paper issued by the [Wistar institute] Bibliographic service, January 16." "Private edition, distributed by the University of Chicago Libraries, Chicago, Illinois." "Reprinted from the Journal of comparative neurology, vol. 34, no. 1, February, 1922." Includes bibliographical references (p. 169-171).

The role of the forebrain commissures in the developing and developed kindled amygdaloid seizure in the rat was investigated. In the first two experiments, bisection of the corpus callosum, hippocampal commissure, and anterior commissure prior to kindling caused a significant facilitation in the rate of primary-site kindled seizure development. In the last experiment, bisection of the corpus callosum and hippocampal commissure after primary-site kindling facilitated the subsequent rate of secondary-site kindling. It is evident, that in the intact animal, the nonstimulated hemisphere is able to exert an inhibitory influence over the development of seizure activity in a stimulated hemisphere and that this effect is, in turn, mediated via the forebrain commissures. The corpus callosum and hippocampal commissure were found to participate in the interference phenomenon since bisection of these structures either before or after primary-site kindling caused a facilitation in the rate of primary-site rekindling. In the first two experiments, an extracommissural, possibly brainstem, mechanism is suggested to mediate the transfer effect between the primary and secondary sites since bisection of the forebrain commissures prior to kindling had no effect on the rate of secondary-site kindling. The development of primary generalized motor seizures is in part dependent on the integrity of the corpus callosum and hippocampal commissure. Bisection of these structures after primary-site kindling, however, abolished the subsequent development of primary generalized seizures in a significant number of rats. Therefore, it appears that if the commissures are bisected prior to kindling, alternate pathways able to mediate the development of primary generalized seizures evolve. / Medicine, Faculty of / Graduate

Nestin is a type VI intermediate filament protein that marks proliferative cells in the central and peripheral nervous system of vertebrates during development and adulthood. Nestin is not only expressed in progenitor cells of neuronal tissues but is also present in muscle, heart, lung, pancreas and skin follicle tissues. The goal of this thesis is to investigate and characterize the nestin protein in goldfish and relate nestin expression to neuroregeneration and brain plasticity events in the adult goldfish forebrain. Currently little is known about nestin function and regulation in vertebrates, especially in fish. In this study we used Rapid amplification of cDNA ends PCR (RACE-PCR) to isolate goldfish nestin mRNA. We uncovered several different mRNA transcripts. PCR analysis and sequencing further identified three different nestin transcripts of 4003, 2446, and 2126 nucleotides with a predicted protein length of 860, 274, and 344 amino acids respectively. We next applied a multiple-antigenic peptide (MAP) strategy to generate a polyclonal goldfish-specific nestin antibody against a 23 amino acid sequence located at the N-terminal end of goldfish nestin. Western blotting revealed the existence of three different nestin protein isoforms (nestin A, B and C); the first report of nestin isoforms in teleost species. Nestin expression and distribution in the goldfish brain is complex and revealed both individual and tissue-dependent variations. The most remarkable finding following principal component analysis of the western blot data was the uniqueness of the pituitary, hypothalamus and telencephalon. These tissues are proliferative in nature containing progenitor and proliferative cellular pools that are involved in important biological axes such as the motor and reproductive axis. Interestingly, all three tissues were able to change their proliferative cellular profile of nestin protein expression to alleviate the detrimental effects of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) upon administration. The toxin MPTP destroys dopamine neurons in the fish brain leading to motor deficits and reproductive difficulties. The incorporation of 5-bromo-2’-deoxyuriding (BrdU) into newly synthesized DNA revealed an upregulation of BrdU immunolabeling following MPTP administration in the area telencephali pars dorsalis (Vd) and along the ventricular surface area of the telencephalon suggesting the generation of new neurons in the adult central nervous system. This thesis reports novel nestin isoforms and illustrates regenerative events occurring in the goldfish telencephalon following a neurotoxic insult. This work provides a framework for future investigations of the differential roles and regulation of the nestins to better understand seasonal neuronal plasticity, neuronal regeneration and neuronal circuitry in teleost.

Autism is a severe childhood neuropsychiatric condition characterized by impairments in socialization and communication, and by restricted and repetitive behaviours. Autism spectrum disorder (ASD) is a complex and largely unknown disease with a strong genetic basis, multiple genes involved and environmental factors determining its phenotype. Interestingly, the DLX1/DLX2 and DLX5/DLX6 bigene clusters are located in autism susceptibility loci and Dlx genes are involved in GABAergic interneurons differentiation and migration to the cortex during forebrain development. Dlx gene expression is controlled by different cis-regulatory elements. Of these, 4 are active in the forebrain, URE2, I12b, I56ii and I56i. In order to determine the role of the DLX genes in ASD, variants were found in gene exons and in cis-regulatory elements in autistic individuals. A single nucleotide polymorphism (SNP), a change of an adenine for a guanine, was identified in I56i enhancer. Finding a SNP in I56i was very surprising considering that it is located in a Dlx binding motif highly conserved among >40 species. We showed, using in vitro approaches, that the presence of this SNP affects the affinity of Dlx for their binding site and reduces the transcriptional activation of the enhancer. The SNP also affects activity of the I56i enhancer in transgenic mice. In order to determine the real impact of the SNP in vivo, mutant mice harboring the SNP in their I56i enhancer were produced. That involved the insertion of the I56i enhancer with the SNP, using homologous recombination in mouse embryonic stem cells to replace the wild type version of the enhancer. With these mutant mice, we demonstrated that, in vivo, this SNP reduces Dlx5 and Dlx6 expression in the forebrain. Furthermore, this decrease in Dlx5/Dlx6 expression could affect the differentiation and/or migration of specific populations of inhibitory interneurons in the forebrain. No distinct iv behavioural phenotypes were observed between wild type mice and those carrying the SNP, during social interaction and anxiety tests. Therefore, these results suggest that even a subtle change in a regulatory element can have an impact in the development of the forebrain and may even contribute to disorders such as autism.

Autism is a severe childhood neuropsychiatric condition characterized by impairments in socialization and communication, and by restricted and repetitive behaviours. Autism spectrum disorder (ASD) is a complex and largely unknown disease with a strong genetic basis, multiple genes involved and environmental factors determining its phenotype. Interestingly, the DLX1/DLX2 and DLX5/DLX6 bigene clusters are located in autism susceptibility loci and Dlx genes are involved in GABAergic interneurons differentiation and migration to the cortex during forebrain development. Dlx gene expression is controlled by different cis-regulatory elements. Of these, 4 are active in the forebrain, URE2, I12b, I56ii and I56i. In order to determine the role of the DLX genes in ASD, variants were found in gene exons and in cis-regulatory elements in autistic individuals. A single nucleotide polymorphism (SNP), a change of an adenine for a guanine, was identified in I56i enhancer. Finding a SNP in I56i was very surprising considering that it is located in a Dlx binding motif highly conserved among >40 species. We showed, using in vitro approaches, that the presence of this SNP affects the affinity of Dlx for their binding site and reduces the transcriptional activation of the enhancer. The SNP also affects activity of the I56i enhancer in transgenic mice. In order to determine the real impact of the SNP in vivo, mutant mice harboring the SNP in their I56i enhancer were produced. That involved the insertion of the I56i enhancer with the SNP, using homologous recombination in mouse embryonic stem cells to replace the wild type version of the enhancer. With these mutant mice, we demonstrated that, in vivo, this SNP reduces Dlx5 and Dlx6 expression in the forebrain. Furthermore, this decrease in Dlx5/Dlx6 expression could affect the differentiation and/or migration of specific populations of inhibitory interneurons in the forebrain. No distinct iv behavioural phenotypes were observed between wild type mice and those carrying the SNP, during social interaction and anxiety tests. Therefore, these results suggest that even a subtle change in a regulatory element can have an impact in the development of the forebrain and may even contribute to disorders such as autism.

Autism is a severe childhood neuropsychiatric condition characterized by impairments in socialization and communication, and by restricted and repetitive behaviours. Autism spectrum disorder (ASD) is a complex and largely unknown disease with a strong genetic basis, multiple genes involved and environmental factors determining its phenotype. Interestingly, the DLX1/DLX2 and DLX5/DLX6 bigene clusters are located in autism susceptibility loci and Dlx genes are involved in GABAergic interneurons differentiation and migration to the cortex during forebrain development. Dlx gene expression is controlled by different cis-regulatory elements. Of these, 4 are active in the forebrain, URE2, I12b, I56ii and I56i. In order to determine the role of the DLX genes in ASD, variants were found in gene exons and in cis-regulatory elements in autistic individuals. A single nucleotide polymorphism (SNP), a change of an adenine for a guanine, was identified in I56i enhancer. Finding a SNP in I56i was very surprising considering that it is located in a Dlx binding motif highly conserved among >40 species. We showed, using in vitro approaches, that the presence of this SNP affects the affinity of Dlx for their binding site and reduces the transcriptional activation of the enhancer. The SNP also affects activity of the I56i enhancer in transgenic mice. In order to determine the real impact of the SNP in vivo, mutant mice harboring the SNP in their I56i enhancer were produced. That involved the insertion of the I56i enhancer with the SNP, using homologous recombination in mouse embryonic stem cells to replace the wild type version of the enhancer. With these mutant mice, we demonstrated that, in vivo, this SNP reduces Dlx5 and Dlx6 expression in the forebrain. Furthermore, this decrease in Dlx5/Dlx6 expression could affect the differentiation and/or migration of specific populations of inhibitory interneurons in the forebrain. No distinct iv behavioural phenotypes were observed between wild type mice and those carrying the SNP, during social interaction and anxiety tests. Therefore, these results suggest that even a subtle change in a regulatory element can have an impact in the development of the forebrain and may even contribute to disorders such as autism.

Autism is a severe childhood neuropsychiatric condition characterized by impairments in socialization and communication, and by restricted and repetitive behaviours. Autism spectrum disorder (ASD) is a complex and largely unknown disease with a strong genetic basis, multiple genes involved and environmental factors determining its phenotype. Interestingly, the DLX1/DLX2 and DLX5/DLX6 bigene clusters are located in autism susceptibility loci and Dlx genes are involved in GABAergic interneurons differentiation and migration to the cortex during forebrain development. Dlx gene expression is controlled by different cis-regulatory elements. Of these, 4 are active in the forebrain, URE2, I12b, I56ii and I56i. In order to determine the role of the DLX genes in ASD, variants were found in gene exons and in cis-regulatory elements in autistic individuals. A single nucleotide polymorphism (SNP), a change of an adenine for a guanine, was identified in I56i enhancer. Finding a SNP in I56i was very surprising considering that it is located in a Dlx binding motif highly conserved among >40 species. We showed, using in vitro approaches, that the presence of this SNP affects the affinity of Dlx for their binding site and reduces the transcriptional activation of the enhancer. The SNP also affects activity of the I56i enhancer in transgenic mice. In order to determine the real impact of the SNP in vivo, mutant mice harboring the SNP in their I56i enhancer were produced. That involved the insertion of the I56i enhancer with the SNP, using homologous recombination in mouse embryonic stem cells to replace the wild type version of the enhancer. With these mutant mice, we demonstrated that, in vivo, this SNP reduces Dlx5 and Dlx6 expression in the forebrain. Furthermore, this decrease in Dlx5/Dlx6 expression could affect the differentiation and/or migration of specific populations of inhibitory interneurons in the forebrain. No distinct iv behavioural phenotypes were observed between wild type mice and those carrying the SNP, during social interaction and anxiety tests. Therefore, these results suggest that even a subtle change in a regulatory element can have an impact in the development of the forebrain and may even contribute to disorders such as autism.

The hippocampus of birds and mammals has been shown to play a crucial role in spatial memory and navigation. The hippocampus exhibits plasticity in adulthood in response to diverse environmental factors associated with spatial demands placed on an animal. The cortical telencephalon of squamate reptiles has been implicated as a functional homologue to the hippocampus. This study sought to experimentally manipulate the navigational demands placed on free-ranging northern Pacific rattlesnakes (Crotalus o. oreganus) to provide direct evidence of the relationship between spatial demands and neuroplasticity in the cortical telencephalon of the squamate brain. Adult male rattlesnakes were radio-tracked for two months, during which one of three treatments was imposed weekly: 225 meter translocation in a random direction, 225 meter walk and release at that day’s capture site (handling control), and undisturbed control. Snakes were then sacrificed and brains were removed and processed for histological analysis of cortical features. The volume of the medial cortex was significantly larger in the translocated group compared to undisturbed controls. No differences in dorsal or lateral cortical volume were detected among the groups. Numbers of 5-Bromo-2’-deoxyuridine (BrdU) -labeled cells in the medial and dorsal cortices three weeks after BrdU injection were not affected by treatment. The activity range was larger in the translocated group compared to handled and undisturbed controls. A causal relationship between increased navigation in a free-ranging reptile and changes in brain morphology was established. The use of translocation as a conservation strategy for reptiles is a controversial topic revisited many times. Previous studies have demonstrated the aberrant movement patterns and mortality caused by translocation and have established that short-distance translocation within an animal’s home range is best for the animal. In conjunction with the neuroplasticity study, we examined the physiological impacts that repeated short-distance translocation and handling have on reptiles. This is essential knowledge if the efficacy of the technique is to be properly evaluated. Baseline and stressed concentrations of corticosterone and testosterone were assayed in blood taken immediately upon capture and following one hour of confinement in a bucket. Neither baseline nor stressed concentrations of either hormone were impacted by translocation or handling. Body condition and change in mass were not affected. Translocated animals had larger MCP activity ranges than handled and undisturbed animals at the 95%, but not 100% levels, while an interaction between time and treatment impacted other movement parameters.Treatment had no effect on a number of behaviors observed during visits to each animal. We suggest that rattlesnakes are quite resistant to potential impacts on their physiology and behavior enacted by frequent short-distance translocation or handling. Additionally, studies that require frequent handling of reptilian subjects are not likely to severely alter stress physiology.

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.