An attempt has been made to determine the relationship, if any, between adenosine triphosphate and the transmitter substance responsible for antidromic vasodilatation. Extracts of various areas of the central nervous system have been made by dialyzing boiled, ground brain tissue against distilled water. These extracts were analyzed for; labile phosphate content by the method of Berenblum and Chain (66), adenosine triphosphate content by paper chromatography and by the luciferin-luciferase enzyme method of Strehler and Totter (69). The content of vasodilator activity of extracts from the same areas was determined by the method of Holton (35). The extracts to be tested were injected into the facial artery of a rabbit and allowed to flow into the auricular artery and through the ear. The changes produced in the ear were detected by means of a photoelectric cell which measured differences in the amount of light passing through the ear. Vasodilatation appeared as a decrease in the amount of light transmitted through the ear and vasoconstriction as an increase. It was thought that if there was a relationship between adenozine triphosphate and the transmitter material the areas containing the most ATP should also contain the most vasodilator activity. A comparison of the location and concentration of the two substances revealed no such correlation. The two most important questions to be answered were: 1. is ATP the substance responsible for antidromic vasodilatation? and 2. if so, is it also a sensory synaptic transmitter substance? It was concluded that ATP was unlikely to be the substance responsible for antidromic vasodilatation. If one accepts Dale's hypothesis (48) that a neurone may employ the same transmitter substance at all branches of the axon, then it would also seem to rule out ATP as a sensory synaptic transmitter agent. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

In order to understand molecular mechanisms of neural plasticity, habituation of a simple neural circuit in Drosophila melanogester was investigated. A resistance reflex controlling the femoro-tibial joint angle was identified using electromyogram recording from the tibial extensor muscle. It was shown that typically only one of the two motor neurons innervating the muscle, the slow tibial extensor (SETi), was activated in the reflex response. By targeting the expression of tetanus toxin, which cleaves synaptobrevin and blocks evoked synaptic transmission, to the sensory neurons in the femoral chordotonal organ (feCO) using P (Ga14) insertion lines, it was demonstrated that the feCO was necessary for the resistance reflex. Upon repetitive stimulation of the sensory neurons, the strength of the reflex response decreased. This response decrement conformed to parametric features of habituation: exponential decay, stimulus amplitude and frequency dependent, spontaneous recovery, and dishabituation. Ca$\sp{2+}$/calmodulin-dependent protein kinase II (CaMKII) has been implicated in synaptic plasticity and learning. To test the role of the different activity states of CaMKII in habituation, transgenes coding for an inhibitory peptide of the kinase, a CaMKII subunit incapable of becoming Ca$\sp{2+}$-independent, and a constitutively active kinase was expressed selectively in the presynaptic sensory neurons using a P (Ga14) insertion line. Expression of a Ca$\sp{2+}$-independent form of CaMKII in the sensory neurons increased reflex response and blocked habituation. When a kinase incapable of becoming Ca$\sp{2+}$-independent was expressed in the sensory neurons, the initial reflex response during a habituation trial was reduced and habituation of the reflex was nearly blocked. When the inhibitory peptide was expressed in the sensory neurons, the reflex response was decreased and habituation was also blocked. These results suggested that the Ca$\sp{2+}$-independent CaMKII sets the response level of the neural circuit, allowing the circuit to exhibit appropriate dynamics, and an optimal level of CaMKII in the presynaptic neuron is required for the reflex to show habituation. This system now can be used to dissect the molecular cascade involved in habituation and other forms of neural plasticity.

Availability of oxidizable metabolic fuels is a primary factor affecting fertility, in female mammals. Reductions in metabolic fuel availability caused by food deprivation, treatment with pharmacological inhibitors of glycolysis (2-deoxy-D-glucose, 2DG) and fatty acid oxidation (methyl palmoxirate, MP) or with insulin, inhibit both estrous cyclicity and estrous behavior in Syrian hamsters. In this study, the effects of fuel deprivation on neural estrogen receptors, the pathways by which metabolic fuel status is conveyed in food deprived animals to higher forebrain circuits and the changes in estrogen receptor (ER) mRNA expression after fuel deprivation have been studied. Food deprivation or treatment with 2DG+MP decreased detectable number of estrogen receptor immunoreactive (ERIR) cells in the ventromedial hypothamic nucleus (VMH), increased the detectable number of ERIR in the arcuate nucleus (ARC) and the posterior parvicellular portion of the hypothalamic paraventricular nucleus (PaPo) without any effect on anterior parvicellular portion of the paraventricular nucleus (PaMP) and posterodorsal portion of the medial amygdala (MePD). Insulin treatment significantly reduced sexual receptivity in ovariectomized, steroid treated animals. It also resulted in similar changes in detectable number of ERIR cells in the VMH and medial preoptic area (mPOA) as that caused by food deprivation, cold exposure or treatment with metabolic inhibitors. Area postrema (AP) plays a significant role in transmitting metabolic information to the forebrain along with the nucleus of solitary tract (NTS) and the vagus nerves. The results of the present study clearly show that AP lesions prevent the suppression of estrous behavior, but not the suppression of estrous cyclicity, caused by food deprivation. AP lesions also blocked the suppression of estrous behavior in insulin treated animals. The last part of the project determined the changes in ER mRNA expression in VMH, 12 h after treatment with 2DG and MP. The results showed no differences between the ER mRNA levels between the fed and fuel deprived animals.

The vasopressin (AVP)-expressing neurons in the bed nucleus of the stria terminalis (BST) and centromedial amygdala (CMA) are highly sensitive to gonadal hormones. Gonadectomy eliminates, and testosterone replacement restores, AVP immunoreactivity, and AVP mRNA expression in the BST and CMA. Although AVP-ir cells express both androgen and estrogen receptors, the structure of the AVP gene promoter region suggests that these receptors cannot directly influence AVP gene expression. The AVP gene does not contain the consensus sequences for estrogen and androgen receptor response elements. Here the hypothesis that progestin receptors (PR) may be involved in the regulation of AVP expression by gonadal hormones is explored. Results indicate that virtually all AVP-ir cells in the BST and CMA contained PR in rats. Although, neither gonadectomy nor hormonal treatment influenced the expression of PR immunoreactivity in AVP-ir cells, it did significantly reduce the intensity of PR immunoreactivity expressed in AVP-ir cells. The expression of AVP immunoreactivity was examined in male mice that carried a targeted disruption of the PR gene (PRKO). No differences were found in the number of steroid-responsive AVP-ir cells, and the density of AVP-ir fibers between PRKO and wild type mice. This suggests that AVP expression does not critically depend on PR expression. The effect of ZK 98299 on AVP mRNA expression in the BST was studied. Male rats were castrated and implanted with testosterone-filled or empty capsules, or they were sham-operated. Half of the animals in these groups were given daily injections of the PR antagonist ZK 98299 and the other half vehicle for three days. Although castrated rats had fewer AVP mRNA labeled cells than castrated rats that were treated with testosterone or sham-castrated rats, there were no differences in the number of cells that were labeled for AVP mRNA between rats that were treated with ZK 98299 or vehicle. Similarly, castrated rats had less labeling per cell than the other two groups, and again an overall ANOVA did not indicate differences in labeling per cell between rats treated with ZK 98299 or vehicle. However, testosterone-treated and sham-castrated rats that were treated with ZK 98299 had more labeling per cell than their counterparts which were treated with vehicle. These results suggest that if PR influences AVP mRNA expression in the steroid-sensitive AVP cells, it suppresses rather than stimulates AVP mRNA expression.

Discs Large (DLG), a Drosophila member of the Membrane Associated Guanylated Kinases (MAGUKs) family, exists at the pre- and post-synaptic terminals of Drosophila larval Type I neuromuscular junctions (NMJs). To understand the in vivo roles of MAGUKs as synaptic organizers, Drosophila Type I synapses were used as a model system. Analysis of dlg mutants revealed that DLG is important for proper development of synaptic structures. Mutations in dlg result in abnormal growth of the postsynaptic membrane, the subsynaptic reticulum (SSR). Expression of transgenic DLG at synaptic terminals in dlg mutant larvae rescued this SSR phenotype. In addition DLG is important for the proper localization of synaptic components via interaction between PDZ domains 1 and 2 of DLG and S/TXV motifs in specific synaptic proteins such as Fasciclin II (FasII). For example, the localization of FasII, a Drosophila structural homologue of neuronal cell adhesion molecule (N-CAM), at Type I synapses depends on DLG. A severe hypomorph dlg mutant allele (dlg x1-2) showed reduced FasII localization at Type I synapses. Furthermore ultrastructural defects in dlgx1-2 (increased number of active zones at pre-synaptic terminals) resembled abnormalities found in FasII hypomorph mutant boutons. This observation suggests that DLG and FasII are components of the same signal transduction pathway which regulates the development of pre-synaptic structure. Coimmunoprecipitation of body wall muscle extracts, ELISA, and yeast two-hybrid assay have demonstrated a direct interaction between DLG and FasII. These results suggest the role of DLG as a synaptic organizer which regulates the proper localization of synaptic components. Both DLG binding partners, Shaker channels and FasII, regulate or are regulated by neuronal activity. Therefore we attempted to determine if DLG function could also be regulated by synaptic activity. In particular we investigated the functions of Ca2+/Calmodulin-dependent kinase II (CaMKII), an enzyme whose kinase activity is regulated by synaptic activity, and which partially colocalizes with DLG at NMJs and exists in the same protein complex as DLG. We found that increased CaMKII activity phenocopied the defects of dlg mutant synapses and induced the dissociation of DLG from the synaptic membrane. Decreased CaMKII activity induced the opposite effects. In vitro, CaMKII phosphorylated DLG fragments with a stoichiometry close to one. Furthermore expression of transgenic site-directed mutations of dlg that blocked or mimicked phosphorylation had effects similar to those observed upon inhibiting or constitutively activating CaMKII. We conclude that CaMKII-dependent DLG phosphorylation regulates the association of DLG with the synaptic complex during development and plasticity, thus providing a link between synaptic activity and structure.

Multicellular organisms develop from a single cell. The developmental program takes a fertilized egg through cell division, differentiation and morphogensis, to produce the adult. These processes are controlled by the genes present in the fertilized egg. Therefore, transcriptional regulation of these genes plays an essential role in development. Studying the functional mechanisms of transcriptional regulators will gain key insights into the complex processes of development. Transcriptional regulators that control the pattern of gene expression in the developing embryo are essential for the development of a symmetric body. One such gene is fish-hook (fish). Observations of fish mutant embryos indicate Fish has important functions in embryonic segmentation and CNS development (Nambu and Nambu 1996; Russell et al., 1996). The sequencing analyses indicated that fish encodes a Droosphila high mobility group (HMG) Sox protein. Fish protein has a single HMG domain, which shares over 50% identity with other Sox proteins' HMG domains. Sox protein's HMG domain can bind to the consensus DNA sequences and induce strong DNA bending. Some of the Sox proteins have transcriptional activation capability. To reveal Fish functions during Drosophila embryonic development, genetic and molecular approaches were used to (1) characterize Fish protein, including studying the Fish protein expression pattern during embryonic development, its' DNA binding and bending properties, and its transcriptional activation capability; and (2) uncover the gene regulatory functions of Fish in Drosophila embryonic segmentation and CNS midline development. Characterization of Fish protein revealed that Fish protein is initially expressed in a 7 stripe pattern during early blastoderm, which is rapidly replaced by strong neuroectoderm expression. Fish HMG domain, like other Sox protein's HMG domains, can bind to the vertebrate Sox protein consensus DNA binding sequences, AACAAT and AACAAAG, and this binding induces an 85 degree DNA bend. In addition, the NH2-terminal portion of Fish protein has the transcription activation capability (Ma et al., 1998). Gene regulatory functions of Fish were first investigated in embryonic segmentation. Fish directly regulates the expression of the pair rule gene, even-skipped (eve), by binding to multiple sites located in eve downstream regulatory regions that direct formation of eve stripes 1, 4, 5, and 6. Genetic interactions between fish and pdm mutants suggest that Fish may function along with the Drosophila POU domain proteins Pdm1 and Pdm2 to regulate eve transcription (Ma et al., 1998). Studies of Fish functions in embryonic CNS midline development indicated that Fish and Drifter, a POU domain protein, function along with the PAS domain protein Single-minded (Sim) to control CNS midline development and gene expression. Fish, Drifter and Sim are co-expressed in the developing midline cells. Genetic analyses indicate that fish, drifter and sim synergistically regulate CNS midline development and slit midline expression. Both Fish and Drifter interact with the slit 1.0 kb enhancer element, which contains a CNS Midline Element (CME), the Sim binding site. Fish can directly associate with Drifter and Sim proteins. Further more, these three proteins can form a ternary complex. The direct associations between the Sox protein Sox2 and the PAS domain protein Trh or the POU domain protein Oct3 were also detected, and this indicates the interactions between Sox/POU and Sox/PAS proteins are conserved. These conserved interactions may reveal novel gene regulatory mechanisms that involve these three distinct classes of transcription factors.

The purpose of this dissertation was to gain insight into the integration of the locomotory and respiratory systems by studying the coordinative behaviors that emerge in response to changing constraints. This was accomplished by utilizing analytical methods that have recently provided new insights into their adaptive strategies. Three studies were carried out that focused on different specific constraints that influence the interaction of the locomotory and respiratory systems. The first study investigated the relationship between stride frequency and locomotor-respiratory coordination (LRC) and the effect of LRC on the metabolic cost and ventilatory efficiency during running. It was found that stride frequency is a critical parameter affecting LRC and that the strength of LRC was not related to lower metabolic cost during running. In contrast, greater strength of coordination observed during running at preferred speed was associated with lower ventilatory efficiency. The second study examined the influence of mechanical constraints on LRC. The magnitude of mechanical perturbations imposed on the respiratory system by the upper body were not related to the strength of LRC. Instead, the timing within the stride cycle influenced the nature of LRC variability. It is likely that the coupling between these rhythms is not a passive mechanism but is related to active muscular control of the upper body, primarily influencing the variability of the coupling. The third study investigated the relationship between LRC and upper body control under normal and challenged postural and ventilatory conditions. These challenges resulted in significant changes in upper body control strategies including increases in the variability of motion. The postural challenge, however, did not act to further couple the respiratory rhythm to the locomotor rhythm and the ventilatory challenge did not act to decouple the rhythms. Together, the results of the current set of experiments illustrate the importance of variability in LRC and that mechanical constraints do not act to couple respiration to movement during human locomotion, as is typically assumed. Finally, the integration of upper body control and respiration has important implications for the study of gait stability.

Estradiol (E2) stimulates luteinizing hormone-releasing hormone (LHRH) synthesis and release from neurons in the preoptic area (POA). Stimulatory effects of E2 on LHRH neurons are critical for preovulatory surge of luteinizing hormone (LH). The neural mechanisms mediating these events are unclear. Glutamate and γ-aminobutyric acid (GABA), the most prevalent excitatory and inhibitory neurotransmitters in the brain, play important roles in the regulation of LHRH neurons. Activation of the N-methyl, D-aspartate (NMDA) subtype glutamate receptor is required for E2-dependent LHRH and LH surge release. It is unknown whether these actions are direct or relayed indirectly via other systems. NMDA treatment induces increases in LHRH mRNA levels, but it is unclear whether NMDA receptor activation mediates E2-dependent increases LHRH gene expression on the day of the LH surge. Dual-label in situ hybridization histochemistry (ISHH) and pharmacological techniques revealed a subpopulation of LHRH neurons in midline POA nuclei preferentially contains NMDAR1 and NMDAR2 receptor mRNAs and responds preferentially to NMDA with increased LHRH mRNA. Results show NMDA receptor activation is not required for E2-induced increases LHRH mRNA on the day of the LH surge. GABA mediates inhibitory effects on LHRH neurons. GABAergic inhibition must be relieved before LHRH and LH surge release can occur. Evidence indicates neurons in the anteroventral periventricular nucleus (AVPV), a region important in E2-dependent LHRH neural regulation, exhibit a mixed GABAergic/glutamatergic phenotype. Using dual-label ISHH, I showed that vesicular glutamate transporter 2 (VGLUT2) and glutamic acid decarboxylase 65 and 67 mRNAs are colocalized AVPV neurons and in other brain regions. Results demonstrate GABAergic/glutamatergic AVPV neurons in females are more numerous and greater percentages contain estrogen receptor mRNAs than males. Using immunocytochemistry and confocal microscopy, I showed that E2 increases VGLUT2 levels and decreases vesicular GABA transporter levels in double-labeled terminals contacting medial LHRH neurons. Results indicate that medial LHRH neurons are regulated uniquely by preferential expression of NMDA receptors and by dynamic E2-dependent changes in inhibitory and excitatory input from “dual-function” neurons on the day of the LH surge. Results also reveal sexual dimorphisms in AVPV GABAergic/glutamatergic neurons, which may explain the absence of LH surges in males.

Progesterone receptor expression (PR) is regulated by estradiol in most regions of the adult and developing hypothalamus. The ventromedial nucleus (VMN) of the developing female rat appears to be the exception; PR is present in the absence of circulating gonadal hormones. In the present thesis, the circumstances governing this exception in steroid hormone regulation were explored. Sex, developmental and regional differences in the hormone regulation of PR were revealed. A battery of classic endocrine manipulations revealed that PR in the prenatal VMN was not affected by changes in hormone exposure. However, hormonal regulation of PR in the postnatal VMN was sexually dimorphic. In the neonatal VMN, gonadal hormones regulate PR in the male, but not in the female. Conversely, at a later postnatal age, PR becomes independent of testicular hormones, but is partially dependent on ovarian hormones. In contrast, these endocrine manipulations predictably altered PR within the developing MPN of both males and females, demonstrating a regional difference in estradiol-regulation of PR. In addition, the dependence of PR on estradiol is developmentally regulated. Estradiol induced PR in the adult female but did not affect PR in postnatal female VMN. Ontogenetic analysis revealed that estradiol could not induce PR in the postnatal VMN until around postnatal day 35. The transcription factor islet-1can interfere with the transcriptional function of estrogen receptors (ER), suggesting that Islet-1 may contribute to the sex-, age- and regional differences in estradiol regulation of PR. Levels of Islet-1 were significantly higher in the VMN of neonatal females than in adult females and neonatal males, consistent with the idea that Islet-1 could be obstructing the function of ER to a greater extent in the neonatal female VMN compared to the VMN of adult females and neonatal males. There were no age or sex differences in Islet-1 expression in the MPN. In the developing VMN, PR may not simply be dictated by the presence or absence of estradiol. On the contrary, factors such as Islet-1 may act to modify estrogen regulated gene transcription within the brain in age, sex and region specific ways.

Neurons extend processes that navigate to their synaptic target via a continuous sampling of guidance molecules and selective adhesion. In the Drosophila CNS, two large inter-neurons (the giant fibers) send their axons from the brain to the middle of the thorax, where each forms a large lateral synapse with a motor neuron (TTMn) that mediates the jump escape response. This mixed (electro-chemical) synapse is established during the first 30% of metamorphosis and mediates a fast (0.87 ms) and reliable response (100% at 100 Hz) to eye stimulation in adults. We examined the ability of the giant fiber (GF) to properly pathfind and form a synapse while selectively blocking endocytosis in, the GF, TTMn or both for 24 hours during a 100 hour metamorphosis. Through the directed expression of UAS-shibire (UAS-shi), under the control of a GAL4 driver, endocytosis is blocked when the fly is held at 30°C (considered a heat pulse (HP)). Blocking endocytosis in the GF during the first 60% of metamorphosis resulted in dramatic physiological and anatomical phenotypes. In these specimens, the probability of a GF to TTM or DLM connection was low and the connections made were significantly weaker then controls. In addition, the GF anatomy in these specimens was also aberrant; overgrowth resulted from an early HP (0–25%) and the GF was bendless in middle HP specimens (30–60%). Late HP (60% and afterwards) had no effect on GF anatomy or the probability of a connection to TTM or DLM, but weakened the strength of the synapses. When endocytosis was blocked in the postsynaptic TTMn, the critical period was only during the first 32% of metamorphosis. A HP 48% or afterwards had no effect on anatomy or physiology. Finally, a genetic interaction between a weak disruption in endocytosis by UAS- shi and weak over expression of UAS-semaphorin 1A (UAS-sema 1A) indicates a sema mediated repellent signaling aids in axon guidance and GF synaptogenesis. Based on the GF growth cone activity at the time of the HP and the resulting adult physiology and anatomy, three conclusions were drawn from these results. First, in late pupae (>60%) and adults endocytosis in the GF facilitates synaptic transmission and is not necessary to maintain the GF-TTMn synapse. From the beginning of metamorphosis to 60% was a presynaptic GF critical period where blocking endocytosis resulted in severe and permanent defects to TTM and DLM physiology and GF anatomy. In the postsynaptic TTMn, the endocytosis critical period was shorter, 0–32%. Second, once the GF reached the TTMn (30–60%), presynaptic endocytosis was critical for the removal of sema 1A to maintain synapse stability. In the postsynaptic TTMn, endocytosis was not critical for synapse stability. Third, maintaining sema 1A on the GF growth cone surface during pathfinding (0–25%) limited extra axonal branching.