Dynamics of Neuron-Specific Gene Expression During Development and in Response to Selective Lesions of the Rat Central Nervous System: A Dissertation

Synapse development and injury-induced reorganization in the nervous system have been extensively characterized morphologically, although, relatively little is known regarding the molecular and biochemical events that underlie these processes. In an attempt to better understand, at the molecular level, the role of the expression of synaptic proteins during synapse establishment and regeneration, this dissertation examines the dynamics of expression of the neuron-specific gene synapsin I during development and in response to selective lesions of the rat central nervous system. Synapsin I is the best characterized member of a family of nerve-terminal specific phosphoproteins implicated in the regulation of neurotransmitter release. During development, the expression of synapsin I correlates temporally and topographically with synapse formation, and recent physiological studies by Lu et al., (1992) have suggested that synapsin I may participate in the functional maturation of synapses. To better understand the temporal relationship between synapsin I gene expression and particular cellular events during development, we have used in situhybridization histochemistry to localize synapsin I mRNA in the rat central and peripheral nervous systems throughout embryonic and postnatal development, and into the adult period. During development, from the earliest embryonic time point examined (E12), the expression of the synapsin I gene was detectable in both the rat central and peripheral nervous systems. While, in general, levels of synapsin I mRNAs were high in utero, synapsin I cDNA probes revealed specific patterns of hybridization in different regions of the embryonic nervous system. To precisely determine the temporal onset of expression of the synapsin I gene during neuronal development, we examined in detail the appearance of synapsin I mRNA during the well characterized postnatal development of the cerebellum and hippocampus. In both regions, the onset of synapsin I gene expression correlated with the period of stem cell commitment to terminal differentiation. In a second phase, in accord with prior analyses, synapsin I gene expression increases to a maximum for a given neuronal population during synapse formation. In the adult rat brain, our data demonstrates a widespread yet regionally variable pattern of expression of synapsin I mRNA similar to that seen at earlier time points, with noteworthy exceptions. The greatest abundance of synapsin I mRNA was found in the pyramidal neurons of the CA3 and CA4 fields of the hippocampus, and in the mitral and internal granular cell layers of the olfactory bulb. Other areas abundant in synapsin I mRNA were the layer n neurons of the piriform and entorhinal cortices, the granule cell neurons of the dentate gyrus, the pyramidal neurons of hippocampal fields CA1 and CA2, and the cells of the parasubiculum. In general, the pattern of expression of synapsin I mRNA paralleled those encoding other synaptic terminal-specific proteins, such as synaptophysin, VAMP-2, and SNAP-25. Then, to determine specifically how synapsin I mRNA levels are related to levels of synapsin I protein in the adult rat brain, we employed in situhybridization histochemistry and immunohistochemistry to examine in detail the local distribution of both synapsin I mRNA and protein in the hippocampus. In short, these data revealed differential levels of expression of synapsin I mRNA and protein within defined synaptic circuits of the rat hippocampus. Based on these data we hypothesized that locally high levels of synapsin I mRNA in neuronal somata may reflect the ability of the nervous system to respond to select enviromental stimuli and/or injury by producing longterm changes in synaptic circuitry. To test this hypothesis and to better understand the regulation and putative role of synapsin I gene expression in the development of functional synapses in the central nervous system, we first examined the developmental pattern of expression of the synapsin I gene; in dentate granule neurons of the dentate gyrus and their accompaning mossy fibers during the main period of synaptogenic differentiation in the rat hippocampus. The results of these studies indicate a significant difference between the temporal expression of synapsin I mRNA in dentate granule cell somata and the appearence of protein in their mossy fiber terminals during the posmatal development of these neurons. Next, to investigate the regulation and putative role of synapsin I gene expression during the restoration of synaptic contacts in the central nervous system, we examined the expression of the synapsin I mRNA and protein following lesions of hippocampal circuitry. These studies show marked changes in the pattern and intensity of synapsin I immunoreactivity in the dendritic fields of dentate granule cell neurons following perforant pathway transection. In contrast, changes in synapsin I mRNA expression in target neurons, and in those neurons responsible for the reinnervation of this region of the hippocampus, were not found to accompany new synapse formation. On a molecular level, both developmental and lesion data suggest that the expression of the synapsin I gene is tightly regulated in the central nervous system, and that considerable changes in synapsin I protein may occur in neurons without concommitant changes in the levels of its mRNA. From a functional standpoint, our results suggest that the appearance of detectable levels of synapsin I protein in developing and sprouting synapses does not reflect simply synaptogenesis, but coincides with the acquisition of function by those central synapses

Behavioral and neurobiological consequences of social subjugation during puberty in golden hamsters

In golden hamsters, offensive aggression is facilitated by vasopressin and inhibited by serotonin. We tested whether these neurotransmitter systems respond to modifications resulting from the stress of threat and attack (i.e., social subjugation) during puberty. Male golden hamsters were weaned at postnatal day 25 (P25), exposed daily to aggressive adults from P28 to P42, and tested for offensive aggression as young adults (P45). The results showed a context-dependent alteration in aggressive behavior. Subjugated animals were more likely to attack younger and weaker intruders than nonsubjugated controls. Conversely, subjugated animals were less likely to attack animals of similar size and age. After testing, the animals were killed, and their brains were collected to determine whether these behavioral changes are underlined by changes in the vasopressin and serotonin systems. Social subjugation resulted in a 50% decrease in vasopressin levels within the anterior hypothalamus, a site involved in the regulation of aggression. Furthermore, whereas the density of vasopressin-immunoreactive fibers within the area was not significantly altered in subjugated animals, the number of serotonin-immunoreactive varicosities within the anterior hypothalamus and lateral septum was 20% higher in subjugated animals than in their controls. These results establish puberty as a developmental period sensitive to environmental stressors. Furthermore, the results show that changes in the vasopressin and serotonin systems can correlate with behavioral alterations, supporting the role of these two neurotransmitters in the regulation of aggression

Vasopressin/serotonin interactions in the anterior hypothalamus control aggressive behavior in golden hamsters

Studies in several species of rodents show that arginine vasopressin (AVP) acting through a V1A receptor facilitates offensive aggression, i.e., the initiation of attacks and bites, whereas serotonin (5-HT) acting through a 5-HT1B receptor inhibits aggressive responding. One area of the CNS that seems critical for the organization of aggressive behavior is the basolateral hypothalamus, particularly the anterior hypothalamic region. The present studies examine the neuroanatomical and neurochemical interaction between AVP and 5-HT at the level of the anterior hypothalamus (AH) in the control of offensive aggression in Syrian golden hamsters. First, specific V1A and 5-HT1B binding sites in the AH are shown by in vitro receptor autoradiography. The binding for each neurotransmitter colocalizes with a dense field of immunoreactive AVP and 5-HT fibers and putative terminals. Putative 5-HT synapses on AVP neurons in the area of the AH are identified by double-staining immunocytochemistry and laser scanning confocal microscopy. These morphological data predispose a functional interaction between AVP and 5-HT at the level of the AH. When tested for offensive aggression in a resident/intruder paradigm, resident hamsters treated with fluoxetine, a selective 5-HT reuptake inhibitor, have significantly longer latencies to bite and bite fewer times than vehicle-treated controls. Conversely, AVP microinjections into the AH significantly shorten the latency to bite and increase biting attacks. The action of microinjected AVP to increase offensive aggression is blocked by the pretreatment of hamsters with fluoxetine. These data suggest that 5-HT inhibits fighting, in part, by antagonizing the aggression-promoting action of the AVP system

Genome-Wide Association Study in BRCA1 Mutation Carriers Identifies Novel Loci Associated with Breast and Ovarian Cancer Risk

BRCA1-associated breast and ovarian cancer risks can be modified by common genetic variants. To identify further cancer risk-modifying loci, we performed a multi-stage GWAS of 11,705 BRCA1 carriers (of whom 5,920 were diagnosed with breast and 1,839 were diagnosed with ovarian cancer), with a further replication in an additional sample of 2,646 BRCA1 carriers. We identified a novel breast cancer risk modifier locus at 1q32 for BRCA1 carriers (rs2290854, P = 2.7×10-8, HR = 1.14, 95% CI: 1.09-1.20). In addition, we identified two novel ovarian cancer risk modifier loci: 17q21.31 (rs17631303, P = 1.4×10-8, HR = 1.27, 95% CI: 1.17-1.38) and 4q32.3 (rs4691139, P = 3.4×10-8, HR = 1.20, 95% CI: 1.17-1.38). The 4q32.3 locus was not associated with ovarian cancer risk in the general population or BRCA2 carriers, suggesting a BRCA1-specific associat

Temporal onset of synapsin I gene expression coincides with neuronal differentiation during the development of the nervous system

Synapsin I is the best characterized member of a family of nerve terminal-specific phosphoproteins implicated in the regulation of neurotransmitter release. During development, the expression of synapsin I correlates temporally and topographically with synapse formation, and recent physiological studies (Lu et al. [1992] Neuron 8:521-529.) have suggested that synapsin I may participate in the functional maturation of synapses. To better understand the temporal relationship between synapsin I gene expression and particular cellular events during neuronal development, we have used in situ hybridization histochemistry to localize synapsin I mRNA throughout the rat central and peripheral nervous systems during embryonic and postnatal development. From the earliest embryonic time points assayed (E12), the expression of the synapsin I gene was detectable in both the central and peripheral nervous systems. While, in general, levels of synapsin I mRNAs were high in utero, synapsin I cDNA probes revealed specific patterns of hybridization in different regions of the embryonic nervous system. To determine precisely the temporal onset of expression of the synapsin I gene during neuronal development, we examined in detail the appearance of synapsin I mRNA during the well characterized postnatal development of granule cells of the rat cerebellum and hippocampus. In both regions, the onset of synapsin I gene expression correlated with the period of stem cell commitment to terminal differentiation. Finally, our data demonstrate that, in a second phase, synapsin I gene expression increases to a maximum for a given neuronal population during a particular phase of differentiation, i.e., synaptogenesis

Synapsin I gene expression in the adult rat brain with comparative analysis of mRNA and protein in the hippocampus

Synapsin I is the best characterized member of a family of neuron-specific phosphoproteins thought to be involved in the regulation of neurotransmitter release. In this report, we present the first extensive in situ hybridization study detailing the regional and cellular distribution of synapsin I mRNA in the adult rat brain. Both the regional distribution and relative levels of synapsin I mRNA established by in situ hybridization were confirmed by RNA blot analysis. Our data demonstrate the widespread yet regionally variable expression of synapsin I mRNA throughout the adult rat brain. The greatest abundance of synapsin I mRNA was found in the pyramidal neurons of the CA3 and CA4 fields of the hippocampus, and in the mitral and internal granular cell layers of the olfactory bulb. Other areas abundant in synapsin I mRNA were the layer II neurons of the piriform cortex and layer II and V neurons of the entorhinal cortex, the granule cell neurons of the dentate gyrus, the pyramidal neurons of hippocampal fields CA1 and CA2, and the cells of the parasubiculum. In general, the pattern of expression of synapsin I mRNA paralleled those encoding other synaptic terminal-specific proteins, such as synaptophysin, VAMP-2, and SNAP-25, with noteworthy exceptions. To determine specifically how synapsin I mRNA levels are related to levels of synapsin I protein, we examined in detail the local distribution patterns of both synapsin I mRNA and protein in the rat hippocampus. These data revealed differential levels of expression of synapsin I mRNA and protein within defined synaptic circuits of the rat hippocampus