Center for Molecular, Developmental and Behavioral Neuroscience
Molecular mechanism of brain injury: P2Y2 nucleotide receptor in ischemia.
Fernando A. González, Ph.D.
In stroke, cerebral blood vessels contract or become so occluded that blood flow is blocked. The affected brain tissues suffer from a transient event of lack of oxygen, or ischemia, which leads to neuronal injury and the activation of neuronal plasticity mechanisms that limit the extent of the damage. However, activation of astrocytes may enhance the extent of tissue damage in the brain following an ischemic insult. After mechanical or ischemic insult. After mechanical or ischemic trauma to the central nervous system, the release of nucleotides together with neurotransmitters into the extracellular space activate P2Y nucleotide receptors and can act in synergistic combination with growth factors to stimulate astrocyte proliferation. While much is known about the factors that cause the activation of astrocytes, very little is known about the molecular mechanisms that lead to astrocyte activation during ischemic events. To understand these mechanisms and the role of extracellular nucleotides, it will be important to elucidate the molecular determinants by which P2Y2 receptors activate signal transduction pathways in astrocytes. Therefore we propose to characterize the signal transduction pathways coupled to P2Y2 receptor that mediate astrocyte activation using primary astrocytes from neonatal rodent brain, immortalized astrocytes (DITNC cells) and normal human astroctyes. We will test the hypothesis that an Arg-Gly-Asp motif in the first extracellular loop of the P2Y2 receptor promotes efficient coupling to PLC that is dependent upon receptor association with the anb3/b5 integrins. The relevance of P2Y2 receptor/integrin interactions in astrocytes to the activation of intracellular MAPK cascades also will be investigated. Furthermore, we will test the hypothesis that P2Y2 receptor agonists cause trans-activation of the tyrosine kinase activity of the EGF receptor and that this interaction plays a role in the physiological outcomes of P2Y2 receptor activation in astrocytes. Finally, we will probe the hypothesis that cerebral ischemia upregulates P2Y2 receptor mRNAs in astrocytes and/or microglial cells and that reactive gliosis is an important process preceding neuronal cell death. These studies will utilize the rat model of focal ischemia induced by occlusion of the middle cerebral artery (MCA) and the model of global ischemia induced by ligation of the common carotid arteries (CCA). The studies proposed here will provide novel targets for the development of therapeutic strategies for the management of brain injury after stroke, synaptogenesis, as well as other neurological conditions.
Emotional memory: Genomic basis of emotional learning and memory.
Sandra Peña, Ph.D.
The central goal of this project is to characterize the molecular events involved in the acquisition and extinction of emotional memory, using the behavioral paradigms of conditioned taste aversion (CTA) and condition fear (CF), respectively. A number or neuropsychiatric disorders, such as post traumatic stress disorder and schizphrenia, display aberrations in the development of proper emotional associations. Numerous studies suggest that the amygdala and the medial prefrontal cortex (mPFC) are involved in the processing of emotion in the brain, and may play a role in the pathophysiology of schizophrenia and anxiety disorders. CTA and CF involve the development of long-term memory, which depends on gene transcription and protein synthesis in the amygdala. The studies described in this proposal will define the gene regulatory networks in the amygdala and the mPFC that are involved in emotional learning and memory. Specific Aim 1 will define gene regulatory pathways that lead to long-term memory in CTA. These studies are divided in three stages. Initially, amygdala RNA is extracted at different times after one CTA trial and used to prepare 32P-labeled cDNA probes for cDNA microarray analysis. Thus, a time course of gene expression profiles for long-term memory of CTA is defined using cDNA microarrays. Next from the genes that are expressed in the microarray analysis, a group of candidate genes is selected based on specific criteria and subjected to validation studies with Northern blots and in site hybridizations. Finally, the expression data is used for gene cluster analysis, modeling, and the definition of gene regulatory networks. The gene networks and models are tested biologically with antisense knockdown approaches, protein kinase inhibitors, and protein kinase activity assays. Similarly, Specific Aim 2 will use cDNA microarrays to study NMDA receptor dependent changes in gene expression in mPFC and the amygdala as a result of extinction of fear conditioned behavior. Rather than erase the association between the conditioned and unconditioned stimuli, extinction of fear is thought to involve new learning. Previous work on the molecular basis of fear conditioning has solely on the acquisition phase, and has not examined extinction. Projections from the mPFC to the mPFC to the amygdala have been implicated in extinction. Extinction-associated changes in the expression of selected candidate genes in mPFC and amygdala will be confirmed with Northern blots and in situ hybridization. Finally, the role of specific genes in extinction will be assessed using antisense knockdown approaches. These studies will provide important information as to the genetic basis of emotional learning and memory. Identifying the genes activated during emotional learning is the first step in the development of future "gene therapies" for emotional disorder.
Cocaine-seeking behavior: Neural and molecular mechanisms in striatal learning.
Carmen Maldonado, Ph.D.
Previously, an extensive amount of research has reported that the nucleus accumbens (NAS) and the medial prefrontal cortex (mPFC), as part of the dopaminergic mesolimbic system, play a major role in the activating and reinforcing properties of psychostimulants. In addition, biochemical and neuroanatomical studies have indicated that several neuropeptides present within the mesolimbic dopaminergic neurons mediate the reinforcing effects of cocaine. Specifically, within the nucleus accumbens, neurotensin receptors and cholecystokinin (CCK) receptors CCKA and CCKB may be involved in the control of cocaine reward. Also, within the medial prefrontal cortex both of these neuropeptides exert neurochemical actions that seem to modulate cocaine reinforcement. In addition to neuropeptide modulation, recent studies reveal the presence of cellular and molecular neuroadaptations in psychostimulant addiction. In particular, these molecular studies have focused on the role of transcription factors as well as the related changes in gene expression present following chronic exposure to psychostimulants. However, little is known about the role of these neuropeptides and the genetic expression in the motivational learning involved in cocaine-seeking behavior. Experimental results from this proposal may contribute to a more detailed understanding of: 1) the specific roles of neuropeptides within DA neurons in cocaine-seeking behavior 2) to what extent reinstatement of cocaine-seeking behavior elicits molecular and cellular changes that result in specific gene expression and 3) to further characterize the neurochemical and genetic basis of cocaine addition. Specifically our objectives are:
Specific Aim 1: To inivestigate the role of CCK and neurotensin within the mesolimbic dopamine system on environmentally induced cocaine-seeking behavior.
Specific Aim 2: Are genes encoding proteins involved in dopaminergic neurotransmission and neuropeptide activation within the nucleus accumbens and medial prefrontal cortex up-regulated by acquisition and reinstatement of cocaine-seeking behavior?.
Development of maternal behavior: Neurosteroid effects on the structure and function of a sexually dimorphic mammalian brain network.
Juan C. Jorge, Ph.D.
The central goal of this project is to determine the structural, physiological,
This web site is supported by an Institutional Development Award (IDeA), P20RR15565, from the National Center for Research Resources, National Institutes of Health.