Carlos A. Jimenez-Rivera - US grants

APPLICANT'S ABSTRACT: The major goal of this research is to explore whether a net dysfunction of the central noradrenergic system physiology occurs after a paradigm of chronic cocaine administration. A research plan will be developed to study changes in locus coeruleus (LC) function and norepinephrine (NE) neuromodulatory actions in the somatosensory and cerebellar cortices after repeated cocaine administration. An initial series of investigations will examine several parameters of LC physiology in terms of spontaneous LC firing rate and patterns of activity, and in changes in alpha-2-receptor sensitivity to agonists and antagonists compounds. In addition, studies will also investigate alterations in the behavior of LC responses to sensory evoked discharges and iontophoretically-induced excitation by the release of putative neurotransmitters that supposedly mediate LC reaction to sensory stimulation. We hypothesized that cocaine, by virtue of its ability to increase synaptic level of NE or other independent actions, can alter LC neuron activity and thus the input/output relationship of NE cells. A second series of studies will examine changes in NE modulatory actions in the somatosensory and cerebellar cortices, two target structures of LC noradrenergic innervation. Experiments using microiontophoresis application of putative neurotransmitters glutamate, GABA and acetylcholine together with peripheral and central nervous system stimulation procedures will be employed to quantitatively assess the NE modulatory actions on synaptic efficacy within the aforementioned brain circuits. The purpose of these experiments is to identify possible alterations in NE neuromodulatory actions which might form part of the neural substrates responsible for cocaine's psychostimulant effects. Finally, the "in vitro" receptor autoradiography technique will be employed to measure the relative distribution of alpha-2 receptors in the LC in an effort to observe any changes in this important receptor that control normal LC physiology. Overall, this approach is aimed at developing a comprehensive understanding of the effects of chronic cocaine administration on CNS noradrenergic physiology which might form part of the basis of cocaine's addiction.

Norepinephrine (NE) modulates glutamate-mediated excitation in many brain areas. In the prefrontal cortex, glutamate is strongly implicated in some of the long-term effects of cocaine such as sensitization; a persistent behavior similar to panic attack, anxiety and eventually paranoid psychosis. Jimenez-Rivera et. al 1998 (Abstract 7tb PR Neuroscience Conference) have shown that some of the long term effects of cocaine requires activation of alpha noradrenergic receptors. Preliminary evidence from Ortiz et al., suggests that glutamate uptake is increased in synaptosomes from cocaine-treated rats depleted of NE. On the other hand, Ulrich et al. (1998) have identified RNA aptamers that can be displaced by cocaine and presents the unique opportunity of using these aptamers as possible uptake inhibitors and modifiers of cocaine sensitization. Cocaine inhibits the reuptake of dopamine (DA) and norepinephrine with very similar potency. The collaborative research proposed in this project is focused on clarifying the neuromodulatory role of NE on glutamate transmission in the PFC and how this role is modified by cocaine. Neuromodulation of glutamatergic tone by NE in the prefrontal cortex is critical for the development of some of the long-term effects of cocaine (sensitization). This hypothesis will be tested by pharmacologically altering NE input in the PFC, while monitoring the neurochemical changes in glutamate transmission. In parallel, RNA aptamers that could interfere with DA and NE uptake will be tested for their ability to interfere with the modulatory effects of NE on glutamate transmission in the PFC. This project is the direct result of CMBN's long-term interactions. Dr. Jimenez- Rivera at Universidad Central del Caribe and Dr. Ortiz (Univ. of Puerto Rico) have been interacting with Dr. Hess (Cornell Univ.) for the past three years. The applicants will focus on the behavioral, electrophysiological and neurochemical modifications of NE modulation of g1utamatergic tone in the PFC of cocaine-treated rats. On the other hand, the collaborators will synthesize and screen for RNA aptamers with defined characteristics (ie. cocaine displaceability, among others). The experimental scheme proposed maximizes the expertise of both components and is consistent with a convergent effort towards understanding neuroadaptive changes. This collaboration provides the opportunity for participants at both institutions to further their theoretical and practical approaches exemplified by the work of Ulrich et al., 1998. The results of the proposed experiments are crucial for understanding how NE modulates glutamate neurotransmission in the PFC. Pharmacological manipulations (ie. Cocaine, RNA aptamers) should reveal the relevance of NE modulatory role(s) as well as, provide novel therapeutic strategies.

DESCRIPTION (provided by applicant): Cocaine sensitization is a progressive and long-lasting enhancement of the motor stimulant effect induced by a subsequent cocaine challenge. The goal of this work is to elucidate the role of alpha adrenergic receptors at the VTA and RFC in the development and expression of cocaine sensitization. There are three specific Aims: 1) Assess whether microinjections of alpha noradrenergic receptor agonists and antagonists into the VTA or RFC modulate the process of cocaine sensitization. Using the model of cocaine sensitization, intra-VTA or -RFC microinjections of selective alpha-noradrenergic receptor agonists and antagonists will be used to localize the anatomical structure mediating the pharmacological actions of these drugs. 2) Identify changes in alpha noradrenergic receptors at the VTA and RFC induced by behavioral sensitization. We will investigate alpha-1 and alpha-2 adrenergic receptor mediated G protein activation in cocaine-sensitized rats using agonist-stimulated [35S] GTPyS binding as a functional assay. The hypothesis is that development of cocaine sensitization involves an increase in alpha-1 and a decrease in alpha-2 receptor functional sensitivity in both VTA and RFC areas, which will promote cocaine sensitization. Correlations will be made between changes in functionality and number/affinities of receptors in each structure using an in vitro receptor autoradiography assay. We will also assess whether changes in these noradrenergic receptors play a role in the initiation or expression of behavioral sensitization by comparing animals after a 7days withdrawal period. 3) Determine the role of alpha-2 receptors in the modulation of glutamate release at .the VTA. In vitro whole cell patch clamp recordings will be employed to study the role of alpha-2 receptors in the modulation of glutamate release onto VTA dopamine cells. The effects of alpha-2 adrenoreceptor agonists on glutamate-induced excitatory postsynaptic currents (EPSCs) will be assessed. Paired-pulse ratios and miniature EPSCs will be employed to provide evidence that the EPSC's alterations are localized at the presynaptic terminal. The hypothesis is that presynaptic alpha-2 receptors control glutamate release onto VTA neurons. A sub-hypothesis is that there is a decreased alpha-2 -induced inhibition of glutamate release in cocaine sensitized rats. Changes in alpha adrenoceptor modulation of glutamate release into VTA cells after a withdrawal period will also be determined. Understanding of alpha adrenoceptor modulatory changes in cocaine sensitization will increase our knowledge of the role of the noradrenergic system in cocaine addiction and might provide possible avenues for therapeutic pharmacological interventions..

DESCRIPTION (provided by applicant): Cocaine sensitization is a progressive and long-lasting enhancement of the motor stimulant effect induced by a subsequent cocaine challenge. The goal of this work is to elucidate if the persistent activity of PKM? in the VTA and NAc sustains the development and expression of cocaine sensitization. There are three specific aims: 1) to evaluate the effect of PKM? inhibition in the development and expression of cocaine behavioral sensitization. We will use a behavioral sensitization paradigm with intra VTA and intra NAc microinfusions of ZIP (PKM? selective inhibitor) at different stages of the sensitization process to determine PKM? 's role in the development and expression of behavioral sensitization. The hypothesize is that ZIP administration in the VTA and NAc will block the development and expression of cocaine sensitization 2) To determine the role of PKM? inhibition on AMPA currents and AMPA/NMDA ratios in VTA DA and NAc medium spiny neurons after cocaine sensitization. Using whole cell recordings from VTA DA and NAc medium spiny neurons, we will measure AMPA mediated EPSCs and AMPA/NMDA ratios from rats repeatedly exposed to cocaine. ZIP will be used to assess whether PKM? is necessary for the maintenance of the cocaine-induced potentiation. We hypothesize that ZIP will induce a decrease in AMPA currents and therefore, will diminish AMPA/NMDA ratios to basal levels in cocaine sensitized animals. 3) To assess the cellular substrate by which the PKM? inhibitor exerts its effects on addiction related plasticity. Using western blot analysis we will measure PKM¿ and AMPA receptor subunit levels in the VTA and NAc during development and expression of cocaine sensitization. Second, we will use microinjections of the synthetic peptide Tat-GluR23y and rectification index curves in whole cell patch recordings to examine the contribution of GluR2-dependent and GluR2-lacking AMPA receptors in maintaining PKM? effects in cocaine sensitization. The hypothesis is that cocaine sensitization involves an increase in PKM? levels which is acting by inhibiting GluR2-dependent AMPA receptor removal from postsynaptic sites. This research will provide a better fundamental understanding of drug-induced plasticities in the CNS and might present possible avenues for therapeutic pharmacological interventions.