ABSTRACT NO. 1A MODEL DESCRIBING RAPID RELIEF OF RECTIFICATION OF GLIAL Kir CHANNELS S.M. Antonov1, M.J. Eaton2, J. Krušek3, R.W. Veh4, T. Pannicke5, A. Reichenbach5 and S.N. Skatchkov2 Inst. Evol. Physiol. and Biochem., St. Petersburg1, Dept. Biochem., Univ. Cental del Caribe, Bayamon2, Inst. Physiol., Prague3, Inst. Anat., Berlin4, Inst. Brain Res., Leipzig, Germany5
Although, (i) ODC is poorly expressed in healthy glial cells (Prog. Neurobiol 57(1999) 485; Glia 23(1998)209), (ii) the highest level of spermidine/spermine (SD/SP) is found in glia (Glia 19(1997)171; Glia 31(2000)84) suggesting that uptake of SD/SP is a major mechanism of accumulation. (iii) Glial (Müller cell) weakly-rectifying K+-channels (Kir 6.1/SUR1 and Kir 4.1) (Neuroreport 12 (2001)1437) are co-localized with SD/SP (their blockers) and (iv) the SD/SP content positively correlates with (v) the degree of rectification in cell compartments (Glia 31(2000)84). Rectification declines with (v) membrane depolarization and with (vi) lowering [K+]o (Glia 15(1995)54; Glia 27(1999)171). Here we observed that after gliotoxin-induced depolarization (of 60 mV) the decreased SD/SP content correlates with loss of rectification. Also, we found rapid relief of rectification under physiologically relevant conditions as subsecond changes in [K+]o in repeated sequence of 3-1-3-10-3 mM decreased the ratio of inward to outward current more than twice (from 4.6±0.6 to 1.9±0.2, n=9) at normal membrane potential -90 mV. We propose a model explaining relief of rectification during physiological conditions. Thus, switching from rectifying channels to linear ones may be a feedback mechanism for switching from long-range spatial K+-buffering to local buffering and thus may play a significant role in regulation of neuronal activity. Supported by NIH-MBRS-GM5-340508/09, NIH-RCMI G12RR03035, DFG Ve-187/1-2, RFBS 020449685, and BMB+F (IZKF, 01KS 9504/C5).
ABSTRACT NO. 2Norepinephrine (NE) reduces the amplitude of the hyperpolarizing-activated cationic current Ih of dopamine (DA) cells in the Ventral Tegmental Area (VTA) Francisco Arencibia-Albite, Rafael Vázquez-Torres, Rocío Castro, Mónica Feliú-Mójer, J.T. Williams, Carlos A. Paladini and Carlos A. Jimenez-Rivera Department of Physiology Universidad Central del Caribe Bayamón, Puerto Rico
The Ih current is a cationic depolarizing current that is activated by hyperpolarization. This ionic current promotes cell excitability by reducing the time course of inhibitory synaptic potentials (IPSP). In addition, Ih gives the cell the ability to fire in rhythmic burst (pacemaker activity). Deviations from the normal pattern of cell excitability of dopamine nerve cells are known to play an important role in the pathophysiology of parkinson, schizophrenia and drug abuse. Therefore, it is of great importance to understand the molecular and biophysical mechanisms that modulate cell excitability in DA cells. The goal of the present work was to characterize how NE administration affects the voltage dependence of Ih in DA cells from the VTA. Our study consisted of whole cell voltage clamp recordings performed on VTA DA cells from male Sprague-Dawley rats (15 – 30 days post natal). Only neurons with a robust Ih and medial to the medial terminal nucleus of the accessory optic tract were selected. The Ih current was evoked by hyperpolarizing the cell in 10 mV voltage steps (pulse width 1 sec.) from – 60 mV (holding potential) to –140 mV. Ih was calculated as the difference between the current at the end of the voltage step (Iss) minus the current at the end of the capacitive peak (Iin). Recordings of Ih were made from two groups of VTA DA cells: DA neurons from naïve rats (n = 5) and from rats (n = 6) that received a cocaine injection (15 m/ Kg i.p.) per day for a period of three days. It was found that NE (at 40 mM) reduced the amplitude of Ih by increasing the net membrane conductance in naïve and cocaine treated animals. In naïve rats, NE decreased Ih current amplitude by 33.95 % (n = 5). The ability of NE to inhibit Ih was diminished in DA cells from rats that received the cocaine treatment and showed initiation of behavioral sensitization. For the latter group, the Ih amplitude was reduced by 10.95 % (n = 3). In contrast, the Ih amplitude was reduced by 41.43 % (n = 3) in DA cells from rats that received the cocaine treatment but did not show initiation of behavioral sensitization. A hallmark of behavioral sensitization to cocaine is an increase in burst activity in VTA DA cells. The findings of this study suggest that this increase in burst activity may be, to some extent, caused by the loss of inhibitory action of NE on Ih. Supported by NINDS (RCMI) SNRP Program, grant #NS 39408 and GM-50695 to C.A.J.R.
ABSTRACT NO. 3P2Y2 Receptor Signaling and Membrane Microdomains in 1321N1 Astrocytoma Cells. 1Aquino, E., 2Velázquez, G., 3Maldonado, H., 2Silva, W., and 1González, F.. 1Department of Chemistry, UPR, Río Piedras Campus, PR; 2Department of Physiology, UPR Medical School, PR; 3Department of Pharmacology, UCC Medical School, Bayamón, PR.
Nucleotides, released during ischemia or mechanical injury to nervous tissue, activate P2Y purinergic receptors, that act synergistically with growth factors to stimulate astrocyte proliferation, astrogliosis and the formation of glial scarring. Astrocytic P2Y2 purinergic receptors couple to mitogen-activated protein kinase, ERK1/2, PLC-dependent mobilization of intracellular calcium, activation of focal adhesion kinase (FAK), c-src kinase, and other signaling events. Membrane microdomains and caveolae, are believed to facilitate protein-protein interactions among signaling proteins, thus integrating pathways. Caveolae are specialized membrane microdomains with a demonstrated role in receptor-mediated signaling of receptor tyrosine kinases (i.e. receptors for EGF, NGF, and PDGF) and G-protein coupled receptors (i.e. beta-adrenergic, muscarinic, angiotensin II, and bradykinin). In this study we have explored the participation of caveolae in P2Y2 receptor signaling in 1321N1 astrocytoma cells transfected with the P2Y2 receptor gene. As a first approach, caveolin gene expression was assessed via a combination of experimental approaches in naïve, unstimulated 1321N1 astrocytoma cells. RT-PCR analysis revealed the expression of the caveolin-1 (cav1) mRNA. The expression of cav1 was confirmed by immunoblotting, and indirect immunofluorescence analysis of the cells. Immunofluorescence analysis via laser scanning confocal microscopy revealed a punctuate distribution pattern for the P2Y2 receptor, cav1, and clathrin-coated pits/vesicles marker proteins. These findings suggested the potential localization of the P2Y2 receptor in these membrane microdomains. This possibility was confirmed by cellular fractionation via detergent-free sucrose density gradient centrifugation. These preliminary findings have led us to propose a model of P2Y2 receptor trafficking, integrating both subcellular transport routes or membrane microdomains in astroglial cells. Our expanded knowledge on astroglial cell signaling, and the potential for discovery of novel neuromodulatory interactions in membrane lipid microdomains, shall permit development of novel strategies and therapeutic approaches for the treatment of neurological disorders, where both ATP as a neurotransmitter and lipid membrane microdomains, are emerging as key players, i.e. stroke, Alzheimer’s disease, and various forms of muscular dystrophy. This work was supported in part by NIH grants S06-GM08102 (MBRS-SCORE Program) and RR15565 (NCRR COBRE Program) to FG; and, NIH grants S06-GM08224 (MBRS-SCORE Program) awarded to WS.
ABSTRACT NO. 4SPECIFIC BRAIN REGION EFFECTS OF LITHIUM ON GENE EXPRESSION AND BEHAVIOR IN THE RAT. M. Al Banchaabouchi*; R. Menndez; M. Noel; A. Rodriguez; D. Mejia; H. Ge; S. Peña de Ortiz; C.S. Maldonado-Vlaar Dept Biol, Univ Puerto Rico, San Juan, Puerto Rico
The mood stabilizing effects of lithium (Li) lie on the long-term changes in neuronal synaptic function. We investigated the effects of Li on protein kinase C (PKC) and hzf-3/nurr1 gene expression. In Exp.1, protein levels of PKC isoforms were determined after treating rats with Li carbonate (0.1%, Li2CO3 in food) for 7 weeks. Results show a significant decrease only in PKC gamma levels in the membrane fraction of hippocampus and an increase both in membrane and cytosolic fractions of the amygdala. For Exp. 2, rats were exposed to a 0.1% Li for 1 week followed by 0.2 % Li for 3 weeks and were then used for immunohistochemical or behavioral studies. Results showed that Li caused a significant decrease in HZF-3 protein levels in the CA1 region of the hippocampus and the dorsal endopiriform nucleus, while no significant changes occurred in the CA3, perirhinal cortex or in the lateral amygdala. Additional rats were tested for locomotor activity and spatial learning. In behavioral studies, Li rats showed a significant decrease in rearing activity. Furthermore, in a hippocampal-dependent food reward task, trained Li rats spent significantly less time completing the task than controls, while no differences were observed in total errors between the groups. Also, Li rats were significantly more efficient in a 48 hr retention test. When food was visible, Li rats completed faster than controls the task, suggesting an enhanced motivation and flavor sensitivity. Future experiments will further examine Li effects on hzf-3 expression and neuronal activity due to learning within different brain regions. Supported by: NIDA-DA11665-03; NIH-COBRE 5P20RR15565-02
ABSTRACT NO. 5GENES ENCODING PROTEINS WITHIN THE NUCLEUS ACCUMBENS SUBREGIONS, ARE AFFECTED BY REINSTATEMENT OF COCAINE-SEEKING BEHAVIOR. L.J. Alvarez*; D.L. Ramos; J. Flix; M. Al-Banchaabouchi; R. Menndez; S. Peña de Ortiz; C.S. Maldonado-Vlaar. Department of Biology, Univ Puerto Rico, San Juan, Puerto Rico
Molecular studies have implicated several immediate early genes and neuropeptides in eliciting cocaine reward. These experiments are aimed at further characterizing which genes within the nucleus accumbens subregions (NA) may be involved in eliciting cocaine-seeking behavior induced by environmental stimuli. In Exp.I, nave rats were sacrificed and their brains sectioned and stained with thionin. Slices were examined under a microscope and laser capture microsection (LCM) was utilized to collect cells from specific NA core and shell areas. RNA extraction was conducted followed by Real Time PCR. Primers for neurotensin, dynorphin and other house keeping genes were designed and used. Preliminary results showed good RNA extraction from these cells following LCM, and the presence of rat-GAPDH gene in both core and shell. In Exp.2, rats were implanted with intravenous jugular catheters. Following recovery from surgery, rats were trained to discriminate two different environments with either saline or cocaine intravenous infusions until stable baseline. Then rats went through extinction phase. When extinction criteria (5 presses/5 days) was reached, rats were reinstated only with the presentation of cocaine-paired cue. All animals showed reinstatement behavior. Brains from reinstated rats were processed as in Exp.I. Ongoing studies will examine which genes are being regulated by cocaine seeking-behavior. We hypothesize that neurotensin and dynorphin genes will show an up-regulation following the exposure to environmental stimuli previously associated to cocaine reward. Supported by: NIH-COBRE 5P20 RR15565-02
ABSTRACT NO. 6 Anabolic steroid effects in the brain and behavior: the C57Bl/6 mice model. J Barreto-Estrada, J Barreto, A Fortis, Y Fortis, G Corretjer, JC Jorge Department of Anatomy, Medical Sciences Campus, University of P.R. 00936 Anabolic androgenic steroids (AAS) are derivatives of testosterone that were originally design for therapeutic purposes. However, during the last few decades AAS have become a major class of drugs of abuse, despite the detrimental effects on endocrine function and affect. In this study we investigated the modulation of AAS in locomotor, aggressive, anxiety, social, and sexual behaviors in adult mice (C57Bl/6) from both sexes. Chronic exposure to AAS was achieved using an osmotic pump filled with 17α-methyltestosterone (7.5 mg/kg). After 14 days of treatment an increase in body weight was observed in both sexes accompanied by a decrease in ovarian and testicular weight. The weight of uterine tubes was increased, this correlated with irregular estrous ciclicity as a high percent of mice were locked in estrus. Socio-sexual tests showed activation of male-typical sexual behavior in AAS-treated females. Automated systems to measure light-dark transitions and anxiety-related behaviors revealed an anxiolytic response in AAS-treated males. Specifically, an increase in the number of light-dark transitions in activity chambers, an increase in the percent of time spent in open arms in the elevated plus maze (EPM), and an increase in the number of water licks in a conflict paradigm involving punishment (Vogel Conflict Test) was observed. Taken together, we have found that AAS exposure induced male-typical sexual behaviors in females, whereas in males it induced a female-like profile in affective components of behavior. Further studies will be performed to evaluate the basic mechanisms underlying these changes in neuroendocrine function and behavior through the GABAergic system. Support for this study was provided by a Young Investigator Award to JCJ (BRIN-PR Program- P20 RR16470 from the National Center for Research Resources, NCRR-NIH).
ABSTRACT NO. 7 EXOGENOUS Zn+2 INTERACTIONS WITH THE NMDA
GLUTAMATE RECEPTOR.
Berríos-Cartagena, N., De Cardona-Juliá, E.A.; Román-Sierra, L.;
Feliciano-Bonilla, M.M., Rubio-Dávila, M.M., and Ortiz, J.G. Glutamate (Glu), the most important excitatory amino acid neurotransmitter in the mammalian Central Nervous System, is of great importance due to its involvement in the processes of memory and learning. It is recognized by specialized receptors found in the post-synaptic neurons. There are two types of receptors for glutamate: the ionotropic (NMDA, AMPA, KA) which allow the flow of ions, and the metabotropic (Groups I, II and III) which are coupled to G Proteins and activate second messengers. An excessive activation of these receptors due to an increase in [Glu] (>1-5µM), results in oxidative stress and, eventually, in massive neuronal loss. This excitotoxicity has been implicated in various neurodegenerative conditions like Alzheimer’s, Amyotrophical lateral sclerosis, Epilepsy, Global Ischaemia and Parkinson’s Disease. It is well known that Zinc (Zn2+), one of the endogenous trace elements and the most abundant oligoelements in nervous tissue, is co-released with glutamate during synaptic transmission (Weiss,J.H.; Sensi,S.L.; Koh,J.Y. 2000). Zinc, like glutamate, also has a site on site on the NMDA receptor. Furthermore, Zn2+ intervenes with Glutamate receptors and transporters and it has been found that milimolar concentrations, it can produce excitotoxitity (López-García,C.; Molowny,A.; Ponsoda,X.; Nácher,J.; Sancho-Bielsa,F. 2001). Due to the relationship between Zn2+ and Glutamate in excitotoxicity we conducted dose-response experiments in order to observe the interaction of this metal with the NMDA receptor at different concentrations. Experiments on rat synaptic cortical membranes utilizing [3H]MK-801 binding to NMDA receptors. The results showed that at low nanomolar (1-3 nM) and physiological micromolar (5-50µM) concentrations, Zn2+ significatly increases the binding of [3H]MK-801. On the other hand, at high nanomolar (80nM) concentrations, Zn2+ inhibits this binding with a great significance. This results were confirmed utilizing C6 glioma cell line with normal and elevated K+ (10mM) concentrations. In contrast, experiments with [3H]Methyl-Glutamate binding, a ligand for glutamate excitatory amino acid transporters (EAAT) were achieved. The results showed that the effects of Zn2+ were not direct on the EAAT’s.
Mitochondrial DNA Damage and Repair in Young Caloric Restricted Mice. Berríos Vega, L. W., Torres-Ramos, C. A., and Ayala-Torres, S. Universidad Central del Caribe School of Medicine, Bayamón, Puerto Rico
Caloric restriction (CR) is the most robust intervention of slowing aging and extending life span. Although the molecular basis of caloric restriction-life span extension remains unknown, several studies have suggested that CR acts by reducing the levels of oxidative stress. Radical oxygen species cause damage to DNA, primarily to mitochondrial DNA (mtDNA) and this damage can lead to mitochondrial dysfunction. It has been suggested that oxidative mtDNA damage is a major contributor to age-associated loss of brain function in the mouse. The purpose of this study was to determine if CR reduces oxidative DNA damage in mouse brain mitochondria after induction of oxidative stress by the mitochondrial neurotoxin 3-nitropropionicacid (3-NPA). We applied a quantitative polymerase chain reaction (QPCR) assay to assess the relative amounts of mtDNA damage in striatum obtained from 5-month-old mice fed ad libitum (AL) or fed a caloric restricted diet for 6 weeks. The QPCR assay is based on the principle that oxidative DNA damage such as strand breaks, base modifications and abasic sites block the progression of any thermostable PCR polymerase on the DNA template, resulting in a decrease in amplification of a target DNA sequence. We found a significant increase in mtDNA damage in striatum from AL and CR mice after 6h of 3-NPA treatment (n=6 mice for each time point) as shown by a 34% and 35% decrease in the relative amplification of a 10-kb mtDNA fragment, respectively, as compared to controls (n=6 mice for each diet group). After 24 h of 3-NPA treatment there is still mtDNA damage present in AL and CR mice as shown by a 20% and 15% decrease in amplification of the 10-kb mtDNA fragment, respectively. However, there were no differences between the CR and AL animals, suggesting that CR has no effect in protecting the mtDNA from damage in young mice briefly exposed to CR. Future experiments will examine the effect of CR on the induction of mtDNA damage by 3-NPA in middle age (17-month-old) and aged (27-month-old) mice.
THE ROLE OF ENGRAILED PARALOGUES IN THE CONTROL OF AXON PATHFINDING AND TARGET RECOGNITION. Jonathan M. Blagburn and Bruno MarieInstitute of Neurobiology, Medical Sciences Campus, University of Puerto Rico, San Juan, Puerto Rico 00901, USA.
Engrailed paralogues function in patterning and regionalizing the nervous system as well as determining successive events of neuronal differentiation. What remains unclear is whether those paralogues have different functions. In the mouse, even though engrailed-2 can functionally substitute engrailed-1, mutations in these genes produce different phenotypes because of their divergent expression patterns. Similarly, the differential expression of engrailed genes in the Drosophila central nervous system may indicate subtly different neural functions. We have recently used RNA interference to show that, in the cockroach cercal system, en-1 and en-2 are required in an identified post-mitotic sensory neuron to specify successive stages of its development - choice of axonal pathway, establishment of branching pattern and finally, recognition of synaptic targets. Here we present evidence that en-1 and en-2 are co-expressed within medial cells of the cercal neuroepithelium. No cells appear to express only a single paralogue. However, en-1 expression represents approximately 65% of total Engrailed activity while en-2 represents only 35%. We used RNAi to selectively knock out en-1 and/or en-2 expression. While knockout of en-1 affects both axonal shape and synaptic connectivity of an identified sensory neuron, knockout of en-2 affects only the axon morphology. We also investigated the effects of different doses of Engrailed by diluting a mixture of both dsRNAs. We find that a reduction to 65% total Engrailed activity has similar effects to en-2 knockout on the axon projection, while a reduction to 35% En activity is similar to en-1 knockout. Both dilutions also have effects on the synaptic connections. These results suggest strongly that en-1 and en-2 together control axon guidance, while only en-1 controls synaptic target recognition, thus indicating that they have paralogue-specific functions. Supported by NIH P01 NS07464 and R01 NS45547 to J.M.B.
ABSTRACT NO. 10 Chronic cocaine increases midbrain GABAB stimulated G protein activation in female rats. W.J. Bruckman, M. Febo, A.C. Segarra. Univ of Puerto Rico, Med Sci Campus, San Juan, PR
Our previous results demonstrate that estrogen is essential for the expression of cocaine sensitization in the female rat. However, the estrogenic mechanism of action has not been fully studied. Adult Sprague-Dawley rats were ovariectomized (OVX), half received a Silastic implant containing estradiol benzoate (OVX-EB), the other half received empty implants (OVX). After a 7 day recovery period, rats were administered either 0.9% sterile saline (0.1cc/100 g), or cocaine (15 mg/kg, i.p.) for 5 consecutive days and following 2 drug free days, rats were given a cocaine challenge. This resulted in the following injection groups, equally divided among OVX and OVX-EB rats: Sal-Sal (control), Sal-Coc (acute), and Coc-Coc (chronic). Rats were then sacrificed, brains removed and subsequently cryosectioned for in vitro [35S]GTPS autoradiography to measure GABAB stimulated G protein activation in the substantia nigra (SN) and ventral tegmental area (VTA). Estrogen decreased [35S]GTPS binding in the SN and VTA. Acute and chronic cocaine increased GABAB mediated G protein activation in OVX-EB rats. However, OVX rats devoid of plasma estrogen levels displayed decreased G protein activation in both midbrain regions analyzed. Thus, midbrain GABAB neurotransmission is differentially affected by cocaine treatment in OVX and OVX-EB rats. These results implicate the GABAB receptor in estrogenic regulation of cocaine sensitization in the female rat.
Novel role for P2Y2 nucleotide receptor as a
regulator of cytoskeletal assembly.
Nataliya E. Chorna.(1),
Laurie
Erb(3),Gary
A. Weisman
(3),
Fernando A.Gonzalez (1,2) Acute injury to the nervous system such as that caused by stroke or infection triggers an array of morphologic and metabolic changes to promote repair of the damaged tissue as well as protection against infectious agents. These responses are the products of the proper interaction between proteins of different categories such as integrins, transmembrane G-coupled receptors, protein kinases, protein phosphatases. Recently Erb et al (2001) showed that direct interaction exists between aVb3 integrins and the heptahelical P2Y2 nucleotide receptor via Arg-Gly-Asp (RGD) sequence in its first extracellular loop. Our investigation of the crosstalk between aVb3 integrins and P2Y2 nucleotide receptors revealed a new role for nucleotide receptor in the regulation of cell proliferation and modulation of this event by its interaction with aVb3 integrin. Activated P2Y2 receptor mediates signaling cascade downstream to MAP kinase ERK 1/2 and p38 in conjunction with the aVsubunit of the aVb3 integrins. Apart from P2Y2 dependent p38 activation, coupling of P2Y2 receptor to ERK 1/2 has been shown to play an important role in developing characteristic F-actin stress fibers assembly and forming cytoskeletal rearrangements such as membrane ruffles. Engagement of aVb3 integrins with specific antibodies partially inhibits P2Y2 receptors induced membrane ruffles and new DNA synthesis. These results suggest, the novel role for P2Y2 nucleotide receptor in regulation of cell motility and proliferation. Supported by: NIH-COBRE grant P20 RR 15565
CHRONIC EXPOSURE TO DAMGO, A MU OPIOID, DOWN-REGULATES EXPRESSION OF SODIUM PUMP IN SH-SY5Y CELLS Susan Corey Department of Pharmacology and Toxicology, University of Puerto Rico School of Medicine, San Juan, Puerto Rico
Withdrawal from chronic opioid administration is characterized by hyperactivity of the locus coeruleus, an adrenergic cell group that receives prominent enkephalinergic input. Reduced activity of the Na Pump has been suggested as a possible mechanism for increased neuronal excitability. The present studies employed retinoic acid-differentiated SH-SY5Y human neuroblastoma cells. The cells expressed a1 and a3 isoforms of the Na,K-ATPase. Enzymatic activity of the Na,K-ATPase was stimulated up to 250% by a 10-min treatment with 1 µM [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO), a selective -opioid agonist. The acute stimulatory action of DAMGO was blocked by 10 µM naloxone. Chronic treatment with 1 µM DAMGO for 2-7 days caused down-regulation of the a1 subunit of Na,K-ATPase to less than 50% of basal levels. This result supports the hypothesis that chronic administration of µ–opioids reduces expression of Na,K-ATPase, which may lead to partial depolarization and increased excitability. Supported by NIH grants NS0768, RR-03051, and GM-08224 ABSTRACT NO. 13Changes in mesocorticolimbic D2 receptor function during cocaine sensitization are dependent on plasma estrogen levels in the female rat. I. DeJesús-Alvelo, M. Febo, A.C. Segarra. Department of Physiology, Univ of Puerto Rico Sch of Medicine, San Juan, PR
Mesocorticolimbic D2 receptors are implicated in cocaine addiction, and are subject to estrogenic regulation in female rats. Thus D2 receptors constitute a neural substrate by which estrogen may regulate cocaine sensitization in females. Rats were ovariectomized (OVX) and half received 17-estradiol benzoate Silastic implants (OVX-EB). After a week, rats received daily saline (Sal) or cocaine (Coc) (15 mg/kg) injections for 5 consecutive days, kept drug free for 7 days, and then given 1 injection of cocaine. This resulted in naive, acute and chronic injection groups, equally divided among OVX and OVX-EB rats. Animals were sacrificed an hour later, and the ventral tegmental area (VTA) and nucleus accumbens (NAc) brain areas were collected for [35S]GTPgS autoradiography of D2 receptors. Estrogen treatment produced a 50% increase in D2 stimulated [35S]GTPgS binding only in the cingulate cortex. Acute cocaine injections increased D2 stimulation of G protein activity in the NAc and VTA of OVX-EB rats, whereas no effect was observed in OVX rats. Chronic cocaine injections led to higher levels of [35S]GTPgS in the NAc of OVX rats. OVX-EB rats, which exhibit sensitization to cocaine, show higher [35S]GTPS binding in midbrain regions, such as the VTA and SN. Interestingly, the effect of chronic cocaine injections in the cingulate cortex was dependent on the hormonal status of these animals, increased [35S]GTPgS binding was observed in OVX rats whereas decreased binding was found in OVX-EB rats. Collectively, these data support a mechanism by which estrogen modulates cocaine sensitization in female rats: by altering D2 receptor function in the mesocorticolimbic system.
Activation of p38 MAP Kinase by the P2X7 nucleotide receptor in human HL-60 and human astrocytoma 1321 N1 cells. Díaz-Pimentel, J. A 1., Vivas-Mejía, P., 1 Weisman, G., 2 González, F.A, 1 1 Department of Chemistry, University of Puerto Rico, Río Piedras Campus, 2 Department of Biochemistry, University of Missouri- Columbia. The P2X7 nucleotide receptor is a non-selective ligand-gated ion channel expressed primarily in hematopoietic cells, astrocytes, microglia, parotid acinar cells, and fibroblasts. P2X7 activation opens a channel, which triggers rapid depolarization and calcium influx. Additionally, it induces the formation of non-selective pores able to pass molecules up to 900 Da, a phenomenon not observed in any other P2X receptors. Activation of the P2X7 receptor can lead to apoptosis or necrosis in a variety of cells including lymphocytes and microglia, but the biochemical steps linking channel/pore formation to cell death are poorly understood. Here we described the channel/pore formation upon P2X7 receptor activation in human promyelocitic HL-60 and human 1321N1 astrocytoma cells. In addition we found that exposure of these cells to BzATP (2 min) was sufficient to trigger activation of p38 MAP Kinase. Our results show that BzATP activate p38 MAPK pathway and that the profile of activation is characterized by a strong and transient activation of this kinase. Pretreatment of the cells with the specific inhibitor of p38 MAPK, SB203580, inhibit BzATP-dependent LDH liberation associated with necrotic cell death. We conclude that p38 MAPK activation is involved in the pathway conducing to the P2X7-dependent necrotic cell death, and corroborate other observations that necrosis is not a passive form of cell death. This work is supported by the COBRE program and by the PR GK-12 fellowhip
GABA AND DOPAMINE COLOCALIZATION IN INTERNEURONS OF THE APLYSIA FEEDING CIRCUIT: PRESYNAPTIC ACTIONS OF GABA. M.E. Díaz-Ríos; M.W. Miller* 1. Department of Anatomy and Institute of Neurobiology, University of Puerto Rico, San Juan, Puerto Rico
Previous
studies have shown that GABA-like immunoreactivity is colocalized with
markers for catecholaminergic neurons in five interneurons belonging to the
feeding circuit of Aplysia californica (Díaz-Ríos et al., 1999,
2002). One such interneuron, designated B20, is known to elicit rhythmic
buccal motor programs, which result at least in part from its specific
synaptic connections with identified neurons within the buccal ganglion (Teyke
et al., 1993; Jing and Weiss, 2001). To examine the possible role of GABA in
the mediation or modulation of synaptic transmission of B20, two follower
motor neurons, B8 and B16, were examined. As shown previously, B20 produced
a markedly facilitating direct EPSP in the radula closer motor neurons B8a
and B8b and a weakly facilitating EPSP in B16. Following a train of
impulses, the B20-B16 PSP exhibited a slow depolarizing component that was
not observed in the B20-B8 synaptic response.
Bath
application of baclofen (100 µM), a specific agonist of GABAB receptors,
produced an increase in the amplitude of the B20-B8 EPSP. This effect was
reversible after washout. No consistent changes in the B8 membrane potential
or the temporal characteristics (rise time, decay time) of the PSPs were
detected, suggesting that the observed effects were not likely to be
attributable to postsynaptic actions of baclofen.
Bath
application of GABA (100 µM) produced similar effects. It is proposed that
GABAergic presynaptic autoreceptors on B20 could serve to enhance synaptic
transmission in this system.
. . . . . . .
. . . . . . . . . . . . . . . . . . .
ABSTRACT NO. 16THE MU OPIOID RECEPTOR AND COCAINE INDUCED LOCOMOTOR ACTIVITY IN THE FEMALE RAT. M.Febo*; L.A.Gonzlez-Rodrguez; J.Torres-Plata; J.D.Delgado-Lpez; A.C.Toro-Ortiz; A.C.Segarra Dept Physiol, Univ Puerto Rico Sch Med, San Juan, Puerto Rico
We have previously reported that estrogen and opioids interact to modulate the locomotor response to cocaine. In addition, estrogen has been found to decrease mesolimbic mu-opioid receptors (MOR). The purpose of this study is to investigate the role of naloxonazine, a MOR antagonist, on cocaine-induced locomotor activity in ovariectomized (OVX) rats with or without estradiol benzoate (EB) treatment. Female rats were bilaterally ovariectomized and given subcutaneous Silastic implants with or without EB. A week later, rats were habituated to the testing cage and 30 min later were tested for their locomotor response to 0.9 % saline (SAL; 0.2 cc/i.p.), naloxonazine (NXZ; 15 mg/kg/i.p., 16 hrs prior to testing), cocaine (COC; 15 mg/kg/i.p.) or NXZ prior to COC (NXZ+COC) for 60 min. COC increased locomotor and stereotyped activity equally in OVX rats with and without EB. Blocking MORs prior to cocaine increased locomotion and stereotypy in OVX, but not OVX-EB rats. Rearing increased in response to cocaine in all rats, declining faster in rats injected with naloxonazine prior to cocaine. In summary, blocking the MOR results in a higher locomotor and stereotype response to cocaine in female rats. EB priming reduces naloxonazines effect on cocaine-induced locomotor activity and stereotypy. These results indicate that the role of the MOR in cocaine-induced locomotor and stereotyped activity in females is affected by estrogen.
MODULATION OF THE CALLINECTES HEART BY CRUSTACEAN CARDIOACTIVE PEPTIDE (CCAP): PERIPHERAL MODULATION OF A CENTRAL PATTERN GENERATOR CIRCUIT. T.J. Fort1; V. Brezina2; W.-D. Krenz1*; M.W. Miller1 1. Institute of Neurobiology, University of Puerto Rico, San Juan, Puerto Rico. 2. Department of Physiology & Biophysics, Mount Sinai School of Medicine, New York, NY, USA
The nonapeptide CCAP was originally isolated from the pericardial organs (POs) of the shore crab, Carcinus maenas, and characterized as a cardioacceleratory substance on the heart of the same species. The neurogenic heartbeat of the crab, as in other decapod crustaceans, is controlled by the simple (9 neurons) central pattern generator (CPG) cardiac ganglion (CG) that is embedded within the dorsal wall of the heart. Previous reports have shown that very high concentrations (10-5 to 10-4 M) of CCAP are required to alter the activity of the isolated CG, while cardioexcitatory actions occur at much lower levels (10-9 to 10-8 M). We examined this apparent discrepancy in a semi-intact working heart preparation of the blue crab Callinectes sapidus. Immunohistochemical staining of the Callinectes cardiac system revealed a rich plexus of CCAP-like immunoreactive material in the PO. As in other species, exogenous application of high concentrations (>10-6 M) of the peptide were required to alter the activity of the isolated CG. However, in the semi-intact heart, monitoring ganglionic output in the presence of cardioactive concentrations of CCAP (10-9 to 10-8 M) revealed significant increases in burst duration, impulses per burst, and burst frequency. It is concluded that these effects on CPG activity are primarily mediated via peripheral actions of the peptide. Supported by: NSF IBN9722349, NINDS PO1 NS07464 and NS41497, NIGMS GM61838-03
ABSTRACT NO. 18 Neonatal exposure to Pregnenolone sulfate (PS) produces sex-specific effects on anxiety but not locomotors behaviors in the adult rat. Fortis A1, Rivera JC2, Cruz N3, Barreto J1, Corretjer G4, Jorge JC4 Departments of 1Chemistry, 2Biology, 3Psychology, Rio Piedras Campus; Department of 4Anatomy, Medical Sciences Campus, University of Puerto Rico, San Juan, PR 00936.
PS is a neurosteroid that acts as an allosteric modulator of neurotransmitter receptors in the brain. We wanted to determine if neonatal exposure to PS alters the sexual differentiation of locomotor and anxiety behaviors. Eighty six Sprague Dawley pups from both sexes were injected with PS at 10-6M (s.c., 0.01cc/2g body weight) from postnatal (PN) day 1 to PN14. At PN 30 we monitored the day of vaginal opening, and the pattern of estrous cycle. At PN 75 behavioral tests were performed to measure locomotor and anxiety behaviors (AccuScan Instruments, Ohio). Rats were sacrificed by decapitation, and blood was collected to measure hormone levels. Gonads were collected and weighted. Neonatal exposure to PS produced anxiolytic effects in females but not males, this effect did not correlate with changes in locomotor behaviors. PS effects on anxiety were evident during estrous. The opposite effect was seen in males, suppression of locomotor behaviors without changes on anxiety. These sex-specific modulatory effects on behavior cannot be attributed to permanent changes on hypothalamus-pituitary-gonadal axis function since the day of vaginal opening, gonadal weight, pattern of estrous cycle, and plasma levels of sex steroids in adult animals were not altered by neonatal treatment. We suggest that PS may play an organizational role in affective components of behavior. Funding provided by: MBRS-RISE (R25GM61838)-AF and YB, AMP (PRLSAMP-HRD9623943)-JCR and JB. Study funded by NIH-COBRE (RR15565) and RCMI (RR03051)-JCJ.
ABSTRACT NO. 19 Neurogenesis, myogenesis and de-differentiation during holothurian intestinal regeneration. José E. García-Arrarás, José L. Quiñones-Rivera, José L. Roig-López, and Gisela Murray Biology Department, University of Puerto Rico, Río Piedras, PR 00931
Echinoderms comprise the
deuterostome group with the most amazing regeneration capacities. Among
these, holothurians or sea cucumbers, are well known to regenerate body
parts after evisceration or autotomy. Thus, the holothurian system provides
an excellent model to study organogenesis in an adult organism. Our
laboratory has been studying the process of intestinal regeneration in
Holothuria glaberrima for the past few years. We have now focused on the
regeneration of the muscle and neuronal components. Funded by NSF-IBN-0110692, NIH-MBRS and NIH-RCMI.
The cationic residue at position 10 of α-conotoxin MI interacts at close range with γ-tyrosine 111 and δ-arginine 113 on the Torpedo nicotinic acetylcholine receptor. Hann, R.M.*, Valdés, S., Agresar, L., Berolo, A., Bonilla, M.Z., *Guerra, A.Y. and *Vélez-Carrasco, W. Department of Biochemistry and Nutrition and *Center for Molecular and Behavioral Neuroscience, Universidad Central del Caribe, Bayamón PR. The muscle-type nicotinic acetylcholine receptor (ACHR) has two nonidentical agonist-binding domains located at the αγ and αδ subunit interfaces. The α-conotoxins MI, GI and SI are short (13 - 14 amino acid) peptides found in cone snail venoms that act as potent competitive inhibitors of the ACHR. These peptides have homologous sequences and very similar three-dimensional structures. MI and GI display stronger affinity for the αγ agonist site on the ACHR from the Torpedo californica electric organ than for the αδ site, while SI binds with the same affinity to both sites. Arginine (R) at position 9 on GI, homologous to proline (P) 9 on SI and lysine (K) 10 on MI, is the αγ selectivity determinant toward solubilized Torpedo ACHR, while tyrosine (Y) 111 on the γ subunit, as opposed to R113 at the homologous position on the δ subunit, is the main receptor determinant of MI's αγ selectivity on Xenopus oocyte-expressed Torpedo ACHR. The purpose of this study was to determine if the homologous midchain residues on MI, GI and SI interact at close range with Torpedo γY111 and δR113. The affinity constants of these peptides and of several analogues for the two agonist sites on tsA201 cell-expressed Torpedo wild type, γY111R mutant and δR113Y mutant ACHRs were determined and analyzed with double mutant cycles. The results show that K10 is the main determinant of MI’s αγ selectivity for the cell-expressed Torpedo ACHR and interacts at close range with both γY111 and δR113. MI probably binds in the same orientation and, except for γY111/δR113, interacts with equivalent residues at the two sites. In contrast to K10 of MI, R9 of GI does not interact at close range with either γY111 on δR113, although GIR9 and γY111 are both major determinants of GI’s high affinity for the αγ site. Therefore, despite having very similar three-dimensional structures, MI and GI must bind at the high-affinity αγ site with different orientations such that GIR9 interacts with at least one receptor γ subunit residue other than γY111 and γY111 interacts with at least one GI residue other than R9 to produce the high affinity complex. Like MIK10, P9 of SI interacts at close range with δR113 but, unlike MIK10 and like GIR9, does not interact at close range with γY111. Therefore, SI may bind in the αδ site similar to MI and in the αγ site similar to GI. Supported by: NIH-MBRS-SO6-GM50695 and NIH-SNRP-U54-NS39408 to RMH.
Increased phosphorylation of ERK-1,2 might be involved in nicotine-mediated neuroprotection against NMDA excitotoxicity in the CA1 subregion of rat hippocampal slices Jiukuan Hao 1, Keqin Ren2, Jianpeng Wang2, Dinely Perez, Sandra Peña de Ortiz2 and P.A. Ferchmin1 1.Center for Molecular and Behavioral and Neuroscience , Department of Biochemistry, Universidad Central del Caribe, Bayamon, 2Department of Biology, University of Puerto Rico
Signaling through mitogen activated protein kinase (MAPK) cascades is involved in the regulation of neuronal survival, apoptosis, and other adaptive and developmental mechanisms. Specifically, the extracellular signal regulated kinase (ERK-1,2) cascade, involving Ras, Raf-1, MEK-1,2, and ERK-1,2, is often involved in neuroprotection. Several studies have shown that nicotine is neuroprotective, but its mechanism for neuroprotection is still unclear. We have shown that nicotine has neuroprotective activity against NMDA in the CA1 area of rat acute hippocampal slices at concentrations higher than 0.1 µM. Nicotine was applied 1 hour before the application of 0.5 mM NMDA for 10 min. The effect of nicotine is mediated by the a4b2 nicotinic receptor and is inhibited by dihydro-b-erythroidine. The degree of recovery of the capability to produce synaptically elicited population spikes was used as a measure of neuroprotection. The neuroprotective effect of nicotine was also inhibited by PD98059, an inhibitor of MEK-1,2, the kinase that phosphorylates ERK-1,2. In addition to the population spikes, we measured the levels of non-phosphorylated and phosphorylated ERK-1,2 after applications of nicotine, NMDA or both drugs together. NMDA markedly decreased and nicotine increased the phosphorylation of ERK-1. Nicotine partially reversed the decrease in ERK-1phosphorylation by NMDA. These data suggest that an increase in ERK-1 phosphorylation participates in the mechanism of nicotine neuroprotection. To the best of our knowledge, this is the first work to report nicotine-mediated neuroprotection of the capability to synaptically evoke action potentials. Acknowledgement: This work was supported by NINDS and NCRR (RCMI) SNRP Program, grant # NS39408 (PAF) and NINDS-SNRP U54 NS39405 (SPO) and NCRR-NIH 5P20 RR15565-02). Core facilities were supported by NIH-RCMI G-12RR03305.
Nitric Oxide Induces Resensitization of the P2Y2 Nucleotide Receptor in 1321N1 human astrocytoma cells. Melvin G. Hernández-Pérez1, Gary A. Weisman3, Richard C. Garrad4 and Fernando A. González1,2 1Department of Chemistry, Río Piedras Campus & 2Department of Biochemistry, Medical Sciences Campus, University of Puerto Rico 3Department of Biochemistry, University of Missouri-Columbia, 4Department of Biomedical Sciences, Southwest Missouri State University P2Y2 nucleotide receptor is a member of the G-protein coupled receptor (GCPR) superfamily. The P2Y2 receptor is distinguished by the equal potency and efficacy of its activation by the naturally occurring agonists ATP and UTP. Like other members of the GPCR superfamily, P2Y2 receptor undergoes agonist-induced desensitization, which occurs during short term (seconds to minutes) exposure of cells to agonists. The mechanisms that attenuate P2Y2 signaling are considerable importance to regulation of its intracellular signaling and maintenance of their ability to respond to agonist over time. This study evaluates the effect of nitric oxide (NO) on P2Y2 nucleotide receptor resensitization in human 1321N1 astrocytoma cells. Calcium responses to UTP were measured with fura-2 and detected using a dual excitation spectrofluorometric analysis. Potent P2Y2 receptor desensitization was observed after a 5 min exposure to UTP (0.1 mM) which is characterized by lower calcium peak amplitude. The NO precursor, L-arginine (10mM), was added before UTP exposure. L-arginine induced a partial recovery of the [Ca2+]i response. The results indicate that NO can increase the P2Y2 receptor resensitization in human 1321N1 astrocytoma cells. Further studies are currently underway to determine the pathway by which NO is inducing P2Y2 nucleotide receptor resensitization. Supported by: NIH-NIGMS (SCORE), NIH-NCRR (RCMI, COBRE)
Inhibition of corticosterone with metyrapone prevents extinction of the conditioned emotional response (CER). P. Hernández-Poudevida, B.S. McEwen & G.J. Quirk. Dept of Physiology, Ponce School of Medicine, Ponce, Puerto Rico 00732, and Rockefeller University, NY, NY 10021.
Posttraumatic stress disorder (PTSD) is not an inevitable consequence of trauma. It has been suggested that a low level of corticosteroids following a traumatic experience is a predisposing factor for PTSD (Yehuda et al., 1998). It has also been suggested that PTSD may be due to deficits in extinction of conditioned fear (Gorman et al., 2000). Are corticosteroids involved in extinction learning? We addressed this in rats with the corticosterone synthesis inhibitor metyrapone. On day 1, rats received 7 tones paired with footshock (0.5 mA). On day 2, rats were injected with metyrapone (50 mg/kg, s.c, n=12) or vehicle (n=12) and given 15 extinction tones. On day 3, recall of extinction learning was assessed. MET rats showed normal expression of freezing and suppression of bar-pressing for food (CER)at the start of extinction. However, MET impaired within-session extinction especially for conditioned suppression. At the end of extinction, 5/12 MET rats still showed near-maximal suppression, compared to only 1/12 VEH rats. Average freezing levels at the end of extinction were 28% vs. 7% for MET and VEH groups, respectively (t=2.5, df=22,p<0.05). MET did not alter spontaneous press rates, suggesting no locomotor deficits. The following day, half the MET rats remained completely suppressed compared to only 1/12 VEH rats (p<0.05). Our findings suggest that elevated corticosteriod levels are necessary for extinction of conditioned fear. Low cortisol levels may predispose individuals to develop PTSD by compromising extinction learning. Supported by NIH grants MH58883, MH58911, GM08236, and a BRIN Fellowship.
ABSTRACT NO. 24
STRUCTURAL CORRELATES OF AGING RETINA IN RHESUS MONKEYS RAISED IN PUERTO RICO. E. Kicliter, N. Lugo and M. Cotto. Department of Anatomy and Institute of Neurobiology, Univ. Puerto Rico Medical Sciences Campus, San Juan,. Puerto Rico.
Cell density in the ganglion cell (GCL) and optic fiber ( OFL) layers increases during aging in rhesus monkeys raised in Puerto Rico and at least part of this increase is due to proliferation of astrocytes whose cell bodies measure 5-10 μm in diameter (Neurosci. Abstr. 27: 284.16, 2001). To determine whether this proliferation leads to changes in thickness of the GCL/OFL and whether other retinal layers might be affected we measured them. Formalin fixed eyecups from rhesus monkeys aged <1-28.9 yrs were cryostat sectioned. Microscopic measurements were made of the thickness of the GCL/OFL and the remaining retinal layers (inner plexiform layer to photoreceptor outer segments) along a transect parallel to the horizontal meridian through the optic disk at 2 mm intervals up to an eccentricity of 10 mm temporal and nasal to the optic nerve head. Measurements of each series were averaged and plotted against age. The data show no consistent change during aging in average thickness of either the GCL/OFL which ranged 25-44 μm or the remaining retinal layers which ranged 140-210 μm. Using this thickness data and previously acquired data from Nissl stained wholemounts, we have calculated the total volume occupied by each size of cell body per unit volume of GCL/OFL. These calculations indicate that the volume occupied by the smaller cells (5-10 μm diameter) increases while that occupied by larger cells (>15 μm) decreases. The total volume occupied by all cell bodies decreases with age, indicating a growth of cell processes (either neural or glial) during aging. Supported by: NIH Grants AG-16057 to EK, MH-48190 to NL and RR-03640 to the Caribbean Primate Research Center.
ABSTRACT NO. 25Cloning of glutamate receptor subunit genes from crustacean central nervous system and muscle: Tissue-specific alternative splicing. W.D.Krenz12, J.Boulter13, L.Quiñones2, C. Zuazaga2, A.I.Selverston4, S.F.Heinemann1. 1Molecular Neurobiology Lab., The Salk Institute, La Jolla, CA 92037, USA; 2Institute of Neurobiology, Univ. of Puerto Rico, San Juan, PR 00901, USA; 3Department of Psychiatry & Biobehavioral Sciences, UCLA, Los Angeles, CA 90095 USA; 4Institute for Nonlinear Science, UCSD, CA 92093, USA We have cloned cDNAs encoding two splice variants of a crustacean ionotropic glutamate receptor subunit gene from the spiny lobster Panulirus interruptus. The overall protein sequences are 40-57 % identical to non-NMDA receptors of invertebrates and vertebrates. Surprisingly, the alternatively spliced exons comprise membrane region M2. In current topology models this membrane region forms the reentrant p-loop that participates in formation of the ion pore. Alternative splicing of the p-loop has been described in lobster shaker potassium channels but not in any cloned glutamate receptors. The functionally critical amino acid residues in the Q/R editing site, which determines linearity and calcium permeability of channel oligomers, are glutamine and cysteine respectively. No evidence for pre-mRNA editing has been detected, instead, genomic and cDNA sequences are identical. PCR using splice variant-specific primers on cDNAs synthesized from muscle or nervous system mRNA reveals that the two splice forms are expressed in muscle or nervous system tissue respectively. The N-terminal extracellular domain of both isoforms contains two potential Ca++-binding motifs similar to the loop of the EF-hand motif known from a large number of Ca++-binding proteins. These motifs may represent the molecular basis for the dependence on high extracellular Ca++ of EPSPs and responses to glutamate described in crayfish muscle. Supported by RCMI award G12RR-03051 and by NIH awards 28709 and 09322.
A novel functional role of the nicotinic acetylcholine receptor as a regulator of the Na,K-ATPase. I.I. Krivoi1, T.M.Drabkina1, M. Dobretsov2, S.N. Skatchkov3, M.J. Eaton3, R. Meltzer4and F. Mandel4 1St-Petersburg State Univ., St-Petersburg, Russia, 2Dept. Anesthesiology, Univ. of Arkansas for Medical Sciences, Little Rock, AR, USA, 3Dept. Biochem., Univ. Central del Caribe, Bayamon, PR, USA, 4Dept. Molec. Physiol. and Biophysics, Baylor College of Medicine, Houston, TX, USA The nicotinic acetylcholine receptor (nAChR) is well known ligand-operated ion channel in vertebrates. But there are a number of observations that indicate another functional role of nAChR as a regulator of the Na,K-ATPase. We have performed a combined study of this problem using the following experimental models: rat skeletal muscle and erythrocytes; cultured C2C12 myogenic cell line; Xenopus oocytes – intact or as an expression system; nAChR/Na,K-ATPase preparation from the electric organ of Torpedo californica. We found that low (up to 100 nM concentrations) ACh hyperpolarized the rat diaphragm muscle membrane due to activation of the a2, not the a1 isoform of Na,K-ATPase. ACh-induced hyperpolarization was carried out by the nAChR with the extra-high affinity for ACh and d-tubocurarine (36 ± 6 nM and ~ 6 nM, respectively). These affinities were similar to those of nAChR in the desensitized state, supporting the role of desensitized nAChR as a signal transducer for the Na,K-ATPase (a2 isoform). Even after discontinuous and continuous sucrose gradient centrifugations, our nAChR preparation from Torpedo californica contained the Na,K-ATPase. Further purification using affinity chromatography yielded a highly purified preparation; yet, some Na,K-ATPase was still present. Stopped-flow determinations of Dansyl C6 Choline binding to Torpedo nAChR indicate that this binding rate was modulated by ouabain. In Xenopus oocytes with native a1 isoform of Na,K-ATPase, expressing either mouse embryonic muscle nAChR or neuronal a4b2 nAChR, ACh (50 nM – 5 μM) induced only depolarization. The same was observed in cultured myoblasts and myotubes C2C12 natively expressing nAChR and the two (a1 and a2) isoforms of Na,K-ATPase. Taken together, our data suggest the formation of a functional nAChR/Na,K-ATPase(a2) complex which may be formed under certain conditions that exist in adult skeletal muscle. These conditions probably include innervation and/or, perhaps, a highly organized compartmentalization; the last being related to the nAChR ε subunit expressed only in adult muscle. Our data suggest a new functional role of nAChR as a regulator of Na,K-ATPase, presumably unrelated to its function as the ligand-operated ion channel. The physiological role of a functional nAChR - Na,K-ATPase interaction may be the regulation of muscle electrogenesis by low non-quantal ACh via activation of the (2 isoform of the Na,K-ATPase. Supported by RFBR #02-04-48881, NIH MBRS #S06 GM50695 and NSF #9904815.
ABSTRACT NO. 27The role of NMDA receptors in the medial prefrontal cortex during extinction of conditioned fear. L. Lizardi-O’Neill, E. Santini and G.J. Quirk Universidad del Este, SUAGM and Ponce School of Medicine
Pavlovian fear conditioning is a paradigm commonly used to study learning and memory. Pairing a tone with a footshock induces fear responses (e.g. freezing) to the tone. Repeated presentations of the tone alone decreases fear responses, a process known as extinction. While much is known about conditioning, little is known about extinction. Recently, it has been shown that lesions of the medial prefrontal cortex (mPFC) blocked long-term extinction memory but did not prevent extinction learning within a session (Quirk, et al., 2000). Similarly, systemic blockade of NMDA receptors has been shown to block long-term extinction memory without preventing short-term extinction learning, suggesting that consolidation of extinction involves NMDA-dependant synaptic plasticity (Santini et al., 2001). Given the parallel between these two findings, we hypothesized that NMDA receptors in the mPFC might be involve in the formation of long-term extinction memory. To test this hypothesis, a two-day fear conditioning protocol was used. On day 1, rats received 7 conditioning (tone-shock) trials. After an hour break in their home cages, rat received microinfusions of either artificial cerebrospinal fluid (ACSF) or the NMDA receptor antagonist AP5 (5 µgm/µl) into the mPFC, immediately followed by 10 extinction trials (tone alone). On day 2, two extinction tones were given to test for recall of extinction memory. Both groups acquired and extinguished similarly on day 1. On day 2, the ACSF group displayed relatively little freezing (25%). In contrast, the AP5 group showed more freezing (65%) to the tone. Thus, we showed that NMDA receptor blockade at mPFC: 1) did not prevent short-term memory of extinction, and 2) impaired recall of extinction when tested 24 hours later. These data suggest that long-term memory for extinction depends on NMDA receptors in the mPFC. Grant P20RR16470 from the National Center for Research Resources, NCRR-NIH
ABSTRACT NO. 28 Comparison of the effects of different Valeriana officinalis root extracts on GABA and glutamate neurotransmission. Maldonado PM, Ramos I, Iglesias M, González FH, Rassi N and Ortiz JG.
Department of
Pharmacology and Toxicology, School of Medicine, University of Puerto Rico
P.O. Box 365067 San Juan, P.R. 00936-5067.
Supported in part by the NIH-NIGMS MBRS-RISE
Program at the
University of
Puerto Rico,
School
of Medicine (GM61838 and GM08224) and the NIH SNRP Program
(NS39408-03) SELECTIVE BLOCKADE OF IONOTROPIC GLUTAMATE RECEPTORS WITHIN NUCLEUS ACCUMBENS SUBREGIONS DISRUPTS COCAINE-INDUCED CONDITIONING. C.S. Maldonado-Vlaar*; E. Rodríguez; A. Bernardo; Y. Estrada; J.E. Alvarez; M. Burgos Dept Biol, Univ Puerto Rico, San Juan, Puerto Rico
Studies have shown that an environment previously associated with cocaine use elicits cocaine craving. Ionotropic glutamate receptors within the nucleus accumbens (NA) have been implicated in mediating motor learning and reinforcing effects of cocaine. The present study investigated the role of ionotropic glutamate receptors within the NA core and shell in cocaine-induced environment-specific-conditioning. Prior to training, rats were implanted with bilateral cannula into the NA. Rats were assigned to 3 different environment conditions. Experimental condition rats received a cocaine injection (10 mg/kg, i.p.) prior to placing in activity chambers prepared with distinctive odor and visual cues. Control condition included rats receiving saline injections before conditioning exposure. Pseudo-conditioning group received cocaine injections in the home cage. Training lasted 10 days. On test day 12, rats were pre-treated with microinfusions of an NMDA receptor antagonist AP-5 (0,1.0 microg/0.5microl) prior to placing them in their assigned environment without i.p. injections. Control rats on cocaine environment behaved as if they had received a cocaine injection when compared to controls. Blockade of NMDA receptors within the shell and not the NA core disrupted cocaine-induced environmental conditioning. We hypothesize that ionotropic glutamate receptors only within NA shell are involved in the retrieval of important cocaine-related memories essential for the maintenance of strong drug-environment associations. More experiments will be conducted to further characterize this effect. Supported by: S06GM61151-01A1
ABSTRACT NO. 30 ESTROGEN REGULATION OF GABA LEVELS UPON COCAINE EXPOSURE WITHIN ENDOCRINE BRAIN CENTERS. J Marrero1, I Lorenzini1, E García-Corbea2, A Segarra3, JC Jorge4 Departments of Biology1 and Chemistry2- Rio Piedras Campus, Department of Physiology3- Medical Sciences Campus,Department of Anatomy4-Medical Sciences Campus, University of Puerto Rico, San Juan PR- 00936-5067.
There is a strong relationship between hormones, brain, and behavior. They interact to form the neuroendocrine system, which influences physiological and behavioral responses. Sex-specific neuroendocrine function can be correlated with naturally occurring hormonal fluctuations. Gonadal steroids may influence the response of men and women to cocaine. Most studies in the field of drug addiction use males to avoid confounding results that may arise due to female dynamic hormonal changes. Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter that decreases neural activity. This neurotransmitter system is expressed in sexually dimorphic regions of the brain including the medial preoptic area (mPOA), the ventromedial nucleus (VMN) of the hypothalamus and the lateral hypothalamus (LH). Experimental evidence has shown that GABA-mediated neurotransmission is one of multiple systems that is directly influenced by estrogen. Adult female Sprague-Dawley rats were ovariectomized and a 5mm Silastic® tubing was implated subcutaneously. Half of the animals received estradiol benzoate (EB) implants and the other half received empty implants (controls). Female rats were exposed to cocaine to determine whether sex steroid fluctuations have an impact in modulating GABA levels. Brain sections (16μ) of LH and mPOA were stained with antibodies for GABA by immunocytochemistry. Preliminary data suggests that estrogen regulates cocaine-induced changes on GABA levels in the LH and mPOA. These studies will provide critical information to better design sex-specific therapeutic strategies for the management of drug and alcohol abuse where addiction seems to disrupt appropriate GABA function. This work was supported by NIH-COBRE (P20RR15565) and a Young Investigator Award (NIH-BRIN-P20RR16470) to J.C. Jorge. J.Marrero and I. Lorenzini were supported by an undergraduate NIH-BRIN fellowship.
Testosterone enhances paired-pulse facilitation of hippocampal CA1 area in pilocarpine-treated rats. Carlos A. Mejías-Apontea,b, Annabell C. Segarrab, Carlos A. Jiménez-Riverac*. aDepartment of Biology, University of Puerto Rico, Río Piedras Campus, San Juan, Puerto Rico. bDepartment of Physiology, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico. cDepartment of Physiology and Center for Molecular and Behavioral Neuroscience, Universidad Central del Caribe, Bayamón, Puerto Rico.
Previous studies have shown that testosterone enhances the susceptibility to seizures in animal models of temporal lobe epilepsy. In the present study we used the paired-pulse stimulation paradigm to elucidate if testosterone alters the excitability of the CA1 area in rats that exhibited seizures after pilocarpine administration. Hippocampal slices were obtained from gonadally intact, gonadectomized (GDX) and gonadectomized with testosterone replacement (GDX-T) adult male rats 24 hrs after either saline or pilocarpine injections. Our results reveal that testosterone favors the expression of paired-pulse facilitation at the CA1 area in gonadally intact and GDX-T rats in comparison to the GDX rats. Using 20 msec interpulse intervals the GDX-T group exhibited an early expression of facilitation that was not observed in the intact or the GDX groups. On the other hand, a higher expression of facilitation in intact animals in comparison to GDX group was seen at 50 msec interpulse intervals. The duration of the expression of paired-pulse facilitation was also more prolonged in the intact and GDX-T groups compared to GDX males. These results suggest an important role of testosterone in the modulation of the excitatory activity in the CA1 area which may account for the proconvulsive effects of testosterone in animal models of temporal lobe epilepsy. Supported by NINDS and NCRR (RCMI) SNRP Program, grant # NS39408 and GM 50695 to C.A.J.R.
Stimulation of infralimbic cortex simulates memory for extinction of conditioned fear. M.R. Milad* & G.J. Quirk. Dept of Physiology, Ponce School of Medicine, Ponce, PR 00732
During extinction of conditioned fear, rats learn that a tone no longer predicts the occurrence of footshock. Extinction is thought to form a new memory. Lesions of the medial prefrontal cortex (mPFC) including infralimbic (IL) and prelimbic did not block conditioning or extinction learning given on the same day, but blocked extinction recall 24-hr later (Quirk et al., 2000), suggesting that mPFC may store extinction memory. Consistent with this, IL neurons in the same experiment fired to tones only during extinction recall. The latency of extinction-induced responses was 100-400 ms after tone onset. Rats that showed the lowest freezing showed the largest tone responses (Milad & Quirk, submitted). The correlation between freezing and IL tone responses suggests that IL may be critical for inhibiting fear after extinction. To test this, electrical stimulation of IL was paired with conditioned tones in rats that never received extinction, in an attempt to simulate extinction-induced tone responses. On day 1 rats received 5 tone-shock trials. On day 2 rats received 8 extinction tones, each paired with IL stimulation delivered 100-400 ms after tone onset. Control rats received no stimulation or unpaired stimulation. Freezing levels on day 2 were 70%, 73%, and 28%, in unstimulated, stimulated-unpaired, and stimulated-paired rats, respectively. Paired rats acted as if they had received extinction, freezing significantly lower than both controls (p < 0.001). Together these data suggest that IL is a site where long-term extinction memory is stored and IL stimulation can simulate extinction memory. NIH grants F31-MH12818, MH58883, GM08236.
NEUROSTEROID EXPOSURE DURING NEONATAL DEVELOPMENT ALTERS ACCESORY OLFACTORY BULB MORPHOLOGY. Dianelyris Meléndez1, Nidza Lugo1,2 , Juan C Jorge2. Institute of Neurobiology1 and Department of Anatomy2, Medical Sciences Campus, University of Puerto Rico, San Juan- Puerto Rico 00936
Gonadal steroids play a critical role in the sexual differentiation of several brain circuits. However, it is not clear whether neuroactive steroid metabolites, also known as neurosteroids, contribute to the sexual differentiation of the brain and behavior. It has been previously shown that allosteric modulators of GABAA-receptors can alter sex-specific differences in the accessory olfactory bulb (AOB). The aim of this study is to determine the effects of a neurosteroid (3 aDiol) in the neuroanatomical profile of mitral and granular cells from the AOB female rats. Female pups were injected (s.c.) daily from postnatal (PN) day 1 to 14 with the neurosteroid in sesame oil vehicle (1mg/kg at a volume of 0.01cc/2g body weight), controls received vehicle injections alone. On PN 75, rats were decapitated, the brain was removed and post fixed. Saggital sections (16 microns) of the olfactory bulbs were made and stained with cresyl violet. The NIH Image Analysis Program was employed to measure cell body areas. We found that neonatal exposure to 3 aDiol induced a significant change in AOB mitral and granular cell morphology. Specifically, the mean area of mitral cells was reduced from 128 m2 to 64.87m2 (p # 0.05), whereas for granular cells the mean cell area was reduced from 35.82m2 to 17.52 m2 (p # 0.05) These preliminary results suggest, for the first time, that a steroid metabolite during neonatal development alters the morphology of a sexually dimorphic neural network. Experiments in progress aim to determine if these changes are sex- specific, and associated with GABAA-R dependent pathways, androgen receptor dependent-pathways, or a combination of these mechanisms. Study funde |