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Brain spectrin binding to the NMDA receptor is regulated by phosphorylation, calcium and calmodulin
S52 Long-term potentiation (LTP) is a synaptic mechanism thought to be involved in learning and memory. The induction of LTP in the dentate gyrus of the hippocampus is mediated by an N-methyl-D-aspartate (NMDA) receptor-coupled calcium influx. While it is widely accepted that the activation of protein kinases is essential for the induction, and probably also for the maintenance of LTP, the role of phosphatases remains a controversy. In order to examine the role of various phosphatases in synaptic plasticity in vivo, under well-controlled conditions, we applied a method of two recording glass electrodes placed on the different points (about 1 mm apart) in the same region of the dentate and stimulated by the same stimulating electrode. One glass electrode was filled with NaCl (2 M) and served as a control, while the other was filled with NaCl (2 M) + a test drug. Test drugs were either NaCl (2 M) (for baseline control), the NMDA antagonist (CPP, 10 PM) (to test for the localisation of the drugs effects), or okadaic acid, an inhibitor of serine/threonine protein phosphatase 1 (PP1) or 2A (PP2A) (OA, either 10 or 25 PM). After a baseline was recorded, highfrequency stimulation was applied to induce LTP and the magnitude of potentiation of the EPSP slope was evaluated 30 min later. In control rats LTP was equally induced in both electrodes, confirming the validity of the recording and stimulating design. In rats in which the test electrode was filled with CPP, LTP was induced in the control but not in the test electrode, indicating that the drug leak from the electrodes is sufficient and does not spread towards the other (control) electrode. Low concentrations of OA (10 PM) reduced but did not block the induction of LTP. The higher concentration (25 PM) did block the induction of LTP. These findings indicate that synaptic activation of protein phosphatases is important in the regulation of synaptic plasticity. The results also indicate that the experimental design utilized here is a useful method for in vivo pharmacological evaluation of the role of drugs in synaptic activity and plasticity.
BRAIN SPECTRIN BINDING TO THE NMDA RECEPTOR IS REGULATED BY PHOSPHORYLATION, CALCIUM AND CALMODULIN A. Wechsler and V. Teichberg Dept. of Neurobiology, Weizmann Institute of Science, Rehovot, Israel Brain spectrin, a major component of postsynaptic densities (PSDs) [1], links membrane proteins to the actin cytoskeleton [2]. Since the activity of N-methyl-D-aspartate receptor (NMDA-R) channels is dependent on the integrity of actin [3] and leads to calpain-mediated spectrin breakdown [4], we investigated whether the actin-binding spectrin interacts with NMDA-Rs. Spectrin is reported here to interact selectively with the C-terminal cytoplasmic domains of the R1, 2A and 2B subunits of NMDA-R but not with that of AMPA receptor GluR1. Spectrin binds at NR2B sites distinct from those of D-actinin-2 [5] and members of the PSD95/SAP90 family [6]. The spectrin-NR2B interactions are antagonized by Ca2+, fyn-mediated NR2B phosphorylation, but not by Ca2+/calmodulin (CaM) or CaM/kinaseII-mediated NR2B phosphorylation. The spectrin-NR1 interactions are unaffected by Ca2+ but inhibited by CaM and by PKA and PKC-mediated phosphorylations of NR1. The highly regulated linkage of the NMDA-R to spectrin may underlie the morphological changes that occur in neuronal dendrites concurrently with synaptic activity and plasticity [7]. We are currently investigating the spectrin-NMDA-R interaction in their native environment and the physiological role that this interaction might have. [1] Carlin, R.K., Bartelt, D.C. and Siekevitz, P.J. Cell. Biol. 96, 443– 448 (1983). [2] Davis, L.H. and Bennett, V.J. Biol. Chem. 269, 4409–4416 (1994). [3] Rosenmund, C. and Westbrook, G.L. Neuron 10, 805–814 (1993). [4] Di Stasi, A.M.M., Gallo, V., Ceccarini, M. and Petrucci, T.C. Neuron 6, 445–454 (1991). [5] Wyszynski, M. et al. Nature 385, 439–442 (1997).
[6] Kornau, H., Schenker, L., Kennedy, M. and Seeburg, P. Science 269, 1737–1740 (1995). [7] Papa, M. and Segal, M. Neurosci. 71, 1005–1011 (1996). INDIVIDUAL DIFFERENCES IN LATENT INHIBITION IN RATS I. Weiner, R. Barkai, Y. Eitani and J. Feldon Dept. of Psychology, Tel-Aviv University, Tel Aviv, Israel Individual differences in rats’ locomotor activity can predict their response to novelty and psychostimulant drugs such as amphetamine, and these differences seem related to variations in mesolimbic dopamine (DA). The present study tested whether differences in locomotor activity can predict differences in the development of latent inhibition (LI), namely, retardation in conditioning to a stimulus as a consequence of its prior nonreinforced preexposure. LI is sensitive to dopaminergic manipulations, as it is disrupted by the indirect DA agonist amphetamine and facilitated by DA blockers such as haloperidol, and there is a variation between individual subjects in LI magnitude. Rats were screened for their spontaneous activity level and divided into high (20%) and low (20%) activity groups. LI was assessed in a conditioned emotional response procedure in rats licking for water using parameters of preexposure and conditioning which typically do not yield LI. The results showed that: 1. LI was evident in the high- but not in the lowactivity animals; 2. Amphetamine produced LI in low-activity animals while exerting no effect on LI in high-activity animals; 3. Haloperidol produced a robust LI effect in low-activity animals while abolishing LI in high-activity animals. The results show that rats selected as high and low responders on the basis of their locomotor response differ in their propensity to develop LI, as well as in the sensitivity of this phenomenon to amphetamine and haloperidol. THE ROLE OF NUCLEUS ACCUMBENS SUBTERRITORIES IN LATENT INHIBITION I. Weiner, J. Feldon, P. Cassuto and G. Gilad Dept. of Psychology, Tel-Aviv University, Tel-Aviv, Israel Latent inhibition (LI), namely, retarded conditioning to a previously nonreinforced stimulus, is disrupted in amphetamine-treated rats and acute schizophrenic patients. To investigate the role of the nucleus accumbens (NAC) in this phenomenon, electrolytic lesions were made to the shell or core subterritories of the NAC in one experiment, and to the shell or to both shell and core subterritories in another. Using a conditioned emotional response procedure in rats licking for water, it was shown that lesions to the shell abolished LI, whereas lesion to the core as well as the combined shell-core lesion, left LI intact. These results demonstrate a functional dissociation within NAC with respect to LI, and suggest that in the intact brain shell is necessary for LI expression whereas core is necessary for LI disruption. NEUROPROTECTIVE EFFECTS OF AN AChE INHIBITOR AND A SELECTIVE MAO-B INHIBITOR IN ANIMAL MODELS OF ALZHEIMER’S DISEASE M. Weinstock, Y. Chen, E. Shohami Dept. of Pharmacology, School of Pharmacy The Hebrew University of Jerusalem, Jerusalem, Israel A reduction in cholinergic transmission in the basal forebrain occurs in Alzheimer’s disease (AD). Perpetuation of the neuronal damage and its initiation may occur through the production of oxidative free radicals. Rivastigmine is a novel acetylcholinesterase (AChE) inhibitor, currently under clinical investigation for the treatment of AD. Rasagiline is a selective inhibitor of MAO-B with neuroprotective effects against glutamate induced toxicity in vitro. Since head injury is a recognised risk factor for AD, we compared the potential neuroprotective effects of these drugs on the changes in cerebral edema, motor function and spatial memory induced by closed head injury (CHI) un-
BRAIN SPECTRIN BINDING TO THE NMDA RECEPTOR IS REGULATED BY PHOSPHORYLATION, CALCIUM AND CALMODULIN A. Wechsler and V. Teichberg Dept. of Neurobiology, Weizmann Institute of Science, Rehovot, Israel Brain spectrin, a major component of postsynaptic densities (PSDs) [1], links membrane proteins to the actin cytoskeleton [2]. Since the activity of N-methyl-D-aspartate receptor (NMDA-R) channels is dependent on the integrity of actin [3] and leads to calpain-mediated spectrin breakdown [4], we investigated whether the actin-binding spectrin interacts with NMDA-Rs. Spectrin is reported here to interact selectively with the C-terminal cytoplasmic domains of the R1, 2A and 2B subunits of NMDA-R but not with that of AMPA receptor GluR1. Spectrin binds at NR2B sites distinct from those of D-actinin-2 [5] and members of the PSD95/SAP90 family [6]. The spectrin-NR2B interactions are antagonized by Ca2+, fyn-mediated NR2B phosphorylation, but not by Ca2+/calmodulin (CaM) or CaM/kinaseII-mediated NR2B phosphorylation. The spectrin-NR1 interactions are unaffected by Ca2+ but inhibited by CaM and by PKA and PKC-mediated phosphorylations of NR1. The highly regulated linkage of the NMDA-R to spectrin may underlie the morphological changes that occur in neuronal dendrites concurrently with synaptic activity and plasticity [7]. We are currently investigating the spectrin-NMDA-R interaction in their native environment and the physiological role that this interaction might have. [1] Carlin, R.K., Bartelt, D.C. and Siekevitz, P.J. Cell. Biol. 96, 443– 448 (1983). [2] Davis, L.H. and Bennett, V.J. Biol. Chem. 269, 4409–4416 (1994). [3] Rosenmund, C. and Westbrook, G.L. Neuron 10, 805–814 (1993). [4] Di Stasi, A.M.M., Gallo, V., Ceccarini, M. and Petrucci, T.C. Neuron 6, 445–454 (1991). [5] Wyszynski, M. et al. Nature 385, 439–442 (1997).
[6] Kornau, H., Schenker, L., Kennedy, M. and Seeburg, P. Science 269, 1737–1740 (1995). [7] Papa, M. and Segal, M. Neurosci. 71, 1005–1011 (1996). INDIVIDUAL DIFFERENCES IN LATENT INHIBITION IN RATS I. Weiner, R. Barkai, Y. Eitani and J. Feldon Dept. of Psychology, Tel-Aviv University, Tel Aviv, Israel Individual differences in rats’ locomotor activity can predict their response to novelty and psychostimulant drugs such as amphetamine, and these differences seem related to variations in mesolimbic dopamine (DA). The present study tested whether differences in locomotor activity can predict differences in the development of latent inhibition (LI), namely, retardation in conditioning to a stimulus as a consequence of its prior nonreinforced preexposure. LI is sensitive to dopaminergic manipulations, as it is disrupted by the indirect DA agonist amphetamine and facilitated by DA blockers such as haloperidol, and there is a variation between individual subjects in LI magnitude. Rats were screened for their spontaneous activity level and divided into high (20%) and low (20%) activity groups. LI was assessed in a conditioned emotional response procedure in rats licking for water using parameters of preexposure and conditioning which typically do not yield LI. The results showed that: 1. LI was evident in the high- but not in the lowactivity animals; 2. Amphetamine produced LI in low-activity animals while exerting no effect on LI in high-activity animals; 3. Haloperidol produced a robust LI effect in low-activity animals while abolishing LI in high-activity animals. The results show that rats selected as high and low responders on the basis of their locomotor response differ in their propensity to develop LI, as well as in the sensitivity of this phenomenon to amphetamine and haloperidol. THE ROLE OF NUCLEUS ACCUMBENS SUBTERRITORIES IN LATENT INHIBITION I. Weiner, J. Feldon, P. Cassuto and G. Gilad Dept. of Psychology, Tel-Aviv University, Tel-Aviv, Israel Latent inhibition (LI), namely, retarded conditioning to a previously nonreinforced stimulus, is disrupted in amphetamine-treated rats and acute schizophrenic patients. To investigate the role of the nucleus accumbens (NAC) in this phenomenon, electrolytic lesions were made to the shell or core subterritories of the NAC in one experiment, and to the shell or to both shell and core subterritories in another. Using a conditioned emotional response procedure in rats licking for water, it was shown that lesions to the shell abolished LI, whereas lesion to the core as well as the combined shell-core lesion, left LI intact. These results demonstrate a functional dissociation within NAC with respect to LI, and suggest that in the intact brain shell is necessary for LI expression whereas core is necessary for LI disruption. NEUROPROTECTIVE EFFECTS OF AN AChE INHIBITOR AND A SELECTIVE MAO-B INHIBITOR IN ANIMAL MODELS OF ALZHEIMER’S DISEASE M. Weinstock, Y. Chen, E. Shohami Dept. of Pharmacology, School of Pharmacy The Hebrew University of Jerusalem, Jerusalem, Israel A reduction in cholinergic transmission in the basal forebrain occurs in Alzheimer’s disease (AD). Perpetuation of the neuronal damage and its initiation may occur through the production of oxidative free radicals. Rivastigmine is a novel acetylcholinesterase (AChE) inhibitor, currently under clinical investigation for the treatment of AD. Rasagiline is a selective inhibitor of MAO-B with neuroprotective effects against glutamate induced toxicity in vitro. Since head injury is a recognised risk factor for AD, we compared the potential neuroprotective effects of these drugs on the changes in cerebral edema, motor function and spatial memory induced by closed head injury (CHI) un-