Pb via Protein Kinase C Inhibits Nicotinic Cholinergic Modulation of Synaptic Transmission in the Hippocampus

The present study was designed to investigate the effects of Pb on modulation of synaptic transmission by nicotinic receptors (nAChRs) in the rat hippocampus. To this end, inhibitory and excitatory postsynaptic currents (IPSCs and EPSCs, respectively) were recorded by means of the whole-cell mode of the patch-clamp technique from rat hippocampal neurons in culture. Acetylcholine (ACh, 1 mM; 1-s pulses) triggered GABA release via activation of 4 2* and 7* nAChRs. It also triggered glutamate release via activation of 7* nAChRs. Pb (0.1 and 1 M) blocked ACh-triggered transmitter release. Blockade by Pb of ACh-triggered IPSCs was partially reversible upon washing of the neurons. In contrast, even after 30to 60-min washing, there was no reversibility of Pb -induced blockade of ACh-triggered EPSCs. The effects of Pb on GABA release triggered by activation of 7* and 4 2* nACRs were mimicked by the protein kinase C (PKC) activator phorbol12-myristate-13-acetate (1 M) and blocked by the indolocarbazole Gö 7874 (50 nM) and the bisindolylmaleimide Ro-318425 (150 nM), which are selective PKC inhibitors. After washing of fully functional neuronal networks that had been exposed for 5 min to Pb , the irreversible inhibition by Pb of ACh-triggered glutamate release was partially overridden by a disinhibitory mechanism that is likely to involve 4 2* nAChR activation in interneurons that synapse onto other interneurons synapsing onto pyramidal neurons. Long-lasting inhibition of 7* nAChR modulation of synaptic transmission may contribute to the persistent cognitive impairment that results from childhood Pb intoxication. Childhood Pb poisoning represents a major concern for public health, particularly because of the cognitive deficits that persist throughout the lives of people who are exposed at early ages to low levels of this pervasive environmental pollutant (Bellinger et al., 1987; Stokes et al., 1998). Although exposure of children to low Pb levels is not associated with overt physical signs of toxicity, it causes mental retardation with selective impairments of language, cognition, behavior, and school performance (Lidsky and Schneider, 2003; Needleman, 2004). Intellectual abnormalities can be seen even in children who have blood Pb concentrations well below 10 g/dl (Lanphear et al., 2000), the level of concern for exposure of children to Pb established in 1991 by the Centers for Disease Control and Prevention. The negative impact of low-level Pb intoxication on brain functioning increases with the duration of the exposure and is inversely proportional to the age at which children are exposed to the heavy metal (Lidsky and Schneider, 2003). Currently, treatment of Pb intoxication relies on the use of chelators, which are recommended by the Food and Drug Administration for children with Pb blood levels 45 g/dl (Nightingale, 1991). However, chelation therapy does not protect these children or those with Pb blood levels between 10 and 45 g/dl from the developmental neurological impairments that result from exposure to the heavy metal (Rogan et al., 2001). Poor understanding of the means by which neuronal functions are affected by Pb accounts, at This work was supported by U.S. Public Health Service Grants ES05730 and NS41671 (for E.X.A.). Part of this work was presented as an abstract at the 1999 Meeting of the Society for Neurosciences. According to the current status of the nomenclature for nicotinic receptors (nAChRs) and their subunits (Lukas et al., 1999), the asterisk next to nAChR subunits throughout the text is meant to indicate that the exact receptor subunit composition is not known. 1 Current address: Department of Psychiatry, F. Edward Herbert School of Medicine, Uniformed Services University of Health Sciences, Rockville, MD 20842. 2 Current address: Department of Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.104.070466. ABBREVIATIONS: VGCC, voltage-gated Ca channels; NMDA, N-methyl-D-aspartate; nAChR, nicotinic acetylcholine receptor; PKC, protein kinase C; IPSC, inhibitory postsynaptic current; EPSC, excitatory postsynaptic current; ACh, acetylcholine; PMA, phorbol 12-myristate 13-acetate; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; MLA, methyllycaconitine; DH E, dihydro-erythroidine. 0022-3565/04/3112-700–710$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 311, No. 2 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 70466/1173871 JPET 311:700–710, 2004 Printed in U.S.A. 700 at A PE T Jornals on A ril 9, 2016 jpet.asjournals.org D ow nladed from least in part, for the lack of effective therapeutic approaches to reverse the Pb -induced cognitive deficits. Establishment of neuronal circuitries in the developing central nervous system depends on the pattern of electrical activity going through the synapses. At early stages of brain development, most neurons fire spontaneously, and this spontaneous electrical activity, which ultimately controls synaptic strength, seems to be crucial for axon outgrowth, pruning of synaptic connections, and maturation of neuronal signaling properties (Moody, 1998). Therefore, it is conceivable that learning disabilities in children exposed to low levels of Pb result from direct changes that the heavy metal imparts on synaptic activity particularly in the hippocampus, a major brain area that is involved in cognitive processing and accumulates significant amounts of Pb (Swanson et al., 1997). Pb alters synaptic transmission in numerous preparations by disrupting the activity of Ca -regulated proteins, including protein kinases, voltage-gated Ca channels (VGCC), and Ca -binding proteins that regulate mobilization and docking of synaptic vesicles (Atchison, 2003; Suszkiw, 2004). In primary hippocampal cultures, action potential-independent synaptic transmission is facilitated by nanomolar concentrations of Pb due to an intracellular action of the heavy metal (Braga et al., 1999b). Also in these cultures, action potential-dependent synaptic transmission is inhibited by similar concentrations of Pb due to blockade of VGCC (Braga et al., 1999a). In the hippocampus, however, synaptic transmission is modulated by ionotropic receptors such as N-methyl-D-aspartate (NMDA) receptors and 7* nAChRs (Vizi and Kiss, 1998; Pereira et al., 2002), both of which are sensitive to inhibition by micromolar concentrations of Pb (Guilarte and Miceli, 1992; Ujihara and Albuquerque, 1992; Ishihara et al., 1995; Zwart et al., 1995; Marchioro et al., 1996; Mike et al., 2000b; Si and Lee, 2003). To date, very little is known regarding signal transduction mechanisms that link nAChR activation to modulation of action potential-dependent transmitter release in the brain, and no studies have addressed how this modulatory process could be affected by Pb . Thus, the present study was designed to 1) determine whether Pb affects modulation by nAChRs of GABAergic or glutamatergic transmission, and, if so, by what mechanism(s); 2) compare the sensitivity to Pb of whole-cell currents and transmitter release triggered by nAChR activation; and 3) examine the net effect of Pb on nicotinic modulation of synaptic activity in the hippocampus. Glutamatergic and GABAergic postsynaptic currents were recorded by means of the patch-clamp technique from cultured hippocampal neurons, and the effects of acute exposure to Pb on GABA and glutamate release evoked by nAChR activation were analyzed. Results presented herein demonstrate that at nanomolar concentrations Pb acts via protein kinase C (PKC) to inhibit action potential-dependent transmitter release triggered by 7* and/or 4 2* nAChR activation in hippocampal neurons. This effect, which cannot be easily reversed upon removal of Pb from the extracellular compartment, causes a long-lasting disruption of the activity of large neuronal networks and can, therefore, contribute to the cognitive impairment induced by Pb . These findings and the lack of therapeutic approaches to reverse the complex Pb -PKC interactions emphasize the concept that preventive measures are still by far the best means to protect children against Pb -induced neurological deficits. Materials and Methods Cultures of Hippocampal Neurons. Hippocampal neurons were harvested from 16to 18-day-old fetal Sprague-Dawley rats and cultured according to the procedure described by Ujihara and Albuquerque (1992). Neurons cultured for 10 to 30 days were used in this