Cocaine Administered in Vitro to Brain Slices from Rats Treated with Cocaine Chronically in Vivo Results in a g-Aminobutyric Acid Receptor-Mediated Hyperpolarization Recorded from the Dorsolateral Septum1

Previous reports of membrane hyperpolarizations, associated with acute application of cocaine, have been recorded from brain slice preparations containing aminergic nuclei and have always been attributed to cocaine’s ability to elevate levels of local biogenic amines followed by activation of their receptors. The majority of these studies were conducted with brain slices obtained from rats that had not received prior chronic in vivo treatment with cocaine. We observed that cocaine alone, at 3 mM, could induce a membrane hyperpolarization (COC-HYP) in 100% of rat dorsolateral septal nucleus (DLSN) neurons from brain slices of rats treated chronically with cocaine for either 14 or 28 days in vivo. The DLSN is a nucleus absent of biogenic amine cell bodies, but does contain biogenic amine terminals with GABAergic cell bodies and terminals. Cocaine applied to brain slices from rats not previously administered cocaine or administered cocaine for up to seven days in vivo yielded a maximum incidence of COC-HYPs at only 50%. COC-HYPs recorded from DLSN neurons were not blocked by previous treatment with amine receptor antagonists or by a TTX and zero calcium medium. Based on these results, the ability of DLSN neurons to respond to a cocaine challenge with a COC-HYP did not involve inhibition of amine reuptake/uptake or action potential release of neuroactive substances. Rather, the COC-HYP, with an apparent reversal potential of -80 mV, was reduced by the GABA receptor antagonists-bicuculline and CGP55845A. Lowering extracellular Na or Cl , lowering of temperature, or previous superfusion with the GABA uptake blocker NO-711 could block the COC-HYP. In summary, our data suggest that COC-HYPs, after application of a cocaine challenge to brain slices from rats treated chronically (14 28 days, but not acutely, 7 days) with cocaine are due to cocaine-induced changes in GABA release and/or transporter function. The latter changes in transporter function may involve the reversal of the GABA transporter with release of GABA and subsequent activation of postsynaptic GABAA and GABAB receptors. The majority of studies to determine the cellular actions of cocaine in vitro has used CNS synapses of drug naive rats. These synapses have included both aminergic cell body regions and their terminal fields. There has been a remarkable consistency in terms of the responses obtained after administration of cocaine to aminergic cell body regions. That is, when cocaine alone is applied and recordings are made from cell body areas, a significant inhibitory effect, e.g., a membrane hyperpolarization is recorded from biogenic aminecontaining neurons. This membrane hyperpolarization associated with acute cocaine application results from a potentiation of the typical actions of the transmitter released endogenously (Suprenant and Williams, 1987; Pan and Williams, 1989; Lacey et al., 1990; Bonci and Williams, 1996). In general, the ability of cocaine to potentiate biogenic amine responses has been attributed to its well-known action to bind to the respective amine transporters (Ross and Renyi, 1969; Ritz et al., 1987). Subsequently, inhibition of the transporter would inhibit uptake of biogenic amines applied exogenously to the slice or block reuptake of endogenous biogenic amines released within an aminergic synapse. However, in brain areas lacking biogenic amine-containing somata, i.e., terminal field areas, responses to cocaine adminReceived for publication August 11, 1997. 1 This work was supported by National Institutes of Health, National Institutes of Drug Abuse Grant DA-07190 and Training Grant T32-DA07287 ABBREVIATIONS: DLSN, dorsolateral septal nucleus; COC-HYP, membrane hyperpolarization associated with cocaine challenge; TTX, tetrodotoxin; GABA, g-aminobutyric acid; GABAA, GABAA receptor; GABAB, GABAB receptor; COC-7, chronic in vivo 7-day, twice daily, cocaine treatment; COC-14, chronic in vivo 14-day, twice daily, cocaine treatment; COC-28, chronic in vivo 28-day, twice daily, cocaine treatment; Ri, membrane input resistance; f-ipsp, fast inhibitory synaptic potential; s-ipsp, slow inhibitory synaptic potential; EPSP, excitatory postsynaptic potential; CPT, 8-cyclopentyl-1, 3-dimethylxanthine; ACSF, artificial cerebrospinal fluid; D-AP5, (D)-2-amino-5-phosphonovaleric acid; CNQX, cyano-7-nitroquinoxaline-2,3-dione; MP, membrane potential; DA, dopamine; NE, norepinephrine; 5-HT, serotonin; NIDA, National Institute of Drug Abuse. 0022-3565/98/2861-0509$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 286, No. 1 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 286:509–518, 1998 509 at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from istration have varied from alterations of membrane potential, modification of synaptic activity or no effect (Jahromi et al., 1993; Simms and Gallagher, 1996). Although the effects of cocaine when administered alone have been equivocal, cocaine would potentiate the actions of exogenously applied biogenic amines or prolong evoked aminergic synaptic potentials (Uchimura and North, 1990; Bobker and Williams, 1991; Jahromi et al., 1993; Simms and Gallagher, 1996). The DLSN is a biogenic amine terminal area and one that contains a very high density of cell bodies and terminals for inhibitory (GABA) and excitatory (glutamate) amino acids (Jakab and Leranth, 1994). Because its synaptic pathways contain a diversity of receptors (Gallagher et al., 1995), and its implication in emotion and anxiety (Gray, 1982), we chose the DLSN to investigate the multiple actions of cocaine (Woolverton and Johnson, 1992). Furthermore, because cocaine dependence is a process that is essentially chronic in nature, studies of the effects of repeated cocaine administration in animals are required to suggest a possible interpretation of the clinical effects of chronic cocaine administration in humans. For instance, although cocaine initially produces euphoria and mood elevation, continued abuse can lead to psychiatric problems such as anxiety, depression and psychosis (Fischman, 1987). Accordingly, it has become especially important to study the cellular mechanism(s) by which chronic cocaine in vivo affects neural function both prior to and after an acute “cocaine challenge” or reexposure to cocaine in vitro. We had initially examined the acute in vitro actions of cocaine (Simms and Gallagher, 1993; Simms et al., 1994) applied to brain slice preparations from rats pretreated with saline or not pretreated with cocaine in vivo. Data from these experiments were similar to those reported by Jahromi et al. (1993). These investigators recorded from multiple biogenic amine terminal areas and observed a variety of effects after an acute application of cocaine. However, like us, none of their effects were significant statistically. No consistent changes were reported in any of the following parameters: resting membrane potential, input resistance or spontaneous and evoked inhibitory and excitatory synaptic activity. However, after a 14-day, but not 7-day, in vivo exposure to cocaine (15 mg/kg, twice daily) we observed that all of the above properties were altered significantly and consistently (Shoji et al., 1997: figs. 1 and 2). Furthermore, we also demonstrated (Simms and Gallagher, 1997) that after the same 14-day, but not 7-day, chronic treatment with cocaine, the distribution of cell types within the DLSN (Gallagher et al., 1995) differed with chronic cocaine exposure. All of the changes we have reported before occurred in the absence of any additional acute drug treatment, and thus represented the chronic effects of cocaine to alter the intrinsic electrical and synaptic properties of DLSN neurons (Simms and Gallagher, 1996; 1997; Shoji et al., 1997). Our study was undertaken to characterize the cellular response to a “challenge” dose of cocaine in vitro 16 hr after chronic periods of intermittent cocaine exposure in vivo. Materials and Methods Cocaine treatment regimen. Male Sprague-Dawley rats (Harlan, 75–250 g) were housed three to four per cage with free access to food and water. Each rat received injections with either saline (0.9%) or cocaine HCl [Sigma Chemical Co., St. Louis, MO or National Institute on Drug Abuse (NIDA), Rockville, MD] [15 mg/kg, i.p., twice daily (9:00 A.M. and 4:00 P.M.)] for 7 (COC-7), 14 (COC-14) or 28 (COC-28) consecutive days. It is well established that behavioral sensitization can develop to locomotor activity and stereotyped behavior, specifically, rearing, fast repetitive head and/or foreleg movement, induced by cocaine when it is administered intermittently (Post, 1977). We used the development of behavioral sensitization to cocaine as an indicator of the effectiveness of our cocaine injections. Behavioral sensitization was measured as enhanced exploratory locomotor activity and induced stereotypic behavior in all animals 15 min after twice daily treatment with cocaine for periods of either 7, 14 or 28 days (see Simms and Gallagher, 1996). Because we did not find any appreciable differences in the electrophysiological responses obtained from brain slices derived from saline-injected rats and rats not exposed to cocaine or saline, data from these groups were pooled. All chronic results only represent data collected from rats treated chronically with cocaine and killed 1 hr before their next (and final) scheduled cocaine injection, i.e., at 0800 on days 15 or 29. As a result, this paradigm yielded brain slices having a period (16 hr) of early withdrawal from cocaine. Preparation of brain slice. Rat forebrain coronal slices (500-mm thick) containing the DLSN were prepared using standard techniques (Stevens et al., 1984). Briefly, the rat was decapitated and the brain rapidly removed and immersed in a modified cold ACSF solution. The ACSF solution was maintained at 6°C and bubbled continuously with 95% O2 and 5% CO2 to maintain proper pH (7.3–7.4). The composition of the ACSF solution was as follows: NaCl, 117 mM; KCl, 4.7 mM; NaH2PO4, 1.2 mM; MgCl2, 1.2 mM; CaCl2, 2.5 mM; NaHCO3, 25 mM and glucose, 11.5 mM. In the cold (6°C) solution, the brain was quickly blocked to transverse sections 2-mm thick with the caudal edge at the level of the optic chiasm. Diagonal cuts were then made lateral to the anterior commissure to remove most of the cortex and striatum. The resulting block of tissue was glued (Duro Super Glue, Loctite Corp., Rocky Hill, CT) to a chuck and placed in the bath of a Vibroslice (752 M, Campden Instruments, Ltd., London, England, UK) in similarly treated cold ACSF solution. Serial slices were made rostral to caudal until a section containing medial and lateral septal nuclei was produced. The slice was then placed in a superfusion chamber maintained at 32 6 2°C and superfused at a flow rate of 1 to 1.5 ml/min with ACSF solution bubbled continuously with 95% O2 and 5% CO2. We routinely use the following two criteria as indices of viable slices. First, stable MP of at least -50 mV must be maintained for at least 10 min. Second, the neurons must respond to direct positive current stimulation with a rapid and overshooting sodium spike. Recording from brain slice. Sharp intracellular recordings were obtained using Frederick-Haer standard wall 1.0-mm fiber filled glass microelectrodes pulled to final tip resistances of 70 to 100 MV and filled with 2 M potassium acetate. Ri was routinely measured by passing hyperpolarizing current pulses of known intensities through the recording electrode using a bridge-type circuit. Voltage signals and applied current were recorded with an Axoclamp 2A amplifier (Axon Instruments, Inc., Foster City, CA). The output of the amplifier was D.C. coupled to a storage oscilloscope (Model 5111, Tektronix, Portland, OR) and a dual channel Gould (Cleveland, OH) (Model 220) chart recorder. A Model 4208 Panasonic VCR/Recorder (A.R. Vetter Co., Rebersburg, PA) was used to capture all tracings for storage. The stored signal can be played back and analyzed using a pClamp Version 6.0 Software with a DigiData 1200 interface to a Gateway 2000 4DX2–66V computer. Paper copies of the waveforms were generated with a Hewlett Packard Laserjet 4 printer. Electrical stimulation of brain slice. In some experiments the brain slice was stimulated electrically to yield low frequency induced orthodromic responses via outputs from a Grass (Quincy, MA) S-88 stimulator with isolation units. Focal stimulation was applied through a low resistance concentric bipolar electrode (FrederickHaer) inserted into the dorsolateral aspect of the DLSN nucleus of 510 Shoji et al. Vol. 286 at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from the septum. Stimulus parameters were adjusted to yield consistent responses, e.g., 100-msec duration and 1 to 10 V intensity, at a frequency of 0.17 Hz. We have demonstrated previously that neurons in the DLSN display a series of synaptic potentials in the slice preparation after electrical stimulation of fimbrial afferents and/or local interneurons. These include: 1) an excitatory amino acid(probably glutamate) mediated EPSP that results from activation of both non-NMDA and NMDA (N-methyl-D-aspartic acid) receptors (Gallagher and Hasuo, 1989); 2) a f-IPSP-mediated by GABA acting at GABAA receptors (Stevens et al., 1984) and 3) a s-IPSP-mediated, at least in part, by GABA acting at GABAB receptors (Hasuo and Gallagher, 1988). Drug application. Pharmacological sensitivity and drug testing were carried out by superfusion of known concentrations of substances. Substances were dissolved in the ACSF and entered the recording chamber through a gravity feed inlet of the superfusion system. All drug stock solutions were made up in distilled H2O. The drugs used in the these experiments were as follows: (-)-bicuculline methiodide and TTX from Sigma; (6) sulpiride, idazoxan HCl, CPT; atropine sulfate; p-MPPF dihydrochloride; NO-711-HCl, and D-AP5 from Research Biochemicals Incorporated (RBI, Natick, MA); CGP55845A from Ciba-Geigy (Basel, Switzerland); CNQX from TocrisCookson (Essex, UK); (-)-cocaine HCl from Sigma and the National Institute on Drug Abuse (NIDA). Data analysis. Cocaine was applied by superfusion to the slice and a change in membrane potential was recorded intracellularly. We chose to superfuse routinely and primarily with 3 mM cocaine because our preliminary results showed this concentration of cocaine produced consistent hyperpolarizations and was incapable of local anesthetic effects such as increasing the threshold or width of a sodium spike induced by positive current injection. Furthermore, in vivo microdialysis studies have shown that dialysate cocaine concentrations obtained from brain tissue of chronic cocaine treated rats approaches 3 mM after a cocaine challenge injection (Pettit et al., 1990). Moreover, this concentration of cocaine approximates closely the brain concentrations of cocaine found in users of the drug (Javaid et al., 1978; Van Dyke et al., 1978). All cellular data are expressed as mean 6 S.E.M. Statistical analyses used in these studies were the unpaired one-tailed Student’s t test (SigmaPlot, Windows, Ver. 1.0). Statistical significance was determined at the level of P # .05. Graphs and histograms were generated using SigmaPlot (Windows, Ver.1.0) software (Jandel Scientific Corp., San Rafael, CA). In comparing groups of small sample size (fig. 1, bottom) a Fisher exact test (Sigmastat, Ver. 1.0) was used with statistical significance determined at the level of P # .05.