Introduction

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Cocaine abuse is clearly a major public health problem throughout the world. Currently, although several therapeutic regimens have been proposed, no effective treatment has been substantiated (National Institutes of Health, 1996). This lack of an effective pharmacological treatment is not surprising, without a complete understanding of the mechanism(s) responsible for the abuse liability associated with chronic cocaine administration. Numerous investigations have demonstrated that a prominent mechanism underlying the rewarding properties of acute cocaine exposure involves its ability to inhibit amine uptake by binding to amine transporters, thereby enhancing the endogenous actions of one or more of the three major biogenic amine neurotransmitters/neuromodulators, i.e., DA, serotonin (5-hydroxytryptamine) or NEpi (Wise, 1984). The concentration of cocaine required to inhibit these amine transporters is in the micromolar range (Ritz, et al., 1987), a concentration range comparable to blood levels in humans that are associated with subjective feelings of a "high" (Javaid et al., 1978). However, certain aspects of the behavioral pharmacology associated with cocaine suggest that inhibition of biogenic amine uptake at the specific amine transport sites is not solely responsible for the actions of cocaine (Woods et al., 1987; Balster, 1988). In a recent review, Kleber (1995) stated that, despite the use of DA drugs as potential therapies for treating cocaine addiction, no medications have yet proven to be generally effective. In fact, Wise stated that "hope for the pharmacotherapy of addiction (stimulant abuse, including cocaine) lies in the development of drugs that may act at other stages (non-DA) of the brain's reward circuitryperhaps in the anatomical cascade of GABAergic efferents... " (Wise, 1995, p. 579). Thus, the possibility exists that drugs, in addition to those acting within biogenic amine systems, are available to enhance the current tools used in the pharmacotherapy of cocaine addiction. We demonstrate that chronic cocaine exposure alters the efficacy of presynaptic GABA_B receptors to regulate amino acid release in the brain at a nucleus implicated in reward mechanisms, and we suggest that drugs selective for presynaptic GABA_B receptors may be an effective substitute for cocaine as a replacement therapy in the treatment of chronic cocaine abuse.

Because cocaine abuse is primarily associated with long-term use, we designed experiments to examine the cellular effects of cocaine in vitro after its chronic administration in vivo. Prior in vitro intracellular electrophysiological studies examined only the acute effects of cocaine (Yasuda et al., 1984; Suprenant and Williams, 1987; Pan and Williams, 1989; Lacey et al., 1990; Uchimura and North, 1990; Bobker and Williams, 1991; Smith et al., 1993; Tanaka and North, 1993; Wheeler et al., 1993; Cameron and Williams, 1994). Furthermore, in those prior studies with cocaine, the majority of brain areas investigated consisted of amine cell body areas only, as opposed to amine terminal fields or nonamine sites. The effects of chronic cocaine exposure, in vivo, have been reported only with intracellular recordings from neurons of an in vitro brain slice preparation in two studies, i.e., at the locus ceruleus (Harris and Williams, 1992) and at the VTA (Bonci and Williams, 1996). In those studies, no base-line changes in synaptic transmission or electrical properties were noted before re-exposure to cocaine. However, an interval of 7 to 10 days was interposed after chronic administration was discontinued and before in vitro experiments were initiated; such an interval may have induced phenomena associated with "withdrawal," which could have complicated their results. To avoid such a potential complication, we prepared slices on the day of recording and at a time point (8:00 A.M.) within the standard final interval of our normal dosing schedule (9:00 A.M. and 4:00 P.M.), i.e., the animals had not experienced any discontinuation of their normal twice-daily dosing schedule.

	Methods

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Cocaine treatment regimen. Male Sprague-Dawley rats (75-250 g; Harlan) were housed three or four per cage, with free access to food and water. Each rat was injected with either saline (0.9%) or cocaine HCl (Sigma Chemical Co., St. Louis, MO, or National Institute on Drug Abuse, Rockville, MD) (15 mg/kg i.p. twice daily, at 9:00 A.M. and 4:00 P.M.) for 7, 14 or 28 consecutive days. It is well established that sensitization can develop to the locomotor activity and stereotyped behavior, e.g., rearing and fast, repetitive, head and/or foreleg movement, induced by cocaine when it is administered intermittently (Post, 1977). We thus used the development of "behavioral sensitization" to cocaine as an indicator of the effectiveness of the cocaine injections to modify CNS function. To this end, rat behaviors were rated 15 min after the first and final cocaine injections on a six-point behavior scale, modified by Gifford and Johnson (1992) from that described by Ellinwood and Balster (1974), by an observer unaware of the previous injection schedule for each animal. All rats treated chronically with cocaine and used in these studies exhibited behavioral sensitization before sacrifice. A complete description of this rating scale and the time course required to observe, in our hands, the induction and expression of behavioral sensitization was included in a recent report (Simms and Gallagher, 1996).

Preparation of brain slices. Rats were decapitated, and the brain was rapidly dissected out and placed in cold artificial cerebrospinal fluid of the following composition (in mM): NaCl, 117; KCl, 4.7; MgCl₂, 1.2; CaCl₂, 2.5; NaH₂PO₄, 1.2; glucose, 11.5; NaHCO₃, 25; prebubbled with 95% O₂/5% CO₂, pH 7.4. Transverse blocks of tissue containing the septum were serially cut, on a Vibroslice (model 752 M; Campden Instruments, London, England), into 500-µm sections. A single slice was submerged in a recording chamber (1.0-ml volume) and superfused with the gassed artificial cerebrospinal fluid warmed to 32 ± 1°C. We routinely used the following two criteria as indices of viable slices. First, the resting MPs must be stable for 10 min at a potential level of at least 50 mV. Second, the neurons must respond to direct positive current stimulation with a rapid and overshooting sodium spike.

Recording from brain slices. Conventional intracellular recording methods were used, with glass microelectrodes (75-100 M) filled with 2 M KAc or 2 M KCl (50-70 M). Voltage signals and applied current were generated and recorded with an Axoclamp 2A amplifier (Axon Instruments, Inc., Foster City, CA) containing a bridge-type circuit and were used routinely to measure membrane input resistance. The output of the amplifier was direct current-coupled to a storage oscilloscope (model 5111; Tektronix, Portland, OR) and a dual-channel Gould chart recorder (model 220; Gould, Cleveland, OH). A model 4208 Panasonic videocassette recorder (A.R. Vetter Co., Redersburg, PA) was used to capture all tracings for storage. The stored signal could be played back and analyzed using pClamp (version 6.0) software, with a DigiData 1200 interface to a Gateway 2000 4DX2-66V computer. Orthodromic stimuli were delivered with square-wave pulses (5-20 V, 0.10 msec) via a concentric bipolar electrode placed focally within the DLSN.

Drug application. ()-Cocaine hydrochloride (1-10 µM), CGP-55845A, bicuculline methiodide, TTX, (±)-baclofen and GABA were all applied by bath superfusion to achieve steady-state concentrations within the chamber. All drugs were obtained from Sigma except idazoxan, sulpiride, 6-cyano-7-nitroquinoxaline-2,3-dione and (+)-2-amino-5-phosphonopentanoic acid, which were from RBI (Natick, MA). CGP-55845A and cocaine were kindly supplied by Ciba-Geigy (Basel, Switzerland) and the National Institute on Drug Abuse, respectively. The results reflect the effect of drugs applied to only one neuron from each brain slice.

Data analysis. Three populations of rats were used to examine acute and chronic effects of cocaine. Standard electrophysiological intracellular recording techniques were used with an in vitro rat brain slice preparation containing the DLSN (Stevens et al., 1984). Acute experiments involved one population of naive rats (control) who had never been exposed to cocaine. Concentrations of cocaine (1-10 µM) were applied in vitro by superfusion of brain slices obtained from control rats. Chronic experiments consisted of two different populations of rats, 1) rats given saline (i.p., twice daily) for periods of 7, 14 or 28 days and 2) rats given cocaine in vivo (15 mg/kg i.p., twice daily) for periods of 7, 14 or 28 days. To minimize potential complications that may arise from the induction of phenomena associated with withdrawal, all chronic results represent only data collected from rats treated chronically with cocaine and sacrificed 1 hr before their next (and final) scheduled cocaine injection, i.e., at 8:00 A.M. on day 15 or 29. Additional studies are ongoing to sample brain activity at longer intervals after the last injection of cocaine. Cocaine or other drugs were also administered in vitro to brain slices prepared from saline-treated rats, and data from cocaine- and saline-treated rats were compared. Statistical analyses in these studies used unpaired, one-tailed, Student's t test software (SigmaPlot, Windows version 1.0; Jandel Scientific Corp., San Rafael, CA). Statistical significance was determined at the level of P  .05. Graphs and diagrams were generated using SigmaPlot (Windows version 1.0) and Harvard Graphics (Windows version 2.0) software (SPC Software, Santa Clara, CA), respectively, with a Hewlett Packard Laserjet 4 printer. Results are expressed as mean ± S.E.M.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Comparison of sp-IPSP frequency measured from control rat brain neurons with the increased frequency measured from chronically cocaine-treated preparations. *P  .05, unpaired t test, statistically different from control.

	Results

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Chronic cocaine treatment enhances spontaneous inhibitory post-synaptic potentials (sp-IPSPs). We analyzed sp-IPSPs in DLSN neurons from control and cocaine-treated animals. sp-IPSPs were isolated from excitatory synaptic activity by recording from brain slices superfused continuously with excitatory amino acid and biogenic amine antagonists. In the presence of these antagonists, brains from control rats typically exhibit a very low frequency of hyperpolarizing sp-IPSPs when recorded with KAc-filled electrodes (figs. 1A, left, and 2). Recording with KCl-filled electrodes reverses sp-IPSP polarity and increases the signal-to-electrode noise ratio (fig. 1B-D) of the recorded depolarizing sp-IPSPs. As with normals rats (Otis and Mody, 1992; Mody et al., 1994), sp-IPSPs recorded from DLSN neurons in brain slices obtained from cocaine-treated rats appeared to be mediated through activation of GABA_A receptors, because only depolarizing sp-IPSPs were observed in recordings made with KCl-filled electrodes (fig. 1B-D), rather than KAc-filled electrodes (fig. 1A). Application of bicuculline, a selective GABA_A receptor antagonist, eliminated all sp-IPSPs (fig. 3A). DLSN neurons recorded from brain slices obtained from cocaine-treated rats exhibited a significant increase in frequency of sp-IPSPs, from 0.65 to 2.05/sec (fig. 2). Depression of sp-IPSP frequency, due to activation of the GABA_B autoreceptor, became apparent after application of GABA (50 µM) (fig. 1D) or baclofen (0.05 µM) (data not shown); these concentrations of GABA and baclofen (fig. 4C) are below those that activate postsynaptic GABA_B receptors.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Enhancement of GABA release, measured as sp-IPSPs, in brains of rats administered cocaine chronically (15 mg/kg i.p., twice daily for 14 days), compared with naive rats (control). All data were collected in the presence of (+)-2-amino-5-phosphonopentanoic acid, 6-cyano-7-nitroquinoxaline-2,3-dione, idazoxan and sulpiride. A, sp-IPSPs recorded with a KAc-filled electrode are hyperpolarizing (downward deflections) from the resting MP (resting MP = 58 mV). Initial rectangular deflection represents an electrotonic potential used to monitor input resistance. Left, brain slice from a naive rat (control), with typical low-frequency (<1/sec) sp-IPSPs. Right, brain slice from a chronically cocaine-treated rat, with higher-frequency (>2/sec) sp-IPSPs. B, sp-IPSPs recorded with a KCl-filled electrode are depolarizing (upward deflections) from the holding MP (75 mV) maintained by direct current. Left, brain from a naive rat (control), with typical low-frequency (<1/sec) sp-IPSPs. Right, brain from a chronically cocaine-treated rat, with higher basal sp-IPSP frequency. C, control (naive) rat brain exhibits typical low-frequency (<1/sec) depolarizing sp-IPSPs. Blockade of presynaptic GABAB autoreceptors with CGP-55845A (0.5 µM) mimics the increased frequency observed with cocaine-treated brains. D, this chronically cocaine-treated rat (from B) exhibits enhanced frequency of depolarizing sp-IPSPs. Frequency is reduced by application of GABA (50 µM), a concentration that induces no postsynaptic membrane changes.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Depolarization of resting MP after application of bicuculline to brain slices from cocaine-treated rats. Bicuculline, up to 50 µM, does not significantly change the MP of brains from control animals. All data were collected in the presence of (+)-2-amino-5-phosphonopentanoic acid, 6-cyano-7-nitroquinoxaline-2,3-dione, idazoxan and sulpiride. A, bicuculline (10 µM) superfusion (between arrows) to brain slices of naive rats (control) does not change MP (59 mV). During bicuculline application, action potentials (upward deflection from horizontal base-line MP) appear and GABAA receptor-mediated (first in a pair of downward deflections from MP) IPSPs are blocked. Dotted line, 5-min break of the continuous recording in all records. B, bicuculline (10 µM) superfusion to brain slices of chronically cocaine-treated rats results in depolarization. a, original MP (63 mV) is depicted as a horizontal base line (or dashed line), from which upward deflections (action potentials) and downward deflections (electrotonic potentials and evoked late IPSPs) are depicted. b, the depolarization after bicuculline persists in calcium-free medium (MP = 65 mV). Calcium was replaced with 10 mM Mg++; 0.5 mM ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) was added to remove any residual calcium. c, the depolarization after bicuculline persists with a combination of TTX and calcium-free medium (MP = 62 mV).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Concentration-response curves depicting presynaptic (A and B) (0.01-0.1 µM) vs. postsynaptic (C) (0.1-10 µM)) actions of (±)-baclofen. Concentrations of baclofen less than 0.1 µM do not activate postsynaptic GABAB receptors in the DLSN. Each data point represents n = 5. A, chronic cocaine treatment caused a parallel rightward shift for baclofen to depress GABA transmission in brain slices taken from cocaine-treated rats, compared with control rats. GABA transmission was assessed as depression of an orthodromically induced IPSP. Data were collected in the presence of (+)-2-amino-5-phosphonopentanoic acid, 6-cyano-7-nitroquinoxaline-2,3-dione, idazoxan and sulpiride. B, similarly, chronic cocaine treatment caused a parallel rightward shift for baclofen to depress glutamate transmission in brain slices taken from cocaine-treated rats. Glutamate transmission was assessed as depression of an orthodromically induced EPSP. Data were collected in the presence of bicuculline, idazoxan and sulpiride. C, the concentration-response curves for baclofen to activate postsynaptic GABAB receptors and hyperpolarize the MP were unchanged in brain slices from chronically cocaine-treated rats (COCAINE) vs. naive rats (CONTROL).

Enhanced spontaneous GABA release is antagonized by bicuculline. If enhanced GABA release is responsible for the more negative MP (table 1) recorded from DLSN neurons in brains from rats treated chronically with cocaine, then application of a GABA receptor antagonist should reduce this enhanced GABA tone by competing for and blocking GABA receptors. In brain slices of control rats, superfusion with the competitive GABA_A receptor antagonist bicuculline (up to 50 µM) did not alter ( = 0.7 ± 0.7 mV, n = 10) MP significantly (fig. 3A). However, bicuculline (10 µM) (fig. 3Ba) applied to brain slices of cocaine-treated rats resulted in depolarization (5.2 ± 0.7 mV, n = 47). The depolarization recorded with bicuculline persisted in the absence of extracellular calcium (fig. 3Bb) and in the presence of TTX (fig. 3Bc), suggesting that the tonic, more hyperpolarizing MP recorded from brains of cocaine-treated rats may result from an action potential-independent, calcium-independent release of GABA (Pin and Bockaert, 1989). Furthermore, the persistence of bicuculline depolarization in the presence of TTX suggests that this enhanced GABA release is a direct effect of cocaine, administered chronically in vivo, on the GABA release and/or uptake process, rather than an effect mediated via action potential-dependent release of another transmitter.

Presynaptic GABA_B receptor function is altered by chronic cocaine treatment. How does chronic cocaine cause changes in GABA release? In general, neurons use a feedback inhibitory mechanism, involving an autoreceptor, to prevent excessive and continuous release of their transmitter (Starke, 1981). Within the DLSN, activation of presynaptic GABA_B autoreceptors and reduction of GABA release occurred over a concentration range lower than that required to activate postsynaptic GABA_B receptors (fig. 4, A and B vs. C). Figure 4, A and B, depicts only concentrations of baclofen less than 0.1 µM, to demonstrate a concentration-dependent selective presynaptic action; higher concentrations of baclofen completely block both sp-IPSPs and evoked IPSPs (S. Shoji, D. Simms and J. P. Gallagher, unpublished observations) while hyperpolarizing the membrane by a direct postsynaptic effect (fig. 4C).

Chronic cocaine treatment also alters glutamate release. In a parallel series of experiments, we examined the ability of baclofen to inhibit orthodromically evoked EPSPs recorded within the DLSN. GABA_B heteroreceptors inhibit release of transmitters from non-GABAergic neurons by a mechanism similar to that of GABA_B autoreceptors (Potashner, 1979; Anderson and Mitchell, 1985). We recorded focally evoked EPSPs in the presence of bicuculline (10 µM) and the absence of any excitatory amino acid antagonists. Typically, low-stimulus intensity/frequency induced-EPSPs are mediated by ionotropic glutamate receptors (Gallagher and Hasuo, 1989) on DLSN neurons. At low concentrations (0.1 µM)), baclofen inhibited EPSPs presynaptically, while having no postsynaptic action (fig. 4, B and C). However, in experiments with brains from cocaine-treated rats, baclofen was less effective in its ability to depress EPSPs presynaptically (fig. 4B). It is apparent that chronic cocaine treatment also diminished, to an even greater degree, the ability of GABA_B heteroreceptors to control glutamate release, in a manner analogous to that seen with GABA_B autoreceptors to control GABA release (fig. 4A). Thus, chronic cocaine treatment diminished the ability of nerve terminal receptors to control release of both inhibitory and excitatory transmission within this nucleus, while having no apparent effect on comparable postsynaptic receptors. A similar selective decrease in the function of GABA_B heteroreceptors at glutamate terminals has been demonstrated in the kindling model of epilepsy (Asprodini et al., 1992), a disease with plastic changes possibly similar to those associated with chronic cocaine abuse.

	Discussion

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In this study, we tested the hypothesis that, in addition to the biogenic amines, other neurotransmitters are involved in the actions of cocaine (Woolverton and Johnson, 1992). The chronic effects of cocaine were investigated at a brain site, the DLSN, that after electrical stimulation results in somatomotor and visceromotor suppression (Thomas, 1988). This area has also been implicated in behavioral reward functions (Olds and Milner, 1954; Rompre and Shizgal, 1986) and has been designated as a "pleasure center," based on experiments demonstrating intracranial self-stimulation at this site. The DLSN contains 1) a very high density of GABA cell bodies and terminals (interneurons), 2) cell bodies and terminals for excitatory amino acids and 3) terminals, but not cell bodies, for numerous other transmitters, including DA (from the VTA), NEpi (from the locus ceruleus), 5-hydroxytryptamine (from the raphe nuclei) and peptides (from the hypothalamus) (Jakab and Leranth, 1994; Gallagher et al., 1995). Thus, the DLSN brain slice is a highly appropriate preparation to examine the multiple actions of cocaine at inhibitory and excitatory synapses.

GABAergic inhibitory transmission within the DLSN is mediated by activation of two postsynaptic GABA receptors, GABA_A and GABA_B (Stevens et al., 1987; Hasuo and Gallagher, 1988). These receptors are activated by GABA released from within the DLSN as a result of input from both GABAergic and glutamatergic neurons (Jakab and Leranth, 1994; Gallagher et al., 1995). The GABA_A receptor is an ionotropic receptor antagonized by bicuculline and associated with a chloride channel. GABA_A receptor activation by spontaneous or evoked GABA release results in fast IPSPs. Later, slower IPSPs result from GABA_B receptor activation. The GABA_B receptor is a metabotropic receptor coupled by a G protein to its effector(s), activated selectively by baclofen (Hill and Bowery, 1981), antagonized by CGP-55845A and associated postsynaptically with a potassium channel. Endogenous GABA acts at both postsynaptic receptor types to hyperpolarize the MP. In addition to postsynaptic GABA receptors, presynaptic GABA_B autoreceptors (on GABA terminals) (Anderson and Mitchell, 1985) and GABA_B heteroreceptors (e.g., on glutamate terminals) (Potashner, 1979) are also present and, when activated, inhibit further GABA or glutamate release, respectively (Misgeld et al., 1995). Because GABA can activate both GABA_A and GABA_B receptors, baclofen (a selective GABA_B agonist) was used to discriminate between these different GABA receptors and mimic only the effects of GABA_B receptor activation.

Our results (figs. 1 and 2) demonstrate that GABA release is enhanced presynaptically in brain slices obtained from cocaine-treated rats, compared with controls. An increase of released GABA could subsequently activate postsynaptic GABA receptors and result in tonic hyperpolarization, which would account for the more negative MP (61 vs. 59 mV) (table 1) and the lower neuronal input resistance (122 vs. 133 m) observed in DLSN neurons from cocaine-treated rats. An increase in sp-IPSP frequency seen with chronic cocaine (figs. 1 and 2) was mimicked by application of the selective GABA_B receptor antagonist (Davies et al., 1993) CGP-55845A (0.5 µM) to brain slices of control rats (fig. 1C). The GABA_B antagonist acts at the autoreceptor to block the normal negative feedback control of GABA release. Also, within the cocaine-treated slices there was no effect on the sodium spike threshold (table 1), suggesting that, at the concentrations used in this study, cocaine was not acting as a local anesthetic.

Multiple cellular mechanisms may contribute to the phenomena we demonstrate as an increase in sp-IPSPs (figs. 1 and 2) and a decreased ability to inhibit evoked IPSPs and EPSPs (fig. 4, A and B) in brains from rats treated chronically with cocaine. Enhanced synaptic levels of endogenous GABA associated with increased sp-IPSPs could, under chronic conditions, down-regulate, desensitize and/or saturate terminal as well as postsynaptic GABA_B receptors. Figure 4 does not support alterations in postsynaptic receptor number, sensitivity or saturation. On the other hand, the presynaptic GABA_B receptors may be down-regulated, desensitized and/or saturated as a result of excess GABA in the synapse. In the case of down-regulation resulting from a decrease in receptor number, the receptors present should still function, but the measured response would not attain the same maximum as when the receptor number is "normal," assuming there are no "spare receptors." Our data in figure 4 do not support such a mechanism, because we see a parallel shift in the dose-response relationship in brain slices obtained from chronically cocaine-treated animals. When excess agonist is present, the receptor may become desensitized. Our data in figure 1C do not support this possibility, because application of GABA (or baclofen) reduced the increase in sp-IPSP frequency observed in brain slices obtained from chronically cocaine-treated rats. If receptors were desensitized, application of agonist would have exacerbated the desensitized state and caused the receptors to be less responsive. Our results suggest that the negative feedback process is functional and can be activated by exogenous application of agonist, despite the presence of excess endogenous agonist. This latter result also argues against the possibility that functional GABA_B terminal receptors are saturated by the high levels of endogenous GABA, because in a saturated state there would be no receptors available to activate.

An alternative experimental approach to that of applying an agonist exogenously (fig. 1D) is to apply an antagonist (fig. 1C). However, our experiments with CGP-55845A have not been definitive. Possibly because the sp-IPSP frequency is very high (i.e., a ceiling effect has already been attained) in brain slices from animals treated chroncially with cocaine (fig. 1), application of the antagonist has not produced any additional increase in frequency (unpublished observations). Furthermore, due to the relative irreversibility of the antagonist (Bon and Galvan, 1996), these results are not conclusive. Nonetheless, this apparent lack of a further increase in sp-IPSP frequency would support, in general, the observation that the effectiveness of the presynaptic GABA_B receptors is reduced. However, these results do not identify a specific cellular mechanism as being responsible for the phenomena we have observed.

An alternative cellular mechanism that warrants consideration to explain how chronic cocaine treatment alters presynaptic GABA_B receptor function involves the G protein coupling of the GABA_B receptor with its effectors. Our present data do not address this issue, but two recent reviews (Hyman, 1996; Hyman and Nestler, 1996) and the work of Nestler et al. (1990, 1993) support such a possibility. Nestler et al. (1990, 1993) demonstrated that a similar chronic, but not acute, cocaine regimen decreased the levels of the specific G protein subunits G_i and G_o in brain nuclei that have been implicated in brain reward mechanisms, including the VTA, nucleus accumbens and locus ceruleus. Moreover, the G_o protein subunit has been associated with GABA_B receptors coupled to voltage-sensitive calcium channels (Menon-Johansson et al., 1993; Campbell et al., 1993, 1995). We propose that chronic cocaine treatment alters GABA_B auto- and heteroreceptor efficacy by decreasing the levels of G_o subunits typically associated with the GABA_B presynaptic receptors, thereby uncoupling or reducing the efficiency with which this receptor is regulated by endogenous GABA. We do not believe the GABA_B receptor itself is altered by chronic cocaine, because the postsynaptic response remains intact and exhibits an identical dose-response effect in DLSN neurons recorded from brain slices of control and cocaine-treated rats (fig. 4C). These results implicating a role for G proteins in the chronic actions of cocaine may also relate to the data presented by Kalivas and co-workers (Steketee and Kalivas, 1991a,b; Steketee et al., 1991), who concluded that pertussis toxin-sensitive G proteins are involved in the behavioral sensitization to cocaine and other stimulants. Thus, although not temporally identical with the induction of behavioral sensitization (Simms and Gallagher, 1996), our electrophysiological experiments suggest that down-regulation of GABA_B terminal, but not postsynaptic GABA_B, receptors may contribute to the maintenance of the phenomenon described as behavioral sensitization to chronic cocaine in intact animals.

Finally, our data also suggest that administration of low doses of baclofen, acting at presynaptic but not postsynaptic sites, may be a possible nonaddictive substitute for cocaine chronically, because it could maintain GABA_B presynaptic receptors in a down-regulated (fig. 4, A and B) state. Chronic treatment with a GABA_B agonist or antagonist has recently been demonstrated to induce down- or up-regulation, respectively, of GABA_B autoreceptor sensitivity within rat brain and spinal cord (Malcangio et al., 1995). Thus, similarly to cocaine, chronic baclofen administration decreased the effectiveness of endogenous GABA to activate the GABA_B autoreceptor and inhibit additional GABA release. Interestingly, recent support for the use of baclofen in the treatment of cocaine addiction has come from the work of Roberts and co-workers (Andrews et al., 1995), who demonstrated that baclofen attenuates the reinforcing effects of cocaine in rats. Our data with chronic cocaine suggest an apparent down-regulation ("desensitization") of GABA_B autoreceptors, possibly resulting from a decrease in 1) their affinity and/or 2) the efficacy of their coupling mechanism(s), with no effect on the postsynaptic GABA_B receptors (fig. 4C).

Because nerve terminal GABA_B receptors are distributed throughout the brain, chronic cocaine may alter CNS neurotransmission and subsequent neuronal activity in multiple CNS pathways, especially, but not only, those associated with drug reward, learned and motor behaviors, e.g., the VTA, nucleus accumbens, striatum, thalamic nuclei, amygdala, hippocampus and spinal cord. We have attempted in figure 5 to extend our results within the DLSN to septal afferents known (Jakab and Leranth, 1994) to terminate in neighboring nuclei, e.g., nucleus accumbens, a nucleus that has a well-known involvement in brain reward mechanisms. In the pathway depicted, down-regulation of GABA_B heteroreceptors on glutamate afferents would enhance glutamate release and subsequent activation of downstream DA neurons. This enhanced DA activity would lead to an even more exaggerated release with loss of negative feedback control via down-regulated GABA_B receptors on DA terminals. Thus, if changes similar to those we have reported for the DLSN are observed in other brain regions, these results could have widespread implications. For instance, two recent studies in which multiple brain areas were monitored reported elevations in turnover rates and extracellular concentrations of excitatory and inhibitory amino acids after chronic or acute exposure to cocaine (Dworkin and Smith, 1995; Smith et al., 1995). We suggest that presynaptic GABA_B receptors, with their associated neuronal substrates and pathways, should be considered when determining a cellular mechanism underlying the addictive and possibly toxic properties of cocaine.

View larger version (53K): [in this window] [in a new window]   Fig. 5.   Diagram depicting the effects of chronic cocaine within the DLSN to down-regulate presynaptic but not postsynaptic GABAB auto- and heteroreceptors at GABA and glutamate terminals, respectively. In addition, the suggestion is made that GABAB presynaptic heteroreceptors on glutamate terminals and DA terminals within nuclei outside of the DLSN, e.g., at glutamate terminals on DA neurons within the nucleus accumbens (N. Accumbens) and/or at DA terminals on DA neurons within the nucleus accumbens, may be similarly down-regulated, resulting in disinhibition and enhanced release of their respective transmitters.