Introduction

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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 amine-containing 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 administration 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

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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).

Preparation of brain slice. Rat forebrain coronal slices (500-µm 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 ± 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 M 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 (Frederick-Haer) inserted into the dorsolateral aspect of the DLSN nucleus of the septum. Stimulus parameters were adjusted to yield consistent responses, e.g., 100-µsec 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 GABA_A receptors (Stevens et al., 1984) and 3) a s-IPSP-mediated, at least in part, by GABA acting at GABA_B 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; (±) sulpiride, idazoxan HCl, CPT; atropine sulfate; p-MPPF dihydrochloride; NO-711-HCl, and D-AP5 from Research Biochemicals Incorporated (RBI, Natick, MA); CGP-55845A from Ciba-Geigy (Basel, Switzerland); CNQX from Tocris-Cookson (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 µM 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 µM 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 ± 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.

	Results

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Expression of the COC-HYP is Dependent upon Duration of in Vivo Treatment

Cocaine (1-10 µM) applied by superfusion to brain slices (n = 63) in vitro obtained from rats treated chronically with cocaine in vivo [15 mg/kg, i.p., twice daily (BID) × 14 or 28 days] resulted in a slow onset hyperpolarization (COC-HYP) of the MP of DLSN neurons (at 3 µ M, MP = -4.9 ± 0.4 mV, n = 10, fig. 1, top). We conclude that COC-HYPs are an all-or-none event with a change of -2 mV from MP being the minimum recorded threshold of occurrence. A change of -2 mV is also biologically relevant, because at -60 mV a DLSN neuron could exhibit spontaneous firing activity, whereas, at -62 mV the same DLSN neuron would exhibit no spontaneous activity (Gallagher et al., 1995). All COC-HYPs were recorded in the presence of biogenic amine antagonists in the superfusion medium.

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Membrane hyperpolarization associated with a cocaine challenge (COC-HYP) recorded in vitro from brain slices of rats treated chronically, in vivo, for 14 or 28 days with cocaine. Top, Superfusion of cocaine (3 µM) results in a slow-onset membrane hyperpolarization from a membrane potential (MP-dotted line) held initially at -60 mV. Upper deflections from MP represent spontaneous action potential activity attenuated by the frequency response of the chart recorder. Downward deflections represent evoked inhibitory postsynaptic potentials (IPSPs) and hyperpolarizing electrotonic potentials; the latter serve as a monitor of input resistance (Ri). Bottom, Incidence of recording COC-HYPs, in vitro, is concentration dependent and associated with the duration of twice daily injections of cocaine, in vivo. At 3 µM cocaine, a 14- or 28-day treatment regimen, resulted in a 100% incidence of COC-HYPs. Numbers above each bar represent the animals tested at each concentration of cocaine and treatment regimen. All recordings were made with sulpiride (1 µM) and idazoxan (10 µM) in the superfusion solution. Neither sulpiride nor idazoxan alone, or in combination, altered MP or Ri. Statistical comparisons were made between groups consisting of saline, 7-day cocaine, 14-day cocaine and 28-day cocaine at each of the three cocaine concentrations. N.S. = Non-statistically significant difference at P  .05 when comparing saline treated animals with 7-day cocaine-treated animals and with any of the three cocaine challenge concentrations using a Fisher exact test. *Statistically significant difference at P  .05 from saline or 7-day-treated animals. **Statistically significant difference at P  .01 from saline or 7-day-treated animals..

While monitoring the incidence and concentration effect of an acute challenge with cocaine, we noted that COC-HYPs were observed in all rats having had a 14-day treatment regimen (fig. 1, bottom). Furthermore, the concentration of cocaine needed to record a consistent COC-HYP became less as the in vivo exposure was increased from 7 to 28 days of chronic cocaine treatment, i.e., the concentration-effect was shifted leftward (fig. 1, bottom). We conclude that a 14-day chronic cocaine regimen resulted in a "cellular sensitization" to an acute cocaine challenge.

We define the state of "cellular sensitization" to an acute cocaine challenge as an increased incidence of observing a COC-HYP after a challenge dose of cocaine (fig. 1, bottom). We observed that following a 14- or 28-day chronic treatment regimen, a cocaine challenge of 3 µM would result in a hyperpolarization from every cell. Thus, "cellular sensitization" represents an increase in the proportion of DLSN neurons exhibiting a hyperpolarization to a cocaine challenge. This conclusion is based on the application of the Fisher exact probability test to the data depicted in figure 1, bottom. This analysis demonstrates that there is no difference between the incidence of observing a hyperpolarization after a cocaine challenge with the three different concentrations of cocaine to neurons treated with saline (control) or cocaine for 7 days. However, after a 14- or 28-day treatment with cocaine there was a significant difference in the incidence of observing a hyperpolarization when recording DLSN neurons from rats receiving a saline (control) or treatment with cocaine for only 7 days.

Non-Amine Mechanism of COC-HYP

Cocaine is well known for its ability to inhibit the transporters responsible for the uptake of biogenic amines within the nervous system (Goeders and Smith, 1986; Ritz et al., 1987). The DLSN contains postsynaptic receptors for each of the biogenic amines: an alpha-2-adrenotropic receptor, a D-2-dopamine receptor and a 5HT-1A-serotonin receptor (for review see Gallagher et al., 1995). Cocaine and other biogenic amine uptake blockers potentiate the hyperpolarizations recorded from DLSN neurons and induced by exogenous application of these biogenic amines (Joëls et al., 1987; Simms and Gallagher, 1996).

In an effort to minimize a potential endogenous biogenic amine-mediated effect, we have included selective dopamine (DA) and norepinephrine (NE) specific receptor antagonists (sulpiride, 1 µM, and idazoxan, 10 µM, respectively, Gallagher et al., 1995) with all cocaine challenges. These biogenic amine antagonists could also block the activation of nerve terminal catecholamine receptors present on glutamate, GABA or other terminals within the slice. Moreover, we demonstrate that the COC-HYP (fig. 2A) persists when a brain slice is superfused with TTX (1 µM, fig. 2B), and, in addition, persists in the presence of zero calcium medium with TTX (fig. 2C). This combination of a voltage-dependent sodium channel antagonist and lack of extracellular calcium will effectively block synaptic transmission within the slice and eliminate action potential dependent, but not spontaneous release of endogenous biogenic amines and other endogenous neuroactive substances. The presence of TTX and zero calcium medium would not alter non-vesicular release or release mediated by reversal of the uptake carrier for GABA or other transmitters, e.g., glutamate, on neurons or glia.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Cocaine hyperpolarizations (COC-HYPs) are not mediated by action potential dependent or synaptic release of substances from within the brain slice. A, COC-HYP in standard superfusion solution with sulpiride (1 µM) and idaxozan (10 µM). B, COC-HYP in same neuron with tetrodotoxin (TTX, 1 µM). C, Same neuron with TTX and zero calcium (calcium replaced with 2.5 mM magnesium). MP held initially at -60 mV.

We have also conducted experiments with six rats, not previously administered cocaine in vivo, but pretreated with reserpine (5 mg/kg, 48 hr before death; Calabresi et al., 1988). When cocaine (3 µM) was applied to brain slices from these reserpinized rats a 50% incidence of membrane hyperpolarizations was observed. This 50% incidence is identical to that obtained with nonreserpinized rats (fig. 1, bottom). These results add additional support to the concept that a COC-HYP recorded from the DLSN is not mediated by biogenic amines.

GABA Dependence of the COC-HYP

Antagonists were applied along with idazoxan (10 µM) and sulpiride (1 µM) to brain slices from rats treated chronically with cocaine. In conjunction with these two antagonists, the coapplication of the adenosine-1 receptor antagonist CPT (10 µM; n = 3), muscarinic antagonist atropine (1 µM; n = 3) or the 5-HT1A antagonist p-MPPF (1 µM; n = 3) did not alter the COC-HYP.

However, coapplication of bicuculline, a GABA_A receptor antagonist, which initially resulted in a depolarization of the MP (Shoji et al., 1994, 1997), subsequently depressed a typical COC-HYP. Similarly, application of a GABA_B receptor antagonist, CGP-55845A, also initially resulted in a depolarization of the MP, but subsequently depressed a COC-HYP. Coapplication of both bicuculline and CGP-55845A suppressed but did not block completely the COC-HYP (fig. 3). A residual hyperpolarization (1.3 ± 0.3 mV, n = 5) persisted, even in the presence of both GABA_A and GABA_B receptor antagonists.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Coapplication of GABA receptor antagonists reduces, but does not block completely, COC-HYPs. A combination of bicuculline (20 µM) and CGP-55845A (1 µM) initially results in a depolarization of MP with the appearance of rhythmic action potential firing. Downward deflections from the MP consist of an initial hyperpolarizing electrotonic potential followed by a slower pair of inhibitory postsynaptic potentials (IPSPs). During the continued application of the GABA antagonists the IPSPs are blocked and the typical COC-HYP is depressed. MP originally held at -62 mV to reduce spontaneous action potential activity.

Ionic mechanism of COC-HYP. Figure 1, top, 2A and 4A demonstrate the time required for onset and typical decrease of cellular input resistance associated with a COC-HYP. Note that a relatively long (5-10 min) continuous superfusion with cocaine is required to observe a COC-HYP. Shorter superfusion times were without effect. Figure 5A plots an estimation of the reversal potential of the COC-HYP. The apparent reversal potential for the COC-HYP obtained in this manner is -80 ± 1.4 mV (n = 5), a value not commonly attributed to a single ion. A combination of ions that could explain such a reversal potential is potassium and chloride, with E_K ~-90 mV and E_Cl ~-70 mV in this preparation and extracellular media.

View larger version (51K): [in this window] [in a new window]   Fig. 4.   Ion and temperature dependence of COC-HYP. A, Control, COC-HYP in normal solution. B, Lowering Cl blocks COC-HYP. C, Lowering Na+ blocks COC-HYP. D, Lowering temperature blocks COC-HYP.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Estimate of reversal potential and determination of potassium dependence for COC-HYPs. A, COC-HYP reversal potential was -78 mV based on the intersection of I/V curves collected in the absence and presence of cocaine (3 µM). B, Linear K+ dependency of the COC-HYP reversal potential demonstrated by altering [K+ ]o. The slope of this reversal shift (29/10 mV) was less than expected (61/10 mV) if K+ was the only ion responsible for the COC-HYP. n represents the numbers of neurons pooled to generate the three data points.

Blockade of COC-HYPs. When extracellular chloride was substituted with isethionate (117 mM) the COC-HYP was not observed (fig. 4B; n = 4); a result suggesting that chloride ion or a chloride-dependent process contributed significantly to the COC-HYP. To investigate a possible role for chloride, we determined the reversal potential of fast GABA_A-receptor mediated f-IPSPs. F-IPSPs could be isolated by blocking excitatory transmission with a combination of CNQX (20 µ M) and D-AP5 (50 µ M); later, slower GABA_B-receptor mediated IPSPs were blocked with CGP-55845A (1 µM). Under these conditions, the reversal potential of f-IPSPs in DLSN neurons from control brain slices was -70.1 ± 0.5 mV, n = 14, although in brain slices from rats administered cocaine chronically for 14 or 28 days, the reversal potential of the f-IPSP was shifted in a statistically significant manner (P .05) to -67.4 ± 0.3 mV, n = 6. The reversal potential of the f-IPSP is equivalent to the reversal or equilibrium potential for chloride ion (E_Cl-), because the GABA_A-ionotropic receptor is coupled to a chloride (Cl ) channel. This shift in the reversal potential for Cl suggests that the Cl -pump, which maintains Cl concentrations across the cell membrane, has been inhibited by the chronic cocaine treatment. An inhibition of this outwardly directed pump would result in an elevation of both intracellular chloride and sodium.

GABA transport inhibitor, NO-711, blocks COC-HYP. A process that is metabolically dependent, sensitive to a lowering of Na⁺ or Cl , highly active at GABA synapses, and persists in the presence of TTX and zero calcium is GABA transport. Because cocaine inhibits biogenic amine transporters, we considered the possibility that as a genetically related protein (Uhl and Johnson, 1994) the GABA transporter may also be affected after chronic cocaine exposure.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   NO-711, a potent GABA uptake blocker, blocks COC-HYPs. NO-711 (3 µ M), applied continuously, initially, results in a MP hyperpolarization. In the continuous presence of NO-711, cocaine (3 µM) is unable to induce its typical COC-HYP. MP = -59 mV.

	Discussion

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Previous intracellular electrophysiological results of cocaine challenge in vitro after chronic cocaine in vivo. In only one other laboratory have intracellular recording techniques been used to examine the actions of cocaine in vitro with a brain slice preparation following chronic cocaine in vivo (Harris and Williams, 1992). In that study, the effects of repeated cocaine (20 mg/kg/daily i.p., × 14 days) and a 1-wk withdrawal period were examined on norepinephrine neurons of rat brain slices containing the locus ceruleus. No baseline changes of synaptic transmission or electrical properties were noted before reexposure to cocaine. However, neurons from animals administered chronic cocaine exhibited a significant increase in sensitivity to the effects of cocaine which prolonged the time course of the NE-mediated inhibitory postsynaptic potential.

Superfusion of cocaine results in a GABA-dependent hyperpolarization within the DLSN. We have demonstrated in vitro with brain slices from rats treated chronically with cocaine in vivo, for 14 or 28 days that superfusion with cocaine (3 µM) results in a GABA-receptor dependent, biogenic amine independent, MP hyperpolarization of DLSN neurons. Support for the GABA dependence of this hyperpolarization stems from experiments that effectively reduced the COC-HYP; each experiment was conducted in the presence of biogenic amine antagonists. Each experiment points to a number of different mechanisms that may all contribute to the GABA-dependent COC-HYPs.

View larger version (30K): [in this window] [in a new window]   Fig. 7.   Diagram depicting possible mechanisms that contribute to "cellular sensitization" induced and maintained by chronic intermittent cocaine. Control (naive) neurons is depicted to left of vertical dashed line, although chronic cocaine neuron is to the right. Depiction of postsynaptic GABA neurons (or glia) with elevated intracellular sodium [Na+] and possibly [Ca++] induced by inhibition of chloride (Cl )-pump and excessive activation of glutamate receptors on GABAergic neurons. Due to elevated [Na+i], the typically inwardly directly GABA transporter is reversed resulting in "carrier-mediated release" of GABA. Elevated synaptic GABA helps sustain the down-regulation of terminal GABAB-receptors, which also enhances the release of GABA, glutamate or other transmitters, e.g., dopamine.

A combination of mechanisms contribute to the expression of COC-HYPs within the DLSN. Our data suggest the following series of events as a complex mechanism (fig. 7), which underlies the COC-HYP recorded from DLSN neurons. An elevation of intracellular chloride results from inhibition of an outwardly directed chloride pump (Cl -transporter). We provide data that demonstrate a shift in the equilibrium potential of the GABA_A-receptor mediated f-ipsp. Elevation of [Cl^-_I] is associated with movement of Na⁺ into the cell to neutralize the increased intracellular negativity brought about by Cl . Furthermore, an additional increase of [Na⁺_i] and possibly [Ca⁺⁺ _i] results from excessive activation of ionotropic glutamate receptors on DLSN neurons. An excessive release of glutamate is due to a decreased efficacy ("down-regulated") of the GABA-heteroreceptor (fig. 7) on glutamate releasing neurons (Shoji et al., 1997). Our ability to block the COC-HYP by lowering extracellular Na⁺ or Cl , and by lowering temperature both support a cellular mechanism whereby chronic cocaine has resulted in stimulation of the GABA-carrier (reversed transporter), such that when a cocaine challenge is applied, GABA is secreted (released) from neurons and/or glia resulting in a postsynaptic hyperpolarization mediated by GABA_A and GABA_B receptors.