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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Neurosci. Author manuscript; available in PMC 2010 September 13.
Published in final edited form as:
PMCID: PMC2938175
NIHMSID: NIHMS113139

Protracted withdrawal from alcohol and drugs of abuse impairs long-term potentiation of intrinsic excitability in the juxtacapsular bed nucleus of the stria terminalis

Abstract

The juxtacapsular BNST (jcBNST) is activated in response to basolateral amygdala (BLA) inputs through the stria terminalis and projects back to the anterior BLA and to the central nucleus of the amygdala (CeA). Here we show a form of long-term potentiation of the intrinsic excitability (LTP-IE) of jcBNST neurons in response to high-frequency stimulation (HFS) of the stria terminalis. This LTP-IE, which was characterized by a decrease in the firing threshold and increased temporal fidelity of firing, was impaired during protracted withdrawal from self-administration of alcohol, cocaine, and heroin. Such impairment was graded and was more pronounced in rats that self-administered amounts of the drugs sufficient to maintain dependence. Dysregulation of the CRF system has been implicated in manifestation of protracted withdrawal from dependent drug use. Administration of the selective corticotropin-releasing factor receptor 1 (CRF1) antagonist R121919, but not of the CRF2 antagonist astressin2-B (A2-B), normalized jcBNST LTP-IE in animals with a history of alcohol dependence; repeated, but not acute, administration of CRF itself produced a decreased jcBNST LTP-IE. Thus, changes in the integration properties of jcBNST neurons mediated by chronic activation of the CRF system may contribute to the persistent emotional dysregulation associated with protracted withdrawal.

Keywords: Drug Abuse, Excitability, Plasticity, Action Potential, Potassium Channels, metabotropic glutamate receptor, Withdrawal

Introduction

The bed nucleus of the stria terminalis (BNST) has been implicated in the reinforcing effects of alcohol and drugs of abuse (Epping-Jordan et al., 1998; Carboni et al., 2000; Delfs et al., 2000; Eiler et al., 2003; Macey et al., 2003; Dumont et al., 2005) and supports brain self-stimulation in animals with a genetic alcohol preference (Eiler et al., 2007). The BNST has also been shown to be involved in stress-induced reinstatement of drug-seeking behavior during protracted withdrawal (Erb and Stewart, 1999; Shalev et al., 2001; McFarland et al., 2004). Both drug withdrawal (Delfs et al., 2000; Olive et al., 2002; Erb et al., 2004) and stress (Stout et al., 2000) induce neurochemical changes in this brain region.

The juxtacapsular subdivision of the lateral BNST (jcBNST) is the only part of the BNST that receives dense glutamatergic projections from the basolateral nucleus of the amygdala (BLA) and, unlike other subregions of the lateral BNST, does not receive inputs from the central nucleus of the amygdala (CeA) (Larriva-Sahd, 2004). In turn, the jcBNST projects back to the anterior BLA and to the CeA (Dong et al., 2000). The jcBNST also projects to the lateral hypothalamus, striatum, and nucleus accumbens, among other brain regions involved in the actions of drugs of abuse (Dong et al., 2000). The jcBNST, and the dorsolateral BNST in general, contain densely arrayed γ-aminobutyric acid (GABA)-ergic neurons (Sun and Cassell, 1993; Veinante et al., 1997) and abundant corticotropin-releasing factor (CRF)-containing neurons (Sawchenko, 1987). Conversely the jcBNST and the dorsolateral BNST in general, are devoid of glutamatergic neurons (Hur and Zaborszky, 2005).

Neuronal activity can induce persistent modifications in the way a neuron reacts to subsequent inputs, both by changing synaptic efficacy and/or intrinsic excitability. The former has received greater attention in recent years, and it is generally accepted that changes in the strength of synaptic connections underlie memory storage and some of the maladaptive changes in reward processing induced by drugs of abuse (Thomas et al., 2000; Ungless et al., 2001; Melis et al., 2002; Saal et al., 2003; Malenka and Bear, 2004; Dumont et al., 2005). In many brain regions, neuronal activity has also been shown to induce long-term modifications of intrinsic neuronal excitability, i.e., the propensity of neurons to fire action potentials.

Long-term potentiation of the intrinsic excitability (LTP-IE) is a protracted increase in the probability of firing and/or in the efficacy of neuronal circuits (Turrigiano et al., 1994; Armano et al., 2000; Bekkers and Delaney, 2001; Ross and Soltesz, 2001; Daoudal et al., 2002; Egorov et al., 2002; Debanne et al., 2003; Mahon et al., 2003; Smith and Otis, 2003; Cudmore and Turrigiano, 2004; Chen et al., 2007; Desai et al., 1999; Aizenman and Linden, 2000). Here we tested the hypothesis that a history of drug self-administration may induce long-lasting changes in the integrative properties of jcBNST neurons in animal models of alcohol, cocaine, and heroin self-administration. We observed a reduced capacity for synchronously increasing jcBNST neuronal activity during protracted withdrawal that could result in inefficient feedback inhibition to the CeA and increased emotional arousal. These effects were blocked by repeated administration of a CRF1, but not CRF2, receptor antagonists and could be mimicked by repeated, but not acute, administration of CRF.

Methods

Electrophysiological techniques

Acute brain slices were carried out as previously described (Sanna et al., 2000; Sanna et al., 2002b) with minor modifications. Briefly, coronal rat brain slices (350 μm) were collected from the rostral cerebrum of Wistar rats with a Leica VT1000E vibrating microtome (Wetzlar, Germany), at the level shown in Fig. 1A, in oxygenated artificial cerebrospinal fluid (aCSF) [in mM: 130 NaCl, 3.5 KCl, 24 NaHCO3, 1.25 NaH2PO4, 2.2 CaCl2, 10 d-glucose, and 2 MgSO4, pH 7.4], Slices were preincubated in aCSF for 1h at 32° C and then maintained at room temperature for at least 30 min before being transferred to a submerged recording chamber at 31° C. Extracellular field potentials were recorded with microelectrodes filled with aCSF (3–5 MΩ) using an Axoclamp 2B headstage amplifier (Axon Instruments, Foster City, CA). Bipolar stimulating electrodes were placed in the stria terminalis (Fig. 1B). Constant current pulses of 0.08 ms duration were used for stimulation. The amplitude of the second negative component of the field potential (Fig. 1C) was measured at its trough. Acquisition and analyses were performed with the LABVIEW software package (National Instruments, Austin, TX). For field potential recordings, input-output (I/O) curves were performed in each slice, and test stimulation intensity was adjusted to obtain field potentials approximately one-third of the maximum amplitude. The high-frequency stimulation (HFS) paradigm consisted of five trains at 100 Hz for 1 sec at 10 sec intervals at the test stimulation intensity. For occlusion experiments, after bath application of GABAA and GABAB inhibitors or 4-aminopyridine, I/O curves were again determined, and stimulation intensity was adjusted as described to obtain one-third of maximum field potential amplitude before delivering the HFS. Intracellular recordings were made using sharp glass micropipettes filled with 2 M potassium acetate, pH 7.3 (180–200 MΩ) with an Axoclamp 2B headstage amplifier in current-clamp mode. The threshold of the first action potential, evoked in response to single intracellular depolarizing pulses (500 ms at 15 sec intervals) was measured at the sharp transition from prepotential to upstroke (Armano et al., 2000), as shown in Fig. 2E. Input resistance (RN) was measured with sharp electrodes in current clamp mode at the end of 350 ms hyperpolarizing current pulses of −0.2 nA. This current value is in the linear part of the I/V curve. The slope of the depolarizing prepotential was measured over the 10 msec immediately preceding the action potential upstroke and expressed in mV/msec. After 20 min of a stable baseline of threshold recording, HFS was delivered to the stria terminalis. For whole-cell recordings slices of brain tissue containing the BNST were placed in a superfusion chamber mounted on a Olympus microscope stage and were viewed using infra-red differential interference contrast optics and video microscopy. Whole-cell recordings were made using patch pipette with resistances of 3–6MΩ filled with a solution containing (in mM): KMeSO4 120, KCl 10, MgCl2 3, HEPES 10, Phosphocreatine 10, MgATP 2, GTP 0.2; osmolarity 280–290 mOsm, pH 7.2. Whole-cell access resistances were estimated from the peak current produced during voltage clamp steps and were found to be 15–40MΩ. Access and input resistances were monitored throughout the recordings to ensure stability. To isolate the (α-DTX)-sensitive D-Type K+ current (ID), the extracellular solution contained 0.5 μM TTX, 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 100 μM CdCl2 and 10 mM TEA to block interfering K+ and inward currents. For analysis, only the current trace for the +40 mV step was used, and the average current amplitude between 50 and 100 ms after application of the voltage step was measured. For morphological analysis, 1–2 mg ml−1 biocytin (Sigma) was added to the intracellular solution. Immediately after recording, slices were fixed in 0.1 M phosphate-buffered saline (pH 7.4) containing 4% paraformaldehyde and 0.1% picric acid for 24 hr. Slices were then processed for biotinylated horseradish peroxidase conjugated to avidin (ABC-elite, Vectastain, Vector laboratories Inc., Burlingame, CA).

Fig. 1
Field potential in the jcBNST
Fig. 2
LTP-IE in the jcBNST

Alcohol self-administration

Rats were trained to orally self-administer ethanol using a modification of the saccharin-fading procedure, as previously described (Roberts et al., 2000). After successful acquisition of operant responding, the animals were introduced to a free-choice task in which presses on one lever produced 0.1 ml of alcohol solution, while responses at the other lever resulted in delivery of an equal volume of water. Alcohol was alternated daily between the two levers. The criterion for stable baseline intake was ± 20% across three consecutive 30 min sessions with a mean intake of >20 responses. Alcohol vapor chambers were used to induce dependence by intermittent exposure (14h on/10h off) to an air/ethanol mixture for a total of 4 weeks, which reliably produce alcohol dependence in rats (Gilpin et al., 2008). Intermittent alcohol exposure mimics human patterns of alcohol consumption (Slawecki et al., 1999; Sanna et al., 2002a; Repunte-Canonigo et al., 2007) and induces more rapid increases in self-administration than continuous exposure (O’Dell et al., 2004). Blood alcohol levels were measured with an oxygen-rate alcohol analyzer (Analox Instruments, London, UK) and maintained at (M ± SEM) 155 ± 15 mg%.

Intravenous self-administration

Rats were prepared with chronic intravenous catheters as described previously (Ahmed et al., 2005; Chen et al., 2006b). For cocaine self-administration, rats were allowed to self-administer cocaine on a fixed-ratio (FR) 1 schedule (250 μg/injection in a volume of 0.1 ml delivered over 4 s). Each response on the lever resulted in a cocaine injection and was followed by a 20 sec time-out period. Rats were allowed to self-administer cocaine either for 1h short-access (ShA) or 6h long-access (LgA) per day. For heroin self-administration, rats were allowed either 1h (ShA) or 23h (LgA) access on an FR1 schedule (60 μg/kg/0.1 ml infusion, 20 sec time-out) (Chen et al., 2006b).

Analysis of Gene Expression

Target RNA was generated with the BioArray High Yield RNA Transcript Labeling Kit (Enzo, New York, NY). Quality of total RNA was assessed using the NanoDrop ND-1000 spectrophotometer for quantification of small samples and the Agilent Bioanalyzer. The jcBNST was dissected with minimal surrounding tissue from transilluminated vibratome-cut coronal rat brain slices (350 μm) kept cold but not frozen, as described by Cuello and Carson (Cuello and Carson, 1983). All BNST samples used for microarray and qPCR were from animals in which the contralateral BNST was used for electrophysiological experiments. Similarly dissected jcBNST were used for western blotting.

High density microarrays (Affymetrix RAE230A) were hybridized to target cRNA derived from doube IVT as previously described (Sanna et al., 2005) with minor modifications and scanned according to the manufacturer’s protocol. Specifically, pools of 3 samples from alcohol dependent animals and 3 matched control and 3 from cocaine LgA and 3 matched control were tested using RAE 230 Affymetrix arrays. Samples from each experimental group were pooled and run in duplicate. Signal intensities were scaled to a target intensity of 250 using the MAS 5.0 algorithm. Differentially expressed genes were obtained by the Affymetrix Comparison Analysis Algorithm (CAA) and t-test analysis of GeneSpring 7.2-normalized expression values as previously described (Repunte-Canonigo et al., 2007).

Differentially expressed genes were validated by quantitative real-time polymerase chain reaction (qPCR) from individual animals with a history of dependence on the three drugs under study (alcohol, cocaine and heroin) and matched controls. To this aim we tested individual samples from a separate set of animals as previously done (Ahmed et al., 2005; Repunte-Canonigo et al., 2007). Primers were designed using the Beacon Designer Software (Premier Biosoft International). The iQ SYBR Green Supermix (BioRad) was used in 25 μl reaction volume with an iQ5 Real-Time PCR Detection System (BioRad) using 0.2 ml 96-well thin-wall PCR plates (Bio-Rad). Reaction steps were 95°C for 3 min, 40× (95°C for 10 s, 60°C for 20 s, 72°C for 20 sec). For quantification, the relative standard curve method (Livak, 1997) was used as previously described (Ahmed et al., 2005; Repunte-Canonigo et al., 2007). β-actin was used as a reference mRNA for normalization. Standard curves for both the target and reference genes were generated for each qPCR run with 1-log serial dilutions (1 to 10−5) of first-strand cDNAs from a pooled control sample by plotting cycle threshold (Ct) vs. the log of input amount. qPCR standard curves had slopes in the range of −3.5 to −3.0. Linear correlation coefficients (R2) ranged from 0.98 to 0.99. Sample Ct values were interpolated using the formula: (Ct-b)/m, where b = y-intercept of the standard curve line, and m = slope of the standard curve line.

Western blotting was carried out from individual animals as previously described (Sanna et al., 2000; Sanna et al., 2002b) using a rabbit polyclonal specific for AA 417-499 of rat Kv1.2 (APC-010, Alomone Labs) at a dilution of 1:200; for control β-actin was detected with a mouse monoclonal antibody (A1978, Sigma) at a dilution of 1:2000. Quantification of western blot signal was done with the NIH Image software (http://rsb.info.nih.gov/nih-image/).

Double in situ hybridization

Rats were perfused transcardially with 4% (W/V) paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.3. Brains were postfixed in 4% paraformaldehyde for 2h at 4°C, rinsed with PB and transferred sequentially to 12%, 14% and 18% sucrose solutions in PB. In situ hybridization was performed as described previously (Morales et al., 2007). Cryosections were incubated for 10 min in PB containing 0.5% Triton X-100, rinsed 2 × 5 min with PB, treated with 0.2 N HCl for 10 min, rinsed 2 × 5 min with PB and then acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine, pH 8.0 for 10 min. Sections were rinsed 2 × 5 min with PB, post-fixed with 4% paraformaldehyde for 10 min, and after a final rinse with PB, were hybridized for 16h at 55°C in hybridization buffer (50% formamide; 10% dextran sulfate; 5x Denhardt’s solution; 0.62 M NaCl; 50 mM DTT; 10 mM EDTA; 20 mM PIPES, pH 6.8; 0.2% SDS; 250 μg/ml salmon sperm DNA; 250 μg/ml tRNA) containing [35S]-single-stranded antisense rat CRF riboprobes at 107 cpm/ml, together with the 600 ng mix of GAD65 and GAD67 digoxigenin-labeled antisense riboprobes. The antisense digoxigenin GAD65 and GAD67 were obtained by in vitro transcription using digoxigenin-11-UTP labeling mix (Roche Indianapolis IN), and T3 RNA polymerase. Sections were treated with 4 μg/ml RNase A at 37°C for 1h, washed with 1 × SSC, 50% formamide at 55°C for 1h, and with 0.1 × SSC at 68°C for 1h. After the last SSC wash, sections were rinsed with TBS buffer (20 mM Tris.HCl, 0.5 M NaCl, pH 8.2). Afterwards, sections were incubated with an alkaline phosphatase-conjugated antibody against digoxigenin (Roche Applied Science; Indianapolis, IN) overnight at 4°C; the alkaline phosphatase reaction was developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Roche Applied Science; Indianapolis, IN) yielding a purple reaction product. Sections were mounted on slides, air dried and dipped in Ilford K.5 nuclear tract emulsion (Polysciences, Inc., Warrington; 1:1 dilution in double distilled water) and exposed in the dark at 4°C for 4 weeks prior to development.

In vivo drug treatments

The CRF1 antagonist R121919 (Chen et al., 1996) (formerly referred to as NBI 30775) was administered subcutaneous (SC) at a dose of 20 mg/kg, 3 times in 20% hydroxypropyl β-cyclodextrin, pH 4.5, 12h apart because of the extensive data available for this route of administration regarding doses sufficient to occupy brain CRF1 receptors (Gutman et al., 2003) and to produce behavioral effects in models of ethanol or drug dependence (e.g., (Funk et al., 2006; Sabino et al., 2006; Skelton et al., 2007a; Skelton et al., 2007b)). The CRF2 antagonist (Rivier et al., 2002) was administered by intracerebroventricular (ICV) injections (4 μg in 2μl), as previously done (Cottone et al., 2007; Fekete et al., 2007), because as a peptide does not cross the blood-brain barrier. CRF was administered ICV at a dose of 1 μg as previously done (Izzo et al., 2005).

Data analysis

Student’s t test (paired or unpaired as appropriate) or one-way or two way analysis of variance (ANOVA) were used to analyze the behavioral and electrophysiological data using StatView (SAS Institute) or Microsoft Excel. ANOVA was followed by Fisher LSD post-hoc analysis. All results are expressed as mean ± SEM. Cut-off p values of <0.05 were considered to be statistically significant and results are reported as either <0.05 or <0.01.

Results

LTP-IE in the jcBNST

In jcBNST brain slices, stimulation of the region of the stria terminalis (Fig. 1A, B) that conveys glutamatergic inputs from the BLA (Dong et al., 2000) evoked a sharp field potential characterized by two fast negative components followed by a more variable slow positive deflection (Sawada et al., 1980), as shown in Fig. 1C. The second negative and slow positive components of the field potential were abolished by application of the α-hydroxy-5-methyl-4-isoazole-propionic acid (AMPA) receptor inhibitor CNQX either in the bath (20 μM) (Fig. 1C) or locally by diffusion of the inhibitor (25 mM) included in the recording electrode (Fig. 1D). Bath application of the glutamatergic N-methyl-D-aspartate (NMDA) inhibitor D-2-amino-5 phosphonopentanoic acid (D-AP5; 50 μM) had no effect on the field potential (Fig. 1E). Blocking GABAA and GABAB receptors with bicuculline (30 μM) and SCH50911 (20 μM), respectively, considerably increased the size of the second negative components of the field potential without abolishing the positive component or altering the field morphology (Fig 1C). Together, these observations indicate that the second negative and positive components of the field potential are postsynaptic responses mediated by AMPA receptors and originate locally in the jcBNST whereas the first negative component of the field potential represents the presynaptic volley, i.e. the spike activity of the afferent fibers.

Delivery to the stria terminalis of a HFS paradigm consisting of five trains at 100 Hz for 1 sec at 10 sec intervals resulted in protracted potentiation of the amplitude of the negative postsynaptic component of the field potential (184 ± 1.6% at 60 min after HFS; n = 9) (Fig. 1F, G). A transient bath application of either D-AP5 (50 μM) or the mGluR5 inhibitor 2-methyl-6-(phenylethynyl)-pyridine (MPEP; 10 μM) for 20 min around the time of delivery of HFS prevented potentiation of the field potential amplitude (110.9 ± 0.6%, n = 10, and 110.3 ± 1%, n = 5, 60 min after HFS for D-AP5 and MPEP, respectively; not significantly different from baseline before delivery of HFS for D-AP5, t test, t=0.218, p=0.416; and MPEP, paired t test, t=-1.265, jp=0.863) (Fig. 1F). However, HFS during continuous application of the GABAA and GABAB inhibitors bicuculline (30 μM) and SCH50911 (20 μM), respectively, resulted in the same degree of potentiation as controls (167.1 ± 2.7%, n = 6, 60 min after HFS) (Fig. 1F). Bath application of either D-AP5 or the mGluR5 inhibitor MPEP 20 min after HFS was ineffective (Fig. 1H). Thus, tetanization of the stria terminalis resulted in a potentiation of the field potential in the jcBNST that was dependent on NMDA and mGluR5.

To determine the cellular and molecular mechanisms responsible for the potentiation of field potential in the jcBNST, we performed intracellular recordings from jcBNST neurons in current clamp mode with sharp microelectrodes. Delivery of the HFS paradigm only transiently potentiated excitatory postsynaptic potential (EPSP) amplitude in jcBNST neurons, which declined to baseline levels within 16 min (Fig. 2A, B). Thus, the duration of potentiation of field potential amplitude in the jcBNST (Fig. 1F) was much more protracted than the transient EPSP potentiation seen by intracellular recordings (Fig. 2A, B). A potentiation of the field potential without a concomitant potentiation of EPSPs might result from a LTP-IE of jcBNST neurons leading to more cells firing. On anatomical grounds, the multipolar neuronal morphology of jcBNST neurons (Fig. 2C) does not favor the summation of EPSPs into discernable fEPSP. Together, these observations suggested the hypothesis that the post-synaptic component of evoked field potentials in the jcBNST was a manifestation of the summation of synaptically-driven action potentials of jcBNST neurons – i.e. the population spike – as in other neuronal populations characterized by radial dendrites (Misgeld et al., 1979; Zhou and Poon, 2000; Walcott and Langdon, 2002).

Supporting this hypothesis, delivery of HFS induced a protracted increase in firing probability (Fig. 2D) without concomitant increases of the EPSP slope (Fig. 2E). Since activity-dependent LTP-IE of neurons leading to increased firing may be caused by decreased action potential thresholds (Armano et al., 2000; Cudmore and Turrigiano, 2004; Xu et al., 2005; Aizenman and Linden, 2000), we measured the threshold of the first action potential by intracellular recordings in jcBNST neurons before and after HFS. HFS induced a long-lasting shift toward hyperpolarization (4.9 ± 0.7 mV, n=15) of the threshold for action potential generation in response to single intracellular depolarizing pulses selected to evoke one or two action potentials (500 ms at 15 s intervals) (Fig. 2E). Data showed that the action potential threshold measured at different times after HFS delivery was significantly lower than before HFS (at 10 minutes: t=4.26, p<0.01, at 25 minutes: t=2.72, p<0.01, at 40 minutes: t=6.66, p<0.01 n = 14; one sample t test, Fig. 2F). Input resistance measured by injection of a hyperpolarizing and depolarizing current pulses showed no significant changes after delivery of HFS (115.1 ± 8.5 MΩ before and 112.1 ± 10.2 MΩ 60 min after delivery of HFS, t=0.52, NS, n=11). Three different types of jcBNST neurons were recognized based on their rectification properties and rebound depolarization in response to hyperpolarizing current injections, as previously shown in both rat (Hammack et al., 2007) and mouse (Egli and Winder, 2003). A significant shift toward hyperpolarization of the threshold for action potential generation was induced by HFS in all the three types of neurons according to the classification of Hammack et al. (2007): Type I = 5.63 ± 1.4 mV (t=-4.04, p<0.01, n=6), Type II = 5.22 ± 1.0 mV (t=-5.49, p<0.01, n=6), and Type III = 3.52 ± 1.0 mV (t=-3.44, p<0.05, n=4). A transient application of either the NMDA inhibitor D-AP5 (50 μM) or the mGluR5 inhibitor MPEP (10 μM) applied for 20 min around the time of delivery of HFS as in Fig. 1F, blocked the HFS-induced shift of the action potential threshold (Fig. 2G), consistent with the requirement for these glutamatergic receptors for the potentiation of jcBNST field potential (Fig 1F).

Protracted withdrawal from self-administration of alcohol, cocaine, and heroin impairs LTP-IE of jcBNST

To test the hypothesis that LTP-IE in the jcBNST would be altered in rats with a history of alcohol exposure sufficient to produce escalated self-administration, we measured first the amplitude of the field potentials and we then carried out intracellular measures of the threshold for action potential generation.

We used a validated animal model of escalated dependent alcohol intake induced by exposure to alcohol vapors (Roberts et al., 2000; O’Dell et al., 2004). Rats were trained to orally self-administer alcohol in a concurrent, two-lever, free-choice contingency using a modification of the sweet solution fading procedure. Following acquisition of operant responding for ethanol, half of the rats were exposed to intermittent ethanol vapors for 4 weeks to induce dependence (dependent group) while the remaining rats were exposed to air in the same apparatus for the purpose of control (nondependent group). During protracted withdrawal (3–5 weeks after withdrawal) rats were again tested for operant responding for ethanol and used for electrophysiology experiments about 1–2 weeks after testing. The average number of presses for ethanol in the post-dependent group during protracted withdrawal was significantly increased compared to the nondependent group (83.25 ± 6.3 vs. 24.5 ± 7.4, p < 0.01) (Fig. 3A). As shown in Fig. 3B, in dependent rats at 4–6 weeks post-withdrawal, potentiation of the field potential, which as shown above reflects LTP-IE in the jcBNST, was significantly impaired at 60 min after HFS (104.9 ± 1.7%, n = 10, p <0.05 from controls). Conversely, at 4–6 weeks post-withdrawal, in nondependent control rats (161 ± 0.7%, n = 7) field potentials were not significantly different from controls at 60 min after HFS (183.6 ± 1.5%, n = 22). However, in dependent rats tested during acute withdrawal (3–6 h) potentiation of the field potential at 60 min after HFS (208.5 ± 1.9%, n = 7) was not significantly different from controls, while at 1–2 weeks post-withdrawal, a significant impairment was observed in both dependent (127.4 ± 1.9%, n = 8, p < 0.05 from controls and dependents during acute withdrawal) and nondependent rats (129.7 ± 0.9%, n = 8). Thus alcohol dependence induces a protracted impairment of the capacity for plasticity in the jcBNST, while a transient impairment is seen after a history of non-dependent alcohol intake.

Fig. 3
Disruption of LTP-IE in the jcBNST during protracted withdrawal from self-administration of alcohol, cocaine, and heroin

To explore the generality of these findings, we investigated the induction of LTP-IE in rats with or without extended access to cocaine or heroin self-administration sufficient to produce dependence (Koob et al., 2004; Ahmed et al., 2005; Chen et al., 2006a). Rats self-administering cocaine for 1h per day (ShA) showed stable cocaine intake over time (Fig. 3C). Conversely, extended access to cocaine self-administration for 6h per day (LgA) induced an increase in drug intake, previously correlated with a persistent decrease in brain reward function during withdrawal (Koob et al., 2004) (Fig. 3C). Similar to alcohol post-dependent rats, a strong impairment of potentiation of the field potential amplitude was observed in the jcBNST of LgA rats 1–2 weeks after cessation of cocaine self-administration (109.4 ± 3.3%, n = 8, 60 min after HSF) while ShA rats showed a level of potentiation (132.7 ± 5.6%, n = 10) that was intermediate between that of control and LgA rats (Fig. 3D). LTP-IE in LgA heroin self-administering rats 1–2 weeks after cessation of cocaine self-administration was also significantly impaired (110.2 ± 5.9%, 60 min after HFS, n = 5) compared to controls (176.7 ± 9.8%, n = 10) and ShA rats (140 ± 5.2%, n = 10, p < 0.05 from both control and ShA rats) (Fig. 3E, F). Again, ShA rats showed an intermediate level of potentiation impairment (p < 0.01 from controls). Thus, multiple drugs of abuse induced a common disruption of potentiation of the field potential in the jcBNST after delivery of HFS to the stria terminalis.

The threshold for action potential generation in jcBNST neurons is regulated by the ID current

Consistent with the disruption of potentiation of the field potential in the jcBNST of rats with histories of dependence on alcohol or LgA intravenous self-administration of cocaine or heroin during protracted withdrawal, the HFS-induced shift toward hyperpolarization of the threshold for action potential generation that is required for the potentiation of the field potential in the jcBNST was not observed (Fig. 4A). The threshold for action potential in animals with a history of alcohol, cocaine, or heroin dependence before delivery of HFS (control: −50.9 ± 1.5 mV, n = 14; alcohol post-dependent: −50.7 ± 1.9 mV, n = 7; cocaine: −47.5 ± 1.7 mV, n = 9, heroin: −54.1 ± 1.9 mV, n = 7) did not differ from controls (2 tail t test assuming equal variance: control versus alcohol post-dependent: t=0.11, NS; control versus cocaine: t=1.92, NS; control versus heroin: t=−1.25, NS) indicating that the observed lack of shift toward hyperpolarization in post-dependent animals reflects impaired capacity for LTP-IE rather than occlusion by a pre-existing LTP-IE.

Fig. 4
The threshold for action potential generation in jcBNST neurons is regulated by the ID current

As shown in Fig. 4B, using combined DNA microarray and qPCR, we observed increased expression of the potassium channel Kv1.2 in post-dependent rats at the time points indicated in Fig. 3 (4–6 weeks for alcohol dependent; 1–2 weeks for cocaine and heroin LgA). Consistently, Kv1.2 protein was significantly increased in the jcBNST of rats with a history of alcohol dependence by western blotting (Fig. 4C). Kv1.2 is a member of the Kv1 family of potassium channels implicated in mediating the slowly inactivating 4-aminopyridine- and α-dendrotoxin (α-DTX)-sensitive D-type K+ current (ID) (Shen et al., 2004). The α-DTX-sensitive D-type K+ current (ID) is a slow inactivating transient current first identified by Storm in hippocampal CA1 pyramidal neurons by its ability to delay firing action potentials (Storm, 1988). The ID current is selectively blocked by α-DTX and by micromolar concentrations of 4-aminopyridine (Storm, 1988; Coetzee et al., 1999). The ID current is known to be regulated by mGluRs (Wu and Barish, 1999) and controls action potential threshold in various neuronal types (Storm, 1988; Strom, 1993; Coetzee et al., 1999; Bekkers and Delaney, 2001; Kroner et al., 2005; Guan et al., 2007). Consistent with these previous findings, bath application of α-DTX (1μM) or 4-AP (40μM), induced a long-lasting shift of the threshold for action potential generation toward more hyperpolarized membrane potentials in jcBNST neurons (Fig. 4D, E). Delivery of HFS after application of 4-AP did not further shift the AP threshold (from −59.1 ± 2.4 mV before HFS to −60.9 ± 2.8 mV 40 min afr HFS; t=0.21, NS, n=9) (Fig. 4E). A transient application (15 min) of 4-AP induced a protracted shift of the threshold for action potential generation toward hyperpolarization that was unchanged by previous application of inhibitors of synaptic transmission that were maintained for the duration of the recordings (Fig. 4E). This suggests that the persistent shift of the threshold for action potential generation induced by a transient application of 4-AP was due to inhibition of the post-synaptic ID current. A transient application (15 min) of the selective mGluR5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG, 200 μM) also induced a protracted shift of the threshold for action potential generation toward hyperpolarization (Fig. 4E). The expression of the ID current in jcBNST neurons was confirmed in patch whole-cell voltage clamp recordings according to the protocol of Bekkers and Delaney (2001) (Fig. 4F, G, H). As expected, bath application of CHPG (200 μM) reduced the ID current (56.9 ± 4.8%, n = 8) in jcBNST neurons (Fig. 4F, G). Application of α-DTX prior to application of CHPG resulted in whole cell currents and I-V relationships that were identical (Fig 4H), indicating that the effect of CHPG on the whole cell current was mediated exclusively through the ID current. Together, these results indicate that the shift of action potential threshold that characterizes LTP-IE in jcBNST neurons is due to the inhibition of a mGlur5-modulated post-synaptic ID current.

Enhanced temporal fidelity of firing in jcBNST LTP-IE

Using extracellular single unit recordings in the jcBNST in response to stimulation of the stria terminalis, we investigated the variability of the latency to the first spike or “jitter of the spike,” a measure of the temporal fidelity of firing (Pouille and Scanziani, 2001; Sourdet et al., 2003). In the jcBNST of control rats, the jitter of the spike showed a significant and protracted reduction after delivery of HFS of the stria terminalis that lasted for up to 40 min and resulted in increased temporal fidelity of firing (Fig. 5A,B). In the jcBNST of alcohol dependent rats, the HFS-induced reduction of jitter was only transient, returning to control levels by 30 minutes after HFS (Fig. 5C). Since extracellular recordings do not allow one to determine the cellular mechanism behind the reduced jitter of the spike that characterizes LTP-IE in the jcBNST, we carried out concomitant measures of the prepotential slope and jitter in the same jcBNST neurons of normal rats by intracelluar recordings. As shown in Fig. 5D, E, F we observed that HFS induced a significant increase in the slope of the depolarizing prepotential from 0.38 ± 0.04 mV/msec to 0.98 ± 0.3 mV/msec 40 min after HFS (p<0.01; n=9, Fig. 5E), which was associated with a significant decrease of the jitter of the spike (2.64 ± 0.6 msec before vs. 1.56 ± 0.5 msec 40 min after HFS; p<0.05, n= 9, Fig. 5F). Changes in the latency to the first spike as well as in the depolarizing prepotentials may reflect either increases of inward currents, such as Na+ and/or Ca2+-mediated currents, or decreases of outward currents, like the K+-mediated IA or ID (Nisenbaum et al., 1994; Griffin et al., 1996). Therefore, we tested whether bath application of the ID blocker 4-AP at the concentration of 40 μM, which does not block the IA, could mimic both the increase of the slope of the prepotential and the decrease of the jitter. As shown in Fig. 5E, F, the slope of the prepotential of jcBNST neurons was significantly increased from 0.45 ± 0.048 mV/msec to 1.05 ± 0.15 mV/msec (p<0.01, n=9, Fig. 5E) and the jitter was decreased from 2.32 ± 0.3 msec to 0.78 ± 0.43, 40 min after 4-AP (40μM; p<0.01, n=9, Fig. 5F). Conversely, in rats with a history of alcohol, cocaine or heroin dependence, the jitter of the spike measured by intracellular recordings was not significantly changed at 40 min after HFS (Fig. 5G).

Fig. 5
Activity-dependent increase of temporal fidelity of firing of jcBNST neurons

Thus, LTP-IE in the jcBNST is characterized by increased intrinsic excitability due to a decrease in the firing threshold and by increased temporal fidelity of firing both of which depends on the K+-mediated ID and that were not seen in animals with a history of alcohol, cocaine, or heroin dependence.

The impairment of jcBNST LTP-IE in protracted abstinence is associated with activation of the CRF system

The increased anxiety-like behavior and drinking observed during acute withdrawal and protracted abstinence have been attributed to increased CRF activity (Rasmussen et al., 2001; Valdez et al., 2002), including within the BNST (Olive et al., 2002). Drug-seeking behavior previously maintained by various drugs of abuse, including alcohol, cocaine, and heroin, can be reinstated by either exposure to stress or by central administration of CRF in a CRF antagonist-reversible manner (Shaham et al., 2003). Therefore we performed double in situ hybridization for GABA synthetic enzymes GAD65 and GAD67 and for CRF to investigate the distribution of GABA and CRF containing neurons in the jcBNST. We observed that GABAergic neurons are highly represented in the dorsolateral BNST including the jcBNST and show a high degree of colocalization with CRF, especially in the mid section of the dorsolateral BNST and jcBNST (Fig. 6). Previous studies also showed numerous CRF-containing neurons in the lateral BNST (Sawchenko, 1987) as well as a rich plexus of CRF-containing axons and axon collaterals throughout the BNST, suggesting that CRF may regulate neuronal activity in the nucleus. Next, we investigated the potential role of CRF in the impairment of jcBNST LTP-IE during protracted withdrawal in animals with a history of alcohol dependence. Repeated subcutaneous (SC) administration of the selective CRF1 receptor antagonist R121919 (formerly referred to as NBI 30775; (Chen et al., 1996) restored normal jcBNST LTP-IE in alcohol post-dependent rats (190.7 ± 1.4%, n = 8, 60 min after HFS), while vehicle (aCSF contaning 10% DMSO) had no effect (111.6 ± 1.7%, n = 4) (Fig. 7A). Three i.c.v. injections (4 μg in 2μl) of the selective CRF2 receptor antagonist astressin2-B (A2-B (Rivier et al., 2002) every 12 hrs was also ineffective (Fig. 7A). The dose of A2-B was based on previous work (Cottone et al., 2007; Fekete et al., 2007). Repeated intracerebroventricular (ICV) administration of CRF – but not acute administration of CRF - induced a significant impairment of jcBNST LTP in drug-naive rats that resembled the post-dependent phenotype (Fig. 7B). Together, these results suggest that the CRF system may contribute to dependence-associated changes in the integration properties of jcBNST neurons that can be normalized by a CRF1 receptor antagonist, and mimicked by repeated central CRF administration.

Fig. 6
Double in situ hybridization for GAD65 and GAD67 and for CRF in the dorsal BNST
Fig. 7
Disruption of jcBNST LTP in protracted abstinence is due to activation of the CRF system

Discussion

In the present study we have observed that a history of drug exposure sufficient to produce escalated self-administration produced impairment in a novel form of activity-dependent LTP-IE in jcBNST neurons. This form of plasticity was characterized by a decrease in the firing threshold and an increase in the temporal fidelity of firing of jcBNST neurons that were both long-lasting. LTP-IE in the jcBNST was differentially altered in animal models of controlled intake of an abusable drug vs. animal models of dependent use at time points during protracted withdrawal that are characterized by behavioral manifestations of a negative emotional state (Koob et al., 2004), such as increased anxiety-like behavior (Zorrilla et al., 2001; Valdez et al., 2002; Zhao et al., 2007; Sommer et al., 2008) and intracranial self-stimulation reward thresholds (Ahmed et al., 2002). Additionally, rats with a history of heroin self-administration, also show increased stress-induced reinstatement of heroin-seeking behavior at the time point investigated here (Shalev et al., 2001). While mounting evidence suggests that changes in the intrinsic membrane properties of neurons may result from exposure to drugs of abuse (Cooper et al., 2003; Dong et al., 2005; Nasif et al., 2005), changes in the capacity for plasticity of intrinsic excitability have not been previously shown as consequences of drug exposure.

Consistent with classic studies showing that potentiation of the population spike can occur independently of EPSP potentiation (Bliss and Lomo, 1973; Miles et al., 2005), the protracted decrease in the firing threshold and increase in the temporal fidelity of firing of jcBNST neurons observed in LTP-IE were accompanied by only a transient EPSP potentiation. In a previous paper, an LTP of the field potential in the mouse dorsolateral BNST was interpreted as a form of synaptic potentiation (Weitlauf et al., 2004). The present data show that the LTP of the field potential in the jcBNST is an a population spike manifesting the summation of action potentials of jcBNST neurons and cast a new interpretation on the earlier work.

The decrease in the firing threshold of jcBNST neurons observed in LTP-IE was found to be regulated by the ID current. The repertoire, expression, and regulation of K+ channels are believed to be key factors in neuronal integration (Marder and Goaillard, 2006). Previous studies indicate that changes in K+ channel expression levels can alter intrinsic neuronal excitability (Dong et al., 2006) and capacity for plasticity of intrinsic excitability (Wang et al., 2006). Loss of LTP-IE of jcBNST neurons during protracted abstinence from alcohol, cocaine, and heroin self-administration was associated with increased expression of the Kv1.2 K+ channel, a main contributor to the ID current (Shen et al., 2004). Since increased expression of Kv1.2 was correlated with changes in excitability in other neuronal systems (Tsaur et al., 1992), this observation provides a potential mechanism contributing to Kv1.2 regulation. Additionally, a key mechanism of regulation of Kv1.2, appears to be phosphorylation-dependent trafficking of a fraction of the channel (Nesti et al., 2004). If this mechanism mediates the activity-dependent inactivation of Kv1.2 induced by HFS in jcBNST neurons, an increased pool of Kv1.2 consequent to its increased expression may be sufficient to prevent its down-regulation in LTP-IE. Activity- and NMDA-dependent inactivation of another Kv1 channel, Kv1.1, has also been demonstrated in hippocampal CA1 neurons (Raab-Graham et al., 2006).

LTP-IE in the jcBNST was also found to be characterized by increased temporal fidelity of firing of jcBNST neurons, which also depended on the ID current and was impaired in animals with a history of alcohol, cocaine, or heroin dependence. At the circuit level, computational theory predicts that increased temporal fidelity of neuronal firing translates into more efficient use of the capacity of neural connections (Singer, 1999). At the cellular level, the ability to modify the extent and fidelity in which synaptic potentials are translated into activity outputs is believed to be a central feature of the integration properties of neurons (Singer, 1999). Forms of plasticity of intrinsic excitability have been proposed to be adaptive mechanisms directed toward maintaining the homeostasis of network excitability in the central nervous system in response to increases in synaptic inputs (Turrigiano, 1999). The glutamatergic projection to the jcBNST originates in the posterior BLA (Dong et al., 2000). In turn, the jcBNST projects back to the anterior BLA and to the CeA. Its lack of cells expressing glutamatergic markers (Hur and Zaborszky, 2005) and abundance of GABAergic cell bodies (Fig. 6 and N. Sun and M. D. Cassell, 1993;P. Veinante et al., 1997) suggest that the jcBNST is likely to play an inhibitory role on the amygdala. Therefore, the ability of jcBNST neurons to increase their ensemble activity through increased firing rate and temporal fidelity of their firing could provide a more efficient inhibitory influence on the CeA output at times of greater BLA activation. The BLA is activated in humans during drug craving elicited by exposure to drug-related environmental cues (Bonson et al., 2002) and is involved in contextual reinstatement of drug-seeking behavior in experimental animals (Hayes et al., 2003). Thus reduced capacity for LTP-IE and temporal fidelity of firing of jcBNST neurons could be a compensatory response to excessive activation of the BLA in dependent animals and could result in inadequate feedback inhibition of the CeA. Unrestrained CeA activation would be expected to result in increased emotional arousal through its various projection areas, including the paraventricular nucleus of the hypothalamus and the lateral hypothalamus that are believed to be key in the expression of “emotional memories” by interfacing central stress and autonomic systems (LeDoux, 1993). The lateral hypothalamus has been found to undergo structural changes during cocaine escalation (Ahmed et al., 2005). Loss of jcBNST LTP-IE also will directly affect target regions of the jcBNST itself and, indirectly, of other BNST nuclei that in part overlap with those of the CeA (Dong et al., 2000).

Symptoms of a negative emotional state that emerge during drug withdrawal are believed to be due to adaptive changes in the brain reward and stress neurocircuitry that develop during excessive drug intake and persist in a latent state after acute withdrawal (Koob et al., 1998). The alteration of jcBNST plasticity described here was not seen during acute withdrawal in alcohol-dependent rats but became manifest only during protracted withdrawal. Similarly, enhanced long term potentiation (LTP) in the BLA-CeA pathway was seen after 14-day but not 1-day withdrawal from a 7-day cocaine treatment (Fu et al., 2007). Together these data suggest that in addition to acute drug opponent-process adaptations (Koob et al., 1998), drug use also may produce delayed functional changes that are specific to protracted withdrawal. The latter may be especially significant for the neurobiology of vulnerability to relapse to drug-taking after the acute withdrawal syndrome has ceased. Spontaneous anxiety-like behavior in rats with a history of alcohol dependence has recently been found to increase during acute withdrawal, then transiently normalize, but then to resurge during protracted withdrawal and has been linked to changes in the extra-hypothalamic CRF system (Zhao et al., 2007). Similarly, such anxiety-like states can be retriggered by mild stressors that do not affect animals without a history of dependence and these stress-like responses are linked to the extra-hypothalamic CRF system (Valdez et al., 2002). It is particularly noteworthy that in protracted abstinence the HPA shows a blunted response, but the extrahypothalamic CRF system remains activated and may even sensitize (Koob and Kreek, 2007). Together, these observations support the hypothesis that distinct neurobiological mechanisms are recruited during early and protracted withdrawal and suggest that the alteration of the LTP-IE described may be specific to protracted withdrawal. In the rat, exposure to stressors reinstates drug-seeking behavior previously maintained by various drugs of abuse, including alcohol, cocaine, and heroin, and administration of CRF antagonists prevents reinstatement of drug-seeking behavior (Shaham et al., 2003). Extracellular CRF is increased during ethanol withdrawal in the BNST (Olive et al., 2002), which has been implicated in CRF-mediated anxiety-like behavior (Sahuque et al., 2006) and stress-induced reinstatement of drug-seeking behavior depends on extrahypothalamic CRF in the BNST (Erb and Stewart, 1999; Leri et al., 2002; McFarland et al., 2004), Together with our results, these findings support for the hypothesis that extrahypothalamic CRF signaling may regulate neuronal circuits associated with protracted abstinence and stress-induced reinstatement.

In conclusion, our results show that the integration properties of jcBNST neurons are regulated by a novel form of neural plasticity characterized by changes in the intrinsic excitability and temporal fidelity of jcBNST neurons which was altered in a CRF-dependent manner during protracted withdrawal. These results suggest that CRF-mediated impairment of jcBNST neuronal plasticity are associated with the persistent emotional dysregulation following chronic drug exposure and provide a novel neurobiological target for the mechanisms by which stress and protracted abstinence symptoms perpetuate alcohol and drug dependence.

Acknowledgments

Supported by grants AA016587 (WF), DA013821, AA013191 (PS), AA008459, DA004043, DA004398, AA006420 (GFK), and by the Pearson Center for Alcoholism and Addiction Research. We thank Drs. Floyd Bloom and Paul Kenny of The Scripps Research Institute, Elisabeth Walcott of the Neuroscience Institute, and Sascha du Lac of The Salk Institute for critical review of the manuscript, Drs. Bert Weiss and Tamas Bartfai of The Scripps Research Institute, Attila Szücs of the Institute for Nonlinear Science, University of California, San Diego for discussion and Dr. Caroline M. Lanigan for review of statistical analyses, Lena van der Stap for assistance with the analysis of microarray data, and Mike Arends for editorial assistance. A portion of this work was supported by the Intramural Research Programs of the National Institute on Drug Abuse and the NationaljInstitute on Alcohol Abuse and Alcoholism.

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