Introduction

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Peripheral-type nicotinic cholinergic receptors are ligand-gated ion channels that are found at the skeletal neuromuscular junction and in the electric organs of Torpedo californica and Electrephorus electricus (for review, see Lindstrom, 1996). The activation of these receptors results in channel opening leading to an increased flux of Na⁺ into the cell. More than 40 years ago, Katz and Thesleff (1957) reported that prolonged or repeated application of agonists results in a loss of response of the muscle-type nicotinic receptor, and they developed a model that postulated the existence of two forms of the receptor: a ground (activatable) form and a desensitized form. The ground state of the muscle-type receptor has lower affinity for agonists than does the desensitized state and the time course for receptor desensitization parallels the conversion of the receptor to the high-affinity state (Weber et al., 1975; Sine and Taylor, 1979). Heidmann and Changeux (1979, 1980) modified the Katz-Thesleff desensitization model by suggesting that an intermediate state also may exist.

Nicotinic receptors also are found in the brain. Studies done with receptors expressed in oocytes and cell lines indicate that these receptors also desensitize following receptor activation but the rate of desensitization varies depending on receptor subunit composition (Cachelin and Jaggi, 1991; Gross et al., 1991; Séguela et al., 1993; Fenster et al., 1997). In addition, Fenster et al. (1997) found that preexposure of 32-, 34-, 42-, 44-, and homomeric 7-receptors to agonist concentrations lower than those required to evoke reliably measured changes in whole-cell current (subactivating concentrations) resulted in concentration-dependent receptor desensitization. Similarly, the nicotinic receptors found in PC12 cells exhibit desensitization following activation and following preexposure of the PC12 cells to subactivating concentrations of agonists (Boyd, 1987). These cells contain 3-, 5-, 2-, 3-, and 4-subunit mRNA (Rogers et al., 1992), but the precise subunit composition of the nicotinic receptors actually expressed in PC12 cells is unknown, Lester and Dani (1995) found, with whole-cell patch clamp recording techniques, that nicotinic receptors in the rat medial habenula could be desensitized by activating and subactivating concentrations of agonists. The desensitization that develops following receptor activation is faster than desensitization achieved by preexposure of the cells to subactivating concentrations of nicotine.

Several recent studies from our laboratory, with neurochemical assays of receptor function, have demonstrated that mouse brain nicotinic receptors also desensitize following receptor activation and following preexposure to subactivating concentrations of nicotine (Grady et al., 1994; Marks et al., 1994) and other nicotinic agonists (Marks et al., 1996; Grady et al., 1997). The nicotinic receptor(s) that modulate [³H]dopamine release from mouse striatal synaptosomes desensitize following receptor activation but, unlike results obtained with peripheral-type nicotinic receptors, desensitization of the receptor that modulates dopamine release is not complete; ~20% of the maximal response persists in the continued presence of agonist. Rowell (1995) obtained virtually identical results in a study that evaluated [³H]dopamine release from rat striatal synaptosomes. Desensitization also can be achieved by pretreatment with agonist concentrations that do not evoke dopamine release, as demonstrated by the findings that the IC₅₀ values for inhibition of dopamine release produced by pretreatment with low concentrations of nicotine are much lower (two orders of magnitude) than the EC₅₀ values for nicotine-induced activation of dopamine release in rat (Rowell and Hillebrand, 1994) and mouse (Grady et al., 1997) striatal synaptosomes. Marks et al. (1994) measured receptor function more directly with an ion (⁸⁶Rb⁺) efflux assay. The desensitization of the nicotinic receptor that modulates ⁸⁶Rb⁺ efflux is clearly different from the one that modulates [³H]dopamine release because total desensitization of nicotine-stimulated ⁸⁶Rb⁺efflux occurs following exposure to both activating (EC₅₀ value for activation is 600 nM) and subactivating (IC₅₀ value for desensitization is 13 nM) concentrations of nicotine. In addition, every one of nine agonists tested for their desensitization of dopamine release (Grady et al., 1997) and all of the 11 agonists tested for their desensitization of the ⁸⁶Rb⁺ efflux assay (Marks et al., 1996) produced desensitization at concentrations much lower than those required to produce an increase in receptor activity.

Recently, we (Lu et al., 1998) developed a neurochemical assay for nicotinic agonist-stimulated release of [³H]-aminobutyric acid (GABA) from mouse brain synaptosomes. Nicotinic agonists stimulated a concentration-dependent increase in [³H]GABA release that fully desensitized in the continued presence of agonist. The studies reported herein represent an analysis of desensitization of [³H]GABA release produced by preexposure to subactivating concentrations of several nicotinic agonists.

	Experimental Procedures

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Materials. (+)-Anatoxin-a hydrochloride and methylcarbachol chloride were purchased from Research Biochemicals Inc. (Natick, MA). Sucrose and HEPES were obtained from Boehringer Mannheim Corp. (Indianapolis, IN). The following compounds were purchased from Sigma Chemical Co. (St. Louis, MO): nicotine hydrogen ()-tartrate (L-nicotine), (+)-nicotine-(+)-di-p-toluoyltartrate (D-nicotine), acetylcholine iodide (ACh), cytisine, (±)-epibatidine-L-tartrate, carbachol iodide, tetramethylammonium iodide, atropine sulfate, (±)-anabasine, (±)-nornicotine, aminooxyacetic acid, GABA, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, potassium dihydrogen phosphate, D-(+)-glucose, and diisopropyl fluorophosphate (DFP). Dimethylphenylpiperazinium iodide was obtained from Aldrich Chemical Co. (Milwaukee, WI). Econo-safe scintillation cocktail was purchased from Research Products International Corp. (Arlington Heights, IL). [³H]GABA (84-90 Ci/mmol) was purchased from Dupont NEN (Boston, MA).

Animals. Female C57BL/6J mice were used in the study. The animals were bred at the Institute for Behavioral Genetics and were housed five per cage with free access to food and water. The vivarium was maintained on a 12-h light/12-h dark cycle with lights on between 7:00 AM and 7:00 PM. The animals were 60 to 90 days of age at the time of use. Animal care and experimental procedure were in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and were approved by the University of Colorado animal care committee.

Synaptosome Preparation. Crude synaptosomes were prepared from mouse whole brain (forebrain) or dissected regions. Samples were homogenized by hand in 10 volumes of ice-cold 0.32 M sucrose buffered with 5 mM HEPES (pH 7.5) in a glass-Teflon homogenizer. The homogenate was centrifuged at 500g for 10 min. The supernatant was then centrifuged at 12,000g for 20 min. The resulting P2 pellet was resuspended in the perfusion buffer (128 mM NaCl, 2.4 mM KCl, 3.2 CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 25 mM HEPES, pH 7.5, 10 mM glucose). The volume of perfusion buffer used for resuspending the synaptosomes varied between 0.2 and 8 ml depending upon the brain region being studied.

[³H]GABA Uptake. The resuspended synaptosomes were incubated for 10 min at 37°C in buffer containing 1 mM aminooxyacetic acid, an inhibitor of GABA transaminase. [³H]GABA and unlabeled GABA were then added to final concentrations of 0.1 and 0.25 µM, respectively, and the suspension was incubated for an additional 10 min. The unlabeled GABA was used to decrease assay expense. DFP (10 µM) was added to the synaptosomal preparation during the GABA uptake step in those experiments where ACh was used as the test compound. Uptake was terminated by filtration of the 80-µl sample onto 6-mm diameter A/E glass-fiber filters (Gelman Sciences, Inc., Ann Arbor, MI) under gentle vacuum (0.2 atm) followed by two washes with 0.5 ml of buffer. Amount of uptake was determined by filtration of a 5-µl aliquot and averaged 90% under these conditions. Samples to be used with ACh were incubated with 10 µM DFP during the final 10 min of uptake to inhibit acetylcholinesterase.

Perfusion and Release. The 6-mm filter containing the synaptosomes was placed on a 13-mm glass fiber filter mounted on a polypropylene platform and perfused with the buffer containing 0.1% (by weight) BSA at a rate of 1.8 ml/min for 10 to 12 min before fraction collection was started. Fractions were collected in 12-s aliquots. Predesensitization was achieved by adding defined concentrations of agonists to the regular buffer. Atropine (1 µM) was used in the buffer for experiments with ACh and carbachol. This concentration of atropine had no effect on nicotine-evoked [³H]GABA release. The agonist to be tested was present before and after nicotine stimulation. The stimulation of samples was achieved by adding 30 µM nicotine to the perfusate for 12 s. We used 30 µM nicotine because it evokes maximal, or near maximal, [³H]GABA release (Lu et al., 1998). The agonists were tested sequentially. Radioactivity was determined with a Packard liquid scintillation counter. Counting efficiency was 45%.

Data Analysis. To correct for differences in total synaptosomal [³H]GABA content within and between experiments, the amount of [³H]GABA release induced by an agonist stimulation was normalized as follows. The fractions before and after the stimulation that represent basal release were identified and were then fit as the first-order process E_t = E_o * e^kt, where E_t is the actual data obtained at each time (t); E_o is the initial basal release; and k is the rate of decrease of release. This calculation yielded the theoretical basal release for each fraction. The release of [³H]GABA exceeding baseline, which represents the agonist-stimulated release, was then calculated by subtracting the theoretical basal release from the actual data and was finally divided by the average baseline underlying the peak. The data are expressed as "units" of release where 1 unit represents a doubling of the release above baseline. Curve fitting was accomplished with the nonlinear curve fitting algorithm in SigmaPlot 5.0 (Jandel Scientific, Corte Madera, CA). An estimate of the IC₅₀ value for desensitization of release was calculated by fitting the data to the following equation: E = E_o/(1 + C/K), where E is the response to 30 µM nicotine with 10-min preexposure to a concentration (C) of the agonist (E_o) is the response to 30 µM nicotine without any preexposure of agonist; and K is the IC₅₀ value. IC₅₀ values were calculated for each experiment and the means ± S.E. were calculated for each compound with the individual experiment data.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

The time course for desensitization of nicotine-stimulated [³H]GABA release produced by treatment with 50 nM nicotine is shown in Fig. 1. Typical data obtained in an experiment where [³H]GABA-loaded synaptosomes were perfused with buffer containing 50 nM nicotine for 0 to 12 min before stimulation of release are illustrated in Fig. 1A and the mean effect of pretreatment with 50 nM nicotine on total release obtained from six separate experiments is shown in Fig. 1B. Treatment with 50 nM nicotine resulted in a time-dependent decrease in the [³H]GABA release elicited by a 12-s exposure to 30 µM nicotine. The data were best fit by a single exponential with a rate constant of 0.20 ± 0.09 min¹, which yields a T_1/2 for desensitization of 3.46 min.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Time course of desensitization of nicotine-stimulated [3H]GABA release from whole-brain synaptosomes produced by pretreatment with 50 nM nicotine. A, representative normalized [3H]GABA release traces elicited by 12-s stimulation with 30 µM nicotine following preexposure to 50 nM nicotine. Each trace contains 15 fractions (12 s/fraction). The low concentration (50 nM) of nicotine was present before and after the peak. B, time course response curve for 50 nM nicotine-predesensitized [3H]GABA release. Ordinate is percentage of control response that sample was given zero time of preexposure to the low concentration of nicotine. Each point represents mean ± S.E. calculated from six separate synaptosomal preparations. The curve was generated by fitting the data as the first-order process At = Ao * ekt, where At is the release evoked by 30 µM nicotine with time (t) of preexposure to low concentration of agonist; Ao is the release to 30 µM nicotine with zero time of preexposure to agonist; and k is the rate of predesensitization.

The extent of desensitization is dependent on the concentration of nicotine present before stimulation (Fig. 2). Pretreatment for 10 min with nicotine (0-300 nM) produced a concentration-dependent decrease in the total [³H]GABA release evoked by stimulation with 30 µM nicotine (Fig. 2A). The maximal inhibition produced by the highest concentration of nicotine used (300 nM) was >90% and the nicotine concentration required to produce a 50% inhibition of receptor function (IC₅₀) calculated from these data is 26 ± 11 nM (Fig. 2B). This IC₅₀ value is much lower than the EC₅₀ value for activation of [³H]GABA release (1.63 ± 0.50 µM) reported previously (Lu et al., 1998).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent desensitization of [3H]GABA release by pretreatment with nicotine. Whole-brain synaptosomes that had been preloaded with [3H]GABA were preexposed to various low concentrations of nicotine for 10 min before stimulation for 12 s with 30 µM nicotine (activating concentration). A, representative normalized [3H]GABA release traces for 30 µM nicotine after 10-min preexposures of different concentrations of nicotine (nanomolar). B, concentration-effect curve of nicotine-induced predesensitization. The curve was obtained by fitting data to the inhibition equation presented in Experimental Procedures. Each point represents mean ± S.E. calculated from four separate synaptosomal preparations. For other details, see Fig. 1.

The time course for recovery from desensitization produced by a 10-min exposure to 50 nM nicotine is depicted in Fig. 3. The 10-min pretreatment with 50 nM nicotine reduced the response to <15% of control. The data presented in Fig. 3A, which provides typical data from a single series of experiments, illustrate that response recovered following nicotine removal. Figure 3B presents an overall summary of the experiments. Nearly total recovery of function was observed within 12 min; the rate constant for recovery of response calculated from these data is 0.14 ± 0.05 min¹ yielding a T_1/2 for recovery of 4.95 min.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Recovery of [3H]GABA release. Whole-brain synaptosomes were preexposed to 50 nM nicotine for 10 min, then washed with buffer for various times, and finally stimulated with 30 µM nicotine for 12 s. A, representative normalized [3H]GABA release traces. The dashed line presents the control release stimulated by 30 µM nicotine without any preexposure to low (50 nM) concentrations of nicotine. B, time course curve for recovery from desensitization. The curve was obtained by fitting the data as the first-order process At = Ao * (1  ekt), where At is the release elicited by 30 µM nicotine with wash time (t), after the 10-min preexposure to 50 nM nicotine; Ao is the release elicited by 30 µM nicotine without any preexposure to 50 nM nicotine (dotted line); and k is the rate of recovery from desensitization. Data are means ± S.E. calculated from six separate synaptosomal preparations.

The data presented in Fig. 2 were obtained with synaptosomes obtained from whole brain. The effects of pretreatment for 10 min with various concentrations of nicotine also were done with synaptosomes prepared from thalamus, striatum, and cortex (Fig. 4). The data obtained from the three brain regions are presented as a percentage of control. The maximal [³H]GABA release was 0.65, 1.40, and 1.03 units for thalamus, striatum, and cortex, respectively. Pretreatment for 10 min with nicotine (0-300 nM) produced concentration-dependent inhibition of the [³H]GABA release evoked by 30 µM nicotine in all three brain regions. The IC₅₀ values obtained in the three brain regions are 22 ± 9, 24 ± 6, and 21 ± 7 nM, respectively, and are virtually identical with the IC₅₀ value obtained in the whole-brain study (26 ± 11 nM). Consequently, all subsequent studies were done with synaptosomes prepared from whole brain.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Concentration-effect curves of nicotine-induced predesensitization of [3H]-GABA release from synaptosomes prepared from thalamus, striatum, and cortex. Synaptosomes obtained from the three brain regions were pretreated with the designated concentrations of nicotine for 10 min before stimulation for 12 s with 30 µM nicotine. Each data point is mean ± S.E. calculated from four to five separate synaptosomal preparations.

Concentration-dependent desensitization was assessed for 11 other agonists (Fig. 5). The same protocol was used for all of the agonists (10-min pretreatment before a 12-s stimulation with 30 µM nicotine). All of the agonists induced concentration-dependent inhibition of response. Thus, even partial agonists such as cytisine (Lu et al., 1998) can evoke total desensitization of the receptor(s) that modulate [³H]GABA release from mouse brain synaptosomes. The IC₅₀ values calculated from these experiments are listed in Table 1.

View larger version (50K): [in this window] [in a new window]   Fig. 5.   Concentration-effect curves for predesensitization and activation of [3H]GABA release by 12 agonists. For the desensitization experiments, whole-brain synaptosomes were preexposed to various concentrations of the different agonists for 10 min before stimulation for 12 s with nicotine (30 µM). , means ± S.E. calculated from four to six separate synaptosomal preparations. , concentration-dependent stimulation of [3H]GABA release (12-s exposure) (data from Lu et al., 1998).

View this table: [in this window] [in a new window]   TABLE 1 Comparison of agonist inhibition and activation of [3H]GABA release The agonist concentrations nanomolar that produced 50% inhibition of [3H]GABA release (IC50) were determined as described in Experimental Procedures. The agonist concentrations nanomolar that produced 50% of maximal release (EC50) and the maximal release itself (in units) are from Lu et al., 1998. Each value is the mean ± S.E.

Figure 5 also includes, for comparison, the activation data reported in Lu et al. (1998). The EC₅₀ values for stimulation of [³H]GABA release are listed in Table 1. With the exception of cytisine, anabasine, and nornicotine, total or near total desensitization was achieved by agonist concentrations that are at, or below, the concentration required to produce a reliably measured increase in [³H]GABA release. The IC₅₀ and EC₅₀ values were significantly correlated with one another (r = 0.75, p < .01), but the values for cytisine and anabasine clearly fall off the regression line (Fig. 6).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   A comparison of agonist-induced activation and inhibition of [3H]GABA release. A significant correlation (r = 0.75) between the EC50 values for activation (nanomolar) and the IC50 values (nanomolar) for desensitization (inhibition) produced by pretreatment with agonists was obtained. Note that the values for two of the compounds (anabasine and cytisine) do not fall on or near the regression line.

	Discussion

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The experiments reported herein provided clear evidence that pretreatment with agonists resulted in inhibition, or desensitization, of receptor function as measured by nicotine-induced [³H]GABA release. The desensitization that developed was concentration-dependent, and, given an adequate concentration, total inhibition was produced. Desensitization developed slowly and was totally reversible. All of the 12 agonists tested induced desensitization and, for most of these agonists, total or near total desensitization could be achieved by agonist concentrations that failed to evoke a readily measured increase in [³H]GABA release. However, cytisine, anabasine, and nornicotine evoked readily measured increases in [³H]GABA release at agonist concentrations that were less than maximally effective in producing desensitization following pretreatment.

The finding that desensitization can be produced by subactivating concentrations of agonists should not be surprising because both the Katz-Thesleff (Katz and Thesleff, 1957) and Heidmann-Changeux (Heidmann and Changeux, 1980) models of nicotinic receptor function predict that an equilibrium exists between ground-state (low-affinity) and desensitized (high-affinity) conformations of the receptor. These models also predict that ground-state receptors can isomerize to high-affinity, desensitized receptors in the absence of ligand and that ligand binding to predesensitized receptors should reduce the unbound desensitized receptor pool and thereby promote the isomerization of ground-state receptors to the desensitized isoform. One consequence of this isomerization is the pool of activatable receptors should be depleted, which is consistent with the observation that pretreatment with low concentrations of agonist that fail to evoke a measurable effect can produce total receptor desensitization.

One criticism that might be raised concerning the assertion that low concentrations of agonists can induce receptor desensitization without receptor activation is that the assays used in our studies (⁸⁶Rb⁺ efflux, [³H]dopamine, and [³H]GABA release) are not sensitive enough to detect short-term channel opening. However, Tamamizu et al. (1996) recently directly demonstrated, with channel-blocking antibodies, that the peripheral-type nicotinic receptor does, indeed, desensitize without channel opening. In addition, two recent studies with electrophysiological approaches have drawn the same conclusion for neuronal-type nicotinic receptors. Lester and Dani (1995) used whole-cell patch clamp to study desensitization of nicotinic receptor function in the rat medial habenula. This brain region contains the mRNA for nearly all of the nicotinic receptor subtypes and, consequently, it is not clear what receptor subtype Lester and Dani were studying. However, these investigators observed that total desensitization could be produced by agonist concentrations that failed to evoke a measurable change in current flow. Similarly, Fenster et al. (1997) reported that pretreatment with nicotine caused concentration-dependent desensitization of 32-, 34-, 42-, 44-, and 7-receptors expressed in oocytes and that total desensitization was seen (with the exception of 32-receptors) at concentrations less than those required to produce reliably measured changes in whole-cell current. Consequently, it seems likely that desensitization can occur with the ion channel open and closed. If so, this would mimic desensitization of the N-methyl-D-aspartate receptor (Lin and Stevens, 1994).

The nAChR that modulates [³H]GABA release from mouse brain synaptosomes includes a 2-subunit because no nicotine-stimulated release was seen in synaptosomes prepared from 2-null mutant mice (Lu et al., 1998). Definitive assignment of the  subunits involved in modulating GABA release remains to be made, but widespread expression and similarities in pharmacological properties and regional distribution between GABA release and nicotine binding and Rb⁺ efflux suggest that the 4 subunit is included in the nAChR that modulates GABA release (Lu et al., 1998). The receptor that modulates [³H]GABA release is the same one that modulates Rb⁺ efflux and makes up the [³H]nicotine binding site, presumably 42 (Whiting and Lindstrom, 1988; Flores et al., 1992, Picciotto et al., 1995). This assertion is made because of the following findings: 1) the high correlation between the IC₅₀ value for agonist desensitization of GABA release and the K_i value for inhibition of nicotine binding (r = 0.93) and the IC₅₀ values for Rb⁺ efflux (r = 0.98) (Fig. 7); 2) the similarity in the rates of desensitization (K = 0.2 min¹ for onset of desensitization of the [³H]GABA release process and 0.3 min¹ for onset of desensitization of Rb⁺ efflux); and 3) rates of recovery from desensitization (K from recovery is 0.14 min¹ for the [³H]GABA release process and 0.15 min¹ for Rb⁺ efflux).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   A comparison of agonist-induced desensitization of [3H]GABA release with 86Rb+ efflux and inhibition of [3H]nicotine binding. Top, correlational analysis of the relationship between the IC50 values for agonist-induced desensitization of [3H]GABA release (data from Table 1) with the IC50 values for inhibition of 86Rb+ efflux from mouse brain synaptosomes (data from Marks et al., 1996). Bottom, correlational analysis of the relationship between agonist-induced inhibition of [3H]GABA release (data from Table 1) and inhibition of [3H]nicotine binding (Ki values) obtained from Marks et al. (1996).

We (Grady et al., 1994) have shown that nicotine-induced [³H]dopamine release is desensitized following receptor activation as well as by pretreatment with subactivating concentrations of nicotine and several other agonists, but desensitization is incomplete. Approximately 20% of the response persist following both types of desensitization. In contrast, nicotine-evoked [³H]GABA release fully desensitizes following activation (Lu et al., 1998), and, as shown herein, following pretreatment with subactivating concentrations of nicotine and other agonists. These findings argue that the receptors that modulate dopamine and GABA release are different. It is probable, however, that there is overlap in the subunit composition of the nicotinic receptors that modulate the release of these two neurotransmitters because the 2-subunit is required for both dopamine (Picciotto et al., 1998; Grady et al., 1998) and GABA (Lu et al., 1998) release.

A comparison of concentration-dependent inhibition of agonist-stimulated [³H]GABA release and concentration-dependent activation of release revealed that, with the exception of cytisine, anabasine, and nornicotine, nearly total inhibition of response could be achieved by pretreatment with agonist concentrations that failed to produce a readily measured increase in [³H]GABA release. All three of the agonists that showed some overlap in the desensitization-activation concentration-effect curves are partial agonists (Lu et al., 1998), which suggests that the same property that makes a compound a partial agonist also serves to reduce the difference between inhibition and agonist potencies. However, some partial agonists, for example, anatoxin-a, and D-nicotine induce desensitization at concentrations much lower than those required to activate GABA release. It may be that gaining an understanding of the structural features that determine affinity for ground-state and desensitized variants of the receptor could lead to the development of drugs with more pronounced activating or desensitizing properties.

The finding that nicotinic agonists can desensitize the receptor at concentrations lower than those required to activate the receptor predicts that if agonist concentrations rise slowly, as would be the case if a nicotinic agonist is given orally, desensitization, rather than activation, may predominate at this receptor subtype. One consequence of this is behavioral excitation might result from a decrease in GABAergic tone. Thus, it may be that nicotine-induced behaviors, such as increases in acoustic startle seen in the rat (Acri et al., 1991, 1995; Acri, 1994) and mouse (Marks et al., 1989) and nicotine-induced seizures (Miner and Collins, 1989; Stitzel et al., 1998) are due, at least partially, to disinhibition generated by desensitization of the presynaptic nicotinic receptor associated with GABAergic nerve terminals.