Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

The hippocampus (HP) receives its major norepinephrinergic projections from the locus coeruleus (LC) (Aston-Jones et al., 1995). Previous studies have shown that systemic administration of nicotine stimulates hippocampal norepinephrine (NE) release. This NE release was blocked by injecting the nicotinic receptor (NAChR) antagonists mecamylamine (Mec), dihydro--erythroidine (DHE), or methyllycaconitine (MLA) into the cerebral aqueduct immediately upstream from the LC (Fu et al., 1998a) or by directly delivering Mec into the LC (Mitchell, 1993). Thus, NAChRs on or near the norepinephrinergic neurons in the LC are involved in the hippocampal NE response to systemic nicotine. Based on the differential potencies and efficacies of these nicotinic antagonists, 3 subunit-containing NAChRs appeared to be involved (Fu et al., 1998a). In contrast, the selective NAChR antagonist, -bungarotoxin (-BTX), was ineffective. Recently, the NAChR 6 subunit was found to be selectively expressed in the catecholaminergic nuclei of the rat brain, especially within the LC (Le Novere et al., 1996). Although the pharmacology of 6 subunit-containing NAChRs has not been described, it is possible that these receptors also are involved in the hippocampal NE response to systemic nicotine.

As shown in those studies, NAChR antagonists, delivered upstream of the LC, maximally inhibited hippocampal NE release by 63 to 87% (Fu et al., 1998a). These findings suggest that nicotine may directly affect other brain regions, such as the HP itself, to stimulate hippocampal NE release. Indeed, in vitro studies with hippocampal slices or synaptosomes have shown that nicotine can stimulate NE release (Sacaan et al., 1995; Clarke and Reuben, 1996; Sershen et al., 1997). An in vivo microdialysis study, however, reported that Mec (dialyzed into the HP) failed to block hippocampal NE secretion by i.p. nicotine (Mitchell, 1993). In that study, additional NAChR antagonists were not tested.

NAChRs, assembled from various combinations of  (2-7) and  (2-4) subunits, are widely distributed throughout the central nervous system (Karlin and Akabas, 1995; Colquhoun and Patrick, 1997). In the HP, an abundance of 7-containing NAChRs, with high affinity for -BTX, are expressed (Clarke et al., 1985; Harfstrand et al., 1988; Barrantes et al., 1995). The function of 7-containing NAChRs has been established in several ways. Homomeric 7-containing NAChRs expressed by Xenopus oocytes have been shown to conduct Ca²⁺ (Bertrand et al., 1993; Castro and Albuquerque, 1995). Electrophysiological studies of fetal hippocampal neurons in culture have detected a predominant nicotinic current (type IA) that appears to depend on 7-containing NAChRs (Alkondon and Albuquerque, 1993). Finally, presynaptic 7-containing NAChRs appear to modulate glutamate release in rat hippocampal and olfactory bulb neurons (Alkondon et al., 1996; Gray et al., 1996). The present study considered the potential involvement of local -BTX-sensitive NAChRs in the hippocampal NE response to i.v. nicotine. These NAChRs may be relatively insensitive to Mec (Briggs and McKenna, 1996).

Biochemical, physiological, and behavioral responses to nicotine decline after repeated exposure to this drug (Sharp and Beyer 1986; Marks et al., 1995; Fu et al., 1998b). This reflects the desensitization of NAChRs (Dani and Heinemann, 1996). Indeed, prior exposure to nicotine has been reported to differentially affect various NAChR subtypes. In studies with transfected Xenopus oocytes, human 42 and homomeric 7 NAChRs desensitized more readily than receptors containing 3 subunits (Olale et al., 1997). Moreover, much lower concentrations of nicotine were able to induce desensitization than were required to stimulate ion conductance or neurotransmitter release (Lippiello et al., 1995; Marks et al., 1995; Olale et al., 1997).

Systemic nicotine stimulates the secretion of NE in the HP, in part, through NAChRs in the vicinity of the LC. The present study was conducted to determine the role of local (intrahippocampal) NAChRs, particularly those sensitive to -BTX, in this process. To do so, various NAChR antagonists were delivered through the hippocampal microdialysis probe or injected directly into the HP before i.v. nicotine. The role of hippocampal NAChRs was demonstrated further by selectively desensitizing these receptors before the systemic infusion of nicotine. Substimulatory doses of nicotine were dialyzed into the HP, and then nicotine was administered systemically.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Materials. Nicotine sulfate (Pfaltz and Bauer, Inc., Waterbury, CT; all dosages are given as milligrams per kilogram of the free base) was used for i.v. injection. ()-Nicotine-free base (Sigma, St. Louis, MO) was used for intrahippocampal delivery through the microdialysis probe. L-()-[N-methyl-³H]nicotine (60 Ci/mmol) was obtained from NEN Life Sciences (Boston, MA). NE hydrochloride, Mec hydrochloride, DHE hydrobromide, MLA citrate, -BTX, and nomifensine maleate were purchased from RBI (Natick, MA). Sodium dihydrogen phosphate monohydrate, EDTA, acetonitrile and phosphoric acid (Fisher Scientific, Fair Lawn, NJ), 1-octanesulfonic acid sodium salt (J.T. Baker, Phillipsburg, NJ), and triethylamine (Aldrich, Milwaukee, WI) were used to prepare the mobile phase. The alert-rat microdialysis systems and CMA 110 liquid switches were obtained from CMA/Microdialysis (Acton, MA). For constructing dialysis probes, cellulose fiber tubing was obtained from Spectrum (Laguna Hills, CA), and silica tubing (outer diameter, 148 µm; inner diameter, 73 µm; TSP 075150) was from Polymicron Technologies Inc. (Phoenix, AZ).

Animals. Adult male Holtzman rats (250-350 g, HSD, Madison, WI) were given access to standard rat chow and water ad libitum. They were housed individually on a 12-h reversed light cycle (lights off at 9:00 AM, on at 9:00 PM) for 14 days before the microdialysis experiments. This reversed light cycle was used to conduct these experiments during the rat's active (dark) phase. After the rats had been housed under this reversed light/dark cycle for 7 days, they were anesthetized with xylazine-ketamine (5:35 mg/kg b.wt., i.m.; Parke-Davis, Morris Plains, NJ), and chronic guide cannulas (20 gauge) were stereotaxically implanted into the HP, according to the atlas coordinates of Paxinos and Watson (1986). The coordinates were AP, 3.0 mm, DV, 2.6 mm, and ML, 1.4 mm, from bregma with a flat skull. For rats receiving an injection of -BTX into the HP, a double-guide cannula was implanted (20 gauge for the dialysis probe attached to a 23-gauge for microinjection; Fu et al., 1997), with the 20-gauge cannula targeted at the above coordinates. Five days later, rats received jugular cannulas under Innovar Vet anesthesia (3.75 mg/kg droperidol plus 0.08 mg/kg fentanyl, i.m.; Far-Vet, St. Paul, MN) and were allowed to recover for another 2 days. All procedures were conducted in accordance with National Institutes of Health Guidelines Concerning the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of the Minneapolis Medical Research Foundation.

In Vivo Microdialysis. The microdialysis method was described previously (Fu et al., 1997). Briefly, a 2-mm concentric probe (molecular mass cutoff, 13,000 Da; outer diameter, 235 µm) was constructed in our laboratory. The recovery rate of individual probes was determined by in vitro dialysis for 60 min at 22°C in a solution of 200 pg NE/16 µl. The probes we perfused at 1 µl/min with standard perfusate (see below), and three 20-min samples were obtained; the average recovery rate was 7.1% ± 1.0 (n = 10). On the day of microdialysis, rats were moved into the alert-rat microdialysis chambers in an isolated darkroom lit with a red safe-light; all connections were made quickly to minimize stress to the animal. The probe was perfused at 1 µl/min with a solution of Krebs-Ringer Buffer (KRB: 147 mM NaCl, 4.0 mM KCl, and 3.4 mM CaCl₂ in polished water; 0.2-µm filter sterilized and degassed) containing 5 µM nomifensine (NE uptake blocker; Schacht et al., 1982). Two hours after insertion of the probe through the guide cannulas, three consecutive samples were collected to measure basal NE levels before drug administration. Samples were collected over 20 min into vials containing 1 µl of 5% perchloric acid to prevent the degradation of NE. At the end of the experiments, the position of the probe was verified by histological examination; only data obtained from animals with probes identified in the correct location of the HP were used for analysis. The histological verification of probe placement in the HP has been published previously (Fu et al., 1998a).

HPLC-Electrochemical Analysis. Samples (16 µl) were injected immediately by a CMA 200 refrigerated autosampler onto a 150 × 3-mm ODS C18 column (ESA, Bedford, MA) perfused by BAS 200A HPLC pumps (BAS, Inc., West Lafayette, IN) at 0.5 ml/min with a mobile phase containing 80 mM sodium dihydrogen phosphate monohydrate, 2.0 mM 1-octanesulfonic acid sodium salt, 100 µl/liter triethylamine, 5 nM EDTA, and 10% acetonitrile, pH 3.0. Samples were analyzed by an ESA Coulochem II 5200A electrochemical detector with an ESA 5041 high-sensitivity microbore analytical cell and an ESA 5020 guard cell (ESA). Electrochemical detection was performed at 220 mV and 1.0 nA with the guard cell at 350 mV. The limit of detection for NE was 0.5 pg. Representative HPLC chromatograms have been published previously (Fu et al., 1998a).

Experimental Protocols. In all experiments, on day 1 a probe was inserted for 10 min and then removed without further microdialysis. On days 3 and 5, probes were reinserted and the rats received randomized treatments. Previous studies have shown that there were no significant "within rat" changes in basal NE levels nor in NE responses to nicotine when using this protocol of testing on days 3 and 5 in the same rat (Fu et al., 1998a).

Data Analysis and Statistics. Chromatographic data were collected and analyzed with the PowerChrom system (AD Instruments, Castle Hill, NSW, Australia) and expressed either as pg/16-µl sample or as a percentage of basal NE levels. Basal values were defined as the average NE levels of the three samples before nicotine, antagonist, or vehicle administration. Data were analyzed by one-way ANOVA using StatView, and results were considered significant at p < .05. The number shown in parentheses (n) in the text and graphs is the number of rats within a specific treatment group. The calculation of in vivo spread of nicotine was obtained from the percentage of [³H]nicotine in the HP tissue compared with the total amount of radioactivity initially perfused through the probe.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Figure 1 demonstrates that i.v. nicotine-induced NE release in the HP was inhibited by administering -BTX or MLA, but not Mec or DHE, in this region. As shown in Fig. 1A, microinjecting 0.3 nmol -BTX into HP did not affect NE secretion. However, -BTX (0.3 nmol) significantly reduced NE release because of nicotine (0.065 or 0.09 mg/kg i.v.; p < .05). Similarly, MLA (0.4-5.6 mM in dialysate) alone did not affect basal NE levels (data not shown). Figure 1B shows that MLA, at concentrations of 1.4 mM or higher, inhibited the NE response to 0.09 mg/kg nicotine (p < .05-.01). An additional experiment showed that 2.8 mM MLA blocked 62% of the NE response to 0.065 mg/kg nicotine (p < .05). In contrast, Fig. 1C shows that neither Mec (2.9 or 5.8 mM) nor DHE (7 or 14 mM) inhibited nicotine-induced NE release.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Systemic (i.v.) nicotine stimulates hippocampal NE secretion that is blocked by selective administration of NAChR antagonists into the HP. NE levels are expressed as percentage of average basal levels (obtained at 20, 40, and 60 min). Peak NE levels were measured in samples collected 20 min after initiation of i.v. nicotine infusion. A, time course for NE release in the HP after hippocampal injection of 0.3 nmol -BTX or CSF followed by an infusion of nicotine (0.065 mg/kg per 44 s or 0.09 mg/kg per 60 s). Basal level of NE (mean ± S.E.M.) for 0.065 mg/kg CSF/nicotine group was 6.7 ± 0.3 pg/16 µl (n = 6); basal levels for 0.09 mg/kg CSF/nicotine were not different. Peak NE responses to 0.065 mg/kg or 0.09 mg/kg nicotine were reduced significantly by -BTX (F = 4.21, p = 0.028). B, results for similar experiments with MLA in the dialysate. MLA dose dependently blocked the NE response to 0.09 mg/kg nicotine (F = 3.59, p = 0.033). C, lack of blockade by two doses of Mec or DHE administered in the dialysate. A-C, +p < .05 compared with CSF/nicotine 0.065; * or **p < .05 or 0.01, respectively, compared with CSF or KRB/nicotine 0.09 (n = 6 rats per group).

Figure 2 shows the peak NE response to intrahippocampal nicotine administered for 20 min through the microdialysis probe. Enhanced NE secretion was observed only in samples collected during the interval of nicotine perfusion. At concentrations of 2.5 mM (in the dialysis probe) or higher, nicotine produced a significant dose-dependent increase in NE secretion (p < .05-.01).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Hippocampal NE release is dose dependently stimulated by local perfusion of nicotine into the HP. Peak NE levels were measured in samples collected for 20 min during local perfusion of nicotine. Peak NE levels were expressed as a percentage of preinfusion basal levels. Estimated nicotine concentration in the HP is based on data of [3H]nicotine delivered through the probe. Nicotine (2.5-10 mM) significantly increased NE secretion in the HP (F = 5.04, p = 0.026). *p < .05, **p < .01, compared with control (0 mM nicotine) (n = 5 rats per treatment).

After delivering [³H]nicotine through the intrahippocampal microdialysis probe, 0.37 ± 0.05% (n = 3) of the nicotine perfused was detected in the surrounding brain parenchyma. The radial distribution of [³H]nicotine is shown in Table 1. Approximately 80% of the radioactivity was measured within 0.6 mm and 93.4% was measured within 1.0 mm of the central axis of the probe. Based on the assumption of a spherical distribution, 4.2 µm³ was calculated as the volume containing 93.4% of the [³H]nicotine. Therefore, a 1 mM concentration of nicotine in the microdialysate would yield a tissue concentration of 17.6 µM in the HP. The lowest dose of nicotine (2.5 mM in the microdialysate) that induced significant NE release in the HP (Fig. 2) would be expected to yield high tissue concentrations (i.e., 44 µM).

The role of hippocampal NAChRs in the NE response to systemically administered nicotine was investigated further by selectively desensitizing intrahippocampal NAChRs and then delivering i.v. nicotine. Concentrations of nicotine (0.01, 0.1, and 1 mM) that failed to stimulate NE release (Fig. 2) were used to selectively pretreat hippocampal NAChRs by delivering the drug through the microdialysis probe; 20 min thereafter, 0.09 mg/kg nicotine was infused i.v. Figure 3A shows the effect of pretreatment with 0.1 mM nicotine, which was calculated to yield a concentration of nicotine in the HP (1.8 µM) similar to that achieved by i.v. nicotine (0.09 mg/kg/60 s) (Hieda et al., 1998). This pretreatment reduced the NE response to i.v. nicotine (0.09 mg/kg) by 34% (p < .05). Additionally, pre-exposure to 1 mM nicotine resulted in a 40% decrease (p < .01), whereas 0.01 mM nicotine did not result in significant inhibition. Figure 3B shows a linear decrease in responsiveness with increasing pretreatment doses of nicotine (expressed as log dose).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Pretreatment with intrahippocampal nicotine reduced NE secretion in response to i.v. nicotine. A, time course of hippocampal NE response to i.v. nicotine after pretreatment with nicotine. Pretreatment with nicotine, at a dialysate dose of 0.1 mM or 1.0 mM, decreased i.v. nicotine-induced NE release (F = 3.25, p = 0.041). B, a linear decrease in NE secretion with increasing pretreatment doses of nicotine (expressed as log µM). Estimated nicotine concentration in the HP is based on data of [3H]nicotine delivered through the probe. *p < .05, compared with KRB/nicotine 0.09 (n = 5-6 rats per treatment).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The present study shows that the hippocampal NE response to i.v. nicotine was inhibited by selectively administering -BTX or MLA into this region, whereas Mec and DHE were ineffective. These findings suggest that hippocampal NAChRs sensitive to -BTX are involved in NE release by systemic nicotine. Further evidence for the involvement of hippocampal NAChRs in the NE response to systemic nicotine was obtained in studies showing that desensitization of NAChRs in the HP reduced the NE response to i.v. nicotine.

Based on the in vivo diffusion of [³H]nicotine from the microdialysis probe, a dialysate concentration of 0.1 mM nicotine would be expected to yield a peak tissue concentration of 1.8 µM. This level is very close to the concentration predicted in brain parenchyma (approximately 2 µM) after 0.09 mg/kg nicotine i.v. (Hieda et al., 1998). It has been reported that EC₅₀ concentrations of nicotine within the nanomolar range are sufficient to desensitize NAChRs, especially those composed of 7 or 42 subunits (Hsu et al., 1996; Olale et al., 1997). Therefore, a dialysate concentration of 0.1 mM nicotine would be enough to desensitize a large fraction of the NAChRs in rat brain regions containing an abundance of these NAChRs (Clarke et al., 1985; Harfstrand et al., 1988; Wada et al., 1989). Microdialyzing 0.1 mM nicotine into the HP significantly reduced the NE response to i.v. nicotine, further demonstrating that hippocampal NAChRs activated by systemic nicotine modulate the NE secretory response. However, the desensitization of hippocampal NAChRs inhibited NE release by only 40%, which is somewhat less than the 47% reduction by -BTX. The limited efficacy of desensitization is more apparent when compared with the effect of -BTX in animals receiving a lower dose of nicotine (0.065 mg/kg); -BTX abolished the NE response. These observations suggest that nondesensitizing NAChRs also are involved in nicotine-induced NE release. These receptors may be nonhomonomeric 7-containing NAChRs that retain their sensitivity to -BTX and MLA.

-BTX is a specific antagonist of 7-containing NAChRs (Pugh et al., 1995; Colquhoun and Patrick, 1997), and MLA also is specific at relatively low concentrations (Ward et al., 1990; Alkondon et al., 1992). The blockade of hippocampal NE release by both -BTX and MLA suggests that 7-containing NAChRs within the HP are activated by nicotine when it is delivered systemically. Consistent with the findings of Mitchell (1993), Mec and DHE were ineffective in the present investigations. Other studies have shown that Mec has relatively low potency at 7 homonomers, with an IC₅₀ of 1.8 µM (Briggs and McKenna, 1996). However, data on the potency of Mec at nonhomomeric 7-containing NAChRs and on the interaction of DHE with 7-containing NAChRs are unavailable presently. Nonetheless, the lack of effectiveness of Mec and DHE further supports the involvement of 7-containing NAChRs in hippocampal NE secretion. Further evidence for the involvement of 7 NAChRs in the central secretion of NE comes from a recent study showing that NE was secreted in the frontoparietal cortex after the s.c. injection of 3-(2,4-dimethoxybenzylidene)anabaseine, a selective agonist at 7-containing NAChRs (de Fiebre et al., 1995; Summers et al., 1997).

Based on the relative concentrations attained in the HP after the microperfusion of [³H]nicotine in the present study, perfusion with MLA (1.4-5.6 mM in dialysate) was estimated to yield micromolar tissue concentrations (25-99 µM). In contrast, nano-molar MLA is sufficient to block the 7-mediated current in cultured fetal hippocampal neurons (Alkondon and Albuquerque, 1993; Gray et al., 1996). This difference in estimated parenchymal versus in vitro concentrations may reflect the specific subunit composition of the 7-containing NAChRs expressed by mature hippocampal neurons involved in the modulation of NE release by systemic nicotine. Such NAChRs may not be equivalent to the 7 homooligomers expressed by transfected oocytes, which are sensitive to nanomolar concentrations of MLA (Briggs and McKenna, 1996). Similarly, much higher concentrations of -BTX may be required by nonhomomeric a7-containing NAChRs. Indeed, a study of chick embryonic sympathetic neurons showed that 7 antisense oligomers, but not -BTX alone (500 nM), were able to diminish acetylcholine-evoked currents (Listerud et al., 1991). These authors hypothesized that 7-containing NAChRs may include other subunits that influence the -BTX sensitivity of the 7 subunit.

In vitro studies with hippocampal slices and synaptosomes have shown that [³H]NE release was mediated by -BTX-unresponsive NAChRs. Based on data from several laboratories, the EC₅₀ values of nicotine were 34.6 or 91.6 µM for hippocampal slices and 6.5 µM for the synaptosomes (Sacaan et al., 1995; Clarke et al., 1996; Sershen et al., 1997). In the present study, 44 µM intraparenchymal nicotine (estimated from 2.5 mM dialysate concentration) was the minimum concentration required for the direct effects of the drug on hippocampal NE release. In contrast, the concentration of nicotine achieved by i.v. nicotine (0.065-0.09 mg/kg) was estimated to be 1-2 µM in brain parenchyma. In another study, peak brain nicotine levels were 6.9 µM after an s.c. injection of 1.2 mg/kg nicotine twice a day and 1.2 µM after a constant infusion of nicotine at dose of 4.8 mg/kg/day for 10 days (Rowell and Li, 1997). Based on our protocols or those reported by Rowell and Li, systemically administered nicotine would not be expected to achieve the brain concentrations required to induce NE release in the studies with hippocampal preparations in vitro or when microdialyzed into the HP. Therefore, the potential role of the hippocampal -BTX-resistant NAChRs, reported by other laboratories (Sacaan et al., 1995; Sershen et al., 1997), in the hippocampal NE response to systemic nicotine requires further clarification.

Within the HP, 7-containing NAChRs receptors may be located on glutamatergic or other undefined axon terminals. Nicotine (0.5 µM) has been reported to stimulate glutamate release in cultured neonatal hippocampal neurons (Gray et al., 1996). -BTX (50 nM) or 5 nM MLA abolished the nicotine-induced increase in miniature excitatory postsynaptic currents (mEPSC) that were blocked by glutamate receptor antagonists. In a minority of these experiments, the authors noted that nicotine increased the frequency of mEPSCs, but -BTX (50 nM) failed to block. Other studies have reported that glutamate may release NE from hippocampal slices (Puttfarcken et al., 1993). In addition, Toth et al. (1992) observed that nicotine (delivered by microdialysis) induced striatal dopamine release, which was largely dependent on glutamate secretion. Thus, systemic nicotine may activate 7-containing NAChRs located on hippocampal glutamatergic terminals, enhancing the secretion of glutamate, which, in turn, may stimulate NE secretion. 7-Containing NAChRs that are relatively insensitive to -BTX (a subpopulation of the -BTX-responsive NAChRs) appear to be involved.

We have shown previously that NAChRs located near the LC, which gives rise to the noradrenergic cell bodies projecting to the HP, had a significant role in mediating the hippocampal NE response to i.v. nicotine (Fu et al., 1998a). In contrast to the present studies, nanomolar amounts of Mec (and MLA or DHE) blocked 87% of the response when these agents were microinjected into the cerebral aqueduct. However, -BTX was completely ineffective. Therefore, systemic nicotine affects hippocampal NE secretion by activating receptors that are 1) sensitive to MLA and -BTX in the HP and 2) unresponsive to -BTX in the brainstem (Fu et al., 1998a). The incomplete blockade of NE release by MLA delivered through the probe (Fig. 1B) or by the Mec microinjected into the cerebral aqueduct (Fu et al., 1998a) is consistent with the idea that i.v. nicotine stimulates NAChRs located at both LC and HP sites to release NE in the HP. Thus, hippocampal NE release appears to depend, to a high degree, but not completely, on concurrent activation of both sites by systemic nicotine.

A synergistic interaction may exist between these two neuroanatomically and pharmacologically distinct populations of NAChRs. This is supported by differences in the hippocampal nicotine concentrations that are required to induce NE secretion after the i.v. versus direct intrahippocampal administration of nicotine. An estimated tissue concentration of 17.6 µM nicotine, resulting from the intrahippocampal dialysis of 1 mM nicotine, failed to stimulate NE release. In contrast, experiments based on the local desensitization of hippocampal NAChRs or the use of NAChR antagonists both indicated that tissue concentrations of 2 µM nicotine, achieved after i.v. nicotine (0.09 mg/kg for 60 s), were effective. It is likely that the 2 µM concentration was effective because the concurrent delivery of nicotine (i.v.) to the HP and LC allows a synergistic interaction whereby the activation of hippocampal NAChRs potentiates the action of nicotine in the LC.

In summary, these investigations show that i.v. nicotine stimulates NE release in the HP by acting, in part, through hippocampal NAChRs. The hippocampal receptors involved may be nonhomonomeric 7-containing NAChRs, although models that selectively interfere with 7 subunit expression will be required for further validation. These -BTX-sensitive NAChRs modulate hippocampal NE release by systemic nicotine, probably because of a synergistic interaction between pharmacologically distinct populations of NAChRs in the HP and LC.