Introduction

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The binding of nicotine to neuronal nAChRs in the nervous system produces numerous effects (Stolerman et al., 1995). These effects depend on the species studied and the dose administered. In humans, at doses obtained from cigarette smoking, nicotine can improve cognitive function, increase attention, and reduce fatigue and anxiety (Kassel and Shiffman, 1997; Newhouse et al., 1997; Levin and Rezvani, 2000). In laboratory animals, low doses of nicotine affect nociception (Jurna et al., 1993; Damaj et al., 1998; Marubio et al., 1999), cardiovascular responses (Neff et al., 1998; Marano et al., 1999), locomotor activity (Whiteaker et al., 1995; le Novere et al., 1999), thermoregulation (Lupien and Bray, 1988), and learning, memory, and attention (Levin et al., 1994; Stolerman et al., 2000). High doses of nicotine induce clonic-tonic seizures (Miner et al., 1984; Damaj et al., 1999). Understanding how stimulation of certain nicotinic cholinergic brain circuits leads to seizure becomes particularly significant in light of recent studies linking nAChR subunit genes to several forms of idiopathic epilepsies (Bertrand et al., 1998; Neubauer et al., 1998; Steinlein, 2000; Phillips et al., 2001).

Experiments conducted on several inbred mouse strains have indicated a positive correlation between sensitivity to nicotine-induced seizures and the number of -bungarotoxin (-BTX) binding sites in the hippocampus (Miner et al., 1984, 1985; Miner and Collins, 1989), the brain area in which nicotine-induced seizure activity may originate (Stumpf and Gogolak, 1967; Freund and Wehner, 1987). Because 7-containing nicotinic acetylcholine receptors (7* nAChRs) represent the major binding site for -BTX in the mammalian brain (Chen and Patrick, 1997; Orr-Urtreger et al., 1997), it was suggested that 7* nAChRs might contribute to the convulsant effects of nicotine. A recent pharmacological investigation into the mechanisms involved in nicotine-induced seizures has helped support this hypothesis (Damaj et al., 1999).

7-mediated nAChR currents activate very rapidly and show fast desensitization in the presence of high concentrations of nicotine (Radcliffe and Dani, 1998; Alkondon et al., 2000; Ji et al., 2001). Studies of 7 nAChRs expressed in oocytes have shown that a Leu250-to-Thr substitution (L250T) in the channel domain increases agonist affinity and decreases the rate of desensitization, creating a gain-of-function model for this receptor (Revah et al., 1991; Bertrand et al., 1992; Orr-Urtreger et al., 2000). We have demonstrated that neonatal mice homozygous (T/T) for the 7 L250T nAChR "knock-in" mutation have a lethal phenotype (Orr-Urtreger et al., 2000). Furthermore, hippocampal neurons from T/T mice exhibit 7-type evoked currents that have properties consistent with those observed for 7 L250T nAChRs in oocytes. In contrast, heterozygous (+/T) L250T mice survive to adulthood. These mice express a partial gain of function for the 7 nAChR but are not noticeably affected by this alteration. Because of the partial gain of function imparted by the +/T L250T mutation, it was predicted that +/T mice would exhibit a higher sensitivity to nicotine-induced seizures.

To test this hypothesis, we analyzed the behavioral effects of nicotine on +/T L250T adult mice. We found that despite a 60% decline in 7 nAChR protein levels, +/T mice were more sensitive to nicotine at all doses tested than their wild-type littermates. We have recently demonstrated that mice lacking 7 nAChRs display normal sensitivity to the convulsant effects of nicotine (Franceschini et al., 2001). Taken together, these studies indicate that nicotine-induced seizures involve a complex mechanism of action. Whereas 7 nAChRs may not be required, their enhanced functional activity increases the sensitivity to nicotine-induced seizures.

	Materials and Methods

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Animals. All mice used in this study were back-crossed onto a C57BL/6 background for six generations. For seizure studies, male and female mice (2-4 months old; n = 177 for nicotine; n = 20 for dihydro--erythroidine) were housed separately in groups of two to five. One day before seizure induction, mice were weighed, marked, and transferred to the experimentation room for acclimation. Adult male mice aged 2 to 4 months were used for all other analyses. All animals had free access to food and water and were maintained on a 12-h light/dark cycle. Animals were genotyped by standard polymerase chain reaction techniques (Orr-Urtreger et al., 2000). The Institutional Animal Care and Use Committee in accordance with federal guidelines approved all procedures.

Behavioral Characterization of 7 +/T Mice in Basal Conditions. 7 +/T and wild-type littermates were evaluated on a battery of behavioral tests to assess several domains of CNS function as described previously (Paylor et al., 1998). Seventeen 7 +/T and 24 7 +/+ mice were examined on a behavioral battery that included the following tests/assays: 1) neurological screen for simple sensory and motor function; 2) open-field test for exploratory activity and anxiety-related responses; 3) light-dark exploration box for anxiety-related responses; 4) rotarod test for motor coordination and skill learning; 5) acoustic startle response and prepulse inhibition of the startle response; 6) startle habituation; 7) Pavlovian learning using the conditioned fear test; 8) the Morris water task for spatial learning; and 9) hotplate test for analgesia-related responses.

Seizure Testing. Nicotine tartrate (Sigma, St. Louis, MO) was dissolved in physiological saline (0.9% sodium chloride) and administered in a total volume of 10 µl/g of body weight by i.p. injections. Dose groups (0.5-8 mg/kg) were designed so that on any given experimentation day, both a low and a high dose of nicotine were administered to each genotype. After injection, each mouse was placed in a 30 × 18 cm Plexiglas cage with bedding, and observed by two investigators for 5 min. During this period, animals were continuously evaluated for their response to the nicotine challenge. The experimenters were blind to the genotype of the animals tested. The effects of nicotine were dose-dependent and included sedation, Straub tail, tremors, tachypnea, back arching, rapid movement of the limbs, wild running, loss of righting response, and clonic and tonic seizures. The mice were scored independently by two experimenters on a scale from 0 to 5 (Franceschini et al., 2001). Depending on the symptoms presented the animals were scored as follows: 0 = no visible effect; 1 = sedation; 2 = tachypnea, tremors, or back arching; 3 = rapid movement of the limbs or wild run; 4 = loss of equilibrium with tonic-clonic seizure; and 5 = death. The percentage of mice exhibiting a score of 4 or 5 at each dose was calculated, and dose response curves were constructed. The data were analyzed with the Litchfield-Wilcoxon procedure to determine the confidence limits for the ED₅₀. We also examined the latency to seizure and gender susceptibility. Dihydro--erythroidine (DHE; Sigma, St. Louis, MO) was dissolved in 0.9% saline (5 mg/kg) and administered i.p. Mice were observed as described above by two investigators for 10 min.

Slice Preparation and Electrophysiology. Mice (13-20 days old; n = 52) were anesthetized, decapitated, and their brains were immediately removed. Horizontal slices (300 µm thick) of brain were cut in ice-cold cutting solution (220 mM sucrose, 2.5 mM KCl, 30 mM NaHCO₃, 1.25 mM NaH₂PO₄, 10 mM dextrose, 7 mM MgCl₂, and 1 mM CaCl₂, oxygenated with 95% O₂ and 5% CO₂), and then transferred into a holding chamber containing the external solution (125 mM NaCl, 2.5 mM KCl, 25 mM NaHCO₃, 1.25 mM NaH₂PO₄, 25 mM dextrose, 1 mM MgCl₂, and 2 mM CaCl₂, oxygenated with 95% O₂ and 5% CO₂). After a 30-min recovery at 35°C, slices were maintained at room temperature and were used for recordings within the subsequent 5 h. Neurons were visualized by differential interference contrast microscopy and recorded with standard patch-clamp technique in whole-cell configuration at 32 to 34°C. Atropine (1 µM) was always added to the external solution to block muscarinic receptors during the recordings. The internal solution in the recording pipettes contained the following: 115 mM K-gluconate, 20 mM KCl, 10 mM HEPES, 10 mM EGTA, 4 mM ATP (magnesium salt), 0.3 mM GTP (sodium salt), and 7 mM phosphocreatine, adjusted to pH 7.3 to 7.4 with KOH. Interneurons and pyramidal cells were voltage-clamped at 60 mV and granule cells at 80 mV. Acetylcholine (1 mM), dissolved in external solution, was delivered to the soma of interneurons and granule cells and the dendrites of pyramidal cells by a pressure injection pipette controlled by a Picospritzer (Parker Instrumentation, Chicago, IL). The Picospritzer was used to control both the pressure and the duration of the puff. The puffer pipettes had resistances of about 4 M, and a 30-ms to 1-s puff with 5 to 10 psi was most commonly used. The puffer pipettes were mounted on a motorized manipulator controlled by computer. If leaks of ACh from the pipettes caused desensitization, the pipettes were withdrawn by 100 to 200 µm in between pressure applications. Moments before the application, the motorized drive moved the pipette to its final position (about 50-100 µm from the target area), ACh was puffed, and the motorized manipulator immediately withdrew the puffer pipette. This process was all done automatically by computer while visually observed on a video monitor. All genotypes were determined after collection and interpretation of the electrophysiological results.

Western Blot. Tissue for immunoblotting was processed as described previously (Orr-Urtreger et al., 2000). Briefly, 7 nAChR subunit protein was first concentrated from whole-brain homogenates by incubation with an agarose matrix coupled to -cobratoxin (-Cbt). After several washes, bound 7 nAChRs were eluted off the bead matrix with 6× SDS/sample buffer and the entire sample was loaded onto a 9% SDS-polyacrylamide gel. Because of the small volume of eluted 7 receptors obtained from each brain, protein analyses could not be performed. Instead, each gel was loaded with a known amount ( 12 ng) of 7 recombinant protein, which was determined to approximate the amount of the wild-type 7 nAChR sample loaded. Gels were transferred to nitrocellulose membranes, which were then processed for immunoblotting using an affinity-purified 7 antibody (Chen and Patrick, 1997). Proteins recognized by the antibody were visualized with the use of enhanced chemiluminescence and exposure to BioMax film (Kodak, Rochester, NY). Band intensity was quantified from film exposures in the linear range, as determined from the 7 recombinant protein band. Films showing a large discrepancy in intensity between this band and the wild-type band were not included in the final analysis. All data were examined by one-way analysis of variance, followed by Newman-Keuls post hoc comparisons.

Tissue Preparation and Histology. Mice (n = 15) were decapitated and their brains quickly removed and frozen in isopentane (30°C, 20 s). Fresh-frozen brains were cryostat cut (20 µm) and mounted onto either gelatin-coated slides (for receptor binding, histochemical, and histological staining) or slides with an additional coating of poly-L-lysine (for in situ hybridization) kept at 20°C. Slide-mounted sections for receptor binding were stored desiccated at 20°C until use. Sections for in situ hybridization, histochemical, and histological staining were postfixed with 4% paraformaldehyde in 0.1 M phosphate-buffered saline, pH 7.4, for 1 h at 22°C. Sections were then washed in phosphate-buffered saline, air dried, and stored desiccated at 20°C until use. Slide-mounted tissue sections for histological staining were stained with cresyl-violet. Acetylcholinesterase and cytochrome oxidase histochemistry was performed as described previously (Orr-Urtreger et al., 2000).

In Situ Hybridization. Mouse DNA templates encoding the third intracellular loop of each nAChR subunit analyzed were prepared by reverse transcription-polymerase chain reaction amplification of mRNA from a mouse septal neuroblastoma cell line (SN56). Primers were chosen based on published cDNA sequences of the corresponding rat nAChR subunits. The mouse DNA templates were subcloned into pBluescript SK(), sequenced and compared with the rat nAChR sequences for verification. The DNA region and size of each template were as reported in (Franceschini et al., 2001). cRNA riboprobes labeled with [³⁵S]UTP (PerkinElmer Life Sciences, Boston, MA) were synthesized from the mouse DNA templates, and postfixed brain sections for in situ hybridization were processed as described elsewhere (Broide et al., 1996). Briefly, slide-mounted sections were first preincubated with 1 µg/ml proteinase K for 10 min at 22°C, and then incubated for 18 h at 60°C with a hybridization solution containing ³⁵S-UTP-labeled cRNA riboprobes (1 × 10⁷ cpm/ml) in the antisense orientation. Adjacent sections were incubated with riboprobes in the sense orientation to define nonspecific hybridization. Brain sections were then incubated with RNase A (20 µg/ml) for 30 min at 37°C, followed by high-stringency washes of decreasing salinity and a 30-min wash in 0.1× sodium chloride/sodium citrate at 60°C. Sections were dehydrated, dried in a stream of cool air, and apposed to -max film (Amersham Biosciences, Piscataway, NJ) for 1 to 7 days at 4°C.

Receptor Autoradiography. Slide-mounted brain sections for [¹²⁵I]-BTX autoradiography were processed as described previously (Broide et al., 1996). Briefly, sections were incubated at 22°C for 2 h in buffer (50 mM Tris-base, pH 7.4, 120 mM NaCl, and 0.1% bovine serum albumin) containing 5 nM [¹²⁵I]-BTX (specific activity, 10 - 20 µCi/µg; PerkinElmer). Nonspecific binding was defined on adjacent sections in the presence of 10 µM -Cbt). Sections were then washed twice for 10 min each in ice-cold buffer, dipped once in water, air dried, and exposed to -max film (Amersham) for 3 to 7 days.

Data Analysis and Statistics. X-ray films were analyzed, and quantitation was performed using computer-assisted densitometry with NIH Image software (Bethesda, MD). Relative optical densities from protein bands and discrete brain regions were measured and presented as a percentage of readings from wild-type mice. All autoradiographic data were examined by one-way analysis of variance, followed by Newman-Keuls post hoc comparisons.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Heterozygous L250T Mice Are More Sensitive to Nicotine-Induced Seizures. Intraperitoneal injection of nicotine induced seizures in a dose-dependent fashion in both +/T and +/+ mice (Fig. 1), but the potency of nicotine was significantly higher in the +/T mice (ED₅₀ = 2.5 mg/kg, 95% confidence limits: 2.3-2.7 mg/kg) than in their +/+ littermates (ED₅₀ = 5.3 mg/kg, 95% confidence limits: 4.4-6.6 mg/kg). The latency to seizure was significantly shorter (p < 0.01) in the +/T mice (69.3 ± 7.6 s) than in their +/+ littermates (118.6 ± 15.6 s). In addition, the behavioral scores were higher for the +/T mice at each dose of nicotine tested (Fig. 2). No significant difference in seizure sensitivity was observed between male and female mice (data not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Dose response curves for the convulsant effects of nicotine. Intraperitoneal injection of nicotine induced seizure activity in wild-type (+/+) and heterozygous (+/T) 7 L250T mice in a dose-dependent fashion. Data represent the percentage of mice tested that underwent seizure and either survived (score 4), or died (score 5). Twelve to 32 mice were tested for each dose.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Increased sensitivity to nicotine in +/T mice. Heterozygous +/T mice showed increased sensitivity to the behavioral effects of nicotine over the whole range of doses tested. Histograms represent the percentage of animals that was assigned a score from 0 to 5 after intraperitoneal injection of nicotine at 1 mg/kg (A), 2 mg/kg (B), and 3 mg/kg (C), respectively.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Behavioral characterization of 7+/T () and 7+/+ () mutant mice. A, the total distance (in centimeters) traveled and the center distance to total distance ratio in the open-field arena during the 30-min session was similar for 7+/T and 7+/+ mice. B, the total time (in seconds) mice were in the dark side of the light-dark box and the total number of light-dark transitions were similar for 7+/T and 7+/+ mice during the 10-min light-dark test. C, the maximum startle response (peak amplitude) to a 120-dB startle stimulus and the percentage inhibition of the startle response by different prepulse stimuli (74-90 dB) were similar for 7+/T and 7+/+ mice. D, acoustic startle habituation to repeated presentation (100 total presentation) of the 120 dB stimulus was similar 7+/T and 7+/+ mice. Data presented are the average startle response during the first block of 10 stimuli and the last 10 stimuli. D, 7+/T and 7+/+ mice spent similar amounts of time (in seconds) walking on top of a rotating rod during the four trials of the rotarod test. E, levels of fear as assessed by percentage bouts of freezing during the context test and the auditory CS test. 7+/T and 7+/+ mice displayed similar levels of freezing during both tests. F, time to locate the hidden platform during the hidden platform version of the Morris water test. Data are the escape latency (in seconds) for 7+/T and 7+/+ mice during the last block of four trials. The number of platform crossings in the training quadrant during the 60-sec probe trial given after the last training trial. Data indicate that 7+/T and 7+/+ mice used similar spatially-biased search strategies to locate the hidden platform.

Heterozygous L250T Mice Display Normal Sensitivity to Dihydro--erythroidine. The nicotinic antagonist DHE has been shown to act as an agonist and evoke ionic currents from 7 L250T homopentameric nAChRs (Bertrand et al., 1992; Orr-Urtreger et al., 2000). However, whether the compound also acts as an agonist at nAChRs containing both wild-type and 7 L250T subunits is unknown. To determine whether DHE exhibits agonist-like properties at 7 L250T nAChRs in +/T mice by inducing seizures, we injected +/+ (n = 10) and +/T (n = 10) mice i.p. with a single dose of DHE (5 mg/kg). DHE did not induce tonic or clonic seizures in the +/T mice, and both groups of mice displayed the same biphasic behavioral pattern. At first, the mice manifested increased locomotor activity, including restless digging and sniffing, along with an increase in whole body tremors. This was usually followed by a second phase, consisting of partial to full sedation, along with occasional dyspnea. Death occurred in 3 of 10 +/T and 5 of 10 +/+ mice. Because the effects of DHE on +/+ and +/T mice were similar, we did not pursue these experiments further.

Heterozygous L250T Mice Have Altered 7-Type Currents. Hippocampal circuits are thought to participate in the convulsant effects of nicotine (Stumpf and Gogolak, 1967; Freund and Wehner, 1987), and 7* nAChRs are highly expressed throughout the hippocampus (Albuquerque et al., 1997; Radcliffe and Dani, 1998; Ji and Dani, 2000). To examine the potential cellular mechanisms underlying the increased sensitivity of +/T mice to nicotine, we characterized and compared 7-type currents in hippocampal slices from +/T and +/+ mice. Currents were recorded from three different cell types in the hippocampus: CA1 interneurons, pyramidal cells, and granule cells from the dentate gyrus. The cell types were identified by their location and by their membrane properties, which are summarized in Fig. 4A and Table 1. Electrophysiological characteristics of GABA interneurons were as follows (Schwartzkroin and Mathers, 1978; Lacaille et al., 1987; Ji and Dani, 2000): holding potentials more positive than 65 mV, relatively high firing frequency, high input resistance, and no sag in the response to a hyperpolarizing current step. Pyramidal neurons had holding potentials more negative than 65, slower firing rates, lower input resistance, and a sag in the voltage response to an hyperpolarizing current step (Fig. 4A, arrow). Dentate granule neurons had more negative holding potentials, relatively slow firing rates, and showed no sag after a hyperpolarizing current step.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Nicotinic currents in hippocampal neurons from wild-type (+/+) and heterozygous (+/T) L250T mice. A, electrophysiological characteristics were used to identify the three different cell types. The arrow indicates the voltage "sag" seen with pyramidal neurons in response to injection of hyperpolarizing current. The currents that were injected to elicit the responses were as follows (pA): CA1 interneuron, 50, 10, 20 (left), and 180 (right); CA1 pyramidal cell, 200, 50, 80 (left), 300 (right); granule cell, 150, 20, 50 (left), and 150 (right). Membrane properties were the same for both +/+ and +/T mice. B, ACh-induced currents are shown to be sensitive to the 7-specific inhibitor, MLA (20 nM). The ACh-induced currents were recorded from CA1 interneurons, CA1 pyramidal cells, and granule cells from +/+ and +/T mice as indicated. ACh (1 mM) was delivered to the soma of interneurons for 10 ms (arrows above the traces) and to the dendrites of pyramidal neurons and granule cells for 1 s (black bars above the traces). C, the average current amplitudes from +/+ and +/T mice are compared. The number of neurons that expressed MLA-sensitive nAChR currents is indicated above the data bars.

Heterozygous L250T Mice Show Apparently Normal Neuroanatomical Structure. Brains from +/T mice exhibited normal anatomical structure compared with their +/+ littermates. In the hippocampus, histological analysis using Nissl and acetylcholinesterase-staining revealed normal layering within all subregions (Fig. 5, A-D). We were also interested in determining whether adult +/T mice showed defined barrel structures in their somatosensory cortex because T/T L250T neonatal mice exhibit abnormal development in this region of the brain (Orr-Urtreger et al., 2000). Thus, +/T mice were analyzed for the presence of barrels using cytochrome oxidase histochemistry. Sections through the somatosensory cortex of +/T mice showed characteristic barrel-like structures that were indistinguishable from those in +/+ littermates (Fig. 5E, F).

View larger version (131K): [in this window] [in a new window]   Fig. 5.   Heterozygous L250T mice exhibit normal neuroanatomy. A and B, photomicrographs of transverse brain sections magnified to show the hippocampus of wild-type (+/+) and heterozygous (+/T) L250T mice. Sections were histologically stained with cresyl-violet. C and D, adjacent sections histochemically stained for acetylcholinesterase. E and F, magnified brain sections depicting the primary somatosensory cortex were histochemically stained for cytochrome oxidase. Arrowheads point to whisker barrels in layer IV of the cortex. Scale bars, 100 µm.

Heterozygous L250T Mice Have Reduced Levels of 7 nAChRs. Previous results have shown a decrease in [¹²⁵I]-BTX binding and 7 nAChR protein levels in brains of neonatal +/T and T/T mice (Orr-Urtreger et al., 2000). To determine whether a similar reduction occurred in adult +/T mice, we examined and compared the distributions of 7 nAChR mRNA and [¹²⁵I]-BTX binding sites in the brains of +/+ and +/T mice, using in situ hybridization and receptor autoradiography. Strong labeling for 7 mRNA and [¹²⁵I]-BTX binding was observed in such regions as the hippocampus (Hi), hypothalamus (Hy), amygdala (Fig. 6), and the superior (SC) and inferior colliculus (IC) (data not shown). More moderate levels were detected in the cortex (Ctx) and caudate putamen (CPu). Low levels of mRNA and binding were found throughout most of the thalamus (Th). We observed a similar pattern of 7 mRNA and [¹²⁵I]-BTX binding distribution in the brains of +/+ and +/T mice (Fig. 6A, B).

View larger version (36K): [in this window] [in a new window]   Fig. 6.   Heterozygous L250T mouse brains show reduced levels of 7 nAChRs. A and B, autoradiographic images of adjacent transverse brain sections at the level of the hippocampus from wild-type (+/+) and heterozygous (+/T) L250T mice. Sections show the distributions of 7 nAChR mRNA (7 mRNA) and [125I]-BTX binding sites (-BTX). Ctx, cortex; Hi, hippocampus; Hy, hypothalamus; Th, thalamus. Scale bar, 1 mm. C, Western blot analysis of 7 nAChR proteins purified from brains of +/+ and +/T mice. As a control, the binding of proteins to the affinity column was blocked by preincubation with -cobratoxin (-Cbt). Protein size markers are indicated. D, densitometric analysis of Western blots showing levels of 7-subunit protein expression from +/T animals as a percentage of the +/+ controls. Data represent the mean ± S.E.M. for four animals each.

View this table: [in this window] [in a new window]   TABLE 2 Density of [125I]-bungarotoxin and [125I]epibatidine binding, and 4, 5, 6, 7, 2, and 4 mRNA expression in various regions of wild-type (+/+), and heterozygous (+/T) mouse brains Data represent the mean ± S.E.M. for three to five animals and are presented as a percentage of wild-type controls.

Heterozygous L250T Mice Have Normal Levels of Non-7 nAChR Subtypes. Recent studies have suggested that in addition to 7, the 4, 5, and 6 nAChR subunits may play a role in nicotine-induced seizures (Stitzel et al., 1998, 2000). Hence, the increased sensitivity to nicotine in the +/T mice might depend not only on the 7 L250T mutation, but also on changes in expression levels of other nAChR subunits. To test this hypothesis, we performed in situ hybridization experiments to examine the mRNA distribution patterns and levels for the 4, 5, 6, 2, and 4 nAChR subunits (Fig. 7). Levels of 4 mRNA expression were highest throughout the Th, medial habenula (MHb), SN, and ventral tegmental area (VTA), with low to moderate mRNA levels observed in the Ctx, Hi, and Hy. A strong hybridization signal for 5 mRNA was detected in the interpenduncular nucleus (IPn), with more moderate mRNA levels observed in the Ctx, Hi, SN, and VTA. High levels of 6 mRNA expression were seen in the SN and VTA, with moderate mRNA levels in the SC. The strongest signal for 2 mRNA was found throughout the Th, Hi, and MHb, with moderate mRNA levels observed in the Ctx, CPu, SN, and olfactory bulb. Expression of 4 mRNA was restricted to the MHb, IPn, and olfactory bulb. There were no statistically significant differences between +/T and +/+ mice in the relative abundance of 4, 5, 6, 2, and 4 subunit transcripts (Table 2).

View larger version (45K): [in this window] [in a new window]   Fig. 7.   Heterozygous L250T mouse brains show normal mRNA levels of non7 nAChR subunits. Autoradiographic images of adjacent transverse brain sections at the level of the hippocampus and substantia nigra from wild-type (+/+) and heterozygous (+/T) L250T mice. Sections show the distributions of mRNA transcripts for the 4 (A, B), 5 (C, D), 6 (E, F), 2 (G, H), and 4 (I, J) nAChR subunits. Ctx, cortex; Hi, hippocampus; Hy, hypothalamus; MHb, medial habenula; SC, superior colliculus; SNc, substantia nigra compacta; Th, thalamus; VTA, ventral tegmental area. Scale bar, 1 mm.

View larger version (76K): [in this window] [in a new window]   Fig. 8.   Heterozygous L250T mouse brains show normal levels of [125I]epibatidine and [3H]nicotine binding. Autoradiographic images of adjacent transverse brain sections at the level of the hippocampus (A, B, E, F) and substantia nigra (C, D, G, H) from wild-type (+/+) and heterozygous (+/T) L250T mice. Sections show the distributions of [125I]epibatidine (A-D) and [3H]nicotine (E-H) binding sites. Ctx, cortex; Hi, hippocampus; Hy, hypothalamus; IPn, interpenduncular nucleus; MG, medial geniculate; MHb, medial habenula; SC, superior colliculus; SNc, substantia nigra compacta; Th, thalamus. Scale bar, 1 mm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The present study demonstrates that mice heterozygous for the 7 L250T nAChR mutation have a 2-fold higher sensitivity to nicotine-induced seizures than their +/+ littermates. Such increased sensitivity was evident for all behavioral effects of nicotine; +/T mice received higher scores at all nicotine doses tested. In both oocytes and hippocampal cultures, L250T receptors have been shown to have higher affinity for agonists and reduced rates of desensitization (Revah et al., 1991; Orr-Urtreger et al., 2000). We now show that neurons in hippocampal slices from +/T mice express 7-type currents with qualitative properties similar to those found in tissue culture. Because +/T 7-type currents display an intermediate profile between +/+ and T/T currents, it seems that +/T mice maintain a partial gain of function for the 7 nAChR compared with T/T mice (Orr-Urtreger et al., 2000). This hypothesis is supported by the increased sensitivity of +/T mice to nicotine compared with that of their +/+ littermates.

The gain of function in +/T mice is also suggested by the reduction in 7 nAChR expression that was observed in comparison to their +/+ littermates. A negative feedback regulatory response may compensate for the increase in 7 nAChR activity within L250T mutant mice by altering protein synthesis. However, this reduction in 7 expression is only partial (50 to 65% reduction) compared with the reduction (80 to 90% reduction) in T/T mice (Orr-Urtreger et al., 2000) or /T L250T mice (Broide et al., 2001). This result is most likely to arise because of the presence of wild-type subunits in +/T mice that are lacking in T/T and /T mice. Both the T/T and the /T phenotypes are lethal, whereas the presence of a wild-type allele in the +/T mice seems to provide a protective mechanism for these animals (Broide et al., 2001). The expression of wild-type subunits in +/T L250T receptors also confers an intermediate pharmacological profile compared with T/T receptors. In fact, we could not elicit seizures after injections of DHE, an agonist at 7 L250T homopentameric nAChRs. Our in vivo results thus confirm previous in vitro experiments in which DHE evoked currents from T/T hippocampal cultures but not from +/T cultures (Orr-Urtreger et al., 2000).

Role of 7 nAChRs in Nicotine-Induced Seizures. The results of this study show that 7 nAChRs are capable of influencing nicotine-induced seizures. We have previously demonstrated, however, that mice lacking 7 nAChRs show normal sensitivity to nicotine (Franceschini et al., 2001). One possible way to interpret these findings is to consider the desensitization properties of 7 nAChRs. At lower nicotine concentrations, like those found in smokers, the majority of 7 nAChRs are most probably not desensitized. This has recently been demonstrated for 7 nAChRs expressed in both Xenopus laevis oocytes and on rat hippocampal neurons (Alkondon et al., 2000; Fenster et al., 1997). At the high nicotine concentrations required to elicit seizures, 7 nAChRs may exist in a desensitized state (Briggs and McKenna, 1998; Alkondon et al., 2000). Hence, wild-type 7 nAChRs would most probably be inactive during nicotine-induced seizures (Franceschini et al., 2001). Conversely, 7 nAChRs in the +/T mice might still be active in the presence of high doses of nicotine because of the slower desensitization kinetics. Activation of 7 L250T nAChRs might therefore amplify or modify the cellular mechanisms served by 7 nAChRs. This would most probably alter the balance between inhibitory and excitatory circuits in the hippocampus (Ji and Dani, 2000), resulting in a net excitation and increased sensitivity to nicotine-induced seizures.

7 nAChRs in the Hippocampus. Numerous electrophysiological studies have demonstrated the functional expression of 7* nAChRs in the hippocampus. These receptors have been found on presynaptic terminals of pyramidal neurons (Gray et al., 1996) and postsynaptic regions of interneurons (Alkondon et al., 1997; Ji and Dani, 2000), where they regulate neurotransmitter release. Recently, 7 nAChR-mediated currents have been detected on other hippocampal cell types, where they can alter circuit excitability and influence synaptic plasticity (Ji and Dani, 2001). In the present study, we were able to detect 7-type nAChR currents from CA1 interneurons and pyramidal cells as well as dentate gyrus granule cells of +/T hippocampal slices. Furthermore, these currents were much larger in amplitude, with slower desensitization kinetics than the corresponding +/+ 7 currents. These data help to confirm the expression of 7 nAChRs on different cell types in the hippocampus, where they probably modulate both excitatory and inhibitory mechanisms.

Heterozygous L250T Mice Display a Unique Nicotine-Induced Phenotype. Mutant +/T mice were examined in a battery of behavioral tests aimed at assessing several CNS functions. The behavior of +/+ and +/T mice was indistinguishable in basal conditions, but in the presence of nicotine the +/T mice displayed a unique nicotine-induced phenotype. This phenotype was particularly evident at lower doses of nicotine and consisted of head-bobbing and paw-tapping movements. The stereotypic movements sometimes lasted for long periods of time, even after the animal was returned to its home cage. This finding may suggest a role for 7 nAChRs in locomotor activity. Increasing evidence indicates that nicotine stimulates locomotor activity in animals by acting on nAChRs found along the mesostriatal dopaminergic pathway (Whiteaker et al., 1995; Louis and Clarke, 1998; Wonnacott et al., 2000). Although studies have demonstrated the involvement of 6 nAChR subunits in nicotine-elicited locomotion (le Novere et al., 1999), the role of 7 nAChRs seems to be negligible (Grottick et al., 2000; Kempsill and Pratt, 2000). Thus, activation of 7 L250T nAChRs in +/T mouse brains may trigger unique mechanisms that help augment the role of these receptors in the mesostriatal pathway. Identification of these mechanisms may lead to a better understanding of the normal physiological function of 7 nAChRs.