Novel seizure phenotype and sleep disruptions in knock-in mice with hypersensitive alpha 4* nicotinic receptors - PubMed (original) (raw)

Comparative Study

. 2005 Dec 7;25(49):11396-411.

doi: 10.1523/JNEUROSCI.3597-05.2005.

Bruce N Cohen, Raad Nashmi, Paul Whiteaker, Daniel A Wagenaar, Nivalda Rodrigues-Pinguet, Purnima Deshpande, Sheri McKinney, Steven Kwoh, Jose Munoz, Cesar Labarca, Allan C Collins, Michael J Marks, Henry A Lester

Affiliations

Comparative Study

Novel seizure phenotype and sleep disruptions in knock-in mice with hypersensitive alpha 4* nicotinic receptors

Carlos Fonck et al. J Neurosci. 2005.

Abstract

A leucine to alanine substitution (L9'A) was introduced in the M2 region of the mouse alpha4 neuronal nicotinic acetylcholine receptor (nAChR) subunit. Expressed in Xenopus oocytes, alpha4(L9'A)beta2 nAChRs were > or =30-fold more sensitive than wild type (WT) to both ACh and nicotine. We generated knock-in mice with the L9'A mutation and studied their cellular responses, seizure phenotype, and sleep-wake cycle. Seizure studies on alpha4-mutated animals are relevant to epilepsy research because all known mutations linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) occur in the M2 region of alpha4or beta2 subunits. Thalamic cultures and synaptosomes from L9'A mice were hypersensitive to nicotine-induced ion flux. L9'A mice were approximately 15-fold more sensitive to seizures elicited by nicotine injection than their WT littermates. Seizures in L9'A mice differed qualitatively from those in WT: L9'A seizures started earlier, were prevented by nicotine pretreatment, lacked EEG spike-wave discharges, and consisted of fast repetitive movements. Nicotine-induced seizures in L9'A mice were partial, whereas WT seizures were generalized. When L9'A homozygous mice received a 10 mg/kg nicotine injection, there was temporal and phenomenological separation of mutant and WT-like seizures: an initial seizure approximately 20 s after injection was clonic and showed no EEG changes. A second seizure began 3-4 min after injection, was tonic-clonic, and had EEG spike-wave activity. No spontaneous seizures were detected in L9'A mice during chronic video/EEG recordings, but their sleep-wake cycle was altered. Our findings show that hypersensitive alpha4* nicotinic receptors in mice mediate changes in the sleep-wake cycle and nicotine-induced seizures resembling ADNFLE.

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Figures

Figure 1.

Figure 1.

Nicotine- and ACh-induced responses of mouse WT α4β2 and α4(L9′A)β2 receptors expressed in Xenopus oocytes. The L9′A mutation increases nicotine and ACh sensitivity of mouse α4β2 receptors. A, Nicotine-induced voltage-clamp currents recorded from oocytes expressing mouse WT or α4(L9′A)β2 receptors. B, WT and L9′A nicotine concentration-response relationships, normalized to the maximal response of each oocyte. Lines are fits to the Hill equation. WT and L9′A nicotine EC50 values are 7 ± 1 and 0.23 ± 0.03 μ

m

, respectively. Hill coefficients are 1.3 ± 0.1 and 1.1 ± 0.1, respectively. Error bars are ± SEM; n = 8-9. C, ACh-induced voltage-clamp currents recorded from oocytes expressing mouse WT or α4(L9′A)β2 receptors. D, ACh concentration-response relationships and fits to the Hill equation. WT, L9′A, and L9′S ACh EC50 values are 48 ± 8, 0.5 ± 0.06, and 0.02 ± 0.001 μ

m

, respectively. Hill coefficients are 0.8 ± 0.1, 1.2 ± 0.1, and 1.7 ± 0.2, respectively. Error bars are ± SEM; n = 5.

Figure 2.

Figure 2.

Desensitization of mouse WT α4β2 and α4(L9′A)β2 receptors expressed in Xenopus oocytes. A, WT and L9′A responses to 200 n

m

ACh pulses. Each oocyte was exposed to 13 ACh pulses, 2.5 s in duration at intervals of 120 s. Immediately after the third ACh pulse, nicotine was bath applied for 10 min (5 pulses). B, Time course of ACh responses at various nicotine concentrations. Data exemplify responses from three or four oocytes tested at each nicotine concentration. L9′A values were corrected to account for changes in the size of the responses over time in the absence of nicotine (see Materials and Methods). No correction was necessary for WT. C, Concentration dependence of desensitization for WT and L9′A. Regression lines are fits to a sigmoidal inhibitory function. IC50 values are 22 ± 5 and 8.2 ± 0.5 n

m

for WT and L9′A, respectively. Hill coefficients are 0.45 ± 0.06 and 1.13 ± 0.06 for WT and L9′A, respectively. D, Concentration dependence of desensitization and activation (see Fig. 1 B) normalized to maximal response of each oocyte for WT and L9′A.

Figure 3.

Figure 3.

Na+ and Ca2+ permeabilities relative to K+ (_P_Na/_P_K, _P_Ca/_P_K) were measured for mouse WT α4β2 and α4(L9′A)β2 receptors expressed in frog oocytes. WT and L9′A ACh-induced I-V relationships in 98 m

m

K+ (A) and 98 m

m

Na+ (B) between -20 and 20 mV. The reversal potentials of WT and L9′A I-V relationships (_E_r values) were -4 and -2 mV in 98 m

m

K+ and -10 and -11 mV in 98 m

m

Na+, respectively. C, WT and L9′A ACh-induced I-V relationships in 65 m

m

Ca2+ between-30 and 30 mV. WT and mutant _E_r values in 65 m

m

Ca2+ were 9 and 8 mV, respectively. The I-V relationships were obtained from individual oocytes. D, Relative permeabilities, _P_Na/_P_K and _P_Ca/_P_K, for WT and L9′A receptors. Error bars are means ± SEM; n = 4 oocytes per experiment.

Figure 4.

Figure 4.

Functional characteristics of nAChRs in thalamic primary cell cultures from WT, Het, and Hom L9′A mice. Fura-2 ratiometric [Ca2+]i measurements were made at 2 s intervals, starting 20 s before and ending 80 s after a single application of nicotine in WT (A), Het (B), and Hom (C)-derived cell cultures. Each data point is the average of 17 (WT), 22 (Het), or 16 (Hom) cells. D, Responses to various nicotine concentrations were used to compose a concentration-response relationship. Het- and Hom-derived cell cultures were more sensitive and had larger responses to nicotine compared with WT. Whole-cell patch recordings were obtained from L9′A Hom thalamic cell cultures exposed to calcium channel blockers: 750 n

m

calcicludine (E), 30 μ

m

nifedipine (F), and 2 μ

m

ω-conotoxin GVIA (G) (n = 2 or 3 neurons per blocker). Receptor activity was elicited with 3 μ

m

ACh test pulses. After a 10 min wash with extracellular solution, recovery from block was assessed with a final 3 μ

m

ACh pulse. All three calcium blockers reversibly decreased ACh responses.

Figure 5.

Figure 5.

Nicotine-stimulated 86Rb+ efflux and 125I-epibatidine binding in WT, Het, and Hom L9′A mice. A, 86Rb+ efflux from synaptosomes made from cortex and thalamus and stimulated with nicotine. Each point is the mean ± SEM; n = 7. Lines are the best fit to the Hill equation. B, Desensitization and activation curves normalized to maximal response for WT and L9′A synaptosomes. Each point is the mean; n = 6 or 7. C, Concentration dependence of 125I-epibatidine binding in the membrane fraction obtained from cortex and thalamus. Each point is the mean ± SEM; n = 4. D, 125I-Epibatidine binding in cortex and thalamus in the presence or absence of cytisine was used to calculate cytisine-sensitive sites and cytisine-resistant sites. Cytisine-sensitive sites are thought to reflect α4β2 receptors. Saturation binding shows fewer epibatidine binding sites in Het and Hom compared with WT in both brain regions examined (one-way ANOVA, followed by Bonferroni's test; §,*p < 0.01). Each bar represents mean and SEM; n = 4.

Figure 6.

Figure 6.

Nicotine-induced seizures and the effect of nicotine pretreatment on seizures. A, B, Seizures in the absence of pretreatment. Nicotine-induced seizures were expressed as percentage of mice that displayed behavioral seizures (A) and time from injection to seizure onset (B). Mice that did not display seizures 10 min after receiving a nicotine injection were considered nonresponsive and correspond to data points next to the 600 s mark. Het and Hom were more sensitive to nicotine than WT. Each data point in B is the mean ± SEM; n = 6. C, D, Effect of nicotine (nic) pretreatment on nicotine-induced seizures in WT and Het mice. C, Percentage of seizing mice (as in A); D, time to seizure onset (as in B). Mice were injected with a nicotine pretreatment dose 10 min before receiving a second test nicotine injection. Nicotine pretreatment (0.1 mg/kg) completely blocked seizures in Het that received a second dose of 0.2, 0.5, or 1 mg/kg nicotine. Seizures in WT mice were not blocked by nicotine pretreatment. Each data point is the mean ± SEM; n = 5.

Figure 7.

Figure 7.

Traces from EEG recordings of WT, Het, and Hom mice before and after a single nicotine injection. EEG recordings were obtained from screw electrodes placed on the parietal cortex (above the hippocampus). During behavioral seizures, no changes were seen in the EEG traces of Het and Hom mice injected with 2 mg/kg nicotine. Recordings from WT and Hom (10 mg/kg) were obtained from two pairs of screw electrodes bilaterally implanted into left and right hemispheres. Bilateral and synchronous spike-wave traces were observed in the EEG of WT mice injected with 10 mg/kg nicotine during behavioral seizures. Hom mice injected with 10 mg/kg nicotine had two bouts of convulsions separated by an interictal period of 1-3 min (72 s in the example shown). Bilateral and synchronous spike-wave activity was present in the second convulsive burst of Hom mice injected with 10 mg/kg nicotine. Three WT mice were injected with 2 mg/kg nicotine; five animals were tested in each of the remaining treatment groups.

Figure 8.

Figure 8.

Traces from simultaneous EEG and mechanotransducer recordings of WT and Hom mice before and after a single nicotine injection. EEG recordings were obtained from screw electrodes placed on the parietal cortex, on each hemisphere. A mechanotransducer connected to the bottom of the cage was used to record the frequency and intensity of the mouse's movements. Photographs were taken during seizures, within the time period of EEG and mechanotransducer traces shown. During seizures, WT displayed a variety of movements that included jumping, rearing, loss of righting response, and limb clonus; examples are shown in the photographic sequence. L9′A mice seizures were characterized by Straub tail, loss of righting response, and violent and repetitive movements of the forelimbs. n = 4 mice per genotype. WT and Hom mice shown in the photographs are N4 littermates.

Figure 9.

Figure 9.

Chronic EEG recordings in WT, Het, and Hom mice. A, Example of an EEG trace with its corresponding spectrogram from a WT mouse. Power density (in square microvolts per Hertz) changes in the spectrogram are represented by the color intensity of individual pixels: dark coloration means high-power density. The vigilance state of each mouse was determined with an algorithm that analyzed power spectrum changes over time. The blue line above the spectrogram represents the output of the program. B, Brief awakenings were recorded during 13 h with lights on and 11 h with lights off. C, Number of brief awakening events per hour. Het and Hom mice had more brief awakenings than WT (one-way ANOVA, followed by the Tukey's pairwise multiple comparison test, *p < 0.001). Each bar represents the mean and SEM; n = 5 (WT), 5 (Het), and 4 (Hom). D, Wake, NREM sleep, and REM sleep events were quantified in WT, Het, and Hom mice over a 24 h period.

Figure 10.

Figure 10.

Locomotion of L9′A and WT mice. Locomotion was measured for 24 h in activity cages with infrared beams. Each event was defined as two successive beam breaks. A, Locomotion was recorded during 13 h with lights on and 11 h with lights off. B, Locomotion recorded with lights on for 24 h. C, Average locomotion activity was larger in Het and Hom mice compared with WT during lights-on period as measured in A (each bar represents the mean and SEM; n = 8; one-way ANOVA, followed by the Tukey's pairwise multiple comparison test, *p < 0.05). D, Average locomotion activity was larger in Het and Hom mice compared with WT during a 24 h continuous lights-on experiment (each bar represents the mean and SEM; n = 8; Kruskal-Wallis test, followed by the Tukey's pairwise multiple comparison test, *p < 0.01). The same animals were studied in experiments A and B.

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