Sensorimotor gating and spatial learning in α7-nicotinic receptor knockout mice

Authors


Corresponding author: Dr S. Schmid, Department of Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, Medical Sciences Building, Room 470, University of Western Ontario, London, ON, Canada N6A 5C1. E-mail: susanne.schmid@schulich.uwo.ca

Abstract

The role of acetylcholine and specific nicotinic receptors in sensorimotor gating and higher cognitive function has been controversial. Here, we used a commercially available mouse with a null mutation in the Chrna7tm1Bay gene [α7-nicotinic acetylcholine receptor (nAChR) knockout (KO) mouse] in order to assess the role of the α7-nAChR in sensorimotor gating and spatial learning. We examined prepulse inhibition (PPI) of startle and nicotine-induced enhancement of PPI. We also tested short- and long-term habituation of the startle response as well as of locomotor behaviour in order to differentiate the role of this receptor in the habituation of evoked behaviour (startle) vs. motivated behaviour (locomotion). To address higher cognition, mice were also tested in a spatial learning task. Our results showed a mild but consistent PPI deficit in α7-nAChR KO mice. Furthermore, they did not show nicotine-induced enhancement of startle or PPI. Short- and long-term habituation was normal in KO mice for both types of behaviours, evoked or motivated, and they also showed normal learning and memory in the Barnes maze. Thorough analysis of the behavioural data indicated a slightly higher degree of anxiety in α7-nAChR KO mice; however, this could only be partially confirmed in an elevated plus maze test. In summary, our data suggest that α7-nAChRs play a minor role in PPI, but seem to mediate nicotine-induced PPI enhancement. We found no evidence to suggest that they are important for habituation or spatial learning.

Sensorimotor gating refers to the ability of the brain to implicitly filter unnecessary sensory information, preserving its limited neuronal capacity for the processing of salient information. Prepulse inhibition (PPI) and habituation of the acoustic startle response represent two different behavioural measures of sensorimotor filtering (Braff et al. 1978). Prepulse inhibition (PPI) occurs when the presentation of a sensory stimulus (prepulse) reduces the behavioural response to a strong startling stimulus (pulse). Theoretical expositions suggest that the processing of the prepulse actively inhibits the processing of the pulse, resulting in decreased responsiveness (for review, see Koch 1999). Startle can also be used for assessing habituation of an evoked behavioural response. Habituation is defined as the progressive decrease in response amplitude following repeated exposure to the stimulus. There are two forms of habituation: short-term and long-term, which refer to the attenuation of responding within a testing session or across multiple testing sessions, respectively.

Nicotine is well known to enhance PPI (Acri 1994; Acri et al. 1994; Faraday et al. 1999; Gould et al. 2005; Ingram et al. 2005), but the responsible nicotinic receptor subtype is unknown. Pharmacological studies have suggested a role of α7-nicotinic acetylcholine receptors (nAChRs). Positive modulation of α7-nAChRs improves auditory gating in the DBA/2 mouse model of schizophrenia (Simosky et al. 2001), and rescues apomorphine and MK801-induced PPI deficits in rats (Dunlop et al. 2009; Wallace et al. 2011). Surprisingly, previous studies using α7-nAChR knockout (KO) mice have shown that these mice have normal PPI (Paylor et al. 1998; Young et al. 2011). This discrepancy with drug studies may in part be due to methodological considerations, as another study observed a deficit in auditory P50 gating in heterozygous α7-nAChR KO mice (Adams et al. 2008).

In terms of habituation, studies by Williams et al. (1975) have shown that habituation of evoked behaviours, like startle, is mediated by separate mechanisms to those of habituation of motivated behaviours, such as exploratory behaviour and spontaneous locomotion. Pharmacological studies have suggested that acetylcholine is very important for short- and long-term habituation of locomotion (Giovannini et al. 2001; Ikegami 1994; Thiel et al. 1998). In particular, it has been shown that nicotinic receptors in the nucleus accumbens play an important role in early consolidation phases of long-term habituation of locomotion (Schildein et al. 2002). In contrast, reflexive behaviours historically have been suggested to be independent of acetylcholine (Brown 1976; Hughes 1984). Indeed, there is no evidence to suggest that nicotinic receptors are involved in short-term habituation of startle (Brown 1976; Hughes 1984; Paylor et al. 1998). However, a recent study by Schmid et al. (2011) has linked acetylcholine to long-term habituation of startle, as mice with a general knock-down of the vesicular acetylcholine transporter show clear long-term habituation deficits.

In this study, we therefore sought to evaluate the sensory filtering capacities of α7-nAChR KO mice using PPI, short- and long-term habituation of both startle and locomotion. We also injected nicotine to determine whether the enhancement of PPI is dependent on α7-nAChRs. Finally, we performed a spatial learning task in order to test whether sensorimotor gating deficits correlate with impairments in higher cognitive function. Recently, Singer et al. (2013) have demonstrated that in CB57BL/6 mice, PPI correlated with working memory performance in the Morris water maze. We sought to reaffirm and expand on this correlation using the Barnes maze, with an emphasis on spatial learning and memory.

Methods

Subjects

We used a commercially available mutant mouse line (B6.129S7-Chrna7, stock no. 003232; Jackson Laboratories, Bar Harbour, ME, USA) that has a null mutation in the Chrna7tm1Bay gene, which encodes the α7-nAChR protein. The KO was produced by deleting the last three exons (8–10) of the Chrna7 gene. The strain originated on a mixed 129/SvEv and C57BL/6 background and has been backcrossed to the C57BL/6J line for at least eight (N8) generations. Control mice were age-matched wild-type (WT) C57BL/6J counterparts.

Animals were cared for according to the ethical guidelines of the University of Western Ontario Animal Use Subcommittee and Canadian Council on Animal Care (CCAC). Mice were group housed, with a 12-h light–dark cycle with ad libitum food and water. Testing occurred at ages 6–14 weeks during the light phase. For most tests, 18 male KO and WT mice (C57BL/6J from Jackson Laboratories) were used. The order of testing was as follows: startle testing, locomotor box, Barnes maze and elevated plus maze for all animals. Long-term habituation of startle and locomotor testing were tested with a separate batch of mice of both genders.

Testing of startle responses

All startle testing was done using Med Associates sound-proofed startle boxes and associated software (Startle Reflex Version 5.95, St Albans, VT, USA). Figure 1 shows a schematic representation of behavioural protocol. Animals were acclimated to the startle box for 5 min/day for 3 days with just the background noise (65 dB SPL white noise) and all testing sessions began with an acclimation phase (5 min, 65 dB SPL white noise). On the final day of acclimation, animals also underwent an input/output (I/O) test to determine an appropriate gain setting for each individual animal (Fig. 2). An I/O function began with stimulation at 65 dB SPL (20-millisecond duration) and increased in 5 dB SPL steps to 120 dB SPL (for details, see Schmid et al. 2011; Valsamis & Schmid 2011). Once the gain was set it was kept constant throughout all testing days. For the next 5 days, the animals were tested once daily with the behavioural protocol described below.

Figure 1.

Startle testing. As shown in the graph, animals underwent 3 days of acclimation to startle boxes and background noise and an I/O function was measured at the third day. They then underwent five subsequent testing days, where they were exposed to a 5-min acclimation period, a first block with 33 startle stimuli alone for measuring short-term habituation, and a second block with 50 trials, 10 trials each of startle stimuli alone, and any combination of 75 or 85 dB SPL prepulses, administered 30 or 100 milliseconds before the startle pulse. Asterisks indicate the first three startle responses and squares indicate the last three responses in block I that were used to calculate the amount of short-term habituation in each animal. Dots indicate the first three startle responses on day 5 that were used along with the first three responses on day 1 (asterisks) to calculate the amount of long-term habituation. Please note that block II was omitted for testing long-term habituation as displayed in Fig. 5b,d, and a shortened program was used for testing the effects of nicotine injections (see Methods).

Figure 2.

α7-nAChR KO mice show normal baseline startle responses. Representative I/O functions of startle response amplitudes for different startle stimulus intensities. Both genotypes display natural variability of startle magnitudes with very high and very low startler within the group. Based on startle amplitude (low, intermediate or high) a gain was prescribed as indicated in the figure (see also Methods). This allows for accurate signal detection and prevents floor effects in low startler. The I/O function of (a) WT and (b) KO did not differ statistically. The solid black line indicates the average across all animals of the group (n = 18/genotype, all male. Not all mice shown here for clarity).

Eighteen male mice of each genotype underwent PPI and short-term habituation testing. The protocol consisted of two blocks of trials (Fig. 1). Block I assessed habituation by presenting 33 trials of the startle pulse (20-millisecond white noise at 105 dB SPL and 15-second intertrial interval, ITI). Block II assessed PPI. There were five different trial conditions (10 trials per condition) for a total of 50 trials. All trials were presented in a pseudo-randomized order. The trial conditions were as follows: startle pulse alone trials (to determine baseline startle) and combinations of commonly accepted prepulses (75 or 85 dB SPL; 4 milliseconds) at two different interstimulus intervals (ISIs; 30 or 100 milliseconds).

Separate animals were used for long-term habituation experiments (WT: n = 21, 16 males/5 females; KO: n = 14, 9 males/5 females). To examine long-term habituation, we employed the same acclimation schedule, but removed block II to prevent overpresentation of startle stimuli, which can induce sensitization (Plappert et al. 1999).

Nicotine injections

To test the effect of nicotine on PPI, we used a shorter protocol to account for the drug's short half-life in mice blood plasma. Block I was reduced to 3 trials and block II to 30 trials (three different trial conditions, 10 trials per condition): startle pulse alone and 75 or 85 dB SPL prepulse (both with 30-millisecond ISI). Both WT (n = 20, male) and KO (n = 18, male) mice were given a single subcutaneous injection of either nicotine (1 mg/kg free base nicotine, nicotine hydrogen tartrate salt, Sigma Chemical Co., St. Louis, MO, USA, dissolved in phosphate-buffered saline, 0.9% NaCl) or saline immediately before behavioural testing. Each mouse was administered both treatments on separate days. We allotted a 2-day recovery period between treatments and the order of nicotine/saline administration was randomized and counterbalanced across genotypes.

Data and statistical analysis

Startle magnitude was calculated as the maximal displacement of the movement-sensitive platform induced by the startle reflex following the startle pulse (arbitrary units). To detect differences in baseline startle between genotypes, we examined the I/O function, where all animals had the same gain factor, as well as initial startle values (average of the first three trials) on day 1 of testing for the group tested on long-term habituation. We used a two-way repeated measure analysis of variance (anova) (genotype × sound level) for the I/O function and an unpaired Student's t-test for the latter group.

To analyse short-term habituation of startle, we calculated short-term habituation ratios (average of trials 28–30/average of first 3 trials; see Fig. 1, stimuli marked with squares and asterisks, respectively), and compared them using an unpaired Student's t-test. To analyse long-term habituation, we normalized all data points to the average of the first three trials on day 1 for each individual mouse. We then used a three-way repeated measures anova (day × genotype × gender) and post hoc unpaired Student's t-tests. We also calculated a long-term habituation ratio (average of first five trials on day 5/average of first five trials on day 1; corresponding to stimuli marked with asterisks and dots in Fig. 1), and compared ratios between genotypes using an unpaired Student's t-test.

The PPI was expressed as percent of prepulse inhibition (%PPI = [1 − {startle magnitude with prepulse/baseline startle without prepulse} × 100). We determined the average %PPI for each prepulse type and performed two-way anova (trial type × genotype). We also calculated averages for each trial type per day and performed a two-way repeated measures anova (genotype × day) in order to determine if PPI changed over days. When nicotine was administered, we performed a three-way repeated measures anova (drug × genotype × prepulse) to determine changes in %PPI with drug treatment. Additionally, we also subtracted PPI with nicotine administration from PPI with saline (%PPI nicotine − %PPI saline) for each animal, and used t-tests to determine if the difference significantly differed from zero. To determine the effect of nicotine on baseline, we again subtracted baseline startle with saline treatment from baseline with nicotine and compared between genotypes with an unpaired t-test.

Locomotor testing

We used locomotor behaviour to assess habituation of non-reflexive behaviours. To examine short-term habituation of locomotor behaviour, mice (WT: n = 6 males, KO: n = 12 males) were placed in a locomotor box (Versamax, Columbia, OH, USA) to freely explore for 2 h. Distance, rearing, rest time and time spent in each quadrant of the box were measured. Data values were totalled and parsed into 5-min blocks, and a two-way repeated measures anova (blocks of time × genotype) was performed to assess short-term habituation of locomotion.

Long-term habituation of locomotor behaviour was tested in separate mice (WT: n = 15, 10 males/5 females; KO: n = 13, 8 males/5 females), once daily for 15 min for 5 consecutive days. The values of the first 5-min block were analysed using a three-way repeated measures anova (day × genotype × gender). Short-term habituation data were analysed in raw values, but for long-term habituation data were also normalized (activity/activity of first 5 min on day 1). This is for graphical representation and to reduce individual variability in locomotor behaviour as suggested by Thiel et al. (1998).

Barnes maze

The Barnes maze is designed to test spatial learning and memory in rodents. The protocol used for this test has been previously described by Sunyer et al. (2007). Mice (n = 10/genotype, male) completed four trials per day on days 1–4 to ensure acquisition of the task. Trials completed when a mouse entered the target hole, or when 3 min had passed. Intertrial interval between testing was on average 20 min. On days 5 and 12 of testing, the animals completed probe trials to assess short- and long-term spatial memory, with the target hole covered to prevent entrance. Mice were given a single 90-second trial to freely explore the apparatus on probe days.

For all days, holes investigated by mice were tracked by ANY-Maze software (Version 4.82, Stoelting, Wood Dale, IL, USA). Investigation was defined as when a mouse hovered over a hole with their nose (i.e. nose poke). The distance, latency to approach and enter target as well as errors were tracked. In addition, on probe trial days, we measured the location of errors (based on distance to target). We defined two types of errors: a primary error was defined as any time the mouse investigated a hole that was not the target, and a secondary error was defined as the first instance a mouse investigated a non-target hole after investigating the target hole. Total errors were the sum of primary and secondary errors.

Data and statistical analysis

Performance on days 1–4 showed how well the animals were able to learn the task. The values for each of the measures examined were averaged over all four trials/day, in order to give us average performance for each day and analysed using a two-way repeated measures anova (day × genotype). For performance on day 1 only, a separate analysis was completed where latency to approach target was analysed by trial using a two-way repeated measures anova (genotype × trial). This aimed to assess working memory performance, based on improvement across trials. Analysis of only day 1 was appropriate for this measure, to remove mnemonic function as a potential confound.

Performance on days 5 and 12 showed how well animals were able to recall the task in their short- or long-term spatial memory, respectively. To analyse this, separate two-way repeated measures anovas were performed (hole × genotype).

Elevated plus maze

Both WT (n = 14, male) and KO (n = 14, male) were placed in the centre of the elevated plus maze (Stoelting). The apparatus contains four arms: two covered and two uncovered. Animals had 5 min to freely explore the box. The number of entries, latency to enter and time spent in closed and open arms were digitally recorded by ANY-Maze software (Version 4.82, Stoelting). Unpaired Student's t-tests were then used to determine differences between genotypes.

Results

α7-nAChR KO mice have normal startle ability

Critical to our study was the ability of α7-nAChR KO mice to startle normally. We found that startle I/O functions did not differ between genotypes (F11,403 = 3.0, P = 0.1; n = 18/genotype: Fig. 2). Furthermore, baseline startle in block I (average of first three trials on day 1) and in block II (pulse-alone trials) did not differ between the genotypes (t34 = 1.5, P = 0.14; t34 = 0.52, P = 0.6, respectively).

Prepulse inhibition is mildly impaired in α7-nAChR KO mice

Eighteen male WT and KO animals underwent PPI testing. We observed a mild, but consistent impairment of PPI in α7-nAChR KO mice. In WT, with a 75 dB SPL prepulse startle was reduced by about 52.1% and 52.2% at the ISIs of 30 and 100 milliseconds, respectively, whereas in KOs startle was only reduced by 38.5% and 37.2%, respectively. Therefore, PPI was significantly smaller in KO mice than WT (F1,34 = 6.87, P = 0.02, Fig. 3a) with a 75 dB prepulse, regardless of ISI. In all groups, PPI was stable across days of testing as there was no main effect of day at either ISI (30 milliseconds F4,175 = 0.27, P = 0.9; 100 milliseconds F4,175 = 1.0, P = 0.4) or interaction between days and genotype (30 milliseconds F4,700 = 0.87, P = 0.59; 100 milliseconds F4,700 = 0.27, P = 0.9).

Figure 3.

α7-nAChR KO mice have mildly impaired prepulse inhibition. (a) α7-nAChR KO mice cannot suppress startle as effectively as WT across ISIs at a 75 dB SPL prepulse. (b) When an 85 dB SPL prepulse is used, α7-NAChR KO mice have the same ability as WT type to suppress startle across ISIs (n = 18/genotype, male).

When a higher prepulse level of 85 dB SPL was used we did not observe any PPI differences between genotypes. In WT animals, startle was suppressed by 49% and 62.3% at 30- and 100-millisecond ISIs, respectively, and in KO mice by 44% and 53.8%, respectively. While there is still a trend of impaired PPI in KO, it failed to reach statistical significance (F1,34 = 1.5, P = 0.32, Fig. 3b). There was also no main effect of day (30 milliseconds F4,175 = 2.2, P > 0.08; 100 milliseconds F4,175 = 0.48, P = 0.8) or day × genotype interaction (30 milliseconds F4,700 = 1.9, P = 0.11; 100 milliseconds F4,700 = 0.38, P = 0.82).

α7-nAChRs are critical for nicotine-induced enhancement of PPI and startle

Previous studies have shown that acute, systemic nicotine improves PPI. We injected WT (n = 20, male) and α7-nAChR KO mice (n = 18, male) with saline and nicotine (1 mg/kg, Fig. 4) before PPI testing with both prepulse intensities and an ISI of 30 milliseconds (see Methods). We performed a three-way anova (drug × genotype × prepulse) and confirmed an impairment of PPI in KO mice at both prepulses (F1,36 = 5.5, P = 0.025). The anova did not detect a main effect of nicotine treatment (F1,36 = 0.6, P = 0.43) or interaction between genotype × drug × prepulse (F1,36 = 1.1, P = 0.3). However, we did see that the drug tended to act differently according to the prepulse level, but the drug × prepulse interaction just failed to reach significance (F1,36 = 4.05, P = 0.052). Generally, at the 75 dB SPL prepulse, we saw no effect of nicotine, PPI for WT mice was 45.2% with saline and 47% with nicotine administration. A similar trend was observed in the KO mice. When KO mice were administered saline, PPI was 34.6%, and when given nicotine it was 35.3% (Fig. 4a). At the higher prepulse of 85 dB SPL, nicotine seemed to improve PPI in WT mice. When WT mice were administered saline, PPI was 43.6%, and when given nicotine it was 55.6%, whereas in the KO mice PPI was similar in both conditions: PPI with saline was 27.6% and with nicotine it was 30%. When we looked at changes of PPI, we found a significant improvement of PPI in WT (t17 = 2.43, P = 0.03), but not in KO animals (t17 = 0.34, P = 0.73, Fig. 4b). Two WT mice were eliminated from this analysis as outliers (±3 standard deviations from mean).

Figure 4.

α7-nAChR knockout mice do not show nicotine-induced enhancement of PPI and baseline startle. (a) We reconfirmed that KO mice have impaired PPI compared with WT in both drug conditions and prepulses. Nicotine tended to enhance PPI at the 85 dB SPL prepulse in WT mice. We do not see enhancement at lower prepulse levels as PPI is generally weaker and more variable with a 75 dB SPL prepulse. (b) There is a significant effect of nicotine at 85 dB SPL prepulse on changes of PPI in WT, but not in KO mice. The asterisk denotes that the change in PPI (PPI nicotine − PPI saline) between drug conditions is significantly different than zero in WT but not KO mice. (c) The change in baseline (BL) startle amplitude between treatment conditions (nicotine baseline − saline baseline) is displayed for both genotypes. Nicotine enhances baseline startle in WT animals, but not in α7-nAChR KO mice (n = 18/genotype, male).

Nicotine also enhanced baseline startle amplitudes compared with saline treatment in WT animals (t17 = 2.4, P = 0.03), but not in KO mice (t17 = 0.43, P = 0.67). Once again, however, this effect was strongest when the data were analysed for individual changes in each mouse between saline and nicotine conditions. The WT mice showed an increased baseline startle when given nicotine, whereas KO mice showed no changes (mean change around 0, Fig. 4c). Changes of baseline startle were significantly different between genotypes (t34 = 2.05, P = 0.048).

Habituation of both startle and locomotor behaviour is unimpaired in α7-nAChR KO mice

We examined short- and long-term habituation of the startle reflex in male WT and α7-nAChR KO mice over 5 days of testing. Wild-type mice (n = 18, male) showed a decreasing startle amplitude within a testing session on average by about 27% by the end of block I (short-term habituation ratio = 0.73). In KO mice (n = 18, male) responses did also decrease by about 23% (short-term habituation ratio = 0.77). The short-term habituation ratios between genotypes did not differ (t34 = 1.7, P = 0.1, Fig. 5a,b).

Figure 5.

Habituation of startle and locomotion are normal in α7-nAChR knockout mice. (a) Short-term habituation ratios for the startle reflex did not differ between genotypes. (b) Short- and long-term habituation of startle at days 1 and 5. In both genotypes startle amplitudes progressively decrease within a testing session and across testing sessions to a comparable degree. (c) Both genotypes showed significant short-term habituation of locomotor behaviour, with activity greatly attenuated by the end of 2 h. (d) Both WT and KO mice decreased locomotor activity across days, displaying normal long-term habituation of locomotion (WT n = 21, 16 males/5 females; KO n = 14, 9 males/5 females).

We used a separate batch of animals to examine long-term habituation of startle with a shortened behavioural programme in order to avoid overexposure. In WT mice (n = 21, 16 males/5 females) average startle response decreased to 92%, and in KO mice (n = 14, 9 males/5 females) to 91% within 5 days. These long-term habituation ratios did not significantly differ (F1,31 = 1.6, P = 0.22), neither was there any interaction between gender and genotype (F1,31 = 0.6, P = 0.46). When startle was normalized to day 1 of testing, the anova revealed that startle significantly changed across days (F4,31 = 21, P < 0.001). There was no effect of gender (F1,31 = 0.16, P = 0.67) or genotype (F1,31 = 0.01, P = 0.79), or gender × genotype × day interaction (F4,28 = 0.4, P = 0.57; Fig. 5b).

Habituation of reflexive behaviours like the startle response is mediated by mechanisms distinct from habituation of motivated behaviours like locomotion (that reflects exploratory behaviour). Therefore, we also examined short-term habituation of locomotion in WT (n = 9, male) and α7-nAChR KO (n = 12, male) mice. There was a significant decrease of distance travelled within the 2-h test session (F23,529 = 9.9, P < 0.001) with no effect of genotype (F1,23 = 0.3, P = 0.87) or interaction between genotype × time (F23,529 = 1.26, P = 0.2, Fig. 5c). Rearing activity also significantly decreased within a test session (F23,506 = 4.6, P < 0.001), with no significant time × genotype interaction (F23,506 = 1.0, P = 0.49). For rearing analysis, one KO animal was eliminated as it never reared. Rest time tended to increase across time, but this failed to reach significance (F23,529 = 1.3, P = 0.14), with no interaction between time × genotype (F23,529 = 1.2, P = 0.22). This data suggested that α7-nAChR KO mice have normal short-term habituation of locomotor behaviour. However, we observed one difference between genotypes: KO mice spent significantly less time in the centre of the box throughout testing compared with WT (t23 = 1.82, P = 0.04). They also tended to travel less in the centre, although this failed to reach significance (t23 = 1.67, P = 0.11).

To examine long-term habituation of locomotor behaviour, we used separate mice (WT: n = 16, 9 males/5 females; KO: n = 13, 8 males/5 females) and tested them in the locomotor box across 5 days. We found that there was no difference in activity between genotypes (F1,25 = 2.7, P = 0.11) or gender (F1,25 = 0.5, P = 0.51). With normalized data (to day 1 of each animal) we found that the distance travelled significantly decreased across days (F4,100 = 5.3, P = 0.017), with no main effect of genotype (F1,25 = 3.25, P = 0.83), gender (F1,25 = 0.8, P = 0.37) or interaction of day × gender × genotype (F4,100 = 0.7, P = 0.41). This shows that both genotypes showed normal long-term habituation of locomotor behaviour, see Fig. 5d.

α7-nAChR KO mice display normal spatial learning and memory

To date, discrepancies exist whether α7-nAChR KO mice have normal or impaired spatial learning and memory, and spatial memory performance has been linked to PPI performance (Singer et al. 2013). Therefore, we tested male WT and α7-nAChR KO mice in the Barnes maze (n = 10/genotype, male) in order to reassess spatial learning. When we analysed the latency to approach target, we observed a main effect of day, suggesting that learning occurred across days (F5,114 = 18.9, P < 0.001). We found no main effect of genotype (F1,114 = 0.1, P = 0.78) or interaction between day × genotype (F5,570 = 0.4, P = 0.86), suggesting that both genotypes performed similarly (Fig. 6a). Furthermore, both genotypes travelled the same distance across days (F5,114 = 2.6, P ≥ 0.14), suggesting normal activity levels. While there was no difference in the number of primary errors made by genotypes (F5,114 = 4.4, P = 0.07), KO mice tended to make significantly less total and secondary errors (F5,114 = 5.1, P = 0.036; F5,114 = 17.6, P = 0.015, respectively). Consequently, they also took significantly less time to enter the target (as opposed to approach it) than WT mice (F3,76 = 10.2, P = 0.005), despite both genotypes showing improvement across days of testing (F3,76 = 5.1, P = 0.003). Additionally, as Singer et al. (2013) found the strongest correlation between PPI and spatial working memory; we also examined improvements on day 1 across trials. Both genotypes considerably improved across trials (F3,54 = 6.53, P = 0.01) with no difference between genotypes (F1,18 = 0.09, P = 0.78, Fig. 6b).

Figure 6.

α7-nAChR mice show normal spatial learning and memory in the Barnes maze. (a) α7-nAChR mice show normal acquisition of a spatial task, as both genotypes significantly improve performance on training days 1–4. Unchanged performance on days 5 and 12 suggests that both genotypes accurately remember the task. (b) Analysis of spatial working memory during the first training sessions within day 1. Both genotypes show similar times to approach the target on day 1. (c) Number of nose pokes on the different holes on the maze. Both WT and α7-nAChR KO mice show a preference for the target holes on day 5 and (d) day 12, suggesting that α7-nAChR KO mice have normal short- and long-term spatial memory (n = 10/genotype, male).

We found a significant preference for the target hole on days 5 (F19,380 = 39, P < 0.001) and 12 (F19,380 = 23.3, P < 0.001) with no main effect of genotype (F1,18 = 0.01, P = 0.93; F1,18 = 1.1, P = 0.32, days 5 and 12, respectively, Fig. 6c,d). Furthermore, latency to approach target did not differ between genotypes on days 5 or 12 as there was no effect of day (F1,18 = 0.18, P = 0.68), genotype (F1,18 = 0.12, P = 0.73) or interaction between day × genotype (F1,18 = 0.14, P = 0.71), suggesting that α7-nAChR KO mice have normal retention of spatial tasks.

Elevated plus maze

The fact that α7-nAChR KO mice spent less time in the centre of the locomotor box and seemed to have an increased drive to enter the drop box in the Barnes maze may indicate an increased level of anxiety in these mice. Therefore, we decided to directly assess anxiety using the elevated plus maze (n = 14/genotype, all male) We found that total distance travelled (WT = 17.0 ± 0.8 m, KO = 16.5 ± 1.0 m; t26 = 0.3, P = 0.76), latency to enter open arm (WT = 4.5 ± 1.9 seconds, KO = 4.9 ± 2.6 seconds; t24 = 0.3, P = 0.87) and number of entries into closed (WT = 28.0 ± 1.3, KO = 27.4 ± 2.1; t26 = 0.25, P = 0.80) or open arms (WT = 16.1 ± 1.2, KO = 15.4 ± 1.1; t26 = 0.5, P = 0.63) did not differ between genotypes. However, we did find that KO animals spent more time in closed vs. open arms compared with WT (WT = 152 ± 14.9 seconds, KO = 199.1 ± 8.0 seconds; t24 = 2.8, P = 0.001), suggesting that they are more anxious than their WT littermates.

Discussion

The aim of this study was to understand the role of α7-nAChR in sensory filtering mechanisms and how this relates to higher cognition.

Prepulse inhibition

We observed that α7-nAChR KO mice had a mild, but consistent and significant impairment of PPI. The KO mice consistently showed reduced PPI at the 75 dB SPL prepulse, regardless of ISI. At the higher prepulse level of 85 dB SPL, PPI differences failed to reach significance in one of two groups. Generally, PPI is more robust at higher prepulse levels, and we suggest that this impairment is mild and therefore most apparent when PPI is not at its maximum. As pharmacological data suggest that α7-nAChR plays an important role in PPI, the mild deficit may in part be due to compensation by other nicotinic receptors in our KO model, as Adams et al. (2008) observed P50 auditory gating deficits only in heterozygote KO mice.

Our observed PPI deficit does not match with the results of previous α7-nAChR KO mice studies. Both Paylor et al. (1998) and Young et al. (2011) observed normal PPI in KO mice and used a α7-nAChR subunit null mutation, generated in a mixed 129/SvEv C57BL/6J line that was backcrossed onto the C57BL/6J strain for at least six generations. As our line (purchased from Jackson Laboratories) matches this background, genetic differences are unlikely to account for our observed results. The explanation for the discrepancy might lie in the differences between experimental protocols. Prepulse levels and ISIs were the same in all studies; however, both Paylor et al. (1998) and Young et al. (2011) used male and female mice, and found a main effect of gender on PPI and baseline startle. This might have increased the variability of their data and thereby occluded a mild PPI deficit. Reduced variability by using male mice only makes our experiment more apt to detect mild deficits in PPI. Additionally, animals may not have been sufficiently habituated to the startle stimulus prior to PPI testing. Without sufficient prior exposure to startle stimuli alone, short-term habituation interferes especially with the first trials of PPI measurements, thereby further increasing the variability. Finally, Young et al. (2011) did not normalize PPI measurements for each mouse, which greatly increases the inherent variability between mice (see Fig. 2). In fact, they show a higher average baseline startle in KO mice compared with WT, but the same startle levels when a prepulse is present, which may reflect that a disruption of PPI in KO mice had normalized PPI been calculated for each animal. Additionally, studies have shown that differences in baseline startle also influence PPI, particularly when data are not normalized (Csomor et al. 2008).

Nicotine and PPI

Apart from the fact that α7-nAChRs play a minor role in PPI, we also observed that they are critical for nicotine-induced enhancement of startle and PPI. Although the overall anova failed to yield a significant effect of nicotine, we did observe a slight, but significant enhancement of PPI with nicotine when %PPI was normalized to reflect changes from the saline condition (%PPI nicotine − %PPI saline), which is in accordance with the previous studies (Gould et al. 2005). The anova likely failed to reach significance because of the high number of factors involved in the analysis and because of a ceiling effect, as WT mice were already performing well with saline administration. Through reducing the variability by normalizing to the saline condition this effect is strengthened and was able to reach significance.

Where this nicotine effect is mediated is not fully understood yet. Nicotine may simply amplify the contribution of the α7-nAChR to PPI, thereby causing PPI enhancement. Startle-mediating neurons of the caudal pontine reticular nucleus (PnC) receive cholinergic input from the midbrain that is assumed to mediate PPI (Bosch & Schmid 2006, 2008; Fendt & Koch 1999). Potentially, α7-nAChRs in the PnC could directly modulate baseline startle effects and possibly even PPI. Alternatively, many PPI-modulating brain areas are known to express α7-nAChRs, including the prefrontal cortex, hippocampus, ventral tegmental area and nucleus accumbens (Gotti et al. 1997, 2006; Paterson & Nordberg 2000). Future studies should seek to understand where this effect is occurring through localized injections of α7-NAChR agonists and antagonists.

Studies estimate that smoking rates in schizophrenic populations are two to four times greater compared with the normal population (Hughes et al. 1986; Leonard et al. 2000). In both healthy and schizophrenic patients, PPI improves after smoking (Kumari et al. 1997, 2001), which may indicate that schizophrenics are smoking as a form of self-medication (Kumari & Postma 2005). Our study indicates that α7-nAChRs are at least partially mediating aspects of the initial beneficial effects of nicotine. It is important to note, however, that we only provide evidence that α7-nAChRs are critical for acute effects of nicotine. Chronic nicotine is known to alter nicotinic responses and receptor levels; therefore, the situation may be different in smokers.

Habituation of reflexive and motivated behaviour

In accordance with previous literature, we did not find that the α7-nAChR was involved in short- or long-term habituation of the startle reflex. A recent study by Schmid et al. (2011) showed that the neurotransmitter acetylcholine is involved in long-term habituation of startle, but we did not find any influence of the α7-nAChR on long-term habituation, suggesting that the effect may be mediated by other cholinergic receptors. Furthermore, we did not see any evidence for a α7-nAChR involvement in short- or long-term habituation of locomotor behaviour. Previous studies suggested that nAChRs were important for the consolidation of long-term habituation of locomotion (Schildein et al. 2002). Again, our study suggests that a different nicotinic receptor subtype might be responsible for the reported effects. Overall, we found that the α7-nAChRs are not necessary for habituation of reflexive or non-reflexive behaviours, although, as with all constitutive KO mice, compensation by the knockout model cannot be ruled out.

Spatial learning and higher cognition

We tested whether deficits in sensory filtering correlate with deficits in higher cognitive processes, especially in spatial working memory tasks, as previously shown (Erwin et al. 1998; Singer et al. 2013). Studies of spatial learning and memory in the α7-nAChR KO mouse have been inconclusive in the past (Curzon et al. 2006; Fernandes et al. 2006; Paylor et al. 1998). In accordance with the findings of Paylor et al. (1998), we found normal spatial learning and memory in α7-nAChR KO mice. This is rather surprising as the α7-nAChR is known to be highly expressed in the hippocampus (Freedman et al. 1995; Guan et al. 1999). There may be compensation by other nicotinic receptors in our KO model as Curzon et al. (2006) found that a deficit in spatial learning existed in an inducible KO model. However, a recent study by Winterer et al. (2013) also failed to show improvements in P50 sensory gating in schizophrenic patients using an α7-nAChR-positive allosteric modulator, suggesting that the role of α7-nAChR in higher cognition is still unclear.

We found mildly impaired PPI in α7-nAChR KO mice but normal spatial learning and memory, failing to document the hypothesized correlation between sensory filtering deficits and cognitive function. The difference between our and Singer et al.'s (2013) findings could be due to task differences as their protocol emphasized spatial working memory. We assessed improvement of performance on day 1 to assess working memory, but found no differences between genotypes. Conversely, recent data suggest that impaired attention is central in cognitive deficits observed in α7-nAChR KO mice (Young et al. 2007); therefore, the observed PPI deficit might correlate better with disruptions in attention tasks.

Anxiety

Interestingly, we found that KO mice were significantly faster to enter the target during Barnes maze testing, despite no genotype differences in latency to approach target. Accordingly, we also observed that KOs were significantly less likely to make secondary errors. During locomotor testing KO mice spent less time in the centre of the locomotor box. This may indicate an increased level of anxiety in α7-nAChR KO mice, as the mice would be more motivated to enter the target in the Barnes maze instead of exploring other holes, as well as stay closer to the walls in the locomotor box. In the elevated plus maze test, however, most parameters were similar between genotypes, except the time spent in closed vs. open arms. This finding suggested that α7-nAChR KO mice may be slightly more anxious. It should be noted that we also ran light/dark box testing on a separate batch of animals (data not shown), and increased anxiety could not be reconfirmed. Other studies have failed to uncover an anxious phenotype in these mice (Fernandes et al. 2006; Paylor et al. 1998); however, one study observed that α7-nAChR mice had longer freezing times during conditioning tasks, which correlates with heightened anxiety (Davis & Gould 2007). Additionally, a recent study by Pandya and Yakel (2013) found that in rats, high doses of an α7-nAChR agonist (10 mg/kg, PNU-282987) had anxiogenic effects in open field tests that could be rescued by serotonin (5-HT1a) antagonism. Clearly, the role of α7-nAChR in anxiety needs further elucidation.

Conclusions

In summary, we have shown that α7-nAChRs play a (small) role in PPI, and are critical to nicotine-induced enhancement of both PPI and baseline startle. We did not find any evidence to suggest that this receptor is involved in habituation of reflexive or non-reflexive behaviours. We also found that α7-nAChR KO mice had normal spatial learning and memory, consistent with most previous studies, and that they might have slightly different anxiety levels. Future studies will seek to understand the mechanisms underlying the α7-nAChR effects on startle and PPI.

Acknowledgments

This study was sponsored by grants of the Ontario Mental Health Foundation (OMHF), the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institute of Health Research (CIHR) to S.S., and the Ontario Ministry of Training, Colleges and Universities for an Ontario Graduate Scholarship to E.A. The authors declare no conflicts of interests.

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