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" Title: Dose-dependent facilitation of memory for an inhibitory avoidance task by nimodipine Running head: L-type Ca2+ blocker enhances aversive memory Authors: Lynda A. Wilmott & Lucien T. Thompson The University of Texas at Dallas School of Behavioral & Brain Sciences Corresponding & Reprint Request Author: Lucien T. Thompson The University of Texas at Dallas School of Behavioral & Brain Sciences 800 W. Campbell Rd., Richardson TX 75080 USA [email protected] +1 (972) 883-4933 Conflicts of Interest and Source of Funding: No conflicts of interest to declare. Funding was obtained through the Clark Foundation. " " #" " Abstract: Influx of calcium through L-type Ca2+ channels plays a critical role in learning and memory. Increases in cytosolic Ca2+ are associated with impaired learning in aging, and small changes in intracellular calcium concentrations also play an important role in neural plasticity in young brains. In the present study, young adult male and female Fisher 344 x Brown Norway (FBN) hybrid rats were administered different doses of nimodipine (0.0, 0.5, 1.0, 3.0, or 5.0 mg/kg, i.p.), a specific dihydropyridine L-type Ca2+-channel blocker, immediately after a single inhibitory avoidance training trial. When tested 48 hr later, nimodipine dosedependently enhanced memory retention compared to controls (significantly increased latency to enter a dark compartment previously paired with an aversive stimulus). These findings may provide insights into calcium-dependent modulation of aversive memories, and should be considered in the design of effective treatment options for patients with dementia or problems involving memories with negative emotional valence. Key Words: Ca2+ channels; Nimodipine; L-type channels; Inhibitory avoidance task " " $" " Introduction Ca2+ regulation is critical for many neuronal processes, including synaptic plasticity, and for the macroscopic processes based on them, including learning and memory formation. Normally, cytosolic Ca2+ levels are tightly regulated by a multitude of proteins, but during normal aging and dementia, intracellular Ca2+ becomes dysregulated (Foster and Kumar, 2002; Thibault et al., 2007). This dysregulation is associated with impairments in learning and memory (Thibault et al., 2001; Miyashita et al., 2008; Berridge, 2011). ,QSDWLHQWVZLWK$O]KHLPHU¶V disease (AD), cytosolic Ca2+ levels and influx via L-type Ca2+-channels are increased (Mattson et al., 1992; Thibault and Landfield, 1996; Forette et al., 2002). Researchers have used Ca2+-channel blockers, such as nimodipine and nitrendipine, in treatment of memory deficits in dementia (Forette et al., 2002; López-Arrieta and Birks, 2002). Ca2+ regulation is also critically important for young adult synaptic plasticity. N-methyl-D-apartate glutamate receptors (NMDARs) and voltage-dependent Ca2+channels (ie L-type Ca2+-channels) are major transmembrane sources of neuronal calcium influx contributing to neuronal plasticity (Wankerl et al., 2010). Calcium entry via NMDARs are involved in long-term potentiation (LTP) (Bliss and Collingridge, 1993; Nakazawa et al., 2002), and Ca2+ dysregulation impairs this process (Shankar et al., 1998). Additionally, Ca2+ influx is involved in regulation of Ca2+-dependent post-burst afterhyperpolarizations (AHPs); these AHPs are enhanced, while neuronal excitability is decreased, in aging compared to young " " %" " neurons in multiple neuronal populations involved in learning and memory (Thompson et al., 1992; Foster and Kumar, 2002; Oh et al., 2010). Following acquisition of a learning task in young or aging animals, AHPs in the amygdala and hippocampus, two regions involved in multiple forms of learning and memory, including the inhibitory avoidance task used in the present study, are transiently decreased and excitability is increased (Moyer et al., 1996; Thompson et al., 1996b; Farmer and Thompson, 2012). Notably, when L-type Ca2+-channels are blocked with the dihydropyridine nimodipine, AHPs in both young and aging animals are reduced in vitro (Moyer et al., 1992), and hippocampal excitability (neuronal firing activity) is enhanced in young animals in vivo (Thompson et al., 1990). L-type high-voltage activated Ca2+-channels appear to be particularly important for modulating memory (Moyer et al., 1992; Thompson et al., 1992; Moyer and Disterhoft, 1994; Aradi and Holmes, 1999; Borde et al., 2000). L-type calcium-channel blockers, including nilvadipine (Iwasaki et al., 2007; Bachmeier et al., 2011), nifedipine (Quevedo et al., 1998; Barad et al., 2004; Cain et al., 2005; Busquet et al., 2008; McKinney et al., 2008; Walteriet et al., 2008; Davis and Bauer, 2012), isradipine (Gholamipour-Bodie et al., 2013), diltiazem (Quartermain et al., 2001; Barad et al., 2004), verapamil (Davis and Bauer, 2012), and also nimodipine (Levy et al., 1991; Taya et al., 2000; Quartermain et al., 2001) enhance spatial learning in both young (Kane and Robinson, 1999; Quartermain et al., 2001; Rashidy-Pour et al., 2009) and aged rats (McMonagle-Strucko et al., 1993; " " &" " Norris et al., 1998) and rabbits (Deyo et al., 1989; Straube et al., 1990; Moyer et al., 1992). Numerous studies have assessed how L-type Ca2+-channel antagonists affect appetitive spatial learning (which typically requires multiple trials for acquisition) or aversive tasks that again require multiple training trials. None, however, have assessed the impact of L-type blockade on memory for a singletrial task. To assess the role of calcium influx via L-type channels on consolidation of inhibitory avoidance memory, rats were given a single training trial, then dosedependent effects of a single immediate post-trial injection of the L-type calciumchannel blocker nimodipine were assessed on memory retention 48 hr later. The aim was to assess whether nimodipine improved memory consolidation for this task, as has been shown previously in multiple trial learning tasks. " " '" " Methods: Subjects At total of 86 locally bred young (~2-4 mo old) male and female Fisher 344 x Brown Norway hybrid (FBN) rats were socially housed in a temperaturecontrolled environment (~22oC) with a 12 hr light/dark cycle. Care followed established protocols approved by the Institutional Animal Care and Use Committee of the University of Texas at Dallas in accordance with NIH Animal Welfare guidelines. Ad libitum access to both food and water were available in the UDWV¶KRPHFDJHV. The experimenters were blind as to the identity of individual rat¶VGUXJWUHDWPHQWVXQWLOGDWDwas collected and analyzed, with cohorts from each litter pseudo-randomly assigned to each treatment group. Inhibitory avoidance (IA) training The apparatus used was a trough-shaped Plexiglas chamber (91 cm long, 15 cm deep, 20 cm wide at the top, and 6.4 cm wide at the bottom) with a sliding guillotine door to separate a light compartment (30 cm long) from a dark compartment (60 cm long, with two angled and divided metal plates serving as a floor). A lamp (20 W) was placed over the light compartment so as to brightly illuminate the light but not the dark compartment. All rats were handled for 5 d, 5 min/d, prior to undergoing a single training session. For this training session, each rat was placed in the light compartment, facing away from the dark compartment. When the rat turned around and escaped fully into the dark compartment, the door was closed behind and its initial escape " " (" " latency was recorded. When the rat reached the end of the dark compartment and turned around, a moderate footshock (0.18 mA, 1 s) was applied. Any rat that failed to vocalize or jump in response to the footshock was eliminated from further study. After another 15 s, the rat was removed from the apparatus and given an immediate post-training injection of either vehicle or one of the doses of nimodipine (detailed below). The rat was then returned to its home cage. Memory retention testing Forty-eight hr later the rat was returned to the light compartment of the apparatus, and the time it took to fully enter the dark compartment (retention escape latency) was measured. Longer escape latencies served as an indicator of better memory for the previous aversive event. Drug Treatment Nimodipine treatment Male and female FBN rats were divided into 5 treatment groups (see Table 1) and each was given an i.p. injection of either vehicle (65% polyethylene glycol (PEG)/35% 0.9% saline) or nimodipine (0.5, 1.0, 3.0, or 5.0 mg/kg, i.p., dissolved in 65% PEG/35% 0.9% saline; Tokyo Chemical Industry (TCI)). Sensorimotor tests To determine if administration of nimodipine affected rats¶ ability to perform an escape from the light compartment during memory retention testing 48 hr posttrial, sensorimotor function was also assessed immediately following memory retention testing. Each task below was assessed once per rat. " " )" " Wire Hang A wire-hang test was used to measure strength and endurance. The rat was suspended grasping by its forelimbs a wire (200 mm thick) 40 cm above a foam cushion, and the latency to drop was recorded. A greater latency was taken as an indication of better strength or motor endurance. Narrow Beam A narrow beam test was used to measure balance/agility. The rat was placed midway down a 1.2 m long x 2.5 cm beam that had 25 cm2 wooden escape platforms on either end and was 80 cm above a foam cushion. The latency to reach either end of the beam was recorded, with a shorter latency indicating better balance or agility. 45° Incline A 45° incline test measured vestibular processing. The rat was placed facing downward on a 45° incline apparatus (33.5 cm long x 23.5 cm wide x 42 cm high) and the latency to turn facing upward was recorded. Subjects that continuously climbed down the 45϶ incline, rather than turning around, were excluded. A shorter latency was considered a measure of better vestibular processing. Blind Alley Contextual processing was assessed using a blind alley test. The rat was placed face first into an enclosed alley (29 cm long x 9.5 cm wide x 24 cm high) with walls on three sides of the apparatus. The time it took the rat to turn around and face the opening was recorded. Better contextual processing was inferred " " *" " from a short latency. Data analysis Inhibitory avoidance task To compare initial escape latencies to 48 hr retention escape latencies across sex and dose of nimodipine, we used a 2-way ANOVA, followed by an unpaired t-test to compare sex differences and a paired t-test to compare dosedependent differences. To analyze the effect of nimodipine on retention escape latencies, we used a one-way ANOVA with escape latency as the dependent variable and nimodipine dose group as the independent variable (Fig. 2). Significant interactions were further analyzed by Fisher post hoc analysis. Sensorimotor tasks To compare mean latencies on the 4 sensorimotor tasks (narrow beam, wire hang, 45° incline, and blind alley), we used a one-way ANOVA with the task latency as the dependent variable and drug treatment group as the independent variable. Significant interactions were further analyzed by Fisher post hoc test." " " !+" " Results: Memory for inhibitory avoidance learning in male and female FBN rats A 2-way ANOVA for retention latency yielded no significant drug x sex interaction (F4,76=0.217, p=0.9284), and a series of planned comparisons were run using un-paired t-tests for sex but yielded no specific difference when comparing between males and females in any of the drug groups (0.0 mg/kg, t(11)=1.949, p=0.0773; 0.5 mg/kg, t(18)=1.006, p=0.3279; 1.0 mg/kg, t(16)=1.296, p=0.2134; 3.0 mg/kg, t(14)=0.938, p=0.3643; 5.0 mg/kg, t(17)=0.621, p=0.5428); therefore, the data for the two sexes were combined for all further analyses. As seen in Figure 1, when collapsed across conditions and sexes, all rats tested 48 h later exhibited longer retention escape latencies compared to initial escape latencies (t(85)=8.012, p<0.0001), indicating all rats, regardless of sex, exhibited memory for the earlier aversive experience. Drug treatment effects below are collapsed across sexes. Post-training nimodipine enhanced memory As seen in Figure 2, rats in each treatment condition exhibited memory for the aversive foot shocked paired with entry to the dark compartment, with significantly longer retention escape latencies compared to their initial escape latencies (paired t-test: 0.0 mg/kg, t(12)=2.657, p=0.0209; 0.5 mg/kg, t(19)=4.077, p=.0006; 1.0 mg/kg, t(17)=4.317, p=0.0005; 3.0 mg/kg, t(15)=3.609, p=0.0026; 5.0 mg/kg, t(18)=4.689, p=0.0002). " " !!" " The specific L-type Ca2+-channel blocker nimodipine enhanced memory for the aversive IA task (Fig. 2). At all doses tested, nimodipine treatment increased escape latencies, with the dose-response curve taking a general inverted-U shape, typical of nootropic drugs. Forty-eight hr after acquisition of the inhibitory avoidance task, nimodipine dose-dependently increased escape latencies compared to those of control animals, as determined by one-way ANOVAs (F4,81=2.609, p=0.0415). Significant increases in escape latencies were seen at doses of 0.5 mg/kg and 1.0 mg/kg nimodipine (Fisher test; p=0.0077 and p=0.0052, respectively; Fig. 2). Nimodipine had no effect on sensorimotor performance by FBN rats To verify that the nimodipine-induced enhancement of memory (increases in retention latency) were not simply due to impairments in sensorimotor functions, each rat was tested on four sensorimotor tasks immediately after retention testing. Administration of nimodipine had no effect on performance of any of the sensorimotor tasks in male rats, as determined by one-way ANOVAs (wire hang: F4,36=0.66, p=0.62; narrow beam: F4,36=0.38, p=0.82; 45o incline: F4,33=0.67, p=0.61; blind alley: F4,36=0.57, p=0.69). Similarly, administration of nimodipine had no effect on any of the sensorimotor tasks in female rats, as determined by oneway ANOVAs (wire hang: F4,14=1.23, p=0.34; narrow beam: F4,14=0.33, p=0.85; 45o incline: F4,14=0.80, p=0.55; blind alley: F4,14=0.97, p=0.46). " " !#" " Discussion: FBN rats successfully remembered a single-trial aversive event in an inhibitory avoidance task (see Fig. 1), with significantly increased escape latencies when tested 48 hr after a single aversive foot-shock trial compared to initial escape latencies. The specific L-type Ca2+-channel blocker nimodipine (Fig. 2) dose-dependently increased these escape latencies compared to controls when rats were retested 48 hr post-training, an operational measure of enhancement of memory consolidation for the task. Ca2+ regulation is critically important for synaptic plasticity, learning, and memory. Ca2+ influx through voltage-gated Ca2+-channels during action potential firing increases cytosolic Ca2+ (McAfee and Yarowsky, 1979; Freschi, 1983). In hippocampal CA1 neurons, calcium entry via L-type Ca2+-channels can activate sAHPs, which decrease neuronal excitability and impair learning and memory (Landfield and Pitler, 1984; Disterhoft et al., 1996; Thibault et al., 2001; Forette et al., 2002). Learning-dependent reductions in AHP amplitudes and durations have been observed after learning numerous tasks (Moyer et al., 1996; Thompson et al., 1996; Saar et al., 1998; Tombaugh et al., 2005; Farmer and Thompson, 2012). Specifically, nimodipine has been used dose-dependently to enhance multi-trial aversive learning (eyeblink conditioning: Deyo et al., 1989; Straube et al., 1990; Thompson et al., 1992; Kowalska et al., 1994) and to enhance hippocampal pyramidal neuron excitability in vivo (Thompson et al., 1990) and in vitro (Moyer et al., 1992). " " !$" " During normal aging and in dementias (including AD) learning is impaired; this impairment is associated with increases in cytosolic Ca2+ levels (for review see Zündorf and Reiser, 2011; Garwood et al., 2013). L-type Ca2+-channel antagonists have been tested for use in alleviating dementia-like symptoms. When Bachmeier et al. LQWUDFUDQLDOO\DGPLQLVWHUHGKXPDQ$ȕ1-42 into the caudateSXWDPHQRIPLFHWKH\IRXQGDQLQFUHDVHLQ$ȕ1-42 clearance from the brains of mice treated with in vivo administration of the dihydropyridines nitrendipine, nilvadipine, or cilnidipine compared to controls. These mice also showed greater memory for the spatial Morris water maze task than controls (Bachmeier et al., 2011). López-Arrieta and Birks (2002) found that administration of another L-type calcium-channel antagonist, nimodipine, enhanced cognitive function in human patients with dementia. Furthermore, Forette et al. (2002) found that elderly human subjects administered nitrendipine for an average of 3.4 years had a 55% decrease in incidence of dementia or AD compared to those administered a placebo for an average of 3.8 years. When drugs are administered prior to acquiring a behavioral task, both acquisition and memory consolidation are affected, as McGaugh and Petrinovich (1959) detailed in early studies. However, when a drug is administered within 30 minutes after acquisition, only consolidation memory is affected (McGaugh et al., 1962; McGaugh and Krivanek, 1970; for review see McGaugh and Roozendaal, 2009). Similarly, we showed that nimodipine, an L-type specific Ca2+-channel blocker, when administered immediately post-trial, enhanced single-trial aversive " " !%" " memory, as evidenced by longer escape latencies 48 hr following training on a single-trial inhibitory avoidance task. These findings are consistent with numerous other studies demonstrating that the consolidation of learning and memory is enhanced by calcium antagonists, with L-type channel blockers accounting for most of the improvement (Deyo et al., 1989; Fanelli et al., 1994; Quartermain et al., 2001; Schafe, 2008). This enhancement of newly acquired memories, however, is contrary to what has often been reported for extinction of aversive memories. Several studies have found that L-type Ca2+-channel blockers impair extinction (Cain et al., 2005; Busquet et al., 2008; McKinney et al., 2008; Waltereit et al., 2008; Milad et al., 2009; Davis and Bauer, 2012). Waltereit et al. (2008) found that peripheral injections of nifedipine impaired extinction for contextual or auditory fear tasks, and Davis and Bauer (2012) found impaired extinction memory 24 hr after nifedipine was infused into the BLA. While no studies have shown sex-differences in L-type Ca2+-channel blockade on fear extinction, Milad et al. (2009) found that female rats with low estradiol (E2) levels had lower extinction levels compared to females with high E2 levels and males, and when the estrous cycle phases are not taken into consideration, there were no differences in fear extinction in female rats. Our data also indicate no sex-specific differences. Our data are consistent with the hypothesis that L-type Ca2+-channel blockers facilitate memory for an aversive inhibitory avoidance task. We found that post-training injections of nimodipine (Fig. 2) dose-dependently enhanced memory " " !&" " when retested 48 hr later. This is consistent with earlier studies showing that these Ca2+-channel blockers decrease AHP amplitudes (Moyer and Disterhoft, 1994; Borde et al., 2000) and facilitate memory (Deyo et al., 1989b; Shinnick-Gallagher et al., 2003) in other tasks. In our hands, the dihydropryidine nimodipine significantly enhanced escape latencies at doses ranging from 0.5-1.0 mg/kg. While dihydropyridines (Ramdas et al., 2011) can produce side effects, no sensory or motor effects that would affect retest (memory) performance were observed across the range of doses tested here. It is likely that dihydropyridines or other Ltype blockers could have relatively fewer side effects in human studies, as much lower doses would be needed, and should not affect the other voltage-gated Ca2+channel families. L-type blockers also would not interfere with NMDA receptor function. " " !'" " Conclusions: Our data show the L-type Ca2+ channel blocker nimodipine dosedependently enhanced memory for a single-trial inhibitory avoidance task. These results provide further insight into the role of calcium influx in learning and memory." " " !(" " Acknowledgements: This study was funded by the Clark Foundation. We thank Drs. E.J. Donzis and J.R. McReynolds for their advice and commentary; M. Montgomery, A. Partin, and A. Ho for their technical assistance; and Dr. C.K. 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No significant differences in escape latencies were observed comparing between males and females at any of the drug doses tested, so the data for the two sexes were combined. Rats that received immediate post-training injections of nimodipine (0.5-1.0 mg/kg) had significantly longer escape latencies than control rats (p<0.01). Values reported are means ± SEM; *p<0.01. " " 7DEOH Nimodipine treatment number of rats dose (mg/kg) males females ! total 0.0 6 7 13 0.5 9 11 20 1.0 7 11 18 3.0 7 9 16 5.0 9 10 19 total 38 48 86 )LJXUH ! )LJXUH !