Impaired extinction of fear and maintained amygdala-hippocampal theta synchrony in a mouse model of temporal lobe epilepsy

Authors


Address correspondence to Thomas Seidenbecher, Institute for Physiology I, Robert-Koch Strasse 27a, 48149 Muenster, Germany. E-mail: seidenbe@uni-muenster.de

Summary

Purpose: The relationship between epilepsy and fear has received much attention. However, seizure-modulated fear and physiologic or structural correlates have not been examined systematically, and the underlying basics of network levels remain unclear to date. Therefore, this project was set up to characterize the neurophysiologic basis of seizure-related fear and the contribution of the amygdala-hippocampus system.

Methods: The experimental strategy was composed of the following steps: (1) use of the mouse pilocarpine model of temporal lobe epilepsy (TLE); (2) behavioral analyses of anxiety states in the elevated plus maze test, light–dark avoidance test, and Pavlovian fear conditioning; and (3) probing neurophysiologic activity patterns in amygdala-hippocampal circuits in freely behaving mice.

Results: Our results displayed no significant differences in basic anxiety levels comparing mice that developed spontaneous recurrent seizures (SRS) and controls. Furthermore, conditioned fear memory retrieval was not influenced in SRS mice. However, during fear memory extinction, SRS mice showed an extended freezing behavior and a maintained amygdala-hippocampal theta frequency synchronization compared to controls.

Discussion: These results indicate specific alterations in conditioned fear behavior and related neurophysiologic activities in the amygdala-hippocampal network contributing to impaired fear memory extinction in mice with TLE. Clinically, the nonextinguished fear memories may well contribute to the experience of fear in patients with TLE.

A frequent and clinically important comorbid disorder in patients with epilepsy is fear and anxiety (Vazquez & Devinsky, 2003; Beyenburg et al., 2005; Pauli & Stefan, 2009). Up to 50–60% of patients with chronic epilepsy have various mood disorders including depression and anxiety (Beyenburg et al., 2005), and among all types of epilepsy, temporal lobe epilepsy (TLE) is most frequently associated with ictal and interictal fear (Cendes et al., 1994; Feichtinger et al., 2001). The experience of anxiety reported by patients before or in between the occurrence of temporolimbic seizures has been attributed to activation of the amygdala and/or hippocampus (Gloor et al., 1982), which are critically involved in both the pathogenesis of TLE (Löscher, 1998) and fear-related behavior (LeDoux, 2000).

One established experimental paradigm to study fear memory processes is Pavlovian fear conditioning, in which neutral cues (tone or light), paired with aversive outcomes, come to elicit typical fear responses and in which a subject learns to predict danger in the environment (LeDoux, 2000). When conditioned cues no longer predict danger, as can be experimentally modeled through repetitive presentations of nonreinforced cues, fear responses decline: a behavioral phenomenon known as extinction (Maren & Quirk, 2004). On the neural network level, fear-associated stimuli elicit theta activity within the amygdala (Pare et al., 2002) and the hippocampus (Moita et al., 2003), and between these two areas (Seidenbecher et al., 2003; Narayanan et al., 2007a,b), sustaining theta synchrony as a key mechanism underlying sensorimotor integration (Bland & Oddie, 2001).

We propose that the synchronized network oscillations observed in long-term fear memory retrieval could be interconnected with the abnormal fear manifestation in TLE. To investigate the patterns of coherent amygdala-hippocampal theta activities in TLE, we used the pilocarpine mouse model that was first described by Turski et al., (1984). This widely used method, inducing TLE in rodents, leads to a status epilepticus (SE) that can last for several hours. A somewhat variable latent period leads to the chronic phase, characterized by the occurrence of spontaneous seizures (Cavalheiro et al., 1996). Neuropathologic changes, such as cell loss in several hippocampal subfields and mossy fiber sprouting to the supragranular molecular layer, closely resemble those observed in human (Turski et al., 1984; Cavalheiro et al., 1996; Yilmazer-Hanke et al., 2000; Gröticke et al., 2007). Furthermore, histopathologic and molecular changes in temporal lobe brain areas contribute to processes of epileptogenesis (Löscher, 1998).

To characterize neurophysiologic correlates of epilepsy-related alterations in fear behavior and memory, we combined the pilocarpine model of TLE with behavioral analyses of anxiety states, Pavlovian fear conditioning, and probing neurophysiologic activity patterns in amygdala-hippocampal circuits in freely behaving mice.

Material and Methods

Animals

Adult male C57Bl/6N-Crl mice (Charles River Laboratories, Sulzfeld, Germany), single housed in standard light–dark conditions (12/12 h) with access to food and water ad libitum, were used for the experiments. All experiments were carried out in accordance with the European Committees Council Directive (86/609/EEC) for experimentation on animals. Protocols were approved by the Bezirksregierung Münster (AZ 50.0835.1.0, G 53/2005).

Treatment

For the induction of a SE 8- to 12-week-old mice were injected with a single dose of 340 mg/kg pilocarpine subcutaneously (s.c.), after pretreatment with butylscopolamine 1 mg/kg (i.p.) 20 min prior to injection of pilocarpine to reduce the peripheral cholinergic effects. Acute seizures were classified using a modified Racine Scale (Borges et al., 2003). Once SE was reached, it was interrupted by an injection of diazepam (Valium; 4 mg/kg, i.p.). At least for 2 days following SE induction, all mice received oral glucose administration and soaked food. Mortality rate after SE induction was about 50%. Post–pilocarpine injection, animals were video monitored 2 days per week (2 h each) during weeks 8–12 and video monitored (5.5 h) 6 days during weeks 12–14 including field potential recordings in the freely behaving animals (see below and Fig. S1). Classification of animals as the SRS (spontaneous recurrent seizures) group was based on occurrence of spontaneous behavioral seizures of stage III–V (n = 10), supported by high-voltage spike activity recorded in amygdala, hippocampus, or both regions, in a subpopulation of animals (n = 3 of 10). Pilocarpine-treated animals without any observed epileptic-like activities (neither recurrent seizures nor high-voltage spike activity) were grouped as NOS (no observed spontaneous recurrent seizures). Control mice were treated the same manner, except that they received 0.2 ml 0.9% NaCl instead of pilocarpine.

Behavioral protocol

Three months after SE induction, mice were tested for general anxiety using the light–dark avoidance test (L/D) on day 1 followed by the elevated plus maze test (EPM) on day 2. On day 3, mice underwent surgery under maintained and controlled general anesthesia [stainless steel electrodes were implanted in the lateral amygdala (LA) and CA1 region of the dorsal hippocampus in the left hemisphere]. After surgery, the mice had a recovery period of at least 4 and up to 7 days before being taken for further behavioral experiments. The following experiments consisted of a 4-day fear conditioning protocol (see later) during which field potential activity was recorded from the LA and CA1 region in freely behaving mice.

Light–Dark Test

The Light–Dark Test was introduced by Crawley & Goodwin, (1980) to measure anxiety-like behavior. The test depends on the innate aversion of rodents to illuminated open areas. The animals have the choice to stay in the safe area, the dark compartment, or to explore the brightly illuminated aversive area, the light compartment.

Light (30 × 20 × 25 cm, illumination 150 lux) and dark compartments (15 × 20 × 25 cm) were connected by a 4-cm opening door (Fear Conditioning System; TSE Systems GmbH, Bad Homburg, Germany). The experiment started by placing the animal in the light compartment. Locomotor activity, numbers of transitions, total time spent in the dark and light compartment, and latency until the first exit from the light compartment were monitored over a period of 5 min, quantified, and stored on video tape.

Elevated-Plus-Maze test (EPM)

The EPM is one of the most frequently used tests on anxiety-like behavior in rodents and consists of two protected (closed with sidewalls) and two unprotected (open without sidewalls) arms. Mice tend to avoid the open elevated arms. Increased activity and time spent on the open arms reflect less anxiety-like behavior.

The EPM in this study consisted of two open (30 × 5 cm) and two wall-enclosed arms (30 × 5 × 25 cm) connected by a central platform (5 × 5 cm). Light intensity on all arms was 250 lux. The apparatus was elevated 75 cm above the floor. Behavioral testing started by placing the mouse on the central platform facing a closed arm. Exploratory behavior (numbers of entries into open and closed arms, total locomotor activity) and time spent on open and closed arms were monitored (Video-Mot II; TSE Systems GmbH) over a period of 5 min, quantified and stored on video tape. Entries are defined as the body center of an animal entering a new zone.

Surgery

Under deep pentobarbital (50 mg/kg, i.p.) anesthesia, stainless steel electrodes were positioned unilaterally into the left hemisphere at the position AP −1.94 mm, ML 1 mm from, and DV 1.25 mm from bregma (pyramidal cell layer of CA1 of the dorsal hippocampus) and AP −2.06 mm, ML 3.25 mm, and DV 3.2 mm from bregma for the LA (Franklin & Paxinos, 1997). Stainless steel screws connected to silver wires were implanted close to the midline over the nasal and cerebellar regions for reference and ground, respectively.

Fear conditioning

The fear conditioning protocol started at least 4 days after surgery. Animals were adapted twice to the fear conditioning apparatus (TSE Systems GmbH) and six neutral tones (CS−; 2.5 kHz tone, 85 dB, stimulus duration 10 s, interstimulus interval 20 s; intertrial interval 6 h). On the next day, fear conditioning was performed through two trials of three randomly presented CS+ (10 kHz tone, 85 dB, stimulus duration 10 s, randomized interstimulus interval 10–30 s; intertrial interval 6 h); each stimulus was immediately followed by an unconditioned stimulus (US) presentation (scrambled foot shock of 0.4 mA, duration 1 s). Twenty-four hours later (day 3), animals were transferred to the retrieval environment (novel context) and exposed to six retrieval sessions (R1–R6) for extinction training (intertrial interval 30 min), each consisting of a set of 4 CS− and 4 CS+. After 24 h, recall of extinction (E) was tested following the same protocol as for one retrieval session (Fig. S2). Neural activities were simultaneously recorded with animal behavior during the presentation of the first CS− and first CS+.

Behavioral analyses

Freezing, immobility except for respiratory movements, was taken as a behavioral measurement of fear. In each session (R1–R6, E) freezing time was calculated as percentage during the first CS− and CS+ presentations. Furthermore, additional expressed behaviors were monitored, for example, risk-assessment (alert observing and stretched attending), rearing, and exploration.

Field potential recording

During recording, the animals were connected to a swivel commutator that was linked to an amplifier for tracking and to a personal computer for data storage. Local field potential (LFP) waveforms were fed through a differential amplifier (Science Products DPA-2F, Science Products GmbH, Hoffheim, Germany), band pass filtered from 1–30 Hz, transformed by an A/D interface (CED Power 1401, sampling rate: 1 kHz), and stored on a personal computer. LFPs from CA1 and LA were recorded simultaneously during the retrieval (R1–R6) and recall of extinction (E) sessions while monitoring behavior. Color-coded time-frequency spectrograms were calculated via Fourier-wavelet transformation of the time-domain data (The MathWorks, Natick, MA, U.S.A.). In other words, the wavelet transformation is a time-frequency analysis of electroencephalography (EEG) and field potential signals (see Saab et al., 2005). Cross-correlograms during the presentation of the first CS− and CS+ as well as freezing-related correlations during the first CS− and CS+ presentation (at least 3 s of freezing) were calculated using Spike 2 (Cambridge Electronic Design, Cambridge, United Kingdom), and the y-value of the second positive peak, corresponding to the theta frequency peak, was taken to quantify correlation levels between two recording areas. The reference channel was set to CA1 for LA–CA1 cross-correlograms (see Seidenbecher et al., 2003).

Data analyses

One-way analysis of variance (ANOVA) was used to compare the effects of spontaneous recurrent seizures on general anxiety (L/D, EPM) between the three different groups (SRS, NOS, controls). Data are presented as mean with standard error of the mean (±SEM).

To analyze the freezing and correlation results of the fear retrieval protocol, three-way ANOVA with repeated measurements was used. Statistical analyses checked the effects of treatment (control, SRS, NOS) as independent variables and CS presentation (CS−, CS+, two levels) and retrieval session (R1–R6, E, seven levels) as dependent variables, followed by Tukey’s honestly significant difference (HSD) post hoc test analysis for multiple comparisons. For further evaluation, paired and unpaired t-tests were used, as applicable. Freezing related cross-correlograms were compared by using Mann-Whitney U-test. Statistical analysis was computed with STATISTICA 8.0 (StatSoft, Tulsa, OK, U.S.A.). The levels of significance were set at * p < 0.05 and ** p < 0.01.

Results

No effects of epilepsy on anxiety-like behavior

The results of the light–dark avoidance test 3 months after SE during the 5 min test period are illustrated in Fig. 1. No significant differences between mice with SRS, with no observed spontaneous recurrent seizures (NOS) and saline controls were detected. Total time spent in the light, numbers of transitions, and latency until the first exit from the light did not show any significant differences between the groups.

Figure 1.


Anxiety-like behavior is not affected by seizure activity 3 months after SE in the Light–Dark Test. Diagrams of average time spent in the light compartment, transitions between the compartments, and latency to emerge from the light compartment of the Light–Dark Test. Results revealed no significant differences between the groups. Values are mean + SEM.

Similar to the findings in the light–dark avoidance test, no significant differences of anxiety-like behaviors were seen in the EPM (Fig. 2). As shown in Fig. 2, the time spent on the open and closed arms, the entries into the open and closed arms, as well as the total locomotor activity were not significantly different. These observations indicate that in these tests general anxiety is not affected 3 months after status epilepticus.

Figure 2.


Anxiety-like behavior is not affected by seizure activity 3 months after SE in the EPM. Diagrams of average time spent on open/closed arms, entries into open/closed arms, and total locomotor activity of the EPM. Results revealed no significant differences between the groups. Values are mean + SEM.

Extinction of cued fear is impaired in SRS animals

The percentage of time spent freezing during the first CS− and CS+ presentations across retrieval of conditioned fear (R1), extinction training (R2–R6), and recall of extinction (E) was quantified and taken for analysis. Three-way ANOVA with repeated measurements with treatment (control, SRS, NOS) as independent variable and CS presentation (CS−, CS+, two levels) and retrieval session (R1–R6, E, seven levels) as dependent variables revealed the following main effects: The effect of treatment was significant between the three different groups (F(2,30) = 3,51, p < 0.05). Furthermore, CS presentation (F(1,30) = 144.59, p < 0.001) and retrieval session (F(6,180) = 21.76, p < 0.001) showed a highly significant main effect. No significant interaction could be detected. Post hoc analysis with Tukey`s HSD test revealed a significant treatment effect between the control and SRS group (p < 0.05), and a highly significant effect of retrieval session in R3–R6 (R3, p < 0.001; R4, p < 0.001; R5, p < 0.001; R6, p < 0.001) and recall of extinction (E, p < 0.001), always compared to R1.

The freezing response was specific to the cue associated with the foot shock (CS±) and not generalized

To assess fear specificity to the cues, freezing was measured during the first CS− presentation (neutral tone) across extinction training (R1–R6) and recall of extinction (E). All groups showed little freezing to the CS− compared to the CS+ across all sessions with no significant differences between the groups (Fig. 3A), thus demonstrating that their freezing response was specific to the cue associated with the footshock (CS+) and not generalized.

Figure 3.


Effects of epilepsy during retrieval and extinction of conditioned fear. Averages of relative freezing duration upon first CS− (A) and first CS+ (B) presentations during extinction learning (R1–R6) and recall of extinction (E). (A) All groups showed little freezing to the CS− across all sessions compared to CS+ presentation in (B). (B) Control mice showed reduced freezing to the CS+ presentation from R1 to R6, which remained low during E. SRS mice displayed equal levels of freezing in R1 compared to controls (fear memory consolidation is not affected), but showed a pronounced freezing response across all sessions with the exception of R6 (impaired extinction of cued fear). NOS animals did not differ significantly from control and SRS animals. Data are mean ± SEM. Asterisks indicate differences in freezing in a respective session of the SRS group compared to control. Innergroup results (R2–R6, E always compared to R1) are not presented in this Figure. (see Results).

Although fear memory retrieval is not affected, extinction of cued fear is impaired in SRS animals

For the first CS+ presentation, control mice showed a reduction in freezing from R1 to R6, and freezing remained relatively low during E, demonstrating successful extinction of fear (paired t-test compared to R1; R3 p < 0.05, R4 p < 0.001, R5 p < 0.001, R6 p < 0.001, E p < 0.01; Fig. 3B). SRS mice displayed an impaired extinction learning, with high freezing levels across the retrieval sessions with the exception of R6 (paired t-test compared to R1; R6 p < 0.05) as well as an impaired recall of extinction on the next day (E, not significant). When compared to controls, animals of the SRS group showed significantly more freezing at R4 and R5 (unpaired t-test, p < 0.05 and p < 0.01, respectively; Fig. 3B). Therefore, SRS mice are critically impaired in extinction learning (R1–R6) and in recall of extinction (E). NOS animals showed a significant reduction in freezing from R1 to R6 and at E (paired t-test compared to R1; R5 p < 0.05, R6 p < 0.001, E p < 0.05), also demonstrating successful extinction of fear but not significantly different from SRS or controls (Fig. 3B). Retrieval of conditioned fear (R1) revealed no significant differences between the three groups, meaning that, fear memory consolidation is not affected by the experience of SE.

Cue-related theta synchrony between LA and CA1 during extinction

Simultaneous to the behavior, field potential recordings were analyzed from the CA1 region of the hippocampus (CA1) and the lateral amygdala (LA) during the first CS− and CS+ presentations from retrieval 1 (R1) to recall of extinction (E) to assess the synchronized activities of both brain structures. We used cross-correlation analysis to measure the degree of synchronization. Three-way ANOVA with repeated measurements was conducted with treatment (control, SRS, NOS) as independent variable and CS presentation (CS−, CS+, two levels) and retrieval session (R1–R6, E, seven levels) as dependent variables. The main effect of treatment was not significant between the three different groups, whereas CS presentation (F(1,24) = 18.74, p < 0.0001) and retrieval session (F(6,144) = 3.14, p < 0.001) showed a significant main effect. No significant interaction could be detected between the three variables. Because a significant group difference could not be detected, we analyzed in a next step each experimental group separately. Apparently, no significant effect could be shown for all three experimental groups comparing the correlation values of the first CS− in each retrieval session (R2–R6, E) compared to R1 (paired t-test). For the first CS+ presentation LA–CA1 correlation values significantly decreased in control mice during R3–R6 when compared to R1 (paired t-test; R3, p < 0.05, R4, p < 0.05, R5, p < 0.01, R6, p < 0.05; Fig. 4A). SRS mice did not display this decrease, with the LA–CA1 correlation values remaining high across extinction training, paralleling the observed persistence in fear behavior (Fig. 4A). Similar to freezing behavior, LA–CA1 correlation values of the NOS mice are positioned in between control and SRS group. They also showed a reduction in correlated activities from R1 to R6 (paired t-test compared to R1; R3, p < 0.05, R5, p < 0.05, R6, p < 0.01), which parallels the successful extinction of fear (Fig. 4A). Examples of the neural activity and color-coded time-frequency spectograms of the LA and the CA1 and the cross-correlograms between these brain areas of a fear conditioned control and SRS animal are shown in Fig. 5.

Figure 4.


Comparison of averaged CS+ and freezing-related amygdala-hippocampal cross-correlations. Average values of the first CS+ (A) and freezing-related (B) correlations between CA1 and LA. Controls show significant decreased correlation values during extinction learning (R1–R6) in cue (CS+) (A) and freezing-related (B) correlations. During recall of extinction (E), the correlation values receive nearly the initial value of R1. SRS animals do not show any significant changes during extinction learning and recall of extinction in cue (A) and freezing-related (B) correlations. NOS animals showed significant decrease in CS+-related (R3, R5, and R6) but not in freezing-related correlations. Data are mean + SEM. # indicates significant inner group differences compared to R1.

Figure 5.


Neural activity in the LA and CA1 in a fear-conditioned control (A) and SRS (B) animal during presentation of first CS+ in retrieval sessions R1, R6, and recall of extinction E. Original traces of field potential recordings in the LA (upper trace) and the CA1 (bottom trace) during CS+ presentation. (b) Color-coded time-frequency spectrograms calculated via Fourier-wavelet transformation of the original traces in (a). The color-code describes high-power spectral density in red and low power spectral density in blue. Characters indicate the behavior of the animal (e, exploration; r, risk-assessment; f, freezing). (c) Cross-correlations of the activities in the LA and the CA1 during CS+ stimulus presentation (10 s; black curve) and during periods of freezing (freezing-related cross-correlations; red curve). Note the decrease in correlated activity (stimulus and freezing-related) overextinction learning in the control (c, R1 and R6) and the unchanged high freezing-related correlation at R6 of the SRS group.

Freezing-related theta-phase synchronization

All groups showed similar levels of freezing-related correlation during retrieval of conditioned fear (R1). Controls showed significant decreased correlation values during extinction learning (Mann-Whitney U-test compared to R1; R4 p < 0.05, R5 p < 0.05; R6 p < 0.05; Fig. 4B). During recall of extinction (E) the correlation values nearly reach the initial value of R1. SRS and NOS animals did not show significant changes during extinction learning and recall of extinction in freezing related correlations (Fig. 4B).

Discussion

The major results of the present study, revealed after pilocarpine-induced SE in C57BL/6 mice, are as follows. No significant differences in basic anxiety levels were seen when comparing mice that developed spontaneous recurrent seizures (SRS), with mice with no observed recurrent seizures (NOS) and saline controls. In addition, conditioned fear memory retrieval was not affected in all experimental groups. However, during fear memory extinction, SRS mice showed an extended freezing behavior and maintained amygdala-hippocampal theta frequency synchronization compared to controls. These data indicate an impairment of fear memory extinction in mice with temporal lobe epilepsy on the behavioral as well as on the neurophysiologic level.

Anxiety-like behavior in the Light–Dark Test and the EPM was not affected after pilocarpine injection in either group of animals. Previous studies in the pilocarpine mouse model have shown anxiety-like behavior in the open field and Light–Dark Test but not in the EPM in epileptic animals (Gröticke et al., 2007; Müller et al., 2009a). These results are in line with our EPM data but differ regarding the light–dark data. The following might help to explain this difference: First, both previous studies used a ramping-up dosing protocol for pilocarpine treatment compared to a single bolus injection in the present study. We decided to use bolus injection of pilocarpine to keep handling and multiple injection stress-induced changes minimal. In fact, pilot experiments had shown that repeated injection of vehicle alone resulted in generalized fear and impaired conditioned fear extinction compared to single injected controls (Fig. S3). Second, Müller and colleagues described substrain and even subline differences of Bl6 mouse strains in vulnerability to pilocarpine treatment (Müller et al., 2009b). Particularly, sublines of B6NCrl mice differed in SE induction and mortality rate, even if derived only from different barrier rooms of the same vendor.

Previous studies in the pilocarpine rat model (Dos Santos et al., 2005; Szyndler et al., 2005) found a reduction in freezing upon both recall of cued and contextual fear, which was not altered in the present study in mice. These conflicting results might be due to differences in the model (rat vs. mouse pilocarpine model). Differences were evident, however, in responsiveness during fear extinction. In the SRS group, fear responses remained high during fear extinction training and recall of extinction. The cellular correlate of this behavioral deficit may relate to degenerative effects resulting from pilocarpine treatment in the amygdala and/or hippocampus, structures that are critically involved in fear behavior expression and extinction (LeDoux, 2003). Alterations include mossy fiber sprouting, neuronal loss, and cell damage in the CA1, CA3, hilus, and dentate gyrus of the hippocampus as well as morphologic changes of amygdaloid neurons (Turski et al., 1984; Borges et al., 2003). These cellular degenerations might entail changes of the inhibitory and excitatory network activity of the affected brain areas and, therefore, related behaviors.

A decrease in inhibitory γ-aminobutyric acid (GABA)ergic synaptic influence is considered a critical element contributing to hyperexcitability of amygdalar synaptic circuits in a number of experimental models of epilepsy (Gean et al., 1989; Klueva et al., 2003). Pilocarpine treatment in rats has been found to result in a decrease in the number of GABAergic neurons in the amygdala, in particular the somatostatin-containing GABAergic population (Tuunanen et al., 1996). GABAergic interactions in amygdaloid networks appear to be specifically involved in generating response specificity to fear-conditioned stimuli (Ehrlich et al., 2009), and two major populations of GABAergic neurons can be discerned in the amygdala: “local” GABAergic interneurons scattered in the local neuropil and paracapsular GABAergic intercalated cell masses. The latter are prime candidates that mediate medial prefrontal cortex influences during conditioned fear extinction (Pare et al., 2004; Jüngling et al., 2008). Furthermore, cued fear memory extinction is deficient in mice with genetic deletion of the GABA-synthesizing enzyme GAD65 (Sangha et al., 2009) similar to the SRS group of the present study.

Another line of evidence points to glutamatergic mechanisms involved in synaptic hyperexcitability in temporal lobe epilepsy (Gean et al., 1989). Excessive glutamatergic activity plays a key role in the induction and expression of epilepsy (Eid et al., 2008). In vitro patch-clamp recordings of our lab using the same pilocarpine model to induce recurrent spontaneous seizures revealed dynamic changes in glutamatergic transmission within the amygdala. In detail, presynaptic N-methyl-d-aspartate (NMDA) receptors in projection neurons of the lateral amygdala were functionally upregulated in pilocarpine-treated animals (Graebenitz et al., 2010). Several other studies described changes in NMDA receptor (R) associated with the development and maintenance of epileptic seizures (Morimoto et al., 2004; Huo et al., 2006). On the behavioral level it is known that extinction of learned fear is both amygdala- and NMDA-R dependent (Myers & Davis, 2007). Especially the NR2B subunit is required during the acquisition phase of extinction in the lateral amygdala and during consolidation of extinction in the medial prefrontal cortex (mPFC) (Sotres-Bayon et al., 2009). It is tempting to suggest that the observed NMDA-R–dependent increase in glutamatergic transmission in the LA (Graebenitz et al., 2010) relates to the observed extinction deficit of learned fear observed in the present study.

Deficits in fear extinction are also related to mPFC activities. The infralimbic PFC modulates amygdala-dependent fear memories and has become a central element of extinction models (Quirk et al., 2006), but the role of the PFC in temporal lobe epilepsy is only rarely understood. One study described changes of efferent projections of the entorhinal cortex to the PFC after pilocarpine treatment in mice (Ma et al., 2008). We cannot exclude changes on the neural and network levels of the prefrontal cortex that are responsible for the observed impaired extinction of fear in SRS mice.

During fear extinction, control animals showed a decline of synchronized theta activity between the LA and the CA1 region of the hippocampus in conjunction with reduced freezing behavior, whereas SRS mice displayed maintained amygdala-hippocampal theta frequency synchronization. In general, the temporal lobe shows theta frequency oscillations during emotional arousal (Charpak et al., 1995; Pare & Gaudreau, 1996; Pare et al., 2002), and recently changes in theta oscillations were described in pilocarpine induced epilepsy (Chauviere et al., 2009; Marcelin et al., 2009; Garcia-Hernandez et al., 2010; Tejada et al., 2010). In human TLE, patients show an enhancement of temporal synchronization of the theta rhythm within a cerebral network including hippocampus, amygdala, and temporal-occipital neocortex revealed in an auditory verbal learning test (Babiloni et al., 2009). Related to fear learning in mice, it has been shown previously that network activity patterns between LA and hippocampus showed an increase in theta frequency synchronization during conditioned fear responses, particularly with the retrieval of long-term fear memory, but not to short-term and remote fear memory or expression of fear behavior per se (Pape et al., 2005; Narayanan et al., 2007a,b). More recently we found a decline of synchronized theta activity between LA and CA1 during extinction learning in wild types of GAD65-deficient (Sangha et al., 2009) and Bl6 mice (unpublished data). In the present study, in comparison to controls, the theta synchronization decline during extinction learning in SRS mice failed, indicating a putative neurophysiologic correlate of impaired fear memory extinction. As already mentioned, since TLE induces a general decrease in GABAergic function (Tuunanen et al., 1996; Pitkanen et al., 1998; Klueva et al., 2003), this might also influence theta oscillations. A subpopulation of parvalbumin-positive GABAergic neurons in the amygdala is capable of synchronizing synaptically connected principal neurons in the amygdala in the theta range (Rainnie et al., 2006; Woodruff & Sah, 2007).

NOS mice showed a trend of impaired extinction learning compared to controls, whereas extinction learning appeared improved compared to SRS. Although differences were not significant, these data might indicate altered fear responsiveness developing independently of spontaneous seizure frequency, reflecting for instance an outcome syndrome of SE. However, it should be kept in mind that observation time of spontaneous seizure activity was limited in the present study and, therefore, the possibility of seizure activity in absence of observation and recording cannot be excluded.

Taken together, these results indicate specific alterations in conditioned fear behavior and related neurophysiologic activities in the amygdala-hippocampal network contributing to impaired fear memory extinction in mice with TLE. Clinically, the nonextinguished fear memories may well contribute to the experience of fear in patients with TLE.

Acknowledgments

We thank E. Boehning and S. Kiesling for excellent technical assistance and P. Berenbrock, A. Stirnnagel, and H. Bäumer for the animal care. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB-TR3, TPC3 to HCP and TPB7 to TS and HCP; SFB-TRR58, TPA2 to TS and HCP) and the Innovative Medical Research of the University of Münster Medical School (LE210613 to JL).

Disclosure

We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. None of the authors has any conflict of interest to disclose.

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