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Amygdala kindling increases fear responses and decreases glucocorticoid receptor mRNA expression in hippocampal regions
Progress in Neuro-Psychopharmacology & Biological Psychiatry 27 (2003) 1225 – 1234 www.elsevier.com/locate/pnpbp
Amygdala kindling increases fear responses and decreases glucocorticoid receptor mRNA expression in hippocampal regions Lisa E. Kalynchuka,*, Michael J. Meaneyb a
Department of Psychology and Neuroscience Institute, Dalhousie University, 1355 Oxford Street, Halifax, Nova Scotia, Canada B3H 4J1 b Department of Psychiatry, Douglas Hospital Research Center, McGill University, Montreal, Quebec, Canada H4H 1R3 Accepted 9 September 2003
Abstract Amygdala kindling dramatically increases fearful behavior in rats. Because kindling-induced fear increases in magnitude as rats receive more stimulations, kindling provides an excellent model for studying the nature and neural mechanisms of fear sensitization. In the present experiment, we studied whether the development of kindling-induced fear is related to changes in glucocorticoid receptor (GR) mRNA expression in various brain regions. Rats received 20, 60 or 100 amygdala kindling stimulations or 100 sham stimulations. One day after the final stimulation, their fearful behavior was assessed in an unfamiliar open field. Then, the rats were sacrificed and their brains were processed for in situ hybridization of GR mRNA expression. We found that compared with the sham-stimulated rats, the rats that received 60 or 100 kindling stimulations were significantly more fearful in the open field and also had significantly less GR mRNA expression in the dentate gyrus and CA1 subfield of the hippocampus. Importantly, the changes in fearful behavior were significantly correlated with the changes in GR mRNA expression. These results suggest that alterations in GR mRNA expression in hippocampal regions may play a role in the development of kindling-induced fear. D 2003 Published by Elsevier Inc. Keywords: Amygdala; Fear; Glucocorticoid receptor; Hippocampus; Hypothalamic – pituitary – adrenal axis; Kindling; Open field; Stress
1. Introduction Normal fear responses are adaptive in that they serve as protection from potential harm. However, with repeated exposure, fear can become sensitized so that it eventually results in behavioral responses that are maladaptive (Kandel, 1983; Marks and Tobena, 1990). The level of fear experienced by an animal can thus range from ‘‘normal’’ to ‘‘pathological’’ (Rosen and Schulkin, 1998). Studying the progression from normal to pathological fear is a critical approach for identifying the neural mechanisms that underlie the development of affective disorders such as generalized anxiety and panic and for developing new pharmacological treatments that can prevent or reduce these disorders. Abbreviations: CRF, corticotropin-releasing factor; HPA, hypothalamic – pituitary – adrenal; GR, glucocorticoid receptor * Corresponding author. Tel.: +1-902-494-6337; fax: +1-902-4946585. E-mail address: [email protected] (L.E. Kalynchuk). 0278-5846/$ – see front matter D 2003 Published by Elsevier Inc. doi:10.1016/j.pnpbp.2003.09.016
Kindling provides a particularly useful animal model for studying the nature and molecular mechanisms of fear sensitization (Adamec, 1990; Depaulis et al., 1997; Kalynchuk, 2000). Kindling refers to the gradual development and intensification of elicited motor seizures that result from the daily administration of initially subconvulsive stimulations to particular brain regions (Goddard et al., 1969). In addition to its epileptogenic effects, kindling also sensitizes brain circuits responsible for behavioral manifestations of fear (Adamec and Young, 2000; Kalynchuk et al., 2001; Rosen and Schulkin, 1998). For example, amygdala kindling in rats decreases exploratory behavior in an open field (Kalynchuk et al., 1999a; Nieminen et al., 1992), decreases openarm exploration on an elevated plus maze (Adamec and Morgan, 1994; Helfer et al., 1996), increases immobility in a social interaction test (Helfer et al., 1996), increases fearpotentiated startle (Rosen et al., 1996) and increases stressinduced stomach ulcers (Henke and Sullivan, 1985). The kindling model provides an excellent opportunity to study the progression from normal to pathological fear because the magnitude of kindled-induced fear increases
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with the number of stimulations. For example, relative to control rats, rats that receive 20 amygdala stimulations display significant increases in thigmotaxia in an unfamiliar open field, rats that receive 60 amygdala stimulations display significant decreases in open-field activity and increases in resistance to capture from the open field, and rats that receive 100 stimulations display all of these changes plus increases in fleeing behavior in an open-field, escape behavior in an elevated plus maze, and active defensive behaviors in a resident – intruder paradigm (Kalynchuk et al., 1997, 1999a, 2001). Studying the neural mechanisms that underlie the progression of kindling-induced fear may yield important information about the progressive development of affective disorders characterized by high levels of fear (Rosen and Schulkin, 1998). The purpose of the present study was to examine whether changes in glucocorticoid receptors (GR) are related to the progressive intensification of kindling-induced fear. GRs are intimately involved in the physiological stress response. On exposure to an acute stressor, the hypothalamic – pituitary – adrenal (HPA) axis becomes activated, evoking a series of physiological events that culminate in the release of the hormone corticosterone, which then binds to GR in several brain regions. With repeated exposure to stress, GR mRNA expression in the hippocampus is decreased (Herman et al., 1995). Interestingly, repeated HPA axis activation is thought to increase vulnerability toward the development of affective pathology (Francis and Meaney, 1999). For example, both repeated restraint stress and corticosterone administration increase fear-mediated behaviors in rats (Conrad et al., 1999; Corodimas et al., 1994). Similarly, there is some evidence to suggest that the behavioral changes produced by kindling may be mediated by the consequences of repeated HPA axis activation. A single kindling stimulation in animals that have been previously kindled elicits a rapid transient increase in circulating levels of corticosterone (Szafarczyk et al., 1986). Because animals are repeatedly stimulated during kindling, kindling itself may be a form of repeated stress, leading to repeatedly elevated levels of corticosterone, and a compensatory down-regulation of GR in hippocampal regions (Clark et al., 1994). Thus, in the present experiment, we investigated whether GR mRNA expression in limbic brain regions is related to the development of fearful behavior in kindled rats.
2. Methods 2.1. Animals The subjects were 36 male Long– Evans rats (Charles River, Montreal, Canada) weighing between 250 and 350 g at the time of surgery. They were individually housed in steel hanging cages in a colony room with an ambient temperature of about 21 jC and a 12:12-h light/dark cycle (lights on at 8:00 a.m.). Purina rat chow and water were
available continuously. All experimental manipulations were conducted in accordance with the guidelines of the Canadian Council on Animal Care. 2.2. Surgery A single bipolar stimulating electrode (MS-303-2, Plastics One, Roanoke, VA) was implanted in the left basolateral amygdala of each rat under sodium pentobarbital anesthesia (somnotol, 65 mg/kg ip). Each electrode was aimed at a site 2.8 mm posterior, 5.0 mm left and 8.5 mm ventral to the skull surface at bregma (coordinates from Paxinos and Watson, 1998). Each electrode was secured to the skull with four stainless steel screws and dental acrylic. Powdered tetracycline was sprinkled on the incision before suturing to reduce the risk of infection. Left amygdala kindling was employed in this experiment to ensure consistency with our previous experiments (Kalynchuk et al., 1997, 1998a,b, 2001; Wintink et al., 2003). The behavioral consequences of left and right amygdala kindling are not always the same (Adamec and Morgan, 1994), especially for partial and short-term kindling. All our previous experiments have been conducted with rats kindled in the left amygdala. We have thus built up a detailed understanding of the nature of the behavioral changes produced by left amygdala kindling. The purpose of this experiment was to assess whether GRs might be involved in these behavioral changes, so it was logical that we study rats that had been kindled in the left amygdala. 2.3. Kindling phase After a postsurgical recovery period of at least 12 days, the rats were divided into four groups: One group received 80 sham stimulations followed by 20 kindling stimulations (20-stim group, n = 9), one group received 40 sham stimulations followed by 60 kindling stimulations (60-stim group, n = 9), one group received 100 kindling stimulations (100-stim group, n = 10) and one group received 100 sham stimulations (sham-stim group, n = 8). The stimulations were delivered three times per day, 5 days/week, with a minimum of 2 h between consecutive stimulations. A few seconds prior to each stimulation, each rat was removed from its home cage and placed in a plastic box containing a thin layer of commercial bedding. The stimulation lead was attached and the stimulation was then delivered (1 s, 60 Hz, 400 AA square waves). After all convulsive activity had ceased, the rat was returned to its home cage. Rats receiving sham stimulations were treated in exactly the same way except that no current was delivered. The measure of seizure severity was the convulsion class elicited by each stimulation. Convulsion class was scored according to Pinel and Rovner’s (1978) extension of Racine’s (1972) widely used five-class scale (class 1: head nodding only; class 2: head nodding and jaw clonus; class 3: head nodding, jaw clonus and forelimb clonus; class 4: head
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nodding, jaw clonus, forelimb clonus and rearing; class 5: head nodding, jaw clonus, forelimb clonus, rearing and falling once; class 6: a class 5 with multiple rearing and falling episodes; class 7: a class 6 with running fits; class 8: any of the preceding symptoms with periods of tonus). 2.4. Behavioral testing All behavioral testing took place in an open field. The open field was a 60 60 60 cm box with wooden walls and 36 identical squares defined by tape on the Plexiglas floor. It was located in a small brightly lit testing room. The open field, testing room and experimenter were unfamiliar to the rats at the time of testing. The behavioral testing was conducted 1 day after the final stimulation. Each rat was placed individually into a corner of the unfamiliar open field for 5 min, while an experimenter sat quietly in the room out of sight of the rat. The rat’s behavior was videotaped from above. Later, the number of squares crossed by each rat during the open-field test was counted from a video monitor. We limited our analysis of exploration to the first 30 s because we have found that fear-related reductions in exploration between kindled and sham-stimulated rats are greatest during the first 30 s of exposure to an open field, when it is the most unfamiliar (Kalynchuk et al., 1997). After the 5 min, each rat’s resistance to being captured from the open field was assessed. The experimenter, who was wearing a leather glove that was unfamiliar to the rat, attempted to pick up each rat from above. The rat’s resistance to being picked up was scored according to the following seven-point scale:
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0 = easy to pick up, 1 = vocalizes or shies away from hand, 2 = shies away from hand and vocalizes, 3 = runs away from hand, 4 = runs away and vocalizes, 5 = bites or attempts to bite, and 6 = launches a jump attack (Kalynchuk et al., 1997). Some rats were highly resistant to being captured and eluded the gloved hand when the experimenter tried to pick them up. In these cases, the experimenter persisted and the rat was given a score based on the highest degree of resistance to capture that it displayed. The duration of fleeing from the gloved hand before being captured was also recorded for each rat. The open field was cleaned thoroughly with Fantastik solution in between each trial. 2.5. In situ hybridization Each rat was sacrificed f 72 h after the final stimulation, and its brain was rapidly removed and frozen in isopentane maintained on dry ice. Frozen 16-Am coronal sections were cut from each brain on a cryostat, thaw-mounted onto polyL-lysine-coated slides and stored at 80 jC. In situ hybridization of GR mRNA was performed as described previously (O’Donnell et al., 1994) using [35S]UTP-labeled cRNA antisense probes transcribed from a 674-bp PstI – EcoRI fragment of the rat GR cDNA (steroid binding domain: Dr. R. Meisfield, University of Arizona), linearized with AvaI and transcribed with T7 RNA polymerase. Briefly, slides were first removed from the freezer and warmed to room temperature, postfixed in 4% paraformaldehyde in 0.1 phosphate buffer (10 min at pH 7.0) and washed three times in 2 SSC (0.3 M NaCl and 0.03 M sodium citrate)
Fig. 1. The mean F S.E.M. class of convulsions produced by the third stimulation on each of the kindling days in each group of kindled rats. All rats had a minimum of five class 5 convulsions prior to the behavioral testing.
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Fig. 2. Effect of different numbers of kindling stimulations on fearful behavior. Each rat was tested in an unfamiliar open field 1 day after the final kindling stimulation. (A) Mean F S.E.M. number of squares crossed in the first 30 s of the open-field test by the rats in each group. Both 60-stim and 100-stim rats crossed significantly fewer squares during the open-field test than did the sham-stim rats ( P < .01), and the 60-stim rats also crossed significantly fewer squares than the 20-stim rats ( P < .01). (B) Mean F S.E.M. resistance to capture from the open field displayed by the rats in each group. Both 60-stim and the 100-stim rats were significantly more resistant to capture from the open field than the sham-stim rats ( P < .025), and the 100-stim rats were also more resistant to capture than the 20-stim rats ( P < .025). (C) Mean F S.E.M. duration of fleeing displayed by the rats in each group during resistance to capture testing. Both 60-stim and 100-stim rats engaged in a longer duration of fleeing than did the 20-stim or sham-stim rats ( P < .01).
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in sterile water containing 0.2% diethylpryocarbonate. Hybridization was then performed at 37 jC for 18 h in a buffer containing for 50% deionized formamide, 5 M NaCl, 1 M Tris (pH 7.5), 100 Denhardt’s solution, 0.5 M EDTA (pH 8.0), 10 mg/ml denatured salmon sperm DNA, 11 mg/ml yeast tRNA, 10% dextran sulfate, 10 mM dithiothreitol and about 1 107 cpm of [35S]UTP-labeled GR riboprobe. After hybridization, the slides were rinsed in 2 SSC and treated with RNase A for 45 min at 37 jC. Finally, the slides were washed in decreasing salt concentrations to 0.1 SSC at 60 jC, dehydrated in increasing concentrations of ethanol, dried and apposed to Hyperfilm (Amersham) for 21 days. The hybridization signal within the dorsal hippocampus and amygdala was quantified by densitometry with an image analysis system (MCID, Imaging Research, St. Catharines, ON). These brain regions were chosen for analysis based on many previous findings that repeated stress decreases GR mRNA expression in hippocampal regions (Meyer et al., 2001; Kitraki et al., 1999; Herman et al., 1995) and the intriguing recent report that chronic stress has differential morphological effects on hippocampal and amygdaloid neurons (Vyas et al., 2002). Three brain sections per animal for each brain region of interest were quantified. The data are presented as arbitrary optical density units with background subtracted.
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2.6. Statistical analyses The statistical significance of the data was analyzed in four ways. Group differences in open-field exploration and duration of fleeing were assessed using one-way ANOVAs followed by Newman –Keuls post hoc tests. Group differences in resistance to capture were assessed using a Kruskal –Wallis one-way ANOVA by ranks test followed by post hoc multiple comparisons. Group differences in GR mRNA expression were analyzed using a two-way ANOVA (Group Region) followed by Newman – Keuls post hoc tests. Finally, correlations between GR mRNA expression in specific brain regions and each of the three behavioral measures were assessed using Spearman rank order correlations.
3. Results 3.1. Kindling acquisition Fig. 1 shows the mean class of convulsions produced by the third stimulation on each of the kindling days. As expected, the initial kindling stimulations produced little behavioral response other than a momentary behavioral
Fig. 3. Effect of different numbers of amygdala kindling stimulations on GR mRNA expression. GR mRNA was assessed 72 h after the final kindling stimulation. Mean F S.E.M. levels of GR mRNA expression in hippocampal and amygdaloid regions of rats in each group are shown in arbitrary optical density units. Both 60-stim and 100-stim rats had significantly less GR mRNA expression in the dentate gyrus ( P < .01) and CA1 subfield ( P < .05) than did the sham-stim rats. Abbreviations: CeM, central nucleus of the amygdala; BLA, basolateral nucleus of the amygdala.
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arrest; however, within a few days, almost every stimulation elicited a generalized convulsion characterized by facial clonus, forelimb clonus, rearing and loss of equilibrium. The mean number of class 4 or above convulsions experienced by the rats in each group was 91.4 for the 100-stim rats (range = 86– 96), 43.3 for the 60-stim rats (range = 27 – 52) and 12.1 for the 20-stim rats (range = 8 –16). 3.2. Open-field behavior Fig. 2 shows the open-field behaviors displayed by the rats in each group. Kindling had a significant effect on each of these behavioral measures. First, during the first 30 s of the open-field session, both the 60-stim and 100-stim rats crossed significantly fewer squares than did the sham-stim rats [ F(3,35) = 10.146, P < .0001; post hoc P < .01]; the 60stim rats crossed fewer squares than the 20-stim rats ( P < .01). Second, at the end of the open-field session, both the 60-stim and 100-stim rats were significantly more resistant to capture than the sham-stim rats [H(3) = 21.524, P < .0001; post hoc P < .025]; the 100-stim rats were also significantly more resistant to capture than the 20-stim rats ( P < .025). Finally, both the 60-stim and 100-stim rats engaged in a significantly longer duration of fleeing than did the 20-stim or sham-stim rats [ F(3,35) = 12.648, P < .0001; post hoc P < .01].
Table 1 Correlations between behavioral changes and GR mRNA expression Region
Square crosses in first 30 s
Dentate gyrus CA1 CA2 CA3 Central amygdala Basolateral amygdala
.334 * .278 .1 .007 .039 .128
Resistance to capture
Duration of fleeing
.307 .451* * .157 .083 .221 .071
.366 * .376 * .24 .149 .305 .104
* Significant at P < .05. ** Significant at P < .01.
capture and GR mRNA levels in the dentate gyrus narrowly missed our defined level of statistical significance ( P < .07). Finally, the duration of fleeing during the resistance-to-capture testing was significantly correlated to changes in GR mRNA expression in both the dentate gyrus and the CA1 subfield of the hippocampus ( P < .05). None of the open-field behaviors was correlated to changes in GR mRNA levels in any of the other quantified brain regions.
4. Discussion 4.1. Relation between kindling-induced fear and GR mRNA expression
3.3. Glucocorticoid mRNA expression GR mRNA expression in various regions of the dorsal hippocampus and amygdala is shown in Fig. 3. Kindling had significant region-specific effects on GR mRNA expression. An overall two-way ANOVA revealed a significant effect of Group [ F(3,185) = 9.636, P < .0001], a significant effect of Region [ F(6,185) = 153.05, P < .0001] and a significant Group Region interaction [ F(18,185) = 1.796, P < .03]. Post hoc analyses further revealed that both the 60-stim and 100-stim rats had significantly less GR mRNA expression in the dentate gyrus ( P < .01) and CA1 subfield of the hippocampus ( P < .05) than did the sham-stim rats. 3.4. Correlations between behavioral differences and GR mRNA expression To determine whether specific changes in behavior were related to GR mRNA expression in particular brain regions, we calculated the correlation between each behavioral measure and GR mRNA levels in each brain region. These correlations are shown in Table 1. Several observations can be made from these correlations. First, exploration in the first 30 s of the open-field test was significantly correlated to GR mRNA expression in the dentate gyrus ( P < .05). Second, resistance to capture was significantly correlated to GR mRNA levels in the CA1 subfield ( P < .01). The relation between resistance to
In the present study, kindling had significant effects on both fearful behavior and GR mRNA expression. As expected, rats that received 60 or 100 kindling stimulations displayed less open-field exploration, greater resistance to capture from the open field and a longer duration of fleeing before being captured than did rats that received 20 kindling stimulations or sham stimulations. These findings are consistent with many earlier reports of fearful behavior in kindled animals (Adamec and Morgan, 1994; Helfer et al., 1996; Kalynchuk et al., 1998b; Nieminen et al., 1992; McIntyre, 1978; Rosen et al., 1996), and they also confirm our previous observations that the magnitude of kindlinginduced fear depends on the number of stimulations the rats receive (Kalynchuk et al., 1997, 2001; Wintink et al., 2003). In addition to these behavioral differences, rats that received 60 or 100 kindling stimulations also had less GR mRNA expression in the dentate gyrus and CA1 region of the hippocampus compared with the rats that received 20 kindling stimulations or sham stimulations. Importantly, specific changes in fearful behavior in the kindled rats were related to specific changes in GR mRNA expression: increased resistance to capture was significantly correlated to decreased GR mRNA expression in the CA1 region, decreased open-field exploration was related to decreased GR mRNA expression in the dentate gyrus and increased fleeing time was related to decreased GR mRNA in both dentate gyrus and CA1 subfield. These results suggest that decreased GR mRNA expression in particular hippocampal
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regions may be involved in specific aspects of kindlinginduced fear. The results of this experiment extend the previous observations of Clark et al. (1994) who assessed GR mRNA expression at different time points after f 10 amygdala kindling stimulations. They found that kindling decreased GR mRNA expression in the dentate gyrus 24 h but not 96 h after the final stimulation. No significant changes in GR mRNA levels were found in the CA1 subfield. In the present experiment, decreased GR mRNA levels were detected in both the dentate gyrus and CA1 region of the 60-stim and 100-stim rats 72 h after the final kindling stimulation. This suggests that the effect of amygdala kindling on hippocampal GR mRNA expression is longer lasting and more widespread after greater numbers of stimulations. An important next step will be to determine how long the decreased GR mRNA levels persist after 60 or 100 amygdala kindling stimulations. 4.2. Behavioral similarities between kindled rats and those with alterations in GR Kindling exerts complex effects on emotional behavior, and these effects depend on several important parameters, such as the situation in which the rats are tested (Kalynchuk et al., 1999a; Haimovici et al., 2001), the number of stimulations the rats receive (Kalynchuk et al., 1997), the site of kindling within the brain (Kalynchuk et al., 1998a; Adamec, 1998) and the strain of rat being used (Adamec, 1998). In general, rats that have received about 100 amygdala kindling stimulations tend to display panic-like active responses to stressful stimuli (Kalynchuk et al., 2001). They show extreme resistance to capture from an unfamiliar open field (Kalynchuk et al., 1997, 2001), open-arm exploration and escape behavior from an elevated plus maze (Kalynchuk et al., 1997), active defensive postures and defensive jump attacks in a resident – intruder test (Kalynchuk et al., 1999a) and enhanced startle to acoustic stimuli (Menard et al., 2002). In addition, the more novel the testing situation is, the larger the kindling-induced fear response is likely to be. For example, kindled rats show high levels of resistance to capture from an unfamiliar open field but little resistance to capture from their home cages or from a familiar open field (Kalynchuk et al., 1999a). Interestingly, the emotional consequences of kindling appear to be specific to fearrelated responses. Kindling does not increase depressionlike behavior as measured by the forced swim test or sucrose preference tests (Helfer et al., 1996; Wintink et al., 2003). In fact, after 100 stimulations, kindled rats actually show less depression-like behavior than control rats—they engage in less immobility in the forced swim test and show a greater preference for sucrose on the sucrose preference test (Wintink et al., 2003). There are some intriguing similarities between the emotional behaviors displayed by kindled rats and those observed in rodents with decreased GR function. For example,
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transgenic mice with a 50 –70% reduction in central GR levels display increased acoustic startle, increased open-arm exploration in an elevated plus maze, decreased exploration time in an open field and decreased immobility in a forced swim test (Rochford et al., 1997; Beaulieu et al., 1994; Montkowski et al., 1995; Strohle et al., 1998). Decreasing GR function in the dentate gyrus of adult rats with antisense infusions also decreases immobility in the forced swim test (Korte et al., 1996). In addition, rats exposed to maternal separation early in life show decreased GR mRNA expression in the dentate gyrus and CA1 region and decreased exploration time in an open field (Francis et al., 2002). As detailed above, kindled rats display a similar behavioral profile, providing support for the idea that decreased GR mRNA expression in the dentate gyrus and CA1 subfield play an important role in the development of kindlinginduced fear. However, this conclusion must be made tentatively because GR knockout mice appear to be less anxious than wild-type mice (Tronche et al., 1999). One explanation for this discrepancy is that GR is completely absent in the brains of the knockout mice, whereas GR function is only reduced in the brains of kindled, maternally separated and transgenic animals that have increased anxiety-like behavior. This may have functional implications. GR knockout mice have elevated basal corticosterone levels and a normal corticosterone response to acute restraint (Tronche et al., 1999), whereas kindled and maternally separated rats have normal basal corticosterone levels (Francis et al., 2002; Adamec and McKay, 1993) and maternally separated rats and transgenic mice with a 50% reduction of GR have an exaggerated corticosterone response to acute restraint (Francis et al., 2002; Stec et al., 1994) Thus, there are HPA axis differences between animals with impaired GR function and animals with absent GR function that could account for the differences in their emotional behavior. 4.3. Potential mechanisms by which kindling and GR might influence fearful behavior The mechanism by which kindling decreases hippocampal GR mRNA expression is unknown. It is not likely due to kindling-induced loss of hippocampal neurons, because we have previously found increased benzodiazepine receptor binding in the hippocampus of kindled rats subjected to 100 stimulations (Kalynchuk et al., 1999b). One possibility is that the decreased hippocampal GR mRNA expression occurs as a compensatory down-regulation in response to repeated corticosterone release. Szafarczyk et al. (1986) found that the first kindling stimulation in naı¨ve rats evokes a transient increase in circulating corticosterone. This effect was larger in rats that had received about 20 previous stimulations. This suggests that during kindling, the HPA axis is repeatedly activated. Over time, the corticosterone released by each stimulation may result in a progressive down-regulation of hippocampal GR, as observed in the
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present study. This idea is consistent with previous findings that repeated corticosterone administration down-regulates GR mRNA expression in hippocampal regions (Patacchioli et al., 1998). How might decreased GR mRNA expression in hippocampal regions serve to increase fearful behavior in kindled rats? One possibility is that decreased hippocampal GR function alters the transcription of genes controlled by GR in the nucleus of the cell (for a review, see de Kloet et al., 1998). In this way, GR may have downstream effects on many different genes, some of which could be involved in fear regulation. Another possibility is that decreased hippocampal GR in kindled rats leads to a larger and more prolonged HPA axis response to a subsequent stressor. Hippocampal GRs are critically involved in feedback inhibition of the HPA axis: When receptor levels are high, feedback inhibition is enhanced and HPA axis activity is tightly controlled. However, when receptor levels are low, feedback inhibition is inefficient, and stimuli that elicit the release of corticosterone may have stronger effects (de Kloet et al., 1998). Accordingly, when kindled rats are exposed to an unfamiliar open field, they may experience a greater and longer-lasting HPA axis response than normal. A critical question is whether the corticosterone released under conditions of stress acts directly to increase fear or whether another hormone released as part of the HPA axis such as corticotropin-releasing factor (CRF) produces the fear. Certainly, there is evidence for the involvement of both hormones in the regulation of fearful behavior (Korte, 2001; Koob, 1999). For example, high levels of corticosterone at the time of behavioral testing can potentiate fear responses. Korte et al. (1999) found that exposing rats to a conditioned inescapable stressor immediately prior to testing increases fearful behavior on an elevated plus maze. Similarly, reducing corticosterone levels prior to testing by administration of metyrapone, a corticosterone synthesis inhibitor, reduces fear on the elevated plus maze (Roozendaal et al., 1996). However, one problem with this suggestion is that peak plasma corticosterone levels are usually reached about 20 min after the onset of a stressor (Francis et al., 2002). Because our open-field test only lasts 5 min, it seems unlikely that high plasma corticosterone levels triggered by exposure to an open field could affect fearful behavior during that test. It is possible that rats subjected to 60 or 100 kindling stimulations have high basal levels of corticosterone, so that corticosterone levels are already elevated when they are placed in the open field. Testing this possibility will require further experimentation, although Adamec and McKay (1993) have shown that 20 kindling stimulations do not result in elevated basal corticosterone levels. On the other hand, CRF is released very quickly on exposure to a stressor, so CRF levels should be very high while the kindled rats are in the open field. Furthermore, a rather large literature provides evidence that increased brain CRF increases a variety of fearful behaviors (Liang et al., 1992; Takahashi et al., 1989; Skutella et al., 1998; Sajdyk et al.,
1999). The effect of 60 or 100 kindling stimulations on CRF has not been assessed, but 20 stimulations increase CRF mRNA expression in the paraventricular nucleus of the hypothalamus and the dentate gyrus (Greenwood et al., 1997; Smith et al., 1997). Thus, current evidence favors the idea that high levels of CRF may ultimately increase fear in kindled rats, but future studies should investigate the direct involvement of both corticosterone and CRF in the expression of kindling-induced fear.
5. Conclusions In conclusion, the results of the present study demonstrate that the development of fear in kindled rats may be related to decreased GR mRNA expression in the dentate gyrus and CA1 subfield of the hippocampus. Because hippocampal GR are critically involved in negative feedback control of the HPA axis, these findings suggest that kindled rats may experience a stronger and longer-lasting HPA axis response to a stressful stimulus than normal. This is consistent with previous observations that kindling-induced fear is magnified when the rats are tested in a novel situation (Kalynchuk et al., 1999a). Further research is needed to establish the time course of GR mRNA changes in animals subjected to long-term kindling (i.e., 60 or 100 stimulations) and to determine the precise role that stressinduced increases in corticosterone and/or CRF play in the expression of kindling-induced fear. Acknowledgements This research was funded by the Canadian Institutes of Health Research (CIHR) and the Natural Sciences and Engineering Research Council of Canada (NSERC). Lisa E. Kalynchuk is a recipient of a NSERC University Faculty Award and a NARSAD Young Investigator Award. We thank Josie Diorio for her technical assistance and Dr. Tara Perrot-Sinal for helpful comments on an earlier version of this manuscript. References Adamec, R.E., 1990. Does kindling model anything clinically relevant? Biol. Psychiatry 27, 249 – 279. Adamec, R., 1998. Amygdala kindling and rodent anxiety. In: Corcoran, M.E., Moshe, S.L. (Eds.), Kindling, vol. 5. Plenum, New York, pp. 327 – 348. Adamec, R.E., McKay, D., 1993. Amygdala kindling, anxiety, and corticotrophin releasing factor (CRF). Physiol. Behav. 54, 423 – 431. Adamec, R.E., Morgan, H.D., 1994. The effect of kindling of different nuclei in the left and right amygdala on anxiety in the rat. Physiol. Behav. 55, 1 – 12. Adamec, R., Young, B., 2000. Neuroplasticity in specific limbic system circuits may mediate specific kindling induced changes in animal affect—implications for understanding anxiety associated with epilepsy. Neurosci. Biobehav. Rev. 24, 705 – 723.
L.E. Kalynchuk, M.J. Meaney / Progress in Neuro-Psychopharmacology & Biological Psychiatry 27 (2003) 1225–1234 Beaulieu, S., Rousse, I., Gratton, A., Barden, N., Rochford, J., 1994. Behavioral and endocrine impact of impaired type II glucocorticoid receptor function in a transgenic mouse model. Ann. N.Y. Acad. Sci. 746, 338 – 341. Clark, M., Smith, M.A., Weiss, S.R.B., Post, R.M., 1994. Modulation of hippocampal glucocorticoid and mineralocorticoid receptor mRNA expression by amygdaloid kindling. Neuroendocrinology 59, 451 – 456. Conrad, C.D., Magarinos, A.M., LeDoux, J.E., McEwen, B.S., 1999. Repeated restraint stress facilitates fear conditioning independently of causing hippocampal CA3 atrophy. Behav. Neurosci. 113, 902 – 913. Corodimas, K.P., LeDoux, J.E., Gold, P.W., Schulkin, J., 1994. Corticosterone potentiation of conditioned fear in rats. Ann. N.Y. Acad. Sci. 746, 392 – 393. de Kloet, E.R., Vreugdenhil, E., Oitzl, M.S., Joels, M., 1998. Brain corticosteroid receptor balance in health and disease. Endocrinol. Rev. 19, 269 – 301. Depaulis, A., Helfer, V., Deransart, C., Marescaux, C., 1997. Anxiogeniclike consequences in animal models of complex partial seizures. Neurosci. Biobehav. Rev. 21, 767 – 774. Francis, D.D., Meaney, M.J., 1999. Maternal care and the development of stress responses. Curr. Opin. Neurobiol. 9, 128 – 134. Francis, D.D., Diorio, J., Plotsky, P.M., Meaney, M.J., 2002. Environmental enrichment reverses the effects of maternal separation on stress reactivity. J. Neurosci. 22, 7840 – 7843. Goddard, G.V., McIntyre, D.C., Leech, C.K., 1969. A permanent change in brain function resulting from daily electrical stimulation. Exp. Neurol. 25, 295 – 330. Greenwood, R.S., Fan, Z., Meeker, R., 1997. Persistent elevation of corticotrophin releasing factor and vasopressin but not oxytocin mRNA in the rat after kindled seizures. Neurosci. Lett. 224, 66 – 70. Haimovici, A., Wang, Y., Cohen, E., Mintz, M., 2001. Social attraction between rats in open field: long term consequences of kindled seizures. Brain Res. 922, 125 – 134. Helfer, V., Deransart, C., Marescaux, C., Depaulis, A., 1996. Amygdala kindling in the rat: anxiogenic-like consequences. Neuroscience 73, 971 – 978. Henke, P.G., Sullivan, R.M., 1985. Kindling in the amygdala and susceptibility to stress ulcers. Brain Res. Bull. 400, 360 – 364. Herman, J.P., Adams, D., Prewitt, C., 1995. Regulatory changes in neuroendocrine stress-integrative circuitry produced by a variable stress paradigm. Neuroendocrinology 61, 180 – 190. Kalynchuk, L.E., 2000. Long-term amygdala kindling in rats as a model for the study of interictal emotionality in temporal lobe epilepsy. Neurosci. Biobehav. Rev. 24, 691 – 704. Kalynchuk, L.E., Pinel, J.P.J., Treit, D., Kippin, T.E., 1997. Changes in emotional behavior produced by long-term amygdala kindling in rats. Biol. Psychiatry 41, 438 – 451. Kalynchuk, L.E., Pinel, J.P.J., Treit, D., 1998a. Long-term kindling and interictal emotionality in rats: effect of stimulation site. Brain Res. 779, 149 – 157. Kalynchuk, L.E., Pinel, J.P.J., Treit, D., Barnes, S., McEachern, J.C., Kippin, T.E., 1998b. Persistence of the interictal emotionality produced by long-term amygdala kindling in rats. Neuroscience 85, 1311 – 1319. Kalynchuk, L.E., Pinel, J.P.J., Treit, D., 1999a. Characterization of the defensive nature of kindling-induced emotionality in rats. Behav. Neurosci. 113, 766 – 775. Kalynchuk, L.E., Pearson, D.M., Pinel, J.P.J., Meaney, M.J., 1999b. Effect of amygdala kindling on emotional behavior and benzodiazepine receptor binding in rats. Ann. N.Y. Acad. Sci. 877, 737 – 741. Kalynchuk, L.E., Davis, A.C., Gregus, A., Taggart, J., Dodd, C.C., Wintink, A.J., Marchant, E.G., 2001. Hippocampal involvement in the expression of kindling-induced fear in rats. Neurosci. Biobehav. Rev. 25, 687 – 696. Kandel, E.R., 1983. From metapsychology to molecular biology: explorations into the nature of anxiety. Am. J. Psychiatry 140, 1277 – 1293. Kitraki, E., Karandrea, D., Kittas, C., 1999. Long-lasting effects of stress
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tropin-releasing hormone receptor (type I) antisense targeting reduces anxiety. Neuroscience 85, 795 – 805. Smith, M.A., Weiss, S.R.B., Berry, R.L., Zhang, L., Clark, M., Massenburg, G., Post, R.M., 1997. Amygdala-kindled seizures increase the expression of corticotropin-releasing factor (CRF) and CRF-binding protein in GABAergic interneurons of the dentate hilus. Brain Res. 745, 248 – 256. Stec, I., Barden, N., Reul, J.M., Holsboer, F., 1994. Dexamethasone nonsuppression in transgenic mice expressing antisense RNA to the glucocorticoid receptor. J. Psychiatr. Res. 28, 1 – 5. Strohle, A., Poettig, M., Barden, N., Holsboer, F., Montkowski, A., 1998. Age- and stimulus-dependent changes in anxiety-related behaviour of transgenic mice with GR dysfunction. NeuroReport 22, 2099 – 2102. Szafarczyk, A., Caracchini, M., Rondouin, G., Ixart, G., Malaval, F.,
Assenmacher, I., 1986. Plasma ACTH and corticosterone responses to limbic kindling in the rat. Exp. Neurol. 92, 583 – 590. Takahashi, L.K., Kalin, N.H., Vande-Burgst, J.A., Sherman, J.E., 1989. Corticotropin-releasing hormone modulates defensive-withdrawal and exploratory behavior in rats. Behav. Neurosci. 3, 648 – 654. Tronche, F., Kellendonk, C., Kretz, O., Gass, P., Anlag, K., Orban, P.C., Bock, R., Klein, R., Schutz, G., 1999. Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety. Nat. Gen. 23, 99 – 103. Vyas, A., Mitra, R., Shankaranarayana, R., Chattarji, S., 2002. Chronic stress induces contrasting patterns of dendritic remodeling in hippocampal and amygdaloid neurons. J. Neurosci. 22, 6810 – 6818. Wintink, A.J., Young, N.A., Davis, A.C., Gregus, A., Kalynchuk, L.E., 2003. Kindling-induced emotional behavior in male and female rats. Behav. Neurosci. 117, 632 – 640.
Amygdala kindling increases fear responses and decreases glucocorticoid receptor mRNA expression in hippocampal regions Lisa E. Kalynchuka,*, Michael J. Meaneyb a
Department of Psychology and Neuroscience Institute, Dalhousie University, 1355 Oxford Street, Halifax, Nova Scotia, Canada B3H 4J1 b Department of Psychiatry, Douglas Hospital Research Center, McGill University, Montreal, Quebec, Canada H4H 1R3 Accepted 9 September 2003
Abstract Amygdala kindling dramatically increases fearful behavior in rats. Because kindling-induced fear increases in magnitude as rats receive more stimulations, kindling provides an excellent model for studying the nature and neural mechanisms of fear sensitization. In the present experiment, we studied whether the development of kindling-induced fear is related to changes in glucocorticoid receptor (GR) mRNA expression in various brain regions. Rats received 20, 60 or 100 amygdala kindling stimulations or 100 sham stimulations. One day after the final stimulation, their fearful behavior was assessed in an unfamiliar open field. Then, the rats were sacrificed and their brains were processed for in situ hybridization of GR mRNA expression. We found that compared with the sham-stimulated rats, the rats that received 60 or 100 kindling stimulations were significantly more fearful in the open field and also had significantly less GR mRNA expression in the dentate gyrus and CA1 subfield of the hippocampus. Importantly, the changes in fearful behavior were significantly correlated with the changes in GR mRNA expression. These results suggest that alterations in GR mRNA expression in hippocampal regions may play a role in the development of kindling-induced fear. D 2003 Published by Elsevier Inc. Keywords: Amygdala; Fear; Glucocorticoid receptor; Hippocampus; Hypothalamic – pituitary – adrenal axis; Kindling; Open field; Stress
1. Introduction Normal fear responses are adaptive in that they serve as protection from potential harm. However, with repeated exposure, fear can become sensitized so that it eventually results in behavioral responses that are maladaptive (Kandel, 1983; Marks and Tobena, 1990). The level of fear experienced by an animal can thus range from ‘‘normal’’ to ‘‘pathological’’ (Rosen and Schulkin, 1998). Studying the progression from normal to pathological fear is a critical approach for identifying the neural mechanisms that underlie the development of affective disorders such as generalized anxiety and panic and for developing new pharmacological treatments that can prevent or reduce these disorders. Abbreviations: CRF, corticotropin-releasing factor; HPA, hypothalamic – pituitary – adrenal; GR, glucocorticoid receptor * Corresponding author. Tel.: +1-902-494-6337; fax: +1-902-4946585. E-mail address: [email protected] (L.E. Kalynchuk). 0278-5846/$ – see front matter D 2003 Published by Elsevier Inc. doi:10.1016/j.pnpbp.2003.09.016
Kindling provides a particularly useful animal model for studying the nature and molecular mechanisms of fear sensitization (Adamec, 1990; Depaulis et al., 1997; Kalynchuk, 2000). Kindling refers to the gradual development and intensification of elicited motor seizures that result from the daily administration of initially subconvulsive stimulations to particular brain regions (Goddard et al., 1969). In addition to its epileptogenic effects, kindling also sensitizes brain circuits responsible for behavioral manifestations of fear (Adamec and Young, 2000; Kalynchuk et al., 2001; Rosen and Schulkin, 1998). For example, amygdala kindling in rats decreases exploratory behavior in an open field (Kalynchuk et al., 1999a; Nieminen et al., 1992), decreases openarm exploration on an elevated plus maze (Adamec and Morgan, 1994; Helfer et al., 1996), increases immobility in a social interaction test (Helfer et al., 1996), increases fearpotentiated startle (Rosen et al., 1996) and increases stressinduced stomach ulcers (Henke and Sullivan, 1985). The kindling model provides an excellent opportunity to study the progression from normal to pathological fear because the magnitude of kindled-induced fear increases
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with the number of stimulations. For example, relative to control rats, rats that receive 20 amygdala stimulations display significant increases in thigmotaxia in an unfamiliar open field, rats that receive 60 amygdala stimulations display significant decreases in open-field activity and increases in resistance to capture from the open field, and rats that receive 100 stimulations display all of these changes plus increases in fleeing behavior in an open-field, escape behavior in an elevated plus maze, and active defensive behaviors in a resident – intruder paradigm (Kalynchuk et al., 1997, 1999a, 2001). Studying the neural mechanisms that underlie the progression of kindling-induced fear may yield important information about the progressive development of affective disorders characterized by high levels of fear (Rosen and Schulkin, 1998). The purpose of the present study was to examine whether changes in glucocorticoid receptors (GR) are related to the progressive intensification of kindling-induced fear. GRs are intimately involved in the physiological stress response. On exposure to an acute stressor, the hypothalamic – pituitary – adrenal (HPA) axis becomes activated, evoking a series of physiological events that culminate in the release of the hormone corticosterone, which then binds to GR in several brain regions. With repeated exposure to stress, GR mRNA expression in the hippocampus is decreased (Herman et al., 1995). Interestingly, repeated HPA axis activation is thought to increase vulnerability toward the development of affective pathology (Francis and Meaney, 1999). For example, both repeated restraint stress and corticosterone administration increase fear-mediated behaviors in rats (Conrad et al., 1999; Corodimas et al., 1994). Similarly, there is some evidence to suggest that the behavioral changes produced by kindling may be mediated by the consequences of repeated HPA axis activation. A single kindling stimulation in animals that have been previously kindled elicits a rapid transient increase in circulating levels of corticosterone (Szafarczyk et al., 1986). Because animals are repeatedly stimulated during kindling, kindling itself may be a form of repeated stress, leading to repeatedly elevated levels of corticosterone, and a compensatory down-regulation of GR in hippocampal regions (Clark et al., 1994). Thus, in the present experiment, we investigated whether GR mRNA expression in limbic brain regions is related to the development of fearful behavior in kindled rats.
2. Methods 2.1. Animals The subjects were 36 male Long– Evans rats (Charles River, Montreal, Canada) weighing between 250 and 350 g at the time of surgery. They were individually housed in steel hanging cages in a colony room with an ambient temperature of about 21 jC and a 12:12-h light/dark cycle (lights on at 8:00 a.m.). Purina rat chow and water were
available continuously. All experimental manipulations were conducted in accordance with the guidelines of the Canadian Council on Animal Care. 2.2. Surgery A single bipolar stimulating electrode (MS-303-2, Plastics One, Roanoke, VA) was implanted in the left basolateral amygdala of each rat under sodium pentobarbital anesthesia (somnotol, 65 mg/kg ip). Each electrode was aimed at a site 2.8 mm posterior, 5.0 mm left and 8.5 mm ventral to the skull surface at bregma (coordinates from Paxinos and Watson, 1998). Each electrode was secured to the skull with four stainless steel screws and dental acrylic. Powdered tetracycline was sprinkled on the incision before suturing to reduce the risk of infection. Left amygdala kindling was employed in this experiment to ensure consistency with our previous experiments (Kalynchuk et al., 1997, 1998a,b, 2001; Wintink et al., 2003). The behavioral consequences of left and right amygdala kindling are not always the same (Adamec and Morgan, 1994), especially for partial and short-term kindling. All our previous experiments have been conducted with rats kindled in the left amygdala. We have thus built up a detailed understanding of the nature of the behavioral changes produced by left amygdala kindling. The purpose of this experiment was to assess whether GRs might be involved in these behavioral changes, so it was logical that we study rats that had been kindled in the left amygdala. 2.3. Kindling phase After a postsurgical recovery period of at least 12 days, the rats were divided into four groups: One group received 80 sham stimulations followed by 20 kindling stimulations (20-stim group, n = 9), one group received 40 sham stimulations followed by 60 kindling stimulations (60-stim group, n = 9), one group received 100 kindling stimulations (100-stim group, n = 10) and one group received 100 sham stimulations (sham-stim group, n = 8). The stimulations were delivered three times per day, 5 days/week, with a minimum of 2 h between consecutive stimulations. A few seconds prior to each stimulation, each rat was removed from its home cage and placed in a plastic box containing a thin layer of commercial bedding. The stimulation lead was attached and the stimulation was then delivered (1 s, 60 Hz, 400 AA square waves). After all convulsive activity had ceased, the rat was returned to its home cage. Rats receiving sham stimulations were treated in exactly the same way except that no current was delivered. The measure of seizure severity was the convulsion class elicited by each stimulation. Convulsion class was scored according to Pinel and Rovner’s (1978) extension of Racine’s (1972) widely used five-class scale (class 1: head nodding only; class 2: head nodding and jaw clonus; class 3: head nodding, jaw clonus and forelimb clonus; class 4: head
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nodding, jaw clonus, forelimb clonus and rearing; class 5: head nodding, jaw clonus, forelimb clonus, rearing and falling once; class 6: a class 5 with multiple rearing and falling episodes; class 7: a class 6 with running fits; class 8: any of the preceding symptoms with periods of tonus). 2.4. Behavioral testing All behavioral testing took place in an open field. The open field was a 60 60 60 cm box with wooden walls and 36 identical squares defined by tape on the Plexiglas floor. It was located in a small brightly lit testing room. The open field, testing room and experimenter were unfamiliar to the rats at the time of testing. The behavioral testing was conducted 1 day after the final stimulation. Each rat was placed individually into a corner of the unfamiliar open field for 5 min, while an experimenter sat quietly in the room out of sight of the rat. The rat’s behavior was videotaped from above. Later, the number of squares crossed by each rat during the open-field test was counted from a video monitor. We limited our analysis of exploration to the first 30 s because we have found that fear-related reductions in exploration between kindled and sham-stimulated rats are greatest during the first 30 s of exposure to an open field, when it is the most unfamiliar (Kalynchuk et al., 1997). After the 5 min, each rat’s resistance to being captured from the open field was assessed. The experimenter, who was wearing a leather glove that was unfamiliar to the rat, attempted to pick up each rat from above. The rat’s resistance to being picked up was scored according to the following seven-point scale:
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0 = easy to pick up, 1 = vocalizes or shies away from hand, 2 = shies away from hand and vocalizes, 3 = runs away from hand, 4 = runs away and vocalizes, 5 = bites or attempts to bite, and 6 = launches a jump attack (Kalynchuk et al., 1997). Some rats were highly resistant to being captured and eluded the gloved hand when the experimenter tried to pick them up. In these cases, the experimenter persisted and the rat was given a score based on the highest degree of resistance to capture that it displayed. The duration of fleeing from the gloved hand before being captured was also recorded for each rat. The open field was cleaned thoroughly with Fantastik solution in between each trial. 2.5. In situ hybridization Each rat was sacrificed f 72 h after the final stimulation, and its brain was rapidly removed and frozen in isopentane maintained on dry ice. Frozen 16-Am coronal sections were cut from each brain on a cryostat, thaw-mounted onto polyL-lysine-coated slides and stored at 80 jC. In situ hybridization of GR mRNA was performed as described previously (O’Donnell et al., 1994) using [35S]UTP-labeled cRNA antisense probes transcribed from a 674-bp PstI – EcoRI fragment of the rat GR cDNA (steroid binding domain: Dr. R. Meisfield, University of Arizona), linearized with AvaI and transcribed with T7 RNA polymerase. Briefly, slides were first removed from the freezer and warmed to room temperature, postfixed in 4% paraformaldehyde in 0.1 phosphate buffer (10 min at pH 7.0) and washed three times in 2 SSC (0.3 M NaCl and 0.03 M sodium citrate)
Fig. 1. The mean F S.E.M. class of convulsions produced by the third stimulation on each of the kindling days in each group of kindled rats. All rats had a minimum of five class 5 convulsions prior to the behavioral testing.
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Fig. 2. Effect of different numbers of kindling stimulations on fearful behavior. Each rat was tested in an unfamiliar open field 1 day after the final kindling stimulation. (A) Mean F S.E.M. number of squares crossed in the first 30 s of the open-field test by the rats in each group. Both 60-stim and 100-stim rats crossed significantly fewer squares during the open-field test than did the sham-stim rats ( P < .01), and the 60-stim rats also crossed significantly fewer squares than the 20-stim rats ( P < .01). (B) Mean F S.E.M. resistance to capture from the open field displayed by the rats in each group. Both 60-stim and the 100-stim rats were significantly more resistant to capture from the open field than the sham-stim rats ( P < .025), and the 100-stim rats were also more resistant to capture than the 20-stim rats ( P < .025). (C) Mean F S.E.M. duration of fleeing displayed by the rats in each group during resistance to capture testing. Both 60-stim and 100-stim rats engaged in a longer duration of fleeing than did the 20-stim or sham-stim rats ( P < .01).
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in sterile water containing 0.2% diethylpryocarbonate. Hybridization was then performed at 37 jC for 18 h in a buffer containing for 50% deionized formamide, 5 M NaCl, 1 M Tris (pH 7.5), 100 Denhardt’s solution, 0.5 M EDTA (pH 8.0), 10 mg/ml denatured salmon sperm DNA, 11 mg/ml yeast tRNA, 10% dextran sulfate, 10 mM dithiothreitol and about 1 107 cpm of [35S]UTP-labeled GR riboprobe. After hybridization, the slides were rinsed in 2 SSC and treated with RNase A for 45 min at 37 jC. Finally, the slides were washed in decreasing salt concentrations to 0.1 SSC at 60 jC, dehydrated in increasing concentrations of ethanol, dried and apposed to Hyperfilm (Amersham) for 21 days. The hybridization signal within the dorsal hippocampus and amygdala was quantified by densitometry with an image analysis system (MCID, Imaging Research, St. Catharines, ON). These brain regions were chosen for analysis based on many previous findings that repeated stress decreases GR mRNA expression in hippocampal regions (Meyer et al., 2001; Kitraki et al., 1999; Herman et al., 1995) and the intriguing recent report that chronic stress has differential morphological effects on hippocampal and amygdaloid neurons (Vyas et al., 2002). Three brain sections per animal for each brain region of interest were quantified. The data are presented as arbitrary optical density units with background subtracted.
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2.6. Statistical analyses The statistical significance of the data was analyzed in four ways. Group differences in open-field exploration and duration of fleeing were assessed using one-way ANOVAs followed by Newman –Keuls post hoc tests. Group differences in resistance to capture were assessed using a Kruskal –Wallis one-way ANOVA by ranks test followed by post hoc multiple comparisons. Group differences in GR mRNA expression were analyzed using a two-way ANOVA (Group Region) followed by Newman – Keuls post hoc tests. Finally, correlations between GR mRNA expression in specific brain regions and each of the three behavioral measures were assessed using Spearman rank order correlations.
3. Results 3.1. Kindling acquisition Fig. 1 shows the mean class of convulsions produced by the third stimulation on each of the kindling days. As expected, the initial kindling stimulations produced little behavioral response other than a momentary behavioral
Fig. 3. Effect of different numbers of amygdala kindling stimulations on GR mRNA expression. GR mRNA was assessed 72 h after the final kindling stimulation. Mean F S.E.M. levels of GR mRNA expression in hippocampal and amygdaloid regions of rats in each group are shown in arbitrary optical density units. Both 60-stim and 100-stim rats had significantly less GR mRNA expression in the dentate gyrus ( P < .01) and CA1 subfield ( P < .05) than did the sham-stim rats. Abbreviations: CeM, central nucleus of the amygdala; BLA, basolateral nucleus of the amygdala.
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arrest; however, within a few days, almost every stimulation elicited a generalized convulsion characterized by facial clonus, forelimb clonus, rearing and loss of equilibrium. The mean number of class 4 or above convulsions experienced by the rats in each group was 91.4 for the 100-stim rats (range = 86– 96), 43.3 for the 60-stim rats (range = 27 – 52) and 12.1 for the 20-stim rats (range = 8 –16). 3.2. Open-field behavior Fig. 2 shows the open-field behaviors displayed by the rats in each group. Kindling had a significant effect on each of these behavioral measures. First, during the first 30 s of the open-field session, both the 60-stim and 100-stim rats crossed significantly fewer squares than did the sham-stim rats [ F(3,35) = 10.146, P < .0001; post hoc P < .01]; the 60stim rats crossed fewer squares than the 20-stim rats ( P < .01). Second, at the end of the open-field session, both the 60-stim and 100-stim rats were significantly more resistant to capture than the sham-stim rats [H(3) = 21.524, P < .0001; post hoc P < .025]; the 100-stim rats were also significantly more resistant to capture than the 20-stim rats ( P < .025). Finally, both the 60-stim and 100-stim rats engaged in a significantly longer duration of fleeing than did the 20-stim or sham-stim rats [ F(3,35) = 12.648, P < .0001; post hoc P < .01].
Table 1 Correlations between behavioral changes and GR mRNA expression Region
Square crosses in first 30 s
Dentate gyrus CA1 CA2 CA3 Central amygdala Basolateral amygdala
.334 * .278 .1 .007 .039 .128
Resistance to capture
Duration of fleeing
.307 .451* * .157 .083 .221 .071
.366 * .376 * .24 .149 .305 .104
* Significant at P < .05. ** Significant at P < .01.
capture and GR mRNA levels in the dentate gyrus narrowly missed our defined level of statistical significance ( P < .07). Finally, the duration of fleeing during the resistance-to-capture testing was significantly correlated to changes in GR mRNA expression in both the dentate gyrus and the CA1 subfield of the hippocampus ( P < .05). None of the open-field behaviors was correlated to changes in GR mRNA levels in any of the other quantified brain regions.
4. Discussion 4.1. Relation between kindling-induced fear and GR mRNA expression
3.3. Glucocorticoid mRNA expression GR mRNA expression in various regions of the dorsal hippocampus and amygdala is shown in Fig. 3. Kindling had significant region-specific effects on GR mRNA expression. An overall two-way ANOVA revealed a significant effect of Group [ F(3,185) = 9.636, P < .0001], a significant effect of Region [ F(6,185) = 153.05, P < .0001] and a significant Group Region interaction [ F(18,185) = 1.796, P < .03]. Post hoc analyses further revealed that both the 60-stim and 100-stim rats had significantly less GR mRNA expression in the dentate gyrus ( P < .01) and CA1 subfield of the hippocampus ( P < .05) than did the sham-stim rats. 3.4. Correlations between behavioral differences and GR mRNA expression To determine whether specific changes in behavior were related to GR mRNA expression in particular brain regions, we calculated the correlation between each behavioral measure and GR mRNA levels in each brain region. These correlations are shown in Table 1. Several observations can be made from these correlations. First, exploration in the first 30 s of the open-field test was significantly correlated to GR mRNA expression in the dentate gyrus ( P < .05). Second, resistance to capture was significantly correlated to GR mRNA levels in the CA1 subfield ( P < .01). The relation between resistance to
In the present study, kindling had significant effects on both fearful behavior and GR mRNA expression. As expected, rats that received 60 or 100 kindling stimulations displayed less open-field exploration, greater resistance to capture from the open field and a longer duration of fleeing before being captured than did rats that received 20 kindling stimulations or sham stimulations. These findings are consistent with many earlier reports of fearful behavior in kindled animals (Adamec and Morgan, 1994; Helfer et al., 1996; Kalynchuk et al., 1998b; Nieminen et al., 1992; McIntyre, 1978; Rosen et al., 1996), and they also confirm our previous observations that the magnitude of kindlinginduced fear depends on the number of stimulations the rats receive (Kalynchuk et al., 1997, 2001; Wintink et al., 2003). In addition to these behavioral differences, rats that received 60 or 100 kindling stimulations also had less GR mRNA expression in the dentate gyrus and CA1 region of the hippocampus compared with the rats that received 20 kindling stimulations or sham stimulations. Importantly, specific changes in fearful behavior in the kindled rats were related to specific changes in GR mRNA expression: increased resistance to capture was significantly correlated to decreased GR mRNA expression in the CA1 region, decreased open-field exploration was related to decreased GR mRNA expression in the dentate gyrus and increased fleeing time was related to decreased GR mRNA in both dentate gyrus and CA1 subfield. These results suggest that decreased GR mRNA expression in particular hippocampal
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regions may be involved in specific aspects of kindlinginduced fear. The results of this experiment extend the previous observations of Clark et al. (1994) who assessed GR mRNA expression at different time points after f 10 amygdala kindling stimulations. They found that kindling decreased GR mRNA expression in the dentate gyrus 24 h but not 96 h after the final stimulation. No significant changes in GR mRNA levels were found in the CA1 subfield. In the present experiment, decreased GR mRNA levels were detected in both the dentate gyrus and CA1 region of the 60-stim and 100-stim rats 72 h after the final kindling stimulation. This suggests that the effect of amygdala kindling on hippocampal GR mRNA expression is longer lasting and more widespread after greater numbers of stimulations. An important next step will be to determine how long the decreased GR mRNA levels persist after 60 or 100 amygdala kindling stimulations. 4.2. Behavioral similarities between kindled rats and those with alterations in GR Kindling exerts complex effects on emotional behavior, and these effects depend on several important parameters, such as the situation in which the rats are tested (Kalynchuk et al., 1999a; Haimovici et al., 2001), the number of stimulations the rats receive (Kalynchuk et al., 1997), the site of kindling within the brain (Kalynchuk et al., 1998a; Adamec, 1998) and the strain of rat being used (Adamec, 1998). In general, rats that have received about 100 amygdala kindling stimulations tend to display panic-like active responses to stressful stimuli (Kalynchuk et al., 2001). They show extreme resistance to capture from an unfamiliar open field (Kalynchuk et al., 1997, 2001), open-arm exploration and escape behavior from an elevated plus maze (Kalynchuk et al., 1997), active defensive postures and defensive jump attacks in a resident – intruder test (Kalynchuk et al., 1999a) and enhanced startle to acoustic stimuli (Menard et al., 2002). In addition, the more novel the testing situation is, the larger the kindling-induced fear response is likely to be. For example, kindled rats show high levels of resistance to capture from an unfamiliar open field but little resistance to capture from their home cages or from a familiar open field (Kalynchuk et al., 1999a). Interestingly, the emotional consequences of kindling appear to be specific to fearrelated responses. Kindling does not increase depressionlike behavior as measured by the forced swim test or sucrose preference tests (Helfer et al., 1996; Wintink et al., 2003). In fact, after 100 stimulations, kindled rats actually show less depression-like behavior than control rats—they engage in less immobility in the forced swim test and show a greater preference for sucrose on the sucrose preference test (Wintink et al., 2003). There are some intriguing similarities between the emotional behaviors displayed by kindled rats and those observed in rodents with decreased GR function. For example,
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transgenic mice with a 50 –70% reduction in central GR levels display increased acoustic startle, increased open-arm exploration in an elevated plus maze, decreased exploration time in an open field and decreased immobility in a forced swim test (Rochford et al., 1997; Beaulieu et al., 1994; Montkowski et al., 1995; Strohle et al., 1998). Decreasing GR function in the dentate gyrus of adult rats with antisense infusions also decreases immobility in the forced swim test (Korte et al., 1996). In addition, rats exposed to maternal separation early in life show decreased GR mRNA expression in the dentate gyrus and CA1 region and decreased exploration time in an open field (Francis et al., 2002). As detailed above, kindled rats display a similar behavioral profile, providing support for the idea that decreased GR mRNA expression in the dentate gyrus and CA1 subfield play an important role in the development of kindlinginduced fear. However, this conclusion must be made tentatively because GR knockout mice appear to be less anxious than wild-type mice (Tronche et al., 1999). One explanation for this discrepancy is that GR is completely absent in the brains of the knockout mice, whereas GR function is only reduced in the brains of kindled, maternally separated and transgenic animals that have increased anxiety-like behavior. This may have functional implications. GR knockout mice have elevated basal corticosterone levels and a normal corticosterone response to acute restraint (Tronche et al., 1999), whereas kindled and maternally separated rats have normal basal corticosterone levels (Francis et al., 2002; Adamec and McKay, 1993) and maternally separated rats and transgenic mice with a 50% reduction of GR have an exaggerated corticosterone response to acute restraint (Francis et al., 2002; Stec et al., 1994) Thus, there are HPA axis differences between animals with impaired GR function and animals with absent GR function that could account for the differences in their emotional behavior. 4.3. Potential mechanisms by which kindling and GR might influence fearful behavior The mechanism by which kindling decreases hippocampal GR mRNA expression is unknown. It is not likely due to kindling-induced loss of hippocampal neurons, because we have previously found increased benzodiazepine receptor binding in the hippocampus of kindled rats subjected to 100 stimulations (Kalynchuk et al., 1999b). One possibility is that the decreased hippocampal GR mRNA expression occurs as a compensatory down-regulation in response to repeated corticosterone release. Szafarczyk et al. (1986) found that the first kindling stimulation in naı¨ve rats evokes a transient increase in circulating corticosterone. This effect was larger in rats that had received about 20 previous stimulations. This suggests that during kindling, the HPA axis is repeatedly activated. Over time, the corticosterone released by each stimulation may result in a progressive down-regulation of hippocampal GR, as observed in the
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present study. This idea is consistent with previous findings that repeated corticosterone administration down-regulates GR mRNA expression in hippocampal regions (Patacchioli et al., 1998). How might decreased GR mRNA expression in hippocampal regions serve to increase fearful behavior in kindled rats? One possibility is that decreased hippocampal GR function alters the transcription of genes controlled by GR in the nucleus of the cell (for a review, see de Kloet et al., 1998). In this way, GR may have downstream effects on many different genes, some of which could be involved in fear regulation. Another possibility is that decreased hippocampal GR in kindled rats leads to a larger and more prolonged HPA axis response to a subsequent stressor. Hippocampal GRs are critically involved in feedback inhibition of the HPA axis: When receptor levels are high, feedback inhibition is enhanced and HPA axis activity is tightly controlled. However, when receptor levels are low, feedback inhibition is inefficient, and stimuli that elicit the release of corticosterone may have stronger effects (de Kloet et al., 1998). Accordingly, when kindled rats are exposed to an unfamiliar open field, they may experience a greater and longer-lasting HPA axis response than normal. A critical question is whether the corticosterone released under conditions of stress acts directly to increase fear or whether another hormone released as part of the HPA axis such as corticotropin-releasing factor (CRF) produces the fear. Certainly, there is evidence for the involvement of both hormones in the regulation of fearful behavior (Korte, 2001; Koob, 1999). For example, high levels of corticosterone at the time of behavioral testing can potentiate fear responses. Korte et al. (1999) found that exposing rats to a conditioned inescapable stressor immediately prior to testing increases fearful behavior on an elevated plus maze. Similarly, reducing corticosterone levels prior to testing by administration of metyrapone, a corticosterone synthesis inhibitor, reduces fear on the elevated plus maze (Roozendaal et al., 1996). However, one problem with this suggestion is that peak plasma corticosterone levels are usually reached about 20 min after the onset of a stressor (Francis et al., 2002). Because our open-field test only lasts 5 min, it seems unlikely that high plasma corticosterone levels triggered by exposure to an open field could affect fearful behavior during that test. It is possible that rats subjected to 60 or 100 kindling stimulations have high basal levels of corticosterone, so that corticosterone levels are already elevated when they are placed in the open field. Testing this possibility will require further experimentation, although Adamec and McKay (1993) have shown that 20 kindling stimulations do not result in elevated basal corticosterone levels. On the other hand, CRF is released very quickly on exposure to a stressor, so CRF levels should be very high while the kindled rats are in the open field. Furthermore, a rather large literature provides evidence that increased brain CRF increases a variety of fearful behaviors (Liang et al., 1992; Takahashi et al., 1989; Skutella et al., 1998; Sajdyk et al.,
1999). The effect of 60 or 100 kindling stimulations on CRF has not been assessed, but 20 stimulations increase CRF mRNA expression in the paraventricular nucleus of the hypothalamus and the dentate gyrus (Greenwood et al., 1997; Smith et al., 1997). Thus, current evidence favors the idea that high levels of CRF may ultimately increase fear in kindled rats, but future studies should investigate the direct involvement of both corticosterone and CRF in the expression of kindling-induced fear.
5. Conclusions In conclusion, the results of the present study demonstrate that the development of fear in kindled rats may be related to decreased GR mRNA expression in the dentate gyrus and CA1 subfield of the hippocampus. Because hippocampal GR are critically involved in negative feedback control of the HPA axis, these findings suggest that kindled rats may experience a stronger and longer-lasting HPA axis response to a stressful stimulus than normal. This is consistent with previous observations that kindling-induced fear is magnified when the rats are tested in a novel situation (Kalynchuk et al., 1999a). Further research is needed to establish the time course of GR mRNA changes in animals subjected to long-term kindling (i.e., 60 or 100 stimulations) and to determine the precise role that stressinduced increases in corticosterone and/or CRF play in the expression of kindling-induced fear. Acknowledgements This research was funded by the Canadian Institutes of Health Research (CIHR) and the Natural Sciences and Engineering Research Council of Canada (NSERC). Lisa E. Kalynchuk is a recipient of a NSERC University Faculty Award and a NARSAD Young Investigator Award. We thank Josie Diorio for her technical assistance and Dr. Tara Perrot-Sinal for helpful comments on an earlier version of this manuscript. References Adamec, R.E., 1990. Does kindling model anything clinically relevant? Biol. Psychiatry 27, 249 – 279. Adamec, R., 1998. Amygdala kindling and rodent anxiety. In: Corcoran, M.E., Moshe, S.L. (Eds.), Kindling, vol. 5. Plenum, New York, pp. 327 – 348. Adamec, R.E., McKay, D., 1993. Amygdala kindling, anxiety, and corticotrophin releasing factor (CRF). Physiol. Behav. 54, 423 – 431. Adamec, R.E., Morgan, H.D., 1994. The effect of kindling of different nuclei in the left and right amygdala on anxiety in the rat. Physiol. Behav. 55, 1 – 12. Adamec, R., Young, B., 2000. Neuroplasticity in specific limbic system circuits may mediate specific kindling induced changes in animal affect—implications for understanding anxiety associated with epilepsy. Neurosci. Biobehav. Rev. 24, 705 – 723.
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