Aberrant hippocampal neurogenesis after limbic kindling: Relationship to BDNF and hippocampal-dependent memory.

Seizures dramatically increase the number of adult generated neurons in the hippocampus. However, it is not known whether this effect depends on seizures that originate in specific brain regions or whether it is nonspecific to seizure activity regardless of origin. We used kindling of different brain sites to address this question. Rats received 99 kindling stimulations of the basolateral amygdala, dorsal hippocampus, or caudate nucleus over a 6-week period. After kindling, we counted the number of adult generated hippocampal neurons that were birth-dated with the proliferative marker bromodeoxyuridine (BrdU) to evaluate cell proliferation and survival under conditions of repeated seizures. Next, we counted the number of doublecortin immunoreactive (DCX-ir) cells and evaluated their dendritic complexity to determine if limbic and nonlimbic seizures have differential effects on neuronal maturation. We also quantified hippocampal brain-derived neurotrophin factor (BDNF) protein levels using an ELISA kit and assessed memory performance using a hippocampal-dependent fear conditioning paradigm. We found that limbic, but not nonlimbic, seizures dramatically increased hippocampal cell proliferation and the number of hilar-CA3 ectopic granule cells. Further, limbic kindling promoted dendritic outgrowth of DCX-ir cells and the number of DCX-ir cells containing basal dendrites. Limbic kindling also enhanced BDNF protein levels throughout the entire hippocampus and impaired the retrieval of fear memories. Collectively, our results suggest a relationship between limbic seizures, neurogenesis, BDNF protein, and cognition.

Intravenous Reelin rescues despair-like behavior, Reelin cells in the dentate sub-granular zone, and spleen atrophy in the cyclic corticosterone model of recurring depressive episodes.

Novel antidepressants are predominantly evaluated preclinically in rodent models of chronic stress in which animals experience a single prolonged exposure to chronic stress prior to treatment. Rodent models of a single episode of chronic stress translate poorly to human depressive disorders, which are commonly marked by recurring depressive episodes. Intravenous administration of Reelin has previously been shown to resolve immobility in the forced swim test of rats exposed to a single prolonged exposure to chronic stress. To determine whether Reelin has antidepressant-like properties in a model of recurring depressive episodes, Long-Evans rats (N = 57) were exposed to multiple cycles of chronic stress and stress-free periods before the administration of a single injection of Reelin during the final cycle of chronic stress. The animals then performed in the forced swim test and open field test before the post-mortem evaluation of Reelin cell counts in the sub-granular zone of the dentate gyrus to determine the impact of treatment on hippocampal Reelin levels and spleen white pulp to evaluate the role of Reelin treatment in peripheral inflammation. The results show a single Reelin injection reversed elevated levels of immobility in the forced swim test in both male and female subjects exposed to the cyclic chronic stress model of recurring depressive episodes. Treatment with Reelin also restored Reelin-positive cell counts in the dentate gyrus sub-granular zone and reversed atrophy of spleen white pulp. The results shown here indicate that treatment with Reelin could effectively resolve alterations in forced swim test behavior caused by the cyclic corticosterone model of recurring depressive episodes and that Reelin homeostasis is important for regulating stress-related inflammation. Future preclinical antidepressant research should incorporate models of multiple depressive episodes to improve the translation of preclinical rodent research to human depressive disorders.

The effect of amygdala kindling on hippocampal neurogenesis coincides with decreased reelin and DISC1 expression in the adult dentate gyrus.

Temporal lobe seizures can induce the proliferation and abnormal migration of newly generated dentate granule cells, but little is known about the molecular mechanisms that govern these pathological events. Reelin and DISC1 (disrupted-in-schizophrenia 1) are proteins that play a regulatory role in the maturation and integration of new neurons in the developing and adult brain. In this study, we examined whether amygdala kindling results in aberrant neurogenesis and altered expression of reelin and DISC1 in the adult dentate gyrus. Using doublecortin immunohistochemistry, we found that short-term kindling (i.e., 30 electrical stimulations) significantly increased the number of immature neurons in the dentate subgranular zone (SGZ), whereas long-term kindling (i.e., 99 electrical stimulations) did not. However, doublecortin-labeled neurons in long-term kindled rats showed greater dendritic complexity than they did in short-term kindled or control rats. We also found that long-term kindling decreased the number of reelin-positive cells and decreased DISC1 expression in the dentate granule cell layer and subgranular zone. Interestingly, kindling-induced changes in reelin and DISC1 expression coincided with the appearance of ectopically located Prox1-labeled granule cells in the hilus. These effects occurred independently of alterations in granule cell layer length, dentate volume, or the number of hilar neurons. Taken together, these findings suggest a novel role for DISC1 in the pathophysiology of temporal lobe epilepsy and further suggest that changes in reelin and DISC1 expression may contribute to aberrant neurogenesis in the kindling model.

Selective plasticity of hippocampal GABAergic interneuron populations following kindling of different brain regions.

The vulnerability and plasticity of hippocampal GABAergic interneurons is a topic of broad interest and debate in the field of epilepsy. In this experiment, we used the electrical kindling model of epilepsy to determine whether seizures that originate in different brain regions have differential effects on hippocampal interneuron subpopulations. Long-Evans rats received 99 electrical stimulations of the hippocampus, amygdala, or caudate nucleus, followed by sacrifice and immunohistochemical or western blot analyses. We analyzed markers of dendritic (somatostatin), perisomatic (parvalbumin), and interneuron-selective (calretinin) inhibition, as well as an overall indicator (GAD67) of interneuron distribution across all major hippocampal subfields. Our results indicate that kindling produces selective effects on the number and morphology of different functional classes of GABAergic interneurons. In particular, limbic kindling appears to enhance dendritic inhibition, indicated by a greater number of somatostatin-immunoreactive (-ir) cells in the CA1 pyramidal layer and robust morphological sprouting in the dentate gyrus. We also found a reduction in the number of interneuron-selective calretinin-ir cells in the dentate gyrus of hippocampal-kindled rats, which suggests a possible reduction of synchronized dendritic inhibition. In contrast, perisomatic inhibition indicated by parvalbumin immunoreactivity appears to be largely resilient to the effects of kindling. Finally, we found a significant induction in the number of GAD67-cells in caudate-kindled rats in the dentate gyrus and CA3 hippocampal subfields. Taken together, our results demonstrate that kindling has subfield-selective effects on the different functional classes of hippocampal GABAergic interneurons. J. Comp. Neurol. 525:389-406, 2017. © 2016 Wiley Periodicals, Inc.

The effect of chronic corticosterone on fear learning and memory depends on dose and the testing protocol.

Chronic exposure to the stress hormone corticosterone (CORT) is known to alter plasticity within hippocampal and amygdalar circuits that mediate fear learning and memory. The purpose of this experiment was to clarify the effects of chronic CORT on Pavlovian fear conditioning, which is dependent on intact hippocampal and amygdalar activity. In particular, we assessed whether the effect of chronic CORT on fear learning and memory is influenced by two factors-the dose of CORT and the order in which rats are tested for freezing to context versus tone cues. Male Long-Evans rats received low-dose CORT (5mg/kg), high-dose CORT (40mg/kg), or vehicle injections once daily for 21days. On day 22, the rats were trained in a fear-conditioning paradigm. On days 23 and 24, the rats were tested for the retrieval of fear memories to context and tone cues in a counterbalanced way-half the rats received context testing on day 23 and then tone testing on day 24 and half the rats received tone testing on day 23 followed by context testing on day 24. Our results revealed dose-dependent effects of CORT on memory retrieval: Rats injected with high-dose CORT froze significantly more than control rats to both context and tone cues regardless of what testing day these cues were presented. However, rats injected with low-dose CORT froze significantly more than control rats to tone cues only. We also found an order effect in that the effects of CORT on freezing were greater on the second day of testing, regardless of whether that testing was to context or tones cues. This order effect may be due to a lack of extinction in the CORT rats. Overall, these results suggest a relationship between stress intensity and testing conditions that should be taken into account when assessing the effect of stress on fear memories.

Limbic but not non-limbic kindling impairs conditioned fear and promotes plasticity of NPY and its Y2 receptor.

Epileptic seizures negatively affect cognition. However, the mechanisms that contribute to cognitive impairments after seizures are largely unknown. Here, we examined the effects of long-term kindling (i.e., 99 stimulations) of limbic (basolateral amygdala, dorsal hippocampus) and non-limbic (caudate nucleus) brain sites on conditioned fear and hippocampal plasticity. We first showed that kindling had no effect on acquisition of a hippocampal-dependent trace fear-conditioning task but limbic kindling impaired the retrieval of these fear memories. To determine the relationship between memory and hippocampal neuronal activity, we examined the expression of Fos protein 90 min after memory retrieval (i.e., 4 days after the last kindling stimulation). We found that limbic kindling, but not non-limbic kindling, decreased Fos expression in the granule cell layer, hilus, CA3 pyramidal cell layer, and CA1 pyramidal cell layer. Next, to investigate a mechanism that could contribute to dampen hippocampal neuronal activity in limbic-kindled rats, we focused on the endogenous anticonvulsant neuropeptide Y (NPY), which is expressed in a subset of GABAergic interneurons and can prevent glutamate release through interactions with its Y2 receptor. We found that limbic kindling significantly decreased the number of NPY-immunoreactive cells in several hippocampal subfields despite minimal staining of the neurodegenerative marker Fluoro-Jade B. However, we also noted that limbic kindling enhanced NPY immunoreactivity throughout the mossy fiber pathway. In these same regions, we observed limbic kindling-induced de novo expression of the NPY Y2 receptor. These novel findings demonstrate the site-specific effects of kindling on cognition and NPY plasticity, and they provide evidence that altered hippocampal NPY after limbic seizures coincides with dampened neural activity and cognitive impairments.

Long-term amygdala kindling in rats as a model for the study of interictal emotionality in temporal lobe epilepsy.

Temporal lobe epileptics often experience profound interictal (i.e. between seizure) emotional disturbances, such as fear, anxiety, and depression. Although the presence of this interictal emotionality has been well documented, little progress has been made in identifying its precise nature and cause because it is not amenable to experimental analysis in clinical populations. Accordingly, there is much to gain by studying the fundamental nature and neural basis of interictal emotionality using animal models. Kindling is a widely studied animal model of temporal lobe epilepsy in which daily electrical stimulation of certain brain regions results in the gradual progression and intensification of limbic motor seizures. Several investigators have found that partial and short-term kindling produce robust changes in emotional behavior in both cats and rats. Recently, our laboratory has developed a new model to study interictal emotionality using long-term kindling in rats. These long-term kindled rats display profound changes in fearful and defensive behavior which last for at least two months after the final stimulation. We are now beginning to use this model to study the neural mechanisms underlying the development and expression of interictal emotionality.

Hippocampal involvement in the expression of kindling-induced fear in rats.

Kindling dramatically increases fearful behavior in rats. Because kindling-induced fear increases in magnitude as rats receive more stimulations, kindling provides a superb opportunity to study the nature and neural mechanisms of fear sensitization. Interestingly, these changes in behavior are accompanied by increased binding to inhibitory receptors and decreased binding to excitatory receptors in the CA1 and dentate gyrus regions of the hippocampus. This led us to hypothesize that kindling-induced fear may result from an increased inhibitory tone within hippocampal circuits. To test this hypothesis, we investigated FOS protein immunoreactivity in hippocampal and amygdalar regions of kindled rats that were exposed to an unfamiliar open field. We found that FOS immunoreactivity was significantly decreased in the CA1 region, dentate gyrus, and perirhinal cortex of kindled rats compared to sham-stimulated rats. These results support our hypothesis that kindling-induced fear may be produced by inhibition within hippocampal circuits. They also suggest that neural changes within the hippocampus may be important for the sensitization of fear.

Changes in emotional behavior produced by long-term amygdala kindling in rats.

The effects of long-term amygdala kindling on emotional behavior were investigated. In Experiment 1, rats received 99 basolateral amygdala, central amygdala, or sham stimulations. The rats in both kindled groups displayed more resistance to capture from an open field and more open-arm activity on an elevated plus maze than did the sham control rats. In Experiment 2, rats received either 20, 60, or 100 amygdala stimulations or sham stimulations. Compared to the sham controls, the kindled rats explored less during the first 30s in a novel open field, avoided the central area of the open field, resisted being captured from the open field, and engaged in more open-arm activity on the elevated plus maze. The magnitude of these effects was greatest in the 100-stim rats and least in the 20-stim rats. Together, these results suggest that long-term amygdala kindling in rats is a useful model for studying the emotionality associated with temporal lobe epilepsy.

Persistence of the interictal emotionality produced by long-term amygdala kindling in rats.

Long-term amygdala kindling in rats results in large and reliable increases in emotional behaviour that model the interictal emotionality often observed in temporal lobe epileptics [Kalynchuk L. E. et al. (1997) Biol. Psychiat. 41, 438-451; Pinel J. P. J. et al. (1977) Science 197, 1088-1089]. These experiments investigated the persistence of these kindling-induced increases in emotional behaviour after the cessation of the kindling stimulations. In Experiment 1, rats received 99 amygdala or sham stimulations. Then, they were tested on three tests of emotionality (i.e. activity in an unfamiliar open field, resistance to capture from the open field, and activity in an elevated-plus maze) either one day, one week, or one month after the final stimulation. The rats tested one day after the last stimulation displayed substantial decreases in open-field activity, increases in resistance to capture and increases in open-arm activity on the elevated-plus maze; these effects decreased, but not to control levels, in the rats tested one month after the final stimulation. In Experiment 2, rats received 99 amygdala or sham stimulations, and their resistance to capture was assessed one day later. Then, after a 60-day stimulation-free period, the rats received another zero, one, 10, or 30 amygdala stimulations and their resistance to capture was reassessed one day later. The high levels of resistance to capture observed in the rats tested one day after the 99 stimulations declined significantly during the 60-day stimulation-free period, but it remained significantly above control levels. However, the administration of 30 additional stimulations reinstated asymptotic levels of resistance to capture. These results provide the first systematic evidence that kindling-induced increases in emotional behaviour persist at significant levels for at least two months following the termination of kindling stimulations. Thus, they suggest that the neural changes underlying the genesis of interictal emotionality may be closely related to those mediating epileptogenesis itself.

Characterization of the defensive nature of kindling-induced emotionality.

Long-term amygdala kindling produces substantial changes in emotional behavior in rats. The purpose of these experiments was to determine whether kindling-induced emotionality is fundamentally defensive or aggressive in nature. In Experiment 1, amygdala-kindled rats tested as intruders in a resident-intruder paradigm preferred an active defense strategy (i.e., defensive upright stance, jump attacks), whereas the sham-stimulated rats preferred a passive defense strategy (i.e., freezing). In Experiment 2, amygdala-kindled rats explored an unfamiliar open field significantly less than did the sham-stimulated rats, and they were significantly more resistant to capture from the unfamiliar open field than were the sham-stimulated rats. In contrast, there were no significant differences between the kindled and sham-stimulated rats in resistance to capture from their home cages. These results suggest that the emotionality produced by long-term amygdala kindling is fundamentally defensive in nature.

Effect of an ascending dose regimen on the development of tolerance to the anticonvulsant effect of diazepam.

The effect of an ascending dose regimen on the development of tolerance to diazepam's anticonvulsant effect was assessed. During the 22 trials of the tolerance development phase, amygdala-kindled rats received either a series of dosage injections ranging from high (10 mg/kg), to low (1.0 mg/kg), and ascending (1.0 mg/kg and increased by 0.2-mg/kg increments to 3.0 mg/kg) or saline injections. Diazepam was administered by ip injection once every 48 hr, and each injection was followed 1 hr later by a convulsive stimulation. The ascending dose rats displayed significantly more tolerance to the anticonvulsant effect of diazepam than did the high dose, low dose, or saline rats. By contrast, both the ascending and high dose rats displayed a significant withdrawal effect (i.e., increased duration of convulsions) after the cessation of diazepam injections. Results demonstrate that administration of ascending dosages can facilitate the development of tolerance to anticonvulsant drug effects and that tolerance and withdrawal are not necessarily inextricably related.

Contingent tolerance, compensatory responses, and physical dependence in diazepam-treated amygdala-kindled rats.

Three groups of amygdala-kindled rats received 10 bidaily treatment trials: On each trial, the drug-before group received a diazepam (2.5 mg/kg i.p.) injection 1 hr before a convulsive stimulation, the drug-after group received a diazepam injection 1 hr after a stimulation, and the vehicle control group received a vehicle injection either 1 hr before or 1 hr after a stimulation. After treatment, only the drug-before group displayed significantly longer forelimb clonus under the influence of diazepam (that is, they displayed contingent tolerance to diazepam's anticonvulsant effect) and significantly longer forelimb clonus while drug free. Following a 14-day retention period, the rats in the drug-before group retained significant levels of contingent tolerance but did not display significant increases when tested drug free. These data suggest that compensatory responses do not play a causal role in the expression of contingent tolerance.

Object-recognition and spatial learning and memory in rats prenatally exposed to ethanol.

Prenatal ethanol exposure can produce cognitive and behavioral impairments. In the present study, rats from prenatal ethanol (E), pair-fed (PF), and ad libitum-fed control (C) treatment conditions were tested on the object-recognition delayed-nonmatching-to-sample (DNMS) task with nonrecurring items and on the spatial-navigation Morris water maze task. In Experiment 1, there were no significant differences among groups in object-recognition learning and memory, distractibility, or response perseveration on the DNMS task. In Experiment 2, the same rats were tested in the water maze; E rats took significantly longer to learn the task than did the PF or C rats. These data suggest that the mechanisms underlying spatial cognitive abilities are more vulnerable to the teratogenic effects of prenatal ethanol exposure than those underlying object-recognition abilities.

Behavioral analysis of diazepam-induced memory deficits: evidence for sedation-like effects.

Rats were trained on a nonmatching-to-sample task with delays of 2, 5, and 10 min. Subsequently, performance was assessed in three groups of rats following treatment with saline or diazepam (2.0 mg/kg) administered acutely or tested chronically in six administrations. Relative to treatment with saline, diazepam produced a deficit in discrimination performance, which was greater in the acutely treated rats than in those treated chronically. The deficit was not dependent on the length of the delays. Diazepam-treated animals differed from controls in erring on trials in which they failed to investigate both test objects, failed to investigate the test object for a long enough period of time, and displaced the test object on the preferred side of the apparatus. The hypothesis that these effects represented a sedation-like reduction in behavioral variability was also supported by evidence of a diazepam-induced decrease in gross bodily activity, increase in inactivity, and increase in latencies to respond to objects. No support was found for the involvement of diazepam-induced changes in habituation, extinction, or reward effects.

Analysis of the ongoing behavior of rats in non-matching-to-sample: improved acquisition and performance is related to facilitation of investigation.

In a non-matching-to-sample task, rats were trained according to the conventional procedure in which the displacement of the sample object resulted in food reinforcement and termination of the sample period. Compared to these animals, rats given a longer duration sample period, and rats not reinforced with food for displacing the object in the sample period, improved their rate of acquisition and their accuracy of performance. Detailed behavioral observations indicated that improved discrimination was related to increased investigation of the objects in the sample period, reduced side preferences, and an inclination to examine both objects in the test period. The results suggest that more accurate performance was related to the generalization of sampling habits from the sample period to the test period.

Long-term kindling and interictal emotionality in rats: effect of stimulation site.

Long-term amygdala kindling in rats produces increases in emotionality (Kalynchuk et al., Biol. Psychiatry, 41 (1997) 438-451). The present experiment was conducted to investigate whether this hyperemotionality is specific to amygdala kindling or whether it can be produced by kindling other structures. Rats received 99 convulsive or sham stimulations of either the amygdala, the hippocampus, or the caudate nucleus. One day after the stimulation phase, each rat's open-field activity and resistance to capture were assessed; the following day, each rat was tested on an elevated plus maze. The site of stimulation had a significant effect on the results of each of these tests. The amygdala-kindled and hippocampal-kindled rats explored less in the open field, were more resistant to capture from the open field, and engaged in a greater percentage of open-arm activity in the elevated plus maze than did the caudate-kindled rats or the sham-stimulated controls. The caudate-kindled rats were more active in the open field than their sham-stimulated controls, but they did not significantly differ from them in terms of the other measures. These results suggest that kindling-induced emotionality is produced by limbic kindling but not nonlimbic kindling.

Tolerance to the anticonvulsant and ataxic effects of pentobarbital: effect of an ascending-dose regimen.

We assessed the effect of an ascending-dose regimen on the development of tolerance to the anticonvulsant and ataxic effects of pentobarbital in four groups of amygdala-kindled rats. Each rat received 20 bidaily (one every 48 h) trials in which an intraperitoneal (IP) pentobarbital or vehicle injection was delivered 1 h before a convulsive amygdala stimulation. On each trial, the rats in the three pentobarbital groups received either a high dose (50 mg/kg), a low dose (10mg/kg), or ascending doses of pentobarbital that began at 10 mg/kg and increased to as high as 26 mg/kg by 1 mg/kg increments as tolerance developed to its anticonvulsant effect; the rats in the vehicle group received saline. The rats in the ascending-dose condition displayed significantly more tolerance to the anticonvulsant effect of pentobarbital than did the other rats; in contrast, the high-dose rats displayed more tolerance to the ataxic effect of pentobarbital than did the other rats. These findings extend previous reports of the facilitatory effect of ascending-dose regimens on the development of tolerance to the anticonvulsant effect of benzodiazepines, and show that the facilitatory effect of ascending-dose regimens does not extend to all drug effects.

Dissipation of contingent tolerance to the anticonvulsant effect of diazepam: effect of the criterion response.

The effect of convulsive stimulations on the dissipation of tolerance to the anticonvulsant effect of diazepam was investigated using the kindled-convulsion model. Amygdala-kindled rats were rendered tolerant to diazepam's anticonvulsant effect by 25 "bidaily" (one/48 h) diazepam injections (2.5 mg/kg), each followed 1 h later by a convulsive stimulation. They were then divided into nine groups for the tolerance-dissipation phase of the experiment. Of the nine groups, three received bidaily control handling for one trial, three trials, or seven trials; three received bidaily saline injections, each 1 h before a convulsive stimulation, for one, three, or seven trials; and three received bidaily diazepam injections, each 1 h after a convulsive stimulation, for one, three, or seven trials. Finally, each rat received a tolerance-retention test (i.e., a diazepam injection followed 1 h later by a convulsive stimulation) 48 h after its last tolerance-dissipation trial. The tolerance dissipated gradually but completely over the 4-, 8-, and 16-day test intervals in the rats that received a convulsive stimulation before each injection during the tolerance-dissipation phase, whether they were injected with saline or diazepam; in contrast, tolerance did not dissipate in the rats that received saline injections but no stimulations. Remarkably, the discontinuance of the bidaily diazepam injections, even for 16 days, was not sufficient to dissipate the tolerance that had developed to diazepam's anticonvulsant effect; nor was the continuation of the bidaily diazepam injections sufficient to keep tolerance from dissipating.(ABSTRACT TRUNCATED AT 250 WORDS)

Noncontingent drug exposure facilitates the development of contingent tolerance to the anticonvulsant effects of ethanol and diazepam in kindled rats.

Tolerance to anticonvulsant drug effects on kindled convulsions can result from drug exposure alone, but convulsive activity during drug exposure has a substantial facilitatory effect on tolerance development. Tolerance produced by drug exposure in the absence of a criterion response (in this case convulsions) has been termed pharmacologic tolerance (10); tolerance produced by drug exposure with concomitant performance of the criterion response has been termed contingent tolerance (1). The present study examines whether noncontingent drug exposure facilitates the development of contingent tolerance to the anticonvulsant effects of ethanol and diazepam. Amygdala-kindled, Long-Evans rats were treated with either ethanol (5.0 g/kg once daily for 21 days) or diazepam (5.0 mg/kg three times daily for 10 days) in the absence of convulsive stimulation to produce pharmacologic tolerance--control rats received treatments of vehicle. Then, all of the rats were rendered contingently tolerant by a series of "bidaily" (once every 2 days) injections (ethanol 2.0 g/kg or diazepam 2.0 mg/kg), each 1 h prior to a kindled convulsion. The rats that had received noncontingent exposure to ethanol or diazepam developed contingent tolerance significantly faster than the control rats. These results suggest that the mechanisms underlying pharmacologic and contingent tolerance to anticonvulsant drug effects are additive.