Wendie N. Marks

Repeated exposure to corticosterone increases depression-like behavior in two different versions of the forced swim test without altering nonspecific locomotor activity or muscle strength

We have recently shown that repeated high dose injections of corticosterone (CORT) reliably increase depression-like behavior on a modified one-day version of the forced swim test. The main purpose of this experiment was to compare the effect of these CORT injections on our one-day version of the forced swim test and the more traditional two-day version of the test. A second purpose was to determine whether altered behavior in the forced swim test could be due to nonspecific changes in locomotor activity or muscle strength. Separate groups of rats received a high dose CORT injection (40 mg/kg) or a vehicle injection once per day for 21 consecutive days. Then, half the rats from each group were exposed to the traditional two-day forced swim test and the other half were exposed to our one-day forced swim test. After the forced swim testing, all the rats were tested in an open field and in a wire suspension grip strength test. The CORT injections significantly increased the time spent immobile and decreased the time spent swimming in both versions of the forced swim test. However, they had no significant effect on activity in the open field or grip strength in the wire suspension test. These results show that repeated CORT injections increase depression-like behavior regardless of the specific parameters of forced swim testing, and that these effects are independent of changes in locomotor activity or muscle strength.

Impaired recruitment of seizure-generated neurons into functional memory networks of the adult dentate gyrus following long-term amygdala kindling.

Epileptic seizures increase the birth of new neurons in the adult hippocampus. Although the consequences of aberrant neurogenesis on behavior are not fully understood, one hypothesis is that seizure-generated neurons might form faulty circuits that disrupt hippocampal functions, such as learning and memory. In the present study, we employed long-term amygdala kindling (i.e., rats receive 99-electrical stimulations) to examine the effect of repeated seizures on hippocampal neurogenesis and behavior. We labeled seizure-generated cells with the proliferation marker BrdU after 30-stimulations and continued kindling for an additional 4weeks to allow newborn neurons to mature under conditions of repeated seizures. After kindling was complete, rats were tested in a trace fear conditioning task and sacrificed 2h later to examine if 4-week old newborn cells were recruited into circuits involved in the retrieval of emotional memory. Compared to non-kindled controls, long-term kindled rats showed significant impairments in fear memory reflected in a decrease in conditioned freezing to both tone and contextual cues during testing. Moreover, long-term kindling also prevented the activation of 4-week old newborn cells in response to fear memory retrieval. These results indicate that the presence of seizure activity during cell maturation impedes the ability of new neurons to integrate properly into circuits important in memory formation. Together, our findings suggest that aberrant seizure-induced neurogenesis might contribute to the development of learning impairments in chronic epilepsy and raise the possibility that targeting the reduced activation of adult born neurons could represent a beneficial strategy to reverse cognitive deficits in some epileptic patients.

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.

Amygdala kindling disrupts trace and delay fear conditioning with parallel changes in Fos protein expression throughout the limbic brain.

Amygdala kindling is well known to increase unconditioned fear and anxiety. However, relatively little is known about whether this form of kindling causes functional changes within the neural circuitry that mediates fear learning and the retrieval of fear memories. To address this issue, we examined the effect of short- (i.e., 30 stimulations) and long-term (i.e., 99 stimulations) amygdala kindling in rats on trace and delay fear conditioning, which are aversive learning tasks that rely predominantly on the hippocampus and amygdala, respectively. After memory retrieval, we analyzed the pattern of neural activity with Fos, the protein product of the immediate early gene c-fos. We found that kindling had no effect on acquisition of the trace fear conditioning task but it did selectively impair retrieval of this fear memory. In contrast, kindling disrupted both acquisition and retrieval of fear memory in the delay fear conditioning task. We also found that kindling-induced impairments in memory retrieval were accompanied by decreased Fos expression in several subregions of the hippocampus, parahippocampus, and amygdala. Interestingly, decreased freezing in the trace conditioning task was significantly correlated with dampened Fos expression in hippocampal and parahippocampal regions whereas decreased freezing in the delay conditioning task was significantly correlated with dampened Fos expression in hippocampal, parahippocampal, and amygdaloid circuits. Overall, these results suggest that amygdala kindling promotes functional changes in brain regions involved in specific types of fear learning and memory.

The T-type calcium channel antagonist Z944 rescues impairments in crossmodal and visual recognition memory in Genetic Absence Epilepsy Rats from Strasbourg

Childhood absence epilepsy (CAE) is often comorbid with behavioral and cognitive symptoms, including impaired visual memory. Genetic Absence Epilepsy Rats from Strasbourg (GAERS) is an animal model closely resembling CAE; however, cognition in GAERS is poorly understood. Crossmodal object recognition (CMOR) is a recently developed memory task that examines not only purely visual and tactile memory, but also requires rodents to integrate sensory information about objects gained from tactile exploration to enable visual recognition. Both the visual and crossmodal variations of the CMOR task rely on the perirhinal cortex, an area with dense expression of T-type calcium channels. GAERS express a gain-in-function missense mutation in the Cav3.2 T-type calcium channel gene. Therefore, we tested whether the T-type calcium channel blocker Z944 dose dependently (1, 3, 10mg/kg; i.p.) altered CMOR memory in GAERS compared to the non-epileptic control (NEC) strain. GAERS demonstrated recognition memory deficits in the visual and crossmodal variations of the CMOR task that were reversed by the highest dose of Z944. Electroencephalogram recordings determined that deficits in CMOR memory in GAERS were not the result of seizures during task performance. In contrast, NEC showed a decrease in CMOR memory following Z944 treatment. These findings suggest that T-type calcium channels mediate CMOR in both the GAERS and NEC strains. Future research into the therapeutic potential of T-type calcium channel regulation may be particularly fruitful for the treatment of CAE and other disorders characterized by visual memory deficits.

The Genetic Absence Epilepsy Rats from Strasbourg model of absence epilepsy exhibits alterations in fear conditioning and latent inhibition consistent with psychiatric comorbidities in humans

Behavioural, neurological, and genetic similarities exist in epilepsies, their psychiatric comorbidities, and various psychiatric illnesses, suggesting common aetiological factors. Rodent models of epilepsy are used to characterize the comorbid symptoms apparent in epilepsy and their neurobiological mechanisms. The present study was designed to assess Pavlovian fear conditioning and latent inhibition in a polygenetic rat model of absence epilepsy, i.e. Genetic Absence Epilepsy Rats from Strasbourg (GAERS) and the non‐epileptic control (NEC) strain. Electrophysiological recordings confirmed the presence of spike‐wave discharges in young adult GAERS but not NEC rats. A series of behavioural tests designed to assess anxiety‐like behaviour (elevated plus maze, open field, acoustic startle response) and cognition (Pavlovian conditioning and latent inhibition) was subsequently conducted on male and female offspring. Results showed that GAERS exhibited significantly higher anxiety‐like behaviour, a characteristic reported previously. In addition, using two protocols that differed in shock intensity, we found that both sexes of GAERS displayed exaggerated cued and contextual Pavlovian fear conditioning and impaired fear extinction. Fear reinstatement to the conditioned stimuli following unsignalled footshocks did not differ between the strains. Male GAERS also showed impaired latent inhibition in a paradigm using Pavlovian fear conditioning, suggesting that they may have altered attention, particularly related to previously irrelevant stimuli in the environment. Neither the female GAERS nor NEC rats showed evidence of latent inhibition in our paradigm. Together, the results suggest that GAERS may be a particularly useful model for assessing therapeutics designed to improve the emotional and cognitive disturbances associated with absence epilepsy.

The T-type calcium channel antagonist Z944 disrupts prepulse inhibition in both epileptic and non-epileptic rats

The role of T-type calcium channels in brain diseases such as absence epilepsy and neuropathic pain has been studied extensively. However, less is known regarding the involvement of T-type channels in cognition and behavior. Prepulse inhibition (PPI) is a measure of sensorimotor gating which is a basic process whereby the brain filters incoming stimuli to enable appropriate responding in sensory rich environments. The regulation of PPI involves a network of limbic, cortical, striatal, pallidal and pontine brain areas, many of which show high levels of T-type calcium channel expression. Therefore, we tested the effects of blocking T-type calcium channels on PPI with the potent and selective T-type antagonist Z944 (0.3, 1, 3, 10mg/kg; i.p.) in adult Wistar rats and two related strains, the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) and Non-Epileptic Control (NEC). PPI was tested using a protocol that varied prepulse intensity (3, 6, and 12dB above background) and prepulse-pulse interval (30, 50, 80, 140ms). Z944 decreased startle in the Wistar strain at the highest dose relative to lower doses. Z944 dose-dependently disrupted PPI in the Wistar and GAERS strains with the most potent effect observed with the higher doses. These findings suggest that T-type calcium channels contribute to normal patterns of brain activity that regulate PPI. Given that PPI is disrupted in psychiatric disorders, future experiments that test the specific brain regions involved in the regulation of PPI by T-type calcium channels may help inform therapeutic development for those suffering from sensorimotor gating impairments.

Sociability impairments in Genetic Absence Epilepsy Rats from Strasbourg: Reversal by the T-type calcium channel antagonist Z944

&NA; Childhood absence epilepsy (CAE) is associated with interictal co‐morbid symptoms including abnormalities in social behaviour. Genetic Absence Epilepsy Rats from Strasbourg (GAERS) is a model of CAE that exhibits physiological and behavioural alterations characteristic of the human disorder. However, it is unknown if GAERS display the social deficits often observed in CAE. Sociability in rodents is thought to be mediated by neural circuits densely populated with T‐type calcium channels and GAERS contain a missense mutation in the Cav3.2 T‐type calcium channel gene. Thus, the objective of this study was to examine the effects of the clinical stage pan‐T‐type calcium channel blocker, Z944, on sociability behaviour in male and female GAERS and non‐epileptic control (NEC) animals. Female GAERS showed reduced sociability in a three‐chamber sociability task whereas male GAERS, male NECs, and female NECs all showed a preference for the chamber containing a stranger rat. In drug trials, pre‐treatment with 5 mg/kg of Z944 normalized sociability in female GAERS. In contrast, female NECs showed impaired sociability following Z944 treatment. Dose‐dependent decreases in locomotor activity were noted following Z944 treatment in both strains. Treatment with 10 mg/kg of Z944 altered exploration such that only 8 of the 16 rats tested explored both sides of the testing chamber. In those that explored the chamber, significant preference for the stranger rat was observed in GAERS but not NECs. Overall, the data suggest that T‐type calcium channels are critical in regulating sociability in both GAERS and NEC animals. Future research should focus on T‐type calcium channels in the treatment of sociability deficits observed in disorders such as CAE.

T-type calcium channels in the orbitofrontal cortex mediate sensory integration as measured using a spontaneous oddity task in rats

The roles of low-voltage-activated (T-type) calcium channels in brain diseases have been studied extensively. Less is known regarding the involvement of T-type channels in cognition and behavior. Sensory integration (SI) is a cognitive process whereby the brain uses unimodal or multimodal sensory features to create a comprehensive representation of the environment. The multisensory object oddity (MSO) task assesses SI using combinations of sensory features of objects, either in the same or different sensory modalities. The regulation of SI involves the orbitofrontal cortex (OFC), an area which shows high levels of T-type calcium channel expression. We tested the effects of blocking T-type calcium channels on the MSO task with the selective T-type antagonist, Z944 (5 mg/kg; i.p. systemic; 100 or 500 µM OFC infusion), in male Long Evans rats. With systemic treatment, Z944 impaired the visual and visual-olfactory versions of the task. Infusion of 100 and 500 µM Z944 produced deficits in the olfactory version of the task. In addition, only vehicle-infused, but not Z944-infused, rats showed significant performance above chance for all task variants. Thus, the present results suggest that T-type calcium channels in OFC are involved in SI of features in an oddity task. Given that unimodal SI was disrupted by OFC infusions of Z944, the deficits in the multimodal task must be interpreted with caution. As SI is disrupted in psychiatric disorders, further investigations elucidating the brain regions implicated in SI regulation by T-type calcium channels may help inform therapeutic development for those suffering from SI impairments.

Repeated corticosterone enhances the acquisition and recall of trace fear conditioning

Repeated exposure to high levels of stress hormones can enhance contextual and discrete fear conditioning in rats. A common belief is that this enhanced fear memory is largely mediated by the amygdala because both contextual and discrete fear conditioning are dependent on an intact amygdala. However, trace fear conditioning is thought to be amygdala independent, and therefore, it is not clear what impact stress would have on this form of fear learning. Here, we examined whether the stress hormone corticosterone (CORT) would enhance memory in a hippocampal-dependent trace fear conditioning test. Male Long-Evans rats received either 40mg/kg of CORT or vehicle injections for 21 consecutive days. On day 22, rats received either 1, 2, or 5 tone-trace-shock pairings. On day 23, the rats were tested for behavior to the conditioned tone cues in a novel context. We found that CORT significantly increased the acquisition of trace conditioned fear. We also found that CORT significantly increased recall of trace conditioned cues, but only when a 2 trace-pairing protocol was used during training. These results suggest that CORT can enhance non-amygdala forms of fear learning and memory and that high levels of stress hormones modify the physiological substrates that mediate emotionally driven behavior in tasks that are less dependent on amygdala functioning.