Choline acetyltransferase, glutamic acid decarboxylase and somatostatin in the kainic acid model for chronic temporal lobe epilepsy

The aim of the study was to investigate neurochemical changes in a kainic acid (KA; 10 mg/kg, s.c.)-induced spontaneous recurrent seizure model of epilepsy, 6 months after the initial KA-induced seizures. The neuronal markers of cholinergic and gamma-aminobutyric acid (GABA)ergic systems, i.e. choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) activities, and a marker for neuropeptide, i.e. level of somatostatin, have been investigated. The brain regions investigated were the hippocampus, amygdala/piriform cortex, caudate nucleus, substantia nigra and the frontal, parietal, temporal and occipital cortices. Six months after KA injection, reduced ChAT activity was observed in the amygdala/piriform cortex (47% of control; p<0.001), increased ChAT activity in the hippocampus (119% of control; p<0.01) and normal ChAT activity in the other brain regions. The activity of GAD was significantly increased in all analysed cortical regions (between 146 and 171% of control), in the caudate nucleus (144% of control; p<0.01) and in the substantia nigra (126% of control; p<0.01), whereas in the amygdala/piriform cortex, the GAD activity was moderately lowered. The somatostatin level was significantly increased in all cortical regions (between 162 and 221% of control) as well as in the hippocampus (119% of control), but reduced in the amygdala/piriform cortex (45% of control; p<0.01). Six months after KA injection, the somatostatin:GAD ratio was lowered in the amygdala/piriform cortex (49% of control) and in the caudate nucleus (41% of control), whereas it was normal in the hippocampus and moderately increased in the cortical brain regions. A positive correlation was found between seizure severity and the reduction of both ChAT activities and somatostatin levels in the amygdala/piriform cortex. The results show a specific pattern of changes for cholinergic, GABAergic and somatostatinergic activities in the chronic KA model for epilepsy. The revealed data suggest a functional role for them in the new network that follows spontaneous repetitive seizures.     

Persistent impairment of mitochondrial and tissue redox status during lithium-pilocarpine-induced epileptogenesis

Mitochondrial dysfunction and oxidative stress are known to occur following acute seizure activity but their contribution during epileptogenesis is largely unknown. The goal of this study was to determine the extent of mitochondrial oxidative stress, changes to redox status, and mitochondrial DNA (mtDNA) damage during epileptogenesis in the lithium-pilocarpine model of temporal lobe epilepsy. Mitochondrial oxidative stress, changes in tissue and mitochondrial redox status, and mtDNA damage were assessed in the hippocampus and neocortex of Sprague-Dawley rats at time points (24h to 3months) following lithium-pilocarpine administration. A time-dependent increase in mitochondrial hydrogen peroxide (H(2)O(2)) production coincident with increased mtDNA lesion frequency in the hippocampus was observed during epileptogenesis. Acute increases (24-48h) in H(2)O(2) production and mtDNA lesion frequency were dependent on the severity of convulsive seizure activity during initial status epilepticus. Tissue levels of GSH, GSH/GSSG, coenzyme A (CoASH), and CoASH/CoASSG were persistently impaired at all measured time points throughout epileptogenesis, that is, acutely (24-48h), during the 'latent period' (48h to 7days), and chronic epilepsy (21days to 3months). Together with our previous work, these results demonstrate the model independence of mitochondrial oxidative stress, genomic instability, and persistent impairment of mitochondrial specific redox status during epileptogenesis. Lasting impairment of mitochondrial and tissue redox status during the latent period, in addition to the acute and chronic phases of epileptogenesis, suggests that redox-dependent processes may contribute to the progression of epileptogenesis in experimental temporal lobe epilepsy.     

Spastin in the human and mouse central nervous system with special reference to its expression in the hippocampus of mouse pilocarpine model of status epilepticus and temporal lobe epilepsy

In the present in situ hybridization and immunocytochemical studies in the mouse central nervous system (CNS), a strong expression of spastin mRNA and protein was found in Purkinje cells and dentate nucleus in the cerebellum, in hippocampal principal cells and hilar neurons, in amygdala, substantia nigra, striatum, in the motor nuclei of the cranial nerves and in different layers of the cerebral cortex except piriform and entorhinal cortices where only neurons in layer II were strongly stained. Spastin protein and mRNA were weakly expressed in most of the thalamic nuclei. In selected human brain regions such as the cerebral cortex, cerebellum, hippocampus, amygdala, substania nigra and striatum, similar results were obtained. Electron microscopy showed spastin immunopositive staining in the cytoplasma, dendrites, axon terminals and nucleus. In the mouse pilocarpine model of status epilepticus and subsequent temporal lobe epilepsy, spastin expression disappeared in hilar neurons as early as at 2h during pilocarpine induced status epilepticus, and never recovered. At 7 days and 2 months after pilocarpine induced status epilepticus, spastin expression was down-regulated in granule cells in the dentate gyrus, but induced expression was found in reactive astrocytes. The demonstration of widespread distribution of spastin in functionally different brain regions in the present study may provide neuroanatomical basis to explain why different neurological, psychological disorders and cognitive impairment occur in patients with spastin mutation. Down-regulation or loss of spastin expression in hilar neurons may be related to their degeneration and may therefore initiate epileptogenetic events, leading to temporal lobe epilepsy.     

Lithium/pilocarpine status epilepticus-induced neuropathology of piriform cortex and adjoining structures in rats is age-dependent

Distribution of LiCl/pilocarpine status epilepticus-induced neuronal damage was studied in the piriform cortex and in adjoining structures in 12-day-old, 25-day-old and adult rats. No distinct structural and neuronal alterations were detected in the basal telencephalon in 12-day-old rats surviving status epilepticus (SE) for one week or two months. In 25-day-old rats a decrease in Nissl staining was evident. There was also cell loss and gliosis in the caudal 2/3 of the piriform cortex, in the superficial amygdaloid nuclei, in the dorsal and ventral endopiriform nucleus and in the rostrolateral part of the entorhinal cortical area. In adult animals, the topography of neuropathological changes in the basal telencephalon was comparable to those in 25-day-old rats. The damage in the caudal 2/3 or caudal half of the piriform cortex in adult rats with survival times one week or two months was characterized by a marked loss of neurons and striking glial infiltration. The thickness of the piriform cortex and superficial amygdaloid nuclei was significantly reduced. In 25-day-old and in adult animals the sublayer IIb and layer III of the piriform cortex was more affected, while sublayer IIa was less damaged. Parvalbumin (PV) immunocytochemistry revealed a significant decrease in the number of PV-immunoreactive neurons in the rostral piriform cortex and in the dorsal claustrum in animals surviving for two months.     

Amygdala kindling differentially regulates the expression of the elements involved in TRH transmission

Subthreshold electrical stimulation of the amygdala (kindling) activates neuronal pathways increasing the expression of several neuropeptides including thyrotropin releasing-hormone (TRH). Partial kindling enhances TRH expression and the activity or its inactivating ectoenzyme; once kindling is established (stage V), TRH and its mRNA levels are further increased but TRH-binding and pyroglutamyl aminopeptidase II (PPII) activity decreased in epileptogenic areas. To determine whether variations in TRH receptor binding or PPII activity are due to regulation of their synthesis, mRNA levels of TRH receptors (R1, R2) and PPII were semi-quantified by RT-PCR in amygdala, frontal cortex and hippocampus of kindled rats sacrificed at stage II or V. Increased mRNA levels of PPII were found at stage II in amygdala and frontal cortex, and of pro-TRH and TRH-R2, in amygdala and hippocampus. At stage V, pro-TRH mRNA levels increased and those of PPII, decreased in the three regions; TRH-R2 mRNA levels diminished in amygdala and frontal cortex and of TRH-R1 only in amygdala. In situ hybridization analyses revealed, at stage II, enhanced TRH-R1 mRNA levels in dentate gyrus and amygdala while decreased in piriform cortex; those of TRH-R2 increased in amygdala, CA2, dentate gyrus, piriform cortex, thalamus and subiculum and of PPII, in CAs and piriform cortex. In contrast, at stage V decreased expression of TRH-R1 occurred in amygdala, CA2/3, dentate gyrus and piriform cortex; of TRH-R2 in CA2, thalamus and piriform cortex, and of PPII in CA2, and amygdala. The magnitude of changes differed between ipsi and contralateral side. These results support a trans-synaptic modulation of all elements involved in TRH transmission in conditions that stimulate the activity of TRHergic neurons. They show that reported changes in PPII activity or TRH-binding caused by kindling relate to regulation of the expression of TRH receptors and degrading enzyme.     

Alterations in Glutamate Transporter Protein Levels in Kindling-Induced Epilepsy

Abstract: There is increasing evidence that levels of glutamate are elevated in certain brain regions immediately prior to and during induction and propagation of seizures. Modulation of high-affinity glutamate uptake is a potential mechanism responsible for the elevated levels observed with seizures. To date, three distinct Na⁺-dependent glutamate transporters have been cloned from rat and rabbit: GLT-1, GLAST, and EAAC-1. We performed a series of experiments to determine whether levels of these transporters are altered in amygdala-kindled rats. Levels of GLT-1, GLAST, and EAAC-1 were examined in three brain regions (hippocampus, piriform cortex/amygdala, and limbic forebrain) by quantitative immunoblotting using subtype-specific antibodies. GLAST protein was down-regulated in the piriform cortex/amygdala region of kindled rats as early as 24 h after one stage 3 seizure and persisting through multiple stage 5 seizures. In contrast, kindling induced an increase in EAAC-1 levels in piriform cortex/amygdala and hippocampus once the animals had reached the stage 5 level. No changes in GLT-1 were observed in any region examined. Changes in transporter levels could contribute to the changes in glutamate levels seen with kindling.     

Diazepam administration after prolonged status epilepticus reduces neurodegeneration in the amygdala but not in the hippocampus during epileptogenesis

An episode of status epilepticus (SE), if left untreated, can lead to death, or brain damage with long-term neurological consequences, including the development of epilepsy. The most common first-line treatment of SE is administration of benzodiazepines (BZs). However, the efficacy of BZs in terminating seizures is reduced with time after the onset of SE; this is accompanied by a reduced efficacy in protecting the hippocampus against neuronal damage, and is associated with impaired function and internalization of hippocampal GABA_A receptors. In the present study, using Fluoro-Jade C staining, we found that administration of diazepam to rats at 3 h after the onset of kainic acid-induced SE, at a dose sufficient to terminate SE, had no protective effect on the hippocampus, but produced a significant reduction in neuronal degeneration in the amygdala, piriform cortex, and endopiriform nucleus, examined on days 7–9 after SE. Thus, in contrast to the hippocampus, the amygdala and other limbic structures are responsive to neuroprotection by BZs after prolonged SE, suggesting that GABA_A receptors are not significantly altered in these structures during SE.     

Changes in Cholinergic but Not in GABAergic Markers in Amygdala, Piriform Cortex, and Nucleus Basalis of the Rat Brain Following Systemic Administration of Kainic Acid

Three days after systemic administration of kainic acid (15 mg/kg, s.c.), selected cholinergic markers (choline acetyltransferase, acetylcholinesterase, muscarinic acetylcholine receptor, and high-affinity choline uptake) and GABAergic parameters [benzodiazepine and gamma-aminobutyric acid (GABA) receptors] were studied in the frontal and piriform cortex, dorsal hippocampus, amygdaloid complex, and nucleus basalis. Kainic acid treatment resulted in a significant reduction of choline acetyltransferase activity in the piriform cortex (by 20%), amygdala (by 19%), and nucleus basalis (by 31%) in comparison with vehicle-injected control rats. A lower activity of acetylcholinesterase was also determined in the piriform cortex following parenteral kainic acid administration. [3H]Quinuclidinyl benzilate binding to muscarinic acetylcholine receptors was significantly decreased in the piriform cortex (by 33%), amygdala (by 39%), and nucleus basalis (by 33%) in the group treated with kainic acid, whereas such binding in the hippocampus and frontal cortex was not affected by kainic acid. Sodium-dependent high-affinity choline uptake into cholinergic nerve terminals was decreased in the piriform cortex (by 25%) and amygdala (by 24%) after kainic acid treatment. In contrast, [3H]flunitrazepam binding to benzodiazepine receptors and [3H]muscimol binding to GABA receptors were not affected 3 days after parenteral kainic acid application in any of the brain regions studied. The data indicate that kainic acid-induced limbic seizures result in a loss of cholinergic cells in the nucleus basalis that is paralleled by degeneration of cholinergic fibers and cholinoceptive structures in the piriform cortex and amygdala, a finding emphasizing the important role of cholinergic mechanisms in generating and/or maintaining seizure activity.     

Down-Regulation of AMPA Receptor Subunit GluR2 in Amygdaloid Kindling

Abstract: Alterations in glutamatergic transmission are postulated to be important in kindling and epilepsy. The levels of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunits (GluR1, 2, and 4) were compared in amygdalakindled and sham-operated animals using subunit-specific antibodies and quantitative western blotting. Four limbic regions were examined: limbic forebrain, piriform cortex/amygdala, hippocampus, and entorhinal cortex. When subunit levels were examined 24 h after the last stage 5 seizure, levels of GluR2 were found to be selectively reduced in limbic forebrain (30%) and piriform cortex/amygdala (25%), with no changes in other regions examined. In addition, no changes in the other subunits were observed in any region. The decrease in GluR2 that was observed in kindled animals at 24 h was no longer present at 1 week and 1 month after the last stage 5 seizure. Because the GluR2 subunit uniquely determines the calcium permeability of these receptors and because the piriform cortex has been implicated as a source of excitatory drive for limbic seizures, reduced GluR2 expression may be important in increasing neuronal excitability in kindling-induced epilepsy, or may reflect a compensatory mechanism resulting from kindling.     

CCR7, CCR8, CCR9 and CCR10 in the mouse hippocampal CA1 area and the dentate gyrus during and after pilocarpine-induced status epilepticus

The present study showed CCR7, CCR8, CCR9 and CCR10 in the normal Swiss mouse hippocampus at both protein and mRNA levels. CCR7, CCR9 and CCR10 were mainly localized in hippocampal principal cells and some interneurons. CCR9 was also found in the mossy fibres and/or terminals, suggesting an axonal or presynaptic localization, and CCR10 in apical dendrites of pyramidal neurons in the CA1 area. CCR8 was observed in interneurons. Double-labelling immunocytochemistry revealed that most of calbindin (CB)-, calretinin (CR)- and parvalbumin (PV)-immunopositive neurons expressed CCR7-10, except CR-immunopositive cells in which only 10 to 12% expressed CCR8. During and after pilocarpine-induced status epilepticus, progressive changes of each of CCR7, CCR8, CCR9 and CCR10 proteins occurred in different patterns at various time points. Sensitive real-time PCR showed similar change patterns at mRNA level. At the chronic stage, i.e. at 2 months after pilocarpine-induced status epilepticus, significant reduction of CCR7-10 expression in CB-, CR- and PV-immunpositive interneurons may suggest the phenotype change of surviving interneurons. Double labelling of CCR7, CCR8 and CCR9 with glial fibrillary acidic protein (GFAP) at the chronic stage may suggest an induced expression in reactive astrocytes. The present study may, therefore, for the first time, provide evidence that CCR7-10 may be involved in normal hippocampal activity. The demonstration of the progressive changes of CCR7-10 during and after status epilepticus may open a new area to reveal the mechanism of neuronal loss after status epilepticus and of epileptogenesis.     

Down-regulated expression of aquaporin-4 in the cerebellum after status epilepticus

Status epilepticus (SE) is a common neurological condition associated with high rates of mortality and permanent brain injury. SE usually leads to neuronal death which may be accompanied by edema, epileptogenesis and learning impairment. Aquaporin-4 (AQP4), is a transmembrane water channel protein in the neuropil of the central nervous system that has an important role in water transport in the brain; AQP4 expression is altered in many pathological conditions such as changes in the blood- brain barrier and/or astrocytic activation, including seizures. AQP4 was shown to be downregulated in the piriform cortex and the hippocampus after SE. Although it is normally expressed at a high level in the cerebellum, little is known about AQP4 levels in the cerebellum following SE. We addressed this in the present study in a mouse model of pilocarpine-induced SE. We found that AQP4 expression was reduced from 3 h to 3 days after SE, with the levels recovering on day 7. Moreover, mice in the acute post-SE stages exhibited impaired motor coordination and learning. These results indicate that cerebellar damage following SE involves changes in AQP4 expression.     

A Transient Upregulation of Glutamine Synthetase in the Dentate Gyrus Is Involved in Epileptogenesis Induced by Amygdala Kindling in the Rat

Reduction of glutamine synthetase (GS) function is closely related to established epilepsy, but little is known regarding its role in epileptogenesis. The present study aimed to elucidate the functional changes of GS in the brain and its involvement in epileptogenesis using the amygdala kindling model of epilepsy induced by daily electrical stimulation of basolateral amygdala in rats. Both expression and activity of GS in the ipsilateral dentate gyrus (DG) were upregulated when kindled seizures progressed to stage 4. A single dose of L-methionine sulfoximine (MSO, in 2 µl), a selective GS inhibitor, was administered into the ipsilateral DG on the third day following the first stage 3 seizure (just before GS was upregulated). It was found that low doses of MSO (5 or 10 µg) significantly and dose-dependently reduced the severity of and susceptibility to evoked seizures, whereas MSO at a high dose (20 µg) aggravated kindled seizures. In animals that seizure acquisition had been successfully suppressed with 10 µg MSO, GS upregulation reoccurred when seizures re-progressed to stage 4 and re-administration of 10 µg MSO consistently reduced the seizures. GLN at a dose of 1.5 µg abolished the alleviative effect of 10 µg MSO and deleterious effect of 20 µg MSO on kindled seizures. Moreover, appropriate artificial microRNA interference (1 and 1.5×10⁶ TU/2 µl) of GS expression in the ipsilateral DG also inhibited seizure progression. In addition, a transient increase of GS expression and activity in the cortex was also observed during epileptogenesis evoked by pentylenetetrazole kindling. These results strongly suggest that a transient and region-specific upregulation of GS function occurs when epilepsy develops into a certain stage and eventually promotes the process of epileptogenesis. Inhibition of GS to an adequate degree and at an appropriate timing may be a potential therapeutic approach to interrupting epileptogenesis.     

Epileptogenesis after Experimental Focal Cerebral Ischemia

Cerebrovascular diseases are one of the most common causes of epilepsy in adults, and the incidence of stroke-induced epileptogenesis is increasing as the population ages. The mechanisms that lead to stroke-induced epileptogenesis in a subpopulation of patients, however, are still poorly understood. Recent advances in inducing epileptogenesis in rodent focal ischemia models have provided tools that can be used to identify the risk factors and neurobiologic changes leading to development of epilepsy after stroke. Here we summarize data from models in which epileptogenesis has been studied after focal ischemia; photothrombosis, middle cerebral artery (MCA) occlusion with filament, and endothelin-1-induced MCA occlusion. Analysis of the data indicates that neurobiologic changes occurring during stroke-induced epileptogenesis share some similarities to those induced by status epilepticus or traumatic brain injury. Special issue dedicated to Dr. Simo S. Oja     

Mannitol induces selective astroglial death in the CA1 region of the rat hippocampus following status epilepticus

In the present study, we addressed the question of whether treatment with mannitol, an osmotic diuretic, affects astrogliovascular responses to status epilepticus (SE). In saline-treated animals, astrocytes exhibited reactive astrogliosis in the CA1-3 regions 2-4 days after SE. In the mannitol-treated animals, a large astroglial empty zone was observed in the CA1 region 2 days after SE. This astroglial loss was unrelated to vasogenic edema formation. There was no difference in SE-induced neuronal loss between saline- and mannitol-treated animals. Furthermore, mannitol treatment did not affect astroglial loss and vasogenic edema formation in the dentate gyrus and the piriform cortex. These findings suggest that mannitol treatment induces selective astroglial loss in the CA1 region independent of vasogenic edema formation following SE. These findings support the hypothesis that the susceptibility of astrocytes to SE is most likely due to the distinctive heterogeneity of astrocytes independent of hemodynamics. [BMB Reports 2015; 48(9): 507-512]     

Cellular and regional specific changes in multidrug efflux transporter expression during recovery of vasogenic edema in the rat hippocampus and piriform cortex

In the present study, we investigated the characteristics of drug efflux transporter expressions following status epilepticus (SE). In the hippocampus and piriform cortex (PC), vasogenic edema peaked 3-4 days after SE. The expression of breast cancer resistance protein (BCRP), multidrug resistance protein-4 (MRP4), and p-glycoprotein (p-GP) were decreased 4 days after SE when vasogenic edema was peaked, but subsequently increased 4 weeks after SE. Multidrug resistance protein-1 (MRP1) expression gradually decreased in endothelial cells until 4 weeks after SE. These findings indicate that SE-induced vasogenic edema formation transiently reduced drug efflux pump expressions in endothelial cells. Subsequently, during recovery of vasogenic edema drug efflux pump expressions were differentially upregulated in astrocytes, neuropils, and endothelial cells. Therefore, we suggest that vasogenic edema formation may be a risk factor in pharmacoresistent epilepsy. [BMB Reports 2015; 48(6): 348-353]     

Effect of Seizures Induced by Intra-Amygdaloid Kainic Acid on Kainic Acid Binding Sites in Rat Hippocampus and Amygdala

[3H]Kainic acid binding sites with a slow dissociation rate in the rat limbic system were investigated in detail. Extensively washed membranes prepared from the hippocampal formation and from the region comprising the amygdala and the piriform cortex yielded non-linear Scatchard plots. Microdissection showed that the high-affinity component (affinity constant around 1 nM) was present in the hippocampal CA3 region (4.2 fmol/mg wet tissue) and the amygdaloid complex (4.6 fmol/mg wet tissue), whereas the remaining part of the hippocampal formation and the piriform lobe contained the low-affinity component (affinity constant 5-20 nM; 11.6 and 11.3 fmol/mg wet tissue, respectively). In the lateral + medial septum we detected only the low-affinity component. Severe limbic seizures, induced by unilateral injection of 0.7 or 0.8 microgram kainic acid in 0.3 microliter of phosphate-buffered saline into the amygdala, reduced kainic acid binding sites in the ipsilateral amygdala and CA3 region. The decline of kainic acid binding sites in the injected amygdala was followed by a similar effect in the contralateral amygdala ("mirror focus") and later by a moderate loss also in the contralateral CA3 region. Kainic acid receptor autoradiography demonstrated that binding sites were lost from the stratum lucidum in hippocampus. Septal lesion had no effect on kainic acid binding sites in the hippocampus. Comparison with previous results on the histopathological changes after this lesion shows that high-affinity kainic acid binding sites are preferentially located on neurons that undergo selective degenerations after severe kainic acid-induced seizures.     

Chronic inhibition of cortex microsomal Mg2+/Ca2+ ATPase-mediated Ca2+ uptake in the rat pilocarpine model following epileptogenesis

In the rat pilocarpine model, 1 h of status epilepticus caused significant inhibition of Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake in cortex endoplasmic reticulum (microsomes) isolated immediately after the status episode. The rat pilocarpine model is also an established model of acquired epilepsy. Several weeks after the initial status epilepticus episode, the rats develop spontaneous recurrent seizures, or epilepsy. To determine whether inhibition of Ca(2+) uptake persists after the establishment of epilepsy, Ca(2+) uptake was studied in cortical microsomes isolated from rats displaying spontaneous recurrent seizures for 1 year. The initial rate and total Ca(2+) uptake in microsomes from epileptic animals remained significantly inhibited 1 year after the expression of epilepsy compared to age-matched controls. The inhibition of Ca(2+) uptake was not due to individual seizures nor an artifact of increased Ca(2+) release from epileptic microsomes. In addition, the decreased Ca(2+) uptake was not due to either selective isolation of damaged epileptic microsomes from the homogenate or decreased Mg(2+)/Ca(2+) ATPase protein in the epileptic microsomes. The data demonstrate that inhibition of microsomal Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake in the pilocarpine model may underlie some of the long-term plasticity changes associated with epileptogenesis.     

A fear-inducing odor alters PER2 and c-Fos expression in brain regions involved in fear memory

Evidence demonstrates that rodents learn to associate a foot shock with time of day, indicating the formation of a fear related time-stamp memory, even in the absence of a functioning SCN. In addition, mice acquire and retain fear memory better during the early day compared to the early night. This type of memory may be regulated by circadian pacemakers outside of the SCN. As a first step in testing the hypothesis that clock genes are involved in the formation of a time-stamp fear memory, we exposed one group of mice to fox feces derived odor (TMT) at ZT 0 and one group at ZT 12 for 4 successive days. A separate group with no exposure to TMT was also included as a control. Animals were sacrificed one day after the last exposure to TMT, and PER2 and c-Fos protein were quantified in the SCN, amygdala, hippocampus, and piriform cortex. Exposure to TMT had a strong effect at ZT 0, decreasing PER2 expression at this time point in most regions except the SCN, and reversing the normal rhythm of PER2 expression in the amygdala and piriform cortex. These changes were accompanied by increased c-Fos expression at ZT0. In contrast, exposure to TMT at ZT 12 abolished the rhythm of PER2 expression in the amygdala. In addition, increased c-Fos expression at ZT 12 was only detected in the central nucleus of the amygdala in the TMT12 group. TMT exposure at either time point did not affect PER2 or c-Fos in the SCN, indicating that under a light-dark cycle, the SCN rhythm is stable in the presence of repeated exposure to a fear-inducing stimulus. Taken together, these results indicate that entrainment to a fear-inducing stimulus leads to changes in PER2 and c-Fos expression that are detected 24 hours following the last exposure to TMT, indicating entrainment of endogenous oscillators in these regions. The observed effects on PER2 expression and c-Fos were stronger during the early day than during the early night, possibly to prepare appropriate systems at ZT 0 to respond to a fear-inducing stimulus.