Rapid volume pulsations of the extracellular space accompany epileptiform activity in trauma-injured neocortex and depend on the sodium-bicarbonate cotransporter NBCe1.

Post traumatic epilepsy (PTE) is a treatment-resistant consequence of traumatic brain injury (TBI). Recently, it has been revealed that epileptiform activity in acute chemoconvulsant seizure models is accompanied by transient shrinkages of extracellular space (ECS) called rapid volume pulsations (RVPs). Shrinkage of the ECS surrounding neurons and glia may contribute to ictogenic hyperexcitability and hypersynchrony during the chronic phase of TBI. Here, we identify the phenomenon of RVPs occurring spontaneously in rat neocortex at ≥ 3 weeks after injury in the controlled cortical impact (CCI) model for PTE. We further report that blocking the electrogenic action of the astrocytic cotransporter NBCe1 with 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) eliminates both RVPs and epileptiform activity in ex-vivo CCI neocortical brain slices. We conclude that NBCe1-mediated extracellular volume shrinkage may represent a new target for therapeutic intervention in PTE.

4-AP challenge reveals that early intervention with brivaracetam prevents posttraumatic epileptogenesis in rats.

PURPOSE: There are currently no clinical treatments to prevent posttraumatic epilepsy (PTE). Recently, our group has shown that administration of levetiracetam (LEV) or brivaracetam (BRV) shortly after cortical neurotrauma prevents the development of epileptiform activity in rats, as measured ex vivo in neocortical slices. Due to the low incidence of spontaneous seizures in rodent-based models of traumatic brain injury (TBI), chemoconvulsants have been used to test injured animals for seizure susceptibility. We used a low dose of the voltage-gated potassium channel blocker 4-aminopyridine (4-AP) to evaluate posttraumatic epileptogenesis after controlled cortical impact (CCI) injury. We then used this assessment to further investigate the efficacy of BRV as an antiepileptogenic treatment. METHODS: Sprague-Dawley rats aged P24-35 were subjected to severe CCI injury. Following trauma, one group received BRV-21 mg/kg (IP) at 0-2 min after injury and the other BRV-100 mg/kg (IP) at 30 min after injury. Four to eight weeks after injury, animals were given a single, low dose of 4-AP (3.0-3.5 mg/kg, IP) and then monitored up to 90 min for stage 4/5 seizures. RESULTS: The chemoconvulsant challenge revealed that within four to eight weeks, CCI injury led to a two-fold increase in percentage of rats with 4-AP induced stage 4-5 seizures relative to sham-injured controls. Administration of a single dose of BRV within 30 min after trauma significantly reduced injury-induced seizure susceptibility, bringing the proportion of CCI-rats that exhibited evoked seizures down to control levels. CONCLUSIONS: This study is the first to use a low dose of 4-AP as a chemoconvulsant challenge to test epileptogenicity within the first two months after CCI injury in rats. Our findings show that a single dose of BRV administered within 30 min after TBI prevents injury-induced increases in seizure susceptibility. This supports our hypothesis that early intervention with BRV may prevent PTE.

Brivaracetam prevents the development of epileptiform activity when administered early after cortical neurotrauma in rats.

OBJECTIVES: There is no effective therapy to prevent the development of posttraumatic epilepsy (PTE). Recently, we reported that administration of the antiseizure medication (ASM) levetiracetam (LEV) shortly after trauma prevented the development of epileptiform activity in two experimental models of neurotrauma. However, the time window for effective intervention with LEV may be too narrow for most clinical settings. Using the controlled cortical impact (CCI) injury model, the current study tested whether early administration of brivaracetam (BRV), an ASM with 20 times the affinity of LEV for the SV2A synaptic vesicle protein, could improve upon the antiepileptogenic action observed with LEV. METHODS: Rats (postnatal day [P] 24-32) subjected to CCI injury were given a single dose of BRV (21 or 100 mg/kg, i.p.) at one of three post-injury time points: immediately (0-2 minutes), 30 minutes, or 60 minutes. Control animals received only vehicle (0.9% saline). Posttraumatic electrographic epileptiform activity was assayed ex vivo from coronal neocortical slices collected proximal to the injury (four per rat) 3-4 weeks after injury. In this model, ictal-like burst discharges occur spontaneously or can be evoked in an "all or none" manner with applied electrical stimulation within the first 2 weeks after injury. RESULTS: A single dose of BRV administered to rats up to 60 minutes after traumatic brain injury (TBI) significantly reduced the development of posttraumatic epileptiform activity by (1) inhibiting the development of both evoked and spontaneous epileptiform activity, (2) raising the threshold for stimulus-evoked epileptiform discharges, and (3) reducing the intensity of epileptiform bursts that arise after cortical neurotrauma. SIGNIFICANCE: Clinically there has been little success preventing the development of posttraumatic epilepsy. The results of this study support the hypothesis that early intervention with BRV has the potential to prevent or reduce posttraumatic epileptogenesis, and that there may be a limited time window for successful prophylactic intervention.

Early intervention with levetiracetam prevents the development of cortical hyperexcitability and spontaneous epileptiform activity in two models of neurotrauma in rats.

This study examined the antiepileptogenic potential of the antiseizure drug (ASD) levetiracetam (LEV) using the in vitro traumatized-slice and in vivo controlled cortical impact (CCI) models of traumatic brain injury (TBI) in rats when administered early after the injury. For the in vitro model, acute coronal slices (400-450 µm) of rat neocortex (P21-32) were injured via a surgical cut that separated the superficial layers from the deeper regions. Persistent stimulus-evoked epileptiform activity developed within 1-2 h after trauma. In randomly selected slices, LEV (500 µM) was bath-applied for 1 h starting immediately or delayed by 30-80 min after injury. Treated and untreated slices were examined for epileptiform activity via intracellular and extracellular recordings. For the in vivo model, rats (P24-32) were subjected to a non-penetrating, focal, CCI injury targeting the neocortex (5.0 mm diameter; 2.0 mm depth). Immediately after injury, rats were given either a single dose of LEV (60-150 mg/kg, i.p.) or the saline vehicle. At 2-3 weeks after the injury, ex vivo cortical slices were examined for epileptiform activity. The results from the traumatized-slice experiments showed that in vitro treatment with LEV within 60 min of injury significantly reduced (> 50%) the proportion of slices that exhibited stimulus-evoked epileptiform activity. LEV treatment also increased the stimulus intensity required to trigger epileptiform bursts in injured slices by 2-4 fold. Consistent with these findings, LEV treatment of CCI-injured rats (n = 15) significantly reduced the proportion of animals that exhibited spontaneous and stimulus-evoked epileptiform bursts in ex vivo cortical slices compared to saline-treated controls (n = 15 rats), and also significantly increased the stimulus intensity required to evoke epileptiform bursts. These results suggest that early administration of LEV has the potential to prevent or reduce posttraumatic epileptogenesis and that there may be a narrow therapeutic window for successful prophylactic intervention.

Controlled cortical impact-induced neurodegeneration decreases after administration of the novel calpain-inhibitor Gabadur.

One aspect of secondary injury in traumatic brain injury is the marked increase in intracellular calcium and resultant over-activation of the calcium-dependent neutral cysteine protease calpain. Gabadur is a novel protease inhibitor with calpain-inhibition properties formulated from the classic protease inhibitor leupeptin linked to a pregabalin carrier. This construction allows the entire compound to cross the blood-brain barrier after peripheral administration to better target the site of injury. In this study, a single intraperitoneal dose of Gabadur was administered immediately following controlled cortical impact injury in rats. Neocortical slices were examined at 48 h post-injury via Fluoro-Jade B staining, revealing an improvement in cortical neurodegeneration in Gabadur treated rats. Levels of detrimental active calpain-2 measured via western blot were also decreased in rats receiving Gabadur. This data supports the benefit of targeted protease inhibition in the treatment of traumatic brain injury.

Robust training attenuates TBI-induced deficits in reference and working memory on the radial 8-arm maze.

Globally, it is estimated that nearly 10 million people sustain severe brain injuries leading to hospitalization and/or death every year. Amongst survivors, traumatic brain injury (TBI) results in a wide variety of physical, emotional and cognitive deficits. The most common cognitive deficit associated with TBI is memory loss, involving impairments in spatial reference and working memory. However, the majority of research thus far has characterized the deficits associated with TBI on either reference or working memory systems separately, without investigating how they interact within a single task. Thus, we examined the effects of TBI on short-term working and long-term reference memory using the radial 8-arm maze (RAM) with a sequence of four baited and four unbaited arms. Subjects were given 10 daily trials for 6 days followed by a memory retrieval test 2 weeks after training. Multiple training trials not only provide robust training, but also test the subjects' ability to frequently update short-term memory while learning the reference rules of the task. Our results show that TBI significantly impaired short-term working memory function on previously acquired spatial information but has little effect on long-term reference memory. Additionally, TBI significantly increased working memory errors during acquisition and reference memory errors during retention testing 2 weeks later. With a longer recovery period after TBI, the robust RAM training mitigated the reference memory deficit in retention but not the short-term working memory deficit during acquisition. These results identify the resiliency and vulnerabilities of short-term working and long-term reference memory to TBI in the context of robust training. The data highlight the role of cognitive training and other behavioral remediation strategies implicated in attenuating deficits associated with TBI.

Spontaneous epileptiform activity in rat neocortex after controlled cortical impact injury.

A hallmark of severe traumatic brain injury (TBI) is the development of post-traumatic epilepsy (PTE). However, the mechanisms underlying PTE remain poorly understood. In this study, we used a controlled cortical impact (CCI) model in rats to examine post-traumatic changes in neocortical excitability. Neocortical slices were prepared from rats at 7-9 days (week 1) and 14-16 days (week 2) after CCI injury. By week 2, we observed a substantial gray matter lesion with a cavity that extended to the hippocampal structure. Fluoro-Jade B staining of slices revealed active neuronal degeneration during weeks 1 and 2. Intracellular and extracellular recordings obtained from layer V revealed evoked and spontaneous epileptiform discharges in neocortices of CCI-injured rats. At week 1, intracellular recordings from pyramidal cells revealed evoked epileptiform firing that was synchronized with population events recorded extracellularly, suggestive of increased excitability. This activity was characterized by bursts of action potentials that were followed by recurrent, repetitive after-discharges. At week 2, both spontaneous and evoked epileptiform firing were recorded in slices from injured rats. The evoked discharges resembled those observed at week 1, but with longer burst durations. Spontaneous activity included prolonged, ictal-like discharges lasting up to 8-10 sec, and briefer interictal-like burst events (<1 sec). These results indicate that during the first 2 weeks following severe CCI injury, there is a progressive development of neocortical hyperexcitability that ultimately leads to spontaneous epileptiform firing, suggesting a rapid epileptogenic process.

Carbenoxolone modifies spontaneous inhibitory and excitatory synaptic transmission in rat somatosensory cortex.

Gap junction (GJ) coupling between neocortical GABAergic interneurons plays a critical role in the synchronization of activity in cortical networks in physiological and pathophysiological states, e.g., seizures. Past studies have shown that GJ blockers exert anticonvulsant actions in both in vivo and in vitro models of epilepsy. However, the precise mechanisms underlying these antiepileptic effects have not been fully elucidated. This is due, in part, to a lack of information of the influence of GJ blockade on network activity in the absence of convulsant agents or enhanced neuronal excitation. One key question is whether GJ blockers act on excitatory or inhibitory systems, or both. To address this issue, we examined the effects of the GJ blocker carbenoxolone (CarbX, 150 microM) on spontaneous inhibitory postsynaptic currents (sIPSCs) and excitatory postsynaptic currents (sEPSCs) in acute slices of rat somatosensory cortex. Results showed that CarbX decreased the amplitude and frequency of sIPSCs by 30.2% and 25.7%, respectively. CarbX increased the mean frequency of sEPSCs by 24.1%, but had no effect on sEPSC amplitude. During blockade of GABAA-mediated events with picrotoxin (20 microM), CarbX induced only a small increase in sEPSC frequency that was not statistically different from control, indicating CarbX enhancement of sEPECs was secondary to the depression of synaptic inhibition. These findings suggest that in neocortex, blockade of GJs leads to an increase in spontaneous excitation by uncoupling GABAergic interneurons, and that electronic communication between inhibitory cells plays a significant role in regulating tonic synaptic excitation.

Acute injury to superficial cortex leads to a decrease in synaptic inhibition and increase in excitation in neocortical layer V pyramidal cells.

Injury to the superficial layers of cerebral cortex produces alterations in the synaptic responses of local circuits that promote the development of seizures. To further delineate the specific changes in synaptic strength that are induced by this type of cortical injury, whole cell voltage-clamp recordings were used to examine evoked and spontaneous synaptic events from layer V pyramidal cells in coronal slices prepared from surgically traumatized rat neocortices in which the superficial third of the cortex (layers I, II, and part of III) was removed. Slices from intact neocortices were used as controls. Examinations of fast inhibitory postsynaptic currents (IPSCs) indicated that traumatized slices were disinhibited, exhibiting evoked IPSCs (eIPSCs) with lower peak amplitudes. Measurements of spontaneous IPSCs (sIPSCs) revealed no difference in the mean amplitudes of sIPSCs recorded in traumatized versus control slices. However, the mean sIPSC frequency was lower in traumatized slices, indicative of a decrease in GABA release at these inhibitory synapses. Traumatized slices also displayed an increase in synaptic excitation, exhibiting spontaneous EPSCs (sESPCs) with larger peak amplitudes and higher frequencies. Peak-scaled nonstationary fluctuation analysis of sEPSCs and sIPSCs was used to obtain estimates of the unit conductance and number of functional receptor channels. EPSC and IPSC channel numbers and IPSC unit conductance did not differ between traumatized and intact slices. However, the mean unit conductance of EPSCs was higher (+25%) in traumatized slices. These findings suggest that acute injury to the superficial neocortical layers results in a disinhibition of cortical circuits that stems from a decline in GABA release likely due to the loss of superficial inhibitory interneurons and an enhancement of synaptic excitation consequent to an increase in the AMPA receptor unit conductance.

Protein kinase Mzeta enhances excitatory synaptic transmission by increasing the number of active postsynaptic AMPA receptors.

Protein kinase Mzeta (PKMzeta), a constitutively active, atypical PKC isoform, enhances synaptic strength during the maintenance of long-term potentiation (LTP). Here we examine the mechanism by which PKMzeta increases synaptic transmission. Postsynaptic perfusion of PKMzeta during whole-cell recordings of CA1 pyramidal cells strongly potentiated the amplitude of AMPA receptor (AMPAR)-mediated miniature EPSCs (mEPSCs). Nonstationary fluctuation analysis of events recorded before and after PKMzeta enhancement showed that the kinase doubled the number of functional postsynaptic AMPAR channels. After sustained potentiation, application of a PKMzeta inhibitor reversed the increase in functional channel number to basal levels, suggesting that persistent increase of PKMzeta is required to maintain the postsynaptic localization of a mobile subpopulation of receptors. The kinase did not affect other sites of LTP expression, including presynaptic transmitter release, silent synapse conversion, or AMPAR unit conductance. Thus PKMzeta functions specifically to establish and maintain long-term increases in active postsynaptic AMPAR number.

Nootropic agents enhance the recruitment of fast GABAA inhibition in rat neocortex.

It is widely believed that nootropic (cognition-enhancing) agents produce their therapeutic effects by augmenting excitatory synaptic transmission in cortical circuits, primarily through positive modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors (AMPARs). However, GABA-mediated inhibition is also critical for cognition, and enhanced GABA function may be likewise therapeutic for cognitive disorders. Could nootropics act through such a mechanism as well? To address this question, we examined the effects of nootropic agents on excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) recorded from layer V pyramidal cells in acute slices of somatosensory cortex. Aniracetam, a positive modulator of AMPA/kainate receptors, increased the peak amplitude of evoked EPSCs and the amplitude and duration of polysynaptic fast IPSCs, manifested as a greater total charge carried by IPSCs. As a result, the EPSC/IPSC ratio of total charge was decreased, representing a shift in the excitation-inhibition balance that favors inhibition. Aniracetam did not affect the magnitude of either monosynaptic IPSCs (mono-IPSCs) recorded in the presence of excitatory amino acid receptor antagonists, or miniature IPSCs (mIPSCs) recorded in the presence of tetrodotoxin. However, the duration of both mono-IPSCs and mIPSCs was prolonged, suggesting that aniracetam also directly modulates GABAergic transmission. Cyclothiazide, a preferential modulator of AMPAR function, enhanced the magnitude and duration of polysynaptic IPSCs, similar to aniracetam, but did not affect mono-IPSCs. Concanavalin A, a kainate receptor modulator, had little effect on EPSCs or IPSCs, suggesting there was no contribution from kainate receptor activity. These findings indicate that AMPAR modulators strengthen inhibition in neocortical pyramidal cells, most likely by altering the kinetics of AMPARs on synaptically connected interneurons and possibly by modulating GABA(A) receptor responses in pyramidal cells. This suggests that the therapeutic actions of nootropic agents may be partly mediated through enhanced cortical GABAergic inhibition, and not solely through the direct modification of excitation, as previously thought.