Jeffery A. Boychuk

Neural circuit mechanisms of post-traumatic epilepsy

Traumatic brain injury (TBI) greatly increases the risk for a number of mental health problems and is one of the most common causes of medically intractable epilepsy in humans. Several models of TBI have been developed to investigate the relationship between trauma, seizures, and epilepsy-related changes in neural circuit function. These studies have shown that the brain initiates immediate neuronal and glial responses following an injury, usually leading to significant cell loss in areas of the injured brain. Over time, long-term changes in the organization of neural circuits, particularly in neocortex and hippocampus, lead to an imbalance between excitatory and inhibitory neurotransmission and increased risk for spontaneous seizures. These include alterations to inhibitory interneurons and formation of new, excessive recurrent excitatory synaptic connectivity. Here, we review in vivo models of TBI as well as key cellular mechanisms of synaptic reorganization associated with post-traumatic epilepsy (PTE). The potential role of inflammation and increased blood–brain barrier permeability in the pathophysiology of PTE is also discussed. A better understanding of mechanisms that promote the generation of epileptic activity versus those that promote compensatory brain repair and functional recovery should aid development of successful new therapies for PTE.

Distributed Versus Focal Cortical Stimulation to Enhance Motor Function and Motor Map Plasticity in a Rodent Model of Ischemia

Background. Motor rehabilitation after cerebral ischemia can enhance motor performance and induce motor map reorganization. Electrical stimulation of the cortex (CS) during rehabilitative training (CS/RT) augments motor map plasticity and confers gains in motor function beyond those observed with motor rehabilitation alone. However, it is unclear how the distribution of electrical stimulation across the cortex accomplishes these changes. This study examined the behavioral and neurophysiological effects of delivering CS/RT through a distributed versus focal arrangement of electrical contacts. Methods. Adult male rats were given rehabilitative training on a skilled forelimb reaching task following induction of focal ischemic damage within motor cortex. Intracortical microstimulation was used to derive high-resolution maps of forelimb movement representations within motor cortex contralateral to the trained/impaired paw before and after rehabilitation. Results. All animals that received rehabilitation showed greater increases in motor map area and reaching accuracy than animals that received no training. Animals with the distributed configuration performed significantly greater reaching accuracy than animals in both the CS/RT with focused contact arrangement and rehabilitative training alone (RT) conditions on days 3 to 4 and on day 6 through the remainder of the study (P < .05). However, both CS/RT groups exhibited larger motor maps than the RT condition (E1-CS/RT, 4.71 ± 0.66 mm2; E2-CS/RT, 4.64 ± 0.46 mm2; RT, 2.99 ± 0.28 mm2). Conclusion. The results indicate that although both focal and distributed forms of CS/RT promote motor map reorganization only the distributed form of CS/RT enhances motor performance with rehabilitation.

Effects of Rapamycin Treatment on Neurogenesis and Synaptic Reorganization in the Dentate Gyrus after Controlled Cortical Impact Injury in Mice.

Post-traumatic epilepsy (PTE) is one consequence of traumatic brain injury (TBI). A prominent cell signaling pathway activated in animal models of both TBI and epilepsy is the mammalian target of rapamycin (mTOR). Inhibition of mTOR with rapamycin has shown promise as a potential modulator of epileptogenesis in several animal models of epilepsy, but cellular mechanisms linking mTOR expression and epileptogenesis are unclear. In this study, the role of mTOR in modifying functional hippocampal circuit reorganization after focal TBI induced by controlled cortical impact (CCI) was investigated. Rapamycin (3 or 10 mg/kg), an inhibitor of mTOR signaling, was administered by intraperitoneal injection beginning on the day of injury and continued daily until tissue collection. Relative to controls, rapamycin treatment reduced dentate granule cell area in the hemisphere ipsilateral to the injury two weeks post-injury. Brain injury resulted in a significant increase in doublecortin immunolabeling in the dentate gyrus ipsilateral to the injury, indicating increased neurogenesis shortly after TBI. Rapamycin treatment prevented the increase in doublecortin labeling, with no overall effect on Fluoro-Jade B staining in the ipsilateral hemisphere, suggesting that rapamycin treatment reduced posttraumatic neurogenesis but did not prevent cell loss after injury. At later times post-injury (8–13 weeks), evidence of mossy fiber sprouting and increased recurrent excitation of dentate granule cells was detected, which were attenuated by rapamycin treatment. Rapamycin treatment also diminished seizure prevalence relative to vehicle-treated controls after TBI. Collectively, these results support a role for adult neurogenesis in PTE development and suggest that suppression of epileptogenesis by mTOR inhibition includes effects on post-injury neurogenesis.

Enduring changes in tonic GABAA receptor signaling in dentate granule cells after controlled cortical impact brain injury in mice

Changes in functional GABAAR signaling in hippocampus have previously been evaluated using pre-clinical animal models of either diffuse brain injury or extreme focal brain injury that precludes measurement of cells located ipsilateral to injury. As a result, there is little information about the status of functional GABAAR signaling in dentate granule cells (DGCs) located ipsilateral to focal brain injury, where significant cellular changes have been documented. We used whole-cell patch-clamp recordings from hippocampal slices to measure changes in GABAARs in dentate granule cells (DGCs) at 1-2, 3-5, and 8-13 weeks after controlled cortical impact (CCI) brain injury. Synaptic and tonic GABAAR currents (ITonicGABA) were measured in DGCs at baseline conditions and during application of the GABAAR agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol hydrochloride (THIP) to assess in the function of δ subunit-containing GABAARs. DGCs ipsilateral to CCI exhibited no changes in the amplitude of resting ITonicGABA relative to DGCs after sham-injury or contralateral to CCI. In contrast, there was a significant reduction in the THIP-evoked ITonicGABA in DGCs ipsilateral to CCI at both time-points. Tonic GABAergic inhibition of DGCs ipsilateral to injury also exhibited reduced responsiveness to the neurosteroid THDOC. ITonicGABA in DGCs ipsilateral to CCI did not exhibit a change in sensitivity to L655,708, an inverse agonist with selectivity for α5 subunit-containing GABAARs, suggesting a lack of functional change in GABAARs containing this subunit. At the 8-13 week time-point, gene expression of GABAAR subunits expected to contribute to ITonicGABA (i.e., α4, α5 and δ) was not significantly altered by CCI injury in isolated dentate gyrus. Collectively, these results demonstrate enduring functional changes in ITonicGABA in DGCs ipsilateral to focal brain injury that occur independent of altered gene expression.

HCN channels segregate stimulation‐evoked movement responses in neocortex and allow for coordinated forelimb movements in rodents

The present study tested whether HCN channels contribute to the organization of motor cortex and to skilled motor behaviour during a forelimb reaching task. Experimental reductions in HCN channel signalling increase the representation of complex multiple forelimb movements in motor cortex as assessed by intracortical microstimulation. Global HCN1KO mice exhibit reduced reaching accuracy and atypical movements during a single‐pellet reaching task relative to wild‐type controls. Acute pharmacological inhibition of HCN channels in forelimb motor cortex decreases reaching accuracy and increases atypical movements during forelimb reaching.

Differential effects of rapamycin treatment on tonic and phasic GABAergic inhibition in dentate granule cells after focal brain injury in mice.

The cascade of events leading to post-traumatic epilepsy (PTE) after traumatic brain injury (TBI) remains unclear. Altered inhibition in the hippocampal formation and dentate gyrus is a hallmark of several neurological disorders, including TBI and PTE. Inhibitory synaptic signaling in the hippocampus is predominately driven by γ-aminobutyric acid (GABA) neurotransmission, and is prominently mediated by postsynaptic type A GABA receptors (GABAARs). Subsets of these receptors involved in tonic inhibition of neuronal membranes serve a fundamental role in maintenance of inhibitory state, and GABAAR-mediated tonic inhibition is altered functionally in animal models of both TBI and epilepsy. In this study, we assessed the effect of mTOR inhibition on hippocampal hilar inhibitory interneuron loss and synaptic and tonic GABAergic inhibition of dentate gyrus granule cells (DGCs) after controlled cortical impact (CCI) to determine if mTOR activation after TBI modulates GABAAR function. Hilar inhibitory interneuron density was significantly reduced 72h after CCI injury in the dorsal two-thirds of the hemisphere ipsilateral to injury compared with the contralateral hemisphere and sham controls. Rapamycin treatment did not alter this reduction in cell density. Synaptic and tonic current measurements made in DGCs at both 1-2 and 8-13weeks post-injury indicated reduced synaptic inhibition and THIP-induced tonic current density in DGCs ipsilateral to CCI injury at both time points post-injury, with no change in resting tonic GABAAR-mediated currents. Rapamycin treatment did not alter the reduced synaptic inhibition observed in ipsilateral DGCs 1-2weeks post-CCI injury, but further reduced synaptic inhibition of ipsilateral DGCs at 8-13weeks post-injury. The reduction in THIP-induced tonic current after injury, however, was prevented by rapamycin treatment at both time points. Rapamycin treatment thus differentially modifies CCI-induced changes in synaptic and tonic GABAAR-mediated currents in DGCs.

Brain Injury-Induced Synaptic Reorganization in Hilar Inhibitory Neurons Is Differentially Suppressed by Rapamycin

Abstract Following traumatic brain injury (TBI), treatment with rapamycin suppresses mammalian (mechanistic) target of rapamycin (mTOR) activity and specific components of hippocampal synaptic reorganization associated with altered cortical excitability and seizure susceptibility. Reemergence of seizures after cessation of rapamycin treatment suggests, however, an incomplete suppression of epileptogenesis. Hilar inhibitory interneurons regulate dentate granule cell (DGC) activity, and de novo synaptic input from both DGCs and CA3 pyramidal cells after TBI increases their excitability but effects of rapamycin treatment on the injury-induced plasticity of interneurons is only partially described. Using transgenic mice in which enhanced green fluorescent protein (eGFP) is expressed in the somatostatinergic subset of hilar inhibitory interneurons, we tested the effect of daily systemic rapamycin treatment (3 mg/kg) on the excitability of hilar inhibitory interneurons after controlled cortical impact (CCI)-induced focal brain injury. Rapamycin treatment reduced, but did not normalize, the injury-induced increase in excitability of surviving eGFP+ hilar interneurons. The injury-induced increase in response to selective glutamate photostimulation of DGCs was reduced to normal levels after mTOR inhibition, but the postinjury increase in synaptic excitation arising from CA3 pyramidal cell activity was unaffected by rapamycin treatment. The incomplete suppression of synaptic reorganization in inhibitory circuits after brain injury could contribute to hippocampal hyperexcitability and the eventual reemergence of the epileptogenic process upon cessation of mTOR inhibition. Further, the cell-selective effect of mTOR inhibition on synaptic reorganization after CCI suggests possible mechanisms by which rapamycin treatment modifies epileptogenesis in some models but not others.

Enhanced Motor Recovery after Stroke with Combined Cortical Stimulation and Rehabilitative Training Is Dependent on Infarct Location

Background. Cortical electrical stimulation of the motor cortex in combination with rehabilitative training (CS/RT) has been shown to enhance motor recovery in animal models of focal cortical stroke, yet in clinical trials, the effects are much less robust. The variability of stroke location in human patient populations that include both cortical and subcortical brain regions may contribute to the failure to find consistent effects clinically. Objective. This study sought to determine whether infarct location influences the enhanced motor recovery previously observed in response to CS/RT. The efficacy of CS/RT to promote improvements in motor function was examined in 2 different rat models of stroke that varied the amount and location of cortical and subcortical damage. Methods. Ischemic infarctions were induced by injecting the vasoconstricting peptide endothelin-1 either (1) onto the middle cerebral artery (MCA) producing damage to the frontal cortex and lateral striatum or (2) into a subcortical region producing damage to the posterior thalamus and internal capsule (subcortical capsular ischemic injury [SCII]). Daily CS/RT or RT alone was then given for 20 days, during which time performance on a skilled reaching task was assessed. Results. Animals with MCA occlusion infarctions exhibited enhanced improvements on a skilled reaching task in response to CS/RT relative to RT alone. No such enhancement was observed in animals with SCII infarctions across the 20 days of treatment. Conclusions. The efficacy of CS for enhancing motor recovery after stroke may depend in part on the extent and location of the ischemic infarct.

Loss of HCN channel mediated Ih current following seizures accounts for movement dysfunction.

Hyperpolarisation-activated, cyclic nucleotide-gated (HCN) channel mutations are linked to disorders characterized by the occurrence of recurrent seizures (epilepsies) and HCN channel dysfunction has also been observed following evoked seizures in otherwise typical brains. Whether HCN channels contribute to the behavioral co-morbidities associated with epilepsy has received limited attention, despite extensive work to show that they serve as key regulators of neuronal excitability. A recent article, co-authored by us, provides evidence that experimental seizures disrupt HCN channel function and that this type of disruption leads to long-term impairments in skilled motor behavior. Given that interictal motor impairments are observed following experimental seizures and clinical epilepsy, this new study suggests an intriguing possibility that HCN channels represent a novel therapeutic target to treat co-morbidities of this disorder. HCN channels provide a diverse set of contributions to brain excitability. At the level of individual neurons, the current mediated by HCN channels, referred to by many names including Ih, affects integration of synaptic input as well as patterns of action potential firing. It has previously been shown that HCN channels serve as a mechanism to restrict spatial firing fields within entorhinal cortex. HCN channels also restrict hippocampal-dependent spatial memory. In our recent work, we sought to examine the role of HCN channels for networks located in motor cortex. The study manipulated HCN channels using 3 separate approaches. Repeated experimental seizures were used as they reduce Ih in layer 5 pyramidal cells that make up the cortical spinal tract. The pharmacological blocker ZD7288 was locally applied within motor cortex and global HCN1 knockout (HCN1) mice were used as a genetic strategy. In order to examine network function of motor cortex, in vivo studies were performed using standard intracortical microstimulation (ICMS) to systematically measure evoked forelimb movement responses across sites within neocortex. With short-train ICMS parameters, stimulation at individual sites within motor cortex predominantly results in responses on the contralateral side of the body that are characterized by simple flexion or extension across a single joint. With repeated seizures there was a substantial increase in the number of stimulation sites that exhibited complex forelimb movement responses rather than the simple flexion or extension movements. These new complex forelimb responses occurred as combinations of simple movement responses and were present contralateral to stimulation and often bilaterally. This result was also seen after direct application of ZD7228 and in HCN1 mice. Additionally, no further change in ICMS responses occurred when the effects of ZD7228 were tested in HCN1 mice. Since experimental seizures had previously been shown to impair skilled motor behavior, additional experiments tested whether genetic or pharmacological manipulation of HCN channels also affected skilled motor behavior in awake behaving rodents. HCN1 mice exhibited decreased performance and atypical movements on a skilled forelimb-reaching task. Na€ıve rats given local

Nicotine enhances inhibition of mouse vagal motor neurons by modulating excitability of premotor GABAergic neurons in the nucleus tractus solitarii

The caudal nucleus of the solitary tract (NTS) serves as the site of the first synapse for visceral sensory inputs to the central nervous system. The NTS sends functional projections to multiple brain nuclei, with gastric-related projections primarily targeting the dorsal motor nucleus of the vagus (DMV). Previous studies have demonstrated that the majority of caudal NTS neurons that project to the DMV respond robustly to nicotine and express nicotinic acetylcholine receptors (nAChRs). However, the cytochemical identity and relationship with specific viscera of DMV-projecting, nicotine-responsive caudal NTS neurons have not been determined. The present study used transgenic mice that express enhanced green fluorescent protein (EGFP) under a GAD67 promoter in a subset of GABAergic neurons, in vivo retrograde pseudorabies viral labeling to identify gastric-related vagal complex neurons, and patch-clamp electrophysiology in acute brain stem slices to test the hypothesis that gastric-related and GABAergic inhibitory synaptic input to the DMV from the caudal NTS is under a robust modulatory control by nAChRs. Our results suggest that activation of nAChRs in the caudal NTS, but not DMV, potentiates GABAergic, but not glutamatergic, input to the DMV. Gastric-related caudal NTS and DMV neurons are directly involved in this nicotine-sensitive circuitry. Understanding the central patterns of nicotinic modulation of visceral sensory-motor circuitry may help develop therapeutic interventions to restore autonomic homeostasis in patients with autonomic impairments.