Robert F. Hunt

GABA progenitors grafted into the adult epileptic brain control seizures and abnormal behavior.

Impaired GABA-mediated neurotransmission has been implicated in many neurologic diseases, including epilepsy, intellectual disability and psychiatric disorders. We found that inhibitory neuron transplantation into the hippocampus of adult mice with confirmed epilepsy at the time of grafting markedly reduced the occurrence of electrographic seizures and restored behavioral deficits in spatial learning, hyperactivity and the aggressive response to handling. In the recipient brain, GABA progenitors migrated up to 1,500 μm from the injection site, expressed genes and proteins characteristic for interneurons, differentiated into functional inhibitory neurons and received excitatory synaptic input. In contrast with hippocampus, cell grafts into basolateral amygdala rescued the hyperactivity deficit, but did not alter seizure activity or other abnormal behaviors. Our results highlight a critical role for interneurons in epilepsy and suggest that interneuron cell transplantation is a powerful approach to halting seizures and rescuing accompanying deficits in severely epileptic mice.

Posttraumatic epilepsy after controlled cortical impact injury in mice.

Many patients develop temporal lobe epilepsy after trauma, but basic mechanisms underlying the development of chronic seizures after head injury remain poorly understood. Using the controlled cortical impact injury model we examined whether mice developed spontaneous seizures after mild (0.5 mm injury depth) or severe (1.0 mm injury depth) brain injury and how subsequent posttraumatic mossy fiber sprouting was associated with excitability in the dentate gyrus 42-71 d after injury. After several weeks, spontaneous behavioral seizures were observed in 20% of mice with mild and 36% of mice with severe injury. Mossy fiber sprouting was typically present in septal slices of the dentate gyrus ipsilateral to the injury, but not in control mice. In slices with mossy fiber sprouting, perforant path stimulation revealed a significant reduction (P<0.01) in paired-pulse ratios in dentate granule cells at 20 ms and 40 ms interpulse intervals, but not at 80 ms or 160 ms intervals. These slices were also characterized by spontaneous and hilar-evoked epileptiform activity in the dentate gyrus in the presence of Mg(2+)-free ACSF containing 100 microM picrotoxin. In contrast, paired-pulse and hilar-evoked responses in slices from injured animals that did not display mossy fiber sprouting were not different from controls. These data suggest the development of spontaneous posttraumatic seizures as well as structural and functional network changes associated with temporal lobe epilepsy in the mouse dentate gyrus by 71 d after CCI injury. Identifying experimental injury models that exhibit similar pathology to injury-induced epilepsy in humans should help to elucidate the mechanisms by which the injured brain becomes epileptic.

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.

Synaptic Reorganization of Inhibitory Hilar Interneuron Circuitry After Traumatic Brain Injury in Mice

Functional plasticity of synaptic networks in the dentate gyrus has been implicated in the development of posttraumatic epilepsy and in cognitive dysfunction after traumatic brain injury, but little is known about potentially pathogenic changes in inhibitory circuits. We examined synaptic inhibition of dentate granule cells and excitability of surviving GABAergic hilar interneurons 8–13 weeks after cortical contusion brain injury in transgenic mice that express enhanced green fluorescent protein in a subpopulation of inhibitory neurons. Whole-cell voltage-clamp recordings in granule cells revealed a reduction in spontaneous and miniature IPSC frequency after head injury; no concurrent change in paired-pulse ratio was found in granule cells after paired electrical stimulation of the hilus. Despite reduced inhibitory input to granule cells, action potential and EPSC frequencies were increased in hilar GABA neurons from slices ipsilateral to the injury versus those from control or contralateral slices. Furthermore, increased excitatory synaptic activity was detected in hilar GABA neurons ipsilateral to the injury after glutamate photostimulation of either the granule cell or CA3 pyramidal cell layers. Together, these findings suggest that excitatory drive to surviving hilar GABA neurons is enhanced by convergent input from both pyramidal and granule cells, but synaptic inhibition of granule cells is not fully restored after injury. This rewiring of circuitry regulating hilar inhibitory neurons may reflect an important compensatory mechanism, but it may also contribute to network destabilization by increasing the relative impact of surviving individual interneurons in controlling granule cell excitability in the posttraumatic dentate gyrus.

Lhx6 Directly Regulates Arx and CXCR7 to Determine Cortical Interneuron Fate and Laminar Position

Cortical GABAergic interneurons have essential roles for information processing and their dysfunction is implicated in neuropsychiatric disorders. Transcriptional codes are elucidating mechanisms of interneuron specification in the MGE (a subcortical progenitor zone), which regulate their migration, integration, and function within cortical circuitry. Lhx6, a LIM-homeodomain transcription factor, is essential for specification of MGE-derived somatostatin and parvalbumin interneurons. Here, we demonstrate that some Lhx6⁻/⁻ MGE cells acquire a CGE-like fate. Using an in vivo MGE complementation/transplantation assay, we show that Lhx6-regulated genes Arx and CXCR7 rescue divergent aspects of Lhx6⁻/⁻ cell-fate and laminar mutant phenotypes and provide insight into a neonatal role for CXCR7 in MGE-derived interneuron lamination. Finally, Lhx6 directly binds in vivo to an Arx enhancer and to an intronic CXCR7 enhancer that remains active in mature interneurons. These data define the molecular identity of Lhx6 mutants and introduce technologies to test mechanisms in GABAergic interneuron differentiation.

Regionally localized recurrent excitation in the dentate gyrus of a cortical contusion model of posttraumatic epilepsy.

Posttraumatic epilepsy is a frequent consequence of brain trauma, but relatively little is known about how neuronal circuits are chronically altered after closed head injury. We examined whether local recurrent excitatory synaptic connections form between dentate granule cells in mice 8-12 wk after cortical contusion injury. Mice were monitored for behavioral seizures shortly after brain injury and < or = 10 wk postinjury. Injury-induced seizures were observed in 15% of mice, and spontaneous seizures were observed weeks later in 40% of mice. Timms staining revealed mossy fiber sprouting into the inner molecular layer of the dorsal dentate gyrus ipsilateral to the injury in 95% of mice but not contralateral to the injury or in uninjured controls. Whole cell patch-clamp recordings were made from granule cells in isolated hippocampal brain slices. Cells in slices with posttraumatic mossy fiber sprouting had an increased excitatory postsynaptic current (EPSC) frequency compared with cells in slices without sprouting from injured and control animals (P < 0.001). When perfused with Mg(2+)-free artificial cerebrospinal fluid containing 100 microM picrotoxin, these cells had spontaneous bursts of EPSCs and action potentials. Focal glutamate photostimulation of the granule cell layer evoked a burst of EPSCs and action potentials indicative of recurrent excitatory connections in granule cells of slices with mossy fiber sprouting. In granule cells of slices without sprouting from injured animals and controls, spontaneous or photostimulation-evoked epileptiform activity was never observed. These results suggest that a new regionally localized excitatory network forms between dentate granule cells near the injury site within weeks after cortical contusion head injury.

14-3-3ε and ζ Regulate Neurogenesis and Differentiation of Neuronal Progenitor Cells in the Developing Brain

During brain development, neural progenitor cells proliferate and differentiate into neural precursors. These neural precursors migrate along the radial glial processes and localize at their final destination in the cortex. Numerous reports have revealed that 14-3-3 proteins are involved in many neuronal activities, although their functions in neurogenesis remain unclear. Here, using 14-3-3ε/ζ double knock-out mice, we found that 14-3-3 proteins are important for proliferation and differentiation of neural progenitor cells in the cortex, resulting in neuronal migration defects and seizures. 14-3-3 deficiency resulted in the increase of δ-catenin and the decrease of β-catenin and αN-catenin. 14-3-3 proteins regulated neuronal differentiation into neurons via direct interactions with phosphorylated δ-catenin to promote F-actin formation through a catenin/Rho GTPase/Limk1/cofilin signaling pathway. Conversely, neuronal migration defects seen in the double knock-out mice were restored by phosphomimic Ndel1 mutants, but not δ-catenin. Our findings provide new evidence that 14-3-3 proteins play important roles in neurogenesis and neuronal migration via the regulation of distinct signaling cascades.

A novel zebrafish model of hyperthermia-induced seizures reveals a role for TRPV4 channels and NMDA-type glutamate receptors

Febrile seizures are the most common seizure type in children under the age of five, but mechanisms underlying seizure generation in vivo remain unclear. Animal models to address this issue primarily focus on immature rodents heated indirectly using a controlled water bath or air blower. Here we describe an in vivo model of hyperthermia-induced seizures in larval zebrafish aged 3 to 7 days post-fertilization (dpf). Bath controlled changes in temperature are rapid and reversible in this model. Acute electrographic seizures following transient hyperthermia showed age-dependence, strain independence, and absence of mortality. Electrographic seizures recorded in the larval zebrafish forebrain were blocked by adding antagonists to the transient receptor potential vanilloid (TRPV4) channel or N-methyl-d-aspartate (NMDA) glutamate receptor to the bathing medium. Application of GABA, GABA re-uptake inhibitors, or TRPV1 antagonist had no effect on hyperthermic seizures. Expression of vanilloid channel and glutamate receptor mRNA was confirmed by quantitative PCR analysis at each developmental stage in larval zebrafish. Taken together, our findings suggest a role of heat-activation of TRPV4 channels and enhanced NMDA receptor-mediated glutamatergic transmission in hyperthermia-induced seizures.

Interneuron Transplantation as a Treatment for Epilepsy.

Stem-cell therapy has extraordinary potential to address critical, unmet needs in the treatment of human disease. One particularly promising approach for the treatment of epilepsy is to increase inhibition in areas of the epileptic brain by grafting new inhibitory cortical interneurons. When grafted from embryos, young γ-aminobutyric acid (GABA)ergic precursors disperse, functionally mature into host brain circuits as local-circuit interneurons, and can stop seizures in both genetic and acquired forms of the disease. These features make interneuron cell transplantation an attractive new approach for the treatment of intractable epilepsies, as well as other brain disorders that involve increased risk for epilepsy as a comorbidity. Here, we review recent efforts to isolate and transplant cortical interneuron precursors derived from embryonic mouse and human cell sources. We also discuss some of the important challenges that must be addressed before stem-cell-based treatment for human epilepsy is realized.

Posttraumatic mossy fiber sprouting is related to the degree of cortical damage in three mouse strains

Controlled cortical impact injury was used to examine relationships between focal posttraumatic cortical damage and mossy fiber sprouting (MFS) in the dentate gyrus in three mouse strains. Posttraumatic MFS was more robust when cortical injury impinged upon the hippocampus, versus contusions restricted to neocortex, and was qualitatively similar among CD-1, C57BL/6, and FVB/N background strains. Impact parameters influencing injury severity may be critical in reproducing epilepsy-related changes in neurotrauma models.