Ultrastructural and functional changes at the tripartite synapse during epileptogenesis in a model of temporal lobe epilepsy.

The persistent unresponsiveness of many of the acquired epilepsies to traditional antiseizure medications has motivated the search for prophylactic drug therapies that could reduce the incidence of epilepsy in this at risk population. These studies are based on the idea of a period of epileptogenesis that can follow a wide variety of brain injuries. Epileptogenesis is hypothesized to involve changes in the brain not initially associated with seizures, but which result finally in seizure prone networks. Understanding these changes will provide crucial clues for the development of prophylactic drugs. Using the repeated low-dose kainate rat model of epilepsy, we have studied the period of epileptogenesis following status epilepticus, verifying the latent period with continuous EEG monitoring. Focusing on ultrastructural properties of the tripartite synapse in the CA1 region of hippocampus we found increased astrocyte ensheathment around both the presynaptic and postsynaptic elements, reduced synaptic AMPA receptor subunit and perisynaptic astrocyte GLT-1 expression, and increased number of docked vesicles at the presynaptic terminal. These findings were associated with an increase in frequency of the mEPSCs observed in patch clamp recordings of CA1 pyramidal cells. The results suggest a complex set of changes, some of which have been associated with increasingly excitable networks such as increased vesicles and mEPSC frequency, and some associated with compensatory mechanisms, such as increased astrocyte ensheathment. The diversity of ultrastructural and electrophysiological changes observed during epileptogeneiss suggests that potential drug targets for this period should be broadened to include all components of the tripartite synapse.

Hippocampal TNFα Signaling Contributes to Seizure Generation in an Infection-Induced Mouse Model of Limbic Epilepsy.

Central nervous system infection can induce epilepsy that is often refractory to established antiseizure drugs. Previous studies in the Theiler's murine encephalomyelitis virus (TMEV)-induced mouse model of limbic epilepsy have demonstrated the importance of inflammation, especially that mediated by tumor necrosis factor-α (TNFα), in the development of acute seizures. TNFα modulates glutamate receptor trafficking via TNF receptor 1 (TNFR1) to cause increased excitatory synaptic transmission. Therefore, we hypothesized that an increase in TNFα signaling after TMEV infection might contribute to acute seizures. We found a significant increase in both mRNA and protein levels of TNFα and the protein expression ratio of TNF receptors (TNFR1:TNFR2) in the hippocampus, a brain region most likely involved in seizure initiation, after TMEV infection, which suggests that TNFα signaling, predominantly through TNFR1, may contribute to limbic hyperexcitability. An increase in hippocampal cell-surface glutamate receptor expression was also observed during acute seizures. Although pharmacological inhibition of TNFR1-mediated signaling had no effect on acute seizures, several lines of genetically modified animals deficient in either TNFα or TNFRs had robust changes in seizure incidence and severity after TMEV infection. TNFR2-/- mice were highly susceptible to developing acute seizures, suggesting that TNFR2-mediated signaling may provide beneficial effects during the acute seizure period. Taken together, the present results suggest that inflammation in the hippocampus, caused predominantly by TNFα signaling, contributes to hyperexcitability and acute seizures after TMEV infection. Pharmacotherapies designed to suppress TNFR1-mediated or augment TNFR2-mediated effects of TNFα may provide antiseizure and disease-modifying effects after central nervous system infection.

Imaging activity in astrocytes and neurons with genetically encoded calcium indicators following in utero electroporation.

Complex interactions between networks of astrocytes and neurons are beginning to be appreciated, but remain poorly understood. Transgenic mice expressing fluorescent protein reporters of cellular activity, such as the GCaMP family of genetically encoded calcium indicators (GECIs), have been used to explore network behavior. However, in some cases, it may be desirable to use long-established rat models that closely mimic particular aspects of human conditions such as Parkinson's disease and the development of epilepsy following status epilepticus. Methods for expressing reporter proteins in the rat brain are relatively limited. Transgenic rat technologies exist but are fairly immature. Viral-mediated expression is robust but unstable, requires invasive injections, and only works well for fairly small genes (<5 kb). In utero electroporation (IUE) offers a valuable alternative. IUE is a proven method for transfecting populations of astrocytes and neurons in the rat brain without the strict limitations on transgene size. We built a toolset of IUE plasmids carrying GCaMP variants 3, 6s, or 6f driven by CAG and targeted to the cytosol or the plasma membrane. Because low baseline fluorescence of GCaMP can hinder identification of transfected cells, we included the option of co-expressing a cytosolic tdTomato protein. A binary system consisting of a plasmid carrying a piggyBac inverted terminal repeat (ITR)-flanked CAG-GCaMP-IRES-tdTomato cassette and a separate plasmid encoding for expression of piggyBac transposase was employed to stably express GCaMP and tdTomato. The plasmids were co-electroporated on embryonic days 13.5-14.5 and astrocytic and neuronal activity was subsequently imaged in acute or cultured brain slices prepared from the cortex or hippocampus. Large spontaneous transients were detected in slices obtained from rats of varying ages up to 127 days. In this report, we demonstrate the utility of this toolset for interrogating astrocytic and neuronal activity in the rat brain.

The activity within the CA3 excitatory network during Theiler's virus encephalitis is distinct from that observed during chronic epilepsy.

Viral infections of the central nervous system (CNS) are associated with an increased risk for seizures during the acute infection period and the subsequent development of chronic epilepsy that is often difficult to treat. In previous work, we have shown that mice of the C57BL/6 strain infected with Theiler's murine encephalomyelitis virus (TMEV) exhibit a similar sequence, thereby providing a potential useful model of virus-induced epilepsy. The present study examines spontaneous and miniature excitatory postsynaptic currents in CA3 pyramidal cells recorded from brain slices prepared during both the acute phase during encephalitis and 2 months following TMEV infection. Animals that develop chronic epilepsy following TMEV infection exhibit considerable hippocampal sclerosis, directly implicating this brain region in the process of epileptogenesis. There are significant increases in amplitude and frequency of spontaneous and miniature excitatory currents in CA3 cells recorded in brain slices prepared during the acute infection period and 2 months after infection. However, the patterns of changes observed are markedly different during these two periods, suggesting that there are underlying changes in the network over time. These differences have implications for the treatment used during the acute infection and after chronic seizures develop.

Phase-response curves and synchronized neural networks.

We review the principal assumptions underlying the application of phase-response curves (PRCs) to synchronization in neuronal networks. The PRC measures how much a given synaptic input perturbs spike timing in a neural oscillator. Among other applications, PRCs make explicit predictions about whether a given network of interconnected neurons will synchronize, as is often observed in cortical structures. Regarding the assumptions of the PRC theory, we conclude: (i) The assumption of noise-tolerant cellular oscillations at or near the network frequency holds in some but not all cases. (ii) Reduced models for PRC-based analysis can be formally related to more realistic models. (iii) Spike-rate adaptation limits PRC-based analysis but does not invalidate it. (iv) The dependence of PRCs on synaptic location emphasizes the importance of improving methods of synaptic stimulation. (v) New methods can distinguish between oscillations that derive from mutual connections and those arising from common drive. (vi) It is helpful to assume linear summation of effects of synaptic inputs; experiments with trains of inputs call this assumption into question. (vii) Relatively subtle changes in network structure can invalidate PRC-based predictions. (viii) Heterogeneity in the preferred frequencies of component neurons does not invalidate PRC analysis, but can annihilate synchronous activity.

Differences in excitatory transmission between thalamic and cortical afferents to single spiny efferent neurons of rat dorsal striatum.

The striatum is crucially involved in motor and cognitive function, and receives significant glutamate input from the cortex and thalamus. The corticostriatal pathway arises from diverse regions of the cortex and is thought to provide information to the basal ganglia from which motor actions are selected and modified. The thalamostriatal pathway arises from specific thalamic nuclei and is involved in attention and possibly strategy switching. Despite these fundamental functional differences, direct comparisons of the properties of these pathways are lacking. N-methyl-D-aspartate (NMDA) receptors at synapses powerfully affect postsynaptic processing, and incorporation of different NR2 subunits into NMDA receptors has profound effects on the pharmacological and biophysical properties of the receptor. Utilization of different NMDA receptors at thalamostriatal and corticostriatal synapses could allow for afferent-specific differences in information processing. We used a novel rat brain slice preparation preserving corticostriatal and thalamostriatal pathways to medium spiny neurons to examine the properties of NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) recorded using the whole-cell, patch-clamp technique. Within the same neuron, the NMDA/non-NMDA ratio is greater for excitatory responses evoked from the thalamostriatal pathway than for those evoked from the corticostriatal pathway. In addition, reversal potentials and decay kinetics of the NMDA receptor-mediated EPSCs suggest that the thalamostriatal synapse is more distant on the dendritic arbor. Finally, results obtained with antagonists specific for NR2B-containing NMDA receptors imply that NMDA receptors at corticostriatal synapses contain more NR2B subunits. These synapse-specific differences in NMDA receptor content and pharmacology provide potential differential sites of action for NMDA receptor subtype-specific antagonists proposed for the treatment of Parkinson's disease.

The influence of substrate curvature on neurite outgrowth is cell type dependent.

Damage to axonal tracts of the central nervous system results in costly and permanent disability. The observations of aborted neurite outgrowth and disorganized scarring in injured central nervous system tissue have motivated the hypothesis that engineered bridging devices might facilitate regeneration. It is thought that both the shape and surface chemistry are important design parameters, however, their relative importance is poorly understood. Previously, we utilized smooth cylindrical surfaces to demonstrate that surfaces designed with directionally varying curvature bias in a stereotyped way postnatal dorsal root ganglion axonal regeneration in the direction of minimum curvature independent of surface chemistry. In the present study, we extend this analysis to include adult dorsal root ganglion neurons and cerebellar granule cells, cell types more representative of the challenge faced clinically. We found that axonal outgrowth of both the adult neuron and the central neuron was less sensitive to substrate curvature than the outgrowth of the postnatal neurons. These differences were quantified by constructing distributions describing the probability of outgrowth for a defined range of surface curvatures. Both the adult neuron and the central neuron exhibited a higher probability of extension in high-curvature directions compared to the postnatal neuron implying that surface geometry may not be as potent a cue in directing the regeneration of these neurons. A microtubule-stabilizing agent enhanced the sensitivity to curvature of the adult neuron, partially reversing the increased probability of growing in a high-curvature direction. The results suggest novel methods to enhance directed neuron regeneration using bridging substrates.

A rat brain slice preparation for characterizing both thalamostriatal and corticostriatal afferents.

The striatum, the primary input nucleus of the basal ganglia, is crucially involved in motor and cognitive function and receives significant glutamate input from the cortex and thalamus. Increasing evidence suggests fundamental differences between these afferents, yet direct comparisons have been lacking. We describe a slice preparation that allows for direct comparison of the pharmacology and biophysics of these two pathways. Visualization of slices from animals previously injected with BDA into the parafascicular nucleus revealed the presence of axons of thalamic origin in the slice. These axons were especially well-preserved after traversing the reticular nucleus, the location chosen for stimulation of thalamostriatal afferents. Initial characterization of the two pathways revealed both non-NMDA and NMDA receptor-mediated currents at synapses from both afferents and convergence of the afferents in 51% of striatal efferent neurons. Annihilation of action potentials was not observed in collision experiments, nor was current spread from the site of stimulation to striatum found. Differences in short-term plasticity suggest that the probability of release differs for the two inputs. The present work thus provides a novel rat brain slice preparation in which the effects of selective stimulation of cortical versus thalamic afferents to striatum can be studied in the same preparation.

Cyclic strain increases fibroblast proliferation, matrix accumulation, and elastic modulus of fibroblast-seeded polyurethane constructs.

Rapid induction of matrix production and mechanical strengthening is essential to the development of bio-artificial constructs for repair and replacement of load-bearing connective tissues. Toward this end, we describe the development of a mechanical bioreactor and its application to investigate the influence of cyclic strain on fibroblast proliferation, matrix accumulation, and the mechanical properties of fibroblast-seeded polyurethane constructs (FSPC). Human fibroblasts were cultured in 10% serum-containing conditions within three-dimensional, porous elastomeric substrates under static conditions and a model regime of cyclic strain (10% strain, 0.25 Hz, 8 h/day), with and without ascorbic acid supplementation. After one week, the combination of cyclic strain and ascorbic acid resulted in significantly increased construct elastic modulus (>110%) relative to either condition alone. In contrast, cyclic strain alone was sufficient to stimulate significant increases in fibroblast proliferation. Mechanical strengthening of FSPCs was accompanied by increased type I collagen and fibronectin matrix accumulation and distribution, and significantly increased gene expression for type I collagen, TGFbeta-1, and CTGF. These results suggest that strain-induced conditioning in vitro leads to mechanical strengthening of fibroblast/material constructs, most likely resulting from increased collagen matrix deposition, secondary to strain-induced increases in cytokine production.

Substrate curvature influences the direction of nerve outgrowth.

Nerve outgrowth in the developing nervous system utilizes a variety of attractive and repulsive molecules found in the extracellular environment. In addition, physical cues may play an important regulatory role in determining directional outgrowth of nervous tissue. Here, by culturing nerve cells on filamentous surfaces and measuring directional growth, we tested the hypothesis that substrate curvature is sufficient to influence the directional outgrowth of nerve cells. We found that the mean direction of neurite outgrowth aligned with the direction of minimum principle curvature, and the spatial variance in outgrowth direction was directly related to the maximum principle curvature. As substrate size approached the size of an axon, adherent neurons extended processes that followed the direction of the long axis of the substrate similar to what occurs during development along pioneering axons and radial glial fibers. A simple Boltzmann model describing the interplay between adhesion and bending stiffness of the nerve process was found to be in close agreement with the data suggesting that cell stiffness and substrate curvature can act together in a manner that is sufficient to direct nerve outgrowth in the absence of contrasting molecular cues. The study highlights the potential importance of cellular level geometry as a fidelity-enhancing cue in the developing and regenerating nervous system.

Comparison of human fibroblast ECM-related gene expression on elastic three-dimensional substrates relative to two-dimensional films of the same material.

Three-dimensional elastic substrates were fabricated from a commercially available polyurethane with an internal porosity of approximately 70% and elastic modulus of 27.4+/-2.76 KPa and examined for suitability in vocal fold tissue engineering. Using immunohistochemistry, biomechanical testing, and RT-PCR; we examined human fibroblast viability, distribution and extracellular matrix related gene expression within substrates for periods up to 4 weeks. We found that cells were capable of colonizing the entire volume of a 5mm wide x 3mm deep x 20mm long substrate at high viability. Histological cross-sections showed extensive extracellular matrix deposited around the cells and throughout the pore structure of the substrates, which consisted of fibronectin and type I collagen. Cell seeded substrates displayed a significantly higher elastic modulus than unseeded controls similar to native tissue. The transfer of cell growth from two-dimensional to three-dimensional culture resulted in changes in ECM-related gene expression consistent with decreasing cell migration and increasing tissue formation. We found that fibroblasts cultured in three-dimensional substrates expressed significantly higher levels of mRNA for elastin and fibromodulin, while expressing significantly lower levels of mRNA for MMP-1 and hyaluronidase relative to two-dimensional substrates of the same material. The results suggest that three-dimensionally porous, Tecoflex-derived elastic biomaterials may be suitable substrates for engineering vocal fold tissue.