Roy M. Smeal

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.

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.

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.

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.

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.

Contributions of astrocytes to epileptogenesis following status epilepticus: Opportunities for preventive therapy?

Status epilepticus (SE) is a life threatening condition that often precedes the development of epilepsy. Traditional treatments for epilepsy have been focused on targeting neuronal mechanisms contributing to hyperexcitability, however, approximately 30% of patients with epilepsy do not respond to existing neurocentric pharmacotherapies. A growing body of evidence has demonstrated that profound changes in the morphology and function of astrocytes accompany SE and persist in epilepsy. Astrocytes are increasingly recognized for their diverse roles in modulating neuronal activity, and understanding the changes in astrocytes following SE could provide important clues about the mechanisms underlying seizure generation and termination. By understanding the contributions of astrocytes to the network changes underlying epileptogenesis and the development of epilepsy, we will gain a greater appreciation of the contributions of astrocytes to dynamic circuit changes, which will enable us to develop more successful therapies to prevent and treat epilepsy. This review summarizes changes in astrocytes following SE in animal models and human temporal lobe epilepsy and addresses the functional consequences of those changes that may provide clues to the process of epileptogenesis.

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 Parkinsons 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.

Hippocampal TNFα signaling contributes to seizure generation in an infection-induced mouse model of limbic epilepsy

Abstract 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.

Targeted Path Scanning: An Emerging Method for Recording Fast Changing Network Dynamics across Large Distances

Attention is being increasingly focused on the dynamical behavior of large networks of neurons and astrocytes and the changes in these dynamics that occur during the progression of diseases like epilepsy. Recording from large numbers of identified cell types has been traditionally difficult, but the advent of fluorescent indicators capable of detecting changes in the internal calcium levels of cells has led to the ability to visually record the activity of large numbers of cells. However, for most imaging techniques the temporal resolution is sharply limited by the time it takes lasers to traverse the typical raster scan. As network dynamics can evolve quite rapidly, this is a serious limitation. The present paper describes the Targeted Path Scan technique, which dramatically increases the scanning frequency by allowing the user selection of trajectories through cells of interest. TPS is discussed in the context of a study of altered network dynamics in a common rat model of epilepsy. In this study, traveling waves of calcium transients that were frequently encountered in astrocytes imaged in brain slices obtained from control rats were dramatically reduced in astrocytes imaged in brain slices obtained from rats that had experienced status epilepticus. The speed of these traveling waves would have made them impossible to identify using traditional scanning techniques.