Gloster B. Aaron

Internal Dynamics Determine the Cortical Response to Thalamic Stimulation

Although spontaneous activity occurs throughout the neocortex, its relation to the activity produced by external or sensory inputs remains unclear. To address this, we used calcium imaging of mouse thalamocortical slices to reconstruct, with single-cell resolution, the spatiotemporal dynamics of activity of layer 4 in the presence or absence of thalamic stimulation. We found spontaneous neuronal coactivations corresponded to intracellular UP states. Thalamic stimulation of sufficient frequency (>10 Hz) triggered cortical activity, and UP states, indistinguishable from those arising spontaneously. Moreover, neurons were activated in identical and precise spatiotemporal patterns in thalamically triggered and spontaneous events. The similarities between cortical activations indicate that intracortical connectivity plays the dominant role in the cortical response to thalamic inputs. Our data demonstrate that precise spatiotemporal activity patterns can be triggered by thalamic inputs and indicate that the thalamus serves to release intrinsic cortical dynamics.

Differentiation and Functional Incorporation of Embryonic Stem Cell-Derived GABAergic Interneurons in the Dentate Gyrus of Mice with Temporal Lobe Epilepsy

Cell therapies for neurological disorders require an extensive knowledge of disease-associated neuropathology and procedures for generating neurons for transplantation. In many patients with severe acquired temporal lobe epilepsy (TLE), the dentate gyrus exhibits sclerosis and GABAergic interneuron degeneration. Mounting evidence suggests that therapeutic benefits can be obtained by transplanting fetal GABAergic progenitors into the dentate gyrus in rodents with TLE, but the scarcity of human fetal cells limits applicability in patient populations. In contrast, virtually limitless quantities of neural progenitors can be obtained from embryonic stem (ES) cells. ES cell-based therapies for neurological repair in TLE require evidence that the transplanted neurons integrate functionally and replace cell types that degenerate. To address these issues, we transplanted mouse ES cell-derived neural progenitors (ESNPs) with ventral forebrain identities into the hilus of the dentate gyrus of mice with TLE and evaluated graft differentiation, mossy fiber sprouting, cellular morphology, and electrophysiological properties of the transplanted neurons. In addition, we compared electrophysiological properties of the transplanted neurons with endogenous hilar interneurons in mice without TLE. The majority of transplanted ESNPs differentiated into GABAergic interneuron subtypes expressing calcium-binding proteins parvalbumin, calbindin, or calretinin. Global suppression of mossy fiber sprouting was not observed; however, ESNP-derived neurons formed dense axonal arborizations in the inner molecular layer and throughout the hilus. Whole-cell hippocampal slice electrophysiological recordings and morphological analyses of the transplanted neurons identified five basic types; most with strong after-hyperpolarizations and smooth or sparsely spiny dendritic morphologies resembling endogenous hippocampal interneurons. Moreover, intracellular recordings of spontaneous EPSCs indicated that the new cells functionally integrate into epileptic hippocampal circuitry.

Long-Term Seizure Suppression and Optogenetic Analyses of Synaptic Connectivity in Epileptic Mice with Hippocampal Grafts of GABAergic Interneurons

Studies in rodent epilepsy models suggest that GABAergic interneuron progenitor grafts can reduce hyperexcitability and seizures in temporal lobe epilepsy (TLE). Although integration of the transplanted cells has been proposed as the underlying mechanism for these disease-modifying effects, prior studies have not explicitly examined cell types and synaptic mechanisms for long-term seizure suppression. To address this gap, we transplanted medial ganglionic eminence (MGE) cells from embryonic day 13.5 VGAT-Venus or VGAT-ChR2-EYFP transgenic embryos into the dentate gyrus (DG) of adult mice 2 weeks after induction of TLE with pilocarpine. Beginning 3–4 weeks after status epilepticus, we conducted continuous video-electroencephalographic recording until 90–100 d. TLE mice with bilateral MGE cell grafts in the DG had significantly fewer and milder electrographic seizures, compared with TLE controls. Immunohistochemical studies showed that the transplants contained multiple neuropeptide or calcium-binding protein-expressing interneuron types and these cells established dense terminal arborizations onto the somas, apical dendrites, and axon initial segments of dentate granule cells (GCs). A majority of the synaptic terminals formed by the transplanted cells were apposed to large postsynaptic clusters of gephyrin, indicative of mature inhibitory synaptic complexes. Functionality of these new inhibitory synapses was demonstrated by optogenetically activating VGAT-ChR2-EYFP-expressing transplanted neurons, which generated robust hyperpolarizations in GCs. These findings suggest that fetal GABAergic interneuron grafts may suppress pharmacoresistant seizures by enhancing synaptic inhibition in DG neural circuits.

Embryonic stem cell-derived neural precursor grafts for treatment of temporal lobe epilepsy.

SummaryComplex partial seizures arising from mesial temporal lobe structures are a defining feature of mesial temporal lobe epilepsy (TLE). For many TLE patients, there is an initial traumatic head injury that is the precipitating cause of epilepsy. Severe TLE can be associated with neuropathological changes, including hippocampal sclerosis, neurodegeneration in the dentate gyrus, and extensive reorganization of hippocampal circuits. Learning disabilities and psychiatric conditions may also occur in patients with severe TLE for whom conventional anti-epileptic drugs are ineffective. Novel treatments are needed to limit or repair neuronal damage, particularly to hippocampus and related limbic regions in severe TLE and to suppress temporal lobe seizures. A promising therapeutic strategy may be to restore inhibition of dentate gyrus granule neurons by means of cell grafts of embryonic stem cell-derived GABAergic neuron precursors. “Proof-of-concept” studies show that human and mouse embryonic stem cell-derived neural precursors can survive, migrate, and integrate into the brains of rodents in different experimental models of TLE. In addition, studies have shown that hippocampal grafts of cell lines engineered to release GABA or other anticonvulsant molecules can suppress seizures. Furthermore, transplants of fetal GABAergic progenitors from the mouse or human brain have also been shown to suppress the development of seizures. Here, we review these relevant studies and highlight areas of future research directed toward producing embryonic stem cell-derived GABAergic interneurons for cell-based therapies for treating TLE.

Adaptive changes in the somatotopic properties of individual thalamic neurons immediately following microlesions in connected regions of the nucleus cuneatus.

We examined the ability of thalamic neurons in the ventrobasal complex to show adaptive changes in receptive field properties following the loss of projections from the nucleus cuneatus. Thalamic responses to air jet stimulation were tested at multiple peripheral sites before and after making discrete microlesions in topographically‐matched regions of the nucleus cuneatus. Prior to making a lesion, crosscorrelation analysis and orthodromic microstimulation were used to confirm the source of cuneate neurons projecting to the thalamic recording site. A total of 69 thalamic neurons were recorded from 29 rats. Following placement of a microlesion (100–200 μm diameter) in the nucleus cuneatus, 34 thalamic neurons did not show significant changes in stimulus‐induced responses, possibly because the lesion was too small or because critical sites in the receptive field were not tested. The remaining 35 neurons were affected by cuneate microlesions, but the change in responsiveness varied according to stimulation site. When the most responsive site in the receptive field was examined, 24 neurons exhibited significant decreases and three neurons showed significant increases in responsiveness. Cuneate microlesions produced decreases at moderately responsive sites, but the reduction in response magnitude was smaller than at the most responsive site. When responses near the receptive field boundary were examined, 11 neurons displayed significant increases and only four neurons showed significant decreases. For two neurons without well‐defined receptive field boundaries, cuneate microlesions caused new excitatory responses to emerge from sites that had formerly caused a slight inhibition of spontaneous activity. In all cases of increased responsiveness, the changes appeared on only one side of a neurons receptive field. This asymmetry may account for the fact that the probability of detecting receptive field expansion increased from 27% (six of 22 experiments) to 71% (five of seven experiments) when the number of stimulation sites located throughout the receptive field was increased. These results indicate that the receptive field structure of individual neurons shows adaptive properties immediately after loss of the predominant ascending inputs.

Statistical Significance of Precisely Repeated Intracellular Synaptic Patterns

Can neuronal networks produce patterns of activity with millisecond accuracy? It may seem unlikely, considering the probabilistic nature of synaptic transmission. However, some theories of brain function predict that such precision is feasible and can emerge from the non-linearity of the action potential generation in circuits of connected neurons. Several studies have presented evidence for and against this hypothesis. Our earlier work supported the precision hypothesis, based on results demonstrating that precise patterns of synaptic inputs could be found in intracellular recordings from neurons in brain slices and in vivo. To test this hypothesis, we devised a method for finding precise repeats of activity and compared repeats found in the data to those found in surrogate datasets made by shuffling the original data. Because more repeats were found in the original data than in the surrogate data sets, we argued that repeats were not due to chance occurrence. Mokeichev et al. (2007) challenged these conclusions, arguing that the generation of surrogate data was insufficiently rigorous. We have now reanalyzed our previous data with the methods introduced from Mokeichev et al. (2007). Our reanalysis reveals that repeats are statistically significant, thus supporting our earlier conclusions, while also supporting many conclusions that Mokeichev et al. (2007) drew from their recent in vivo recordings. Moreover, we also show that the conditions under which the membrane potential is recorded contributes significantly to the ability to detect repeats and may explain conflicting results. In conclusion, our reevaluation resolves the methodological contradictions between Ikegaya et al. (2004) and Mokeichev et al. (2007), but demonstrates the validity of our previous conclusion that spontaneous network activity is non-randomly organized.

A comparative analysis of coordinated neuronal activity in the thalamic ventrobasal complex of rats and cats

There are substantial differences in the incidence of inhibitory neurons in the ventrobasal complex of rat and cat thalamus. This marked dissimilarity in neuronal composition suggests that there should be corresponding differences in the orchestration of neural activity in these regions during cutaneous stimulation. To explore this possibility, we conducted a cross-correlation analysis of neuronal activity in the ventroposterolateral (VPL) nucleus of anesthetized rats and cats. Pairs of neurons representing hairy skin were recorded simultaneously with one or two electrodes during air jet stimulation of multiple sites throughout the receptive fields. Cross-correlation histograms indicated that correlated activity among adjacent neurons occurred in three distinct patterns. In one pattern, classified as narrow-unimodal, the discharge of one neuron preceded a discharge in the partner neuron over a narrow interval of time (< 5 ms). Narrow-bimodal patterns were characterized by responses in which the temporal order of discharges from the two neurons was variable, but the interspike intervals were always < 5 ms. In wide-unimodal patterns, the discharge of one neuron was correlated with subsequent discharges in the partner neuron over a wide interval of time (> 5 ms). In rat VPL, two-thirds of the 58 neuron pairs showing correlated responses were characterized by narrow-unimodal responses and nearly one-third of the neuron pairs displayed narrow-bimodal patterns. Only one pair of rat VPL neurons were characterized by a wide-unimodal pattern of coordination. By comparison, half of the 61 adjacent neuron pairs with coordinated responses in cat VPL were characterized by narrow-unimodal patterns. Slightly more than one-third of the correlated neuron pairs had narrow-bimodal patterns, while the remainder (13%) were classified as wide-unimodal responses. Pairs of neurons separated by 340-405 microns discharged synchronously in a pattern that was similar to the temporal relationship expressed in the narrow-bimodal patterns found among adjacent neurons. In both species, the wide-unimodal patterns had the strongest coordinated responses as measured by the correlation coefficient. Although inhibitory relationships did not appear in correlation histograms that had been corrected for stimulus coordination, cross-correlation analysis of the raw spike trains revealed brief (10-40 ms) periods of inhibition that were associated with cat VPL neurons exhibiting wide-unimodal coordination patterns. In rat VPL, most inhibition involved longer (30-60 ms) periods of inhibitory oscillations appearing amidst a much larger rhythmic pattern. These results suggest that correlation patterns transpiring over narrow (< 5 ms) time intervals represent the coordination of activity among neighboring thalamocortical relay neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

STEP regulation of seizure thresholds in the hippocampus

Purpose:  To investigate whether striatal enriched protein tyrosine phosphatase (STEP) influences ictogenesis.

Propagation of epileptiform events across the corpus callosum in a cingulate cortical slice preparation.

We report on a novel mouse in vitro brain slice preparation that contains intact callosal axons connecting anterior cingulate cortices (ACC). Callosal connections are demonstrated by the ability to regularly record epileptiform events between hemispheres (bilateral events). That the correlation of these events depends on the callosum is demonstrated by the bisection of the callosum in vitro. Epileptiform events are evoked with four different methods: (1) bath application of bicuculline (a GABA-A antagonist); (2) bicuculline+MK801 (an NMDA receptor antagonist), (3) a zero magnesium extracellular solution (0Mg); (4) focal application of bicuculline to a single cortical hemisphere. Significant increases in the number of epileptiform events, as well as increases in the ratio of bilateral events to unilateral events, are observed during bath applications of bicuculline, but not during applications of bicuculline+MK-801. Long ictal-like events (defined as events >20 seconds) are only observed in 0Mg. Whole cell patch clamp recordings of single neurons reveal strong feedforward inhibition during focal epileptiform events in the contralateral hemisphere. Within the ACC, we find differences between the rostral areas of ACC vs. caudal ACC in terms of connectivity between hemispheres, with the caudal regions demonstrating shorter interhemispheric latencies. The morphologies of many patch clamped neurons show callosally-spanning axons, again demonstrating intact callosal circuits in this in vitro preparation.

Different patterns of synaptic transmission revealed between hippocampal CA3 stratum oriens and stratum lucidum interneurons and their pyramidal cell targets

Stratum lucidum (SL) interneurons likely mediate feedforward inhibition between the dentate gyrus mossy fibers and CA3 pyramidal cells, while stratum oriens (SO) interneurons likely provide both feedforward and feedback inhibition within the CA3 commissural/associational network. Using dual whole-cell patch-clamp recordings between interneurons and CA3 pyramidal cells, we have examined SL and SO interneurons and their synapses within organotypic hippocampal slice cultures. Biocytin staining revealed different morphologies between these interneuron groups, both being very similar to those found previously in acute slices. The kinetics of IPSCs were similar between the two groups, but the reliability of synaptic transmission of SL interneuron (SL-INT) IPSCs was significantly lower than the virtually 100% reliability (non-existent failure rates) of SO-INT IPSCs. The SL-INT IPSCs also had a lower quantal content than the SO-INT IPSCs. In addition, SL-INTs were less likely than SO-INTs to innervate or to be innervated by nearby CA3 pyramidal cells. Paired-pulse stimulation at 100 ms interstimulus intervals produced similar paired-pulse depression in both interneuron synapses, despite the significantly higher failure rate of IPSCs produced by the SL-INTs compared with SO-INTs. CV analysis supported the hypothesis that paired-pulse depression was presynaptic. During repetitive, high frequency stimulation (>10 Hz for 500 ms) the two different synapses exhibited distinctly different forms of short-term plasticity: all SL interneurons displayed significant short-term facilitation (mean 113% facilitation, n=4), while, by contrast, SO interneuron synapses displayed either short-term depression (mean 42% depression, n=5 of 8) or no net facilitation or depression (n=3 of 8). These results indicate that the synaptic properties of interneurons can be quite different for interneurons in different hippocampal circuits.