Amyotrophic Lateral Sclerosis-Associated Persistent Organic Pollutant cis-Chlordane Causes GABAA-Independent Toxicity to Motor Neurons, Providing Evidence toward an Environmental Component of Sporadic Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by the death of upper and lower motor neurons. While causative genes have been identified, 90% of ALS cases are not inherited and are hypothesized to result from the accumulation of genetic and environmental risk factors. While no specific causative environmental toxin has been identified, previous work has indicated that the presence of the organochlorine pesticide cis-chlordane in the blood is highly correlated with ALS incidence. Never before tested on the motor system, here, we show that cis-chlordane is especially toxic to motor neurons in vitro- and in vivo-independent of its known antagonism of the GABAA receptor. We find that human stem-cell-derived motor neurons are more sensitive to cis-chlordane than other cell types and their action potential dynamics are altered. Utilizing zebrafish larvae, we show that cis-chlordane induces motor neuron and neuromuscular junction degeneration and subsequent motor deficits in a touch-evoked escape response. Together, our work points to cis-chlordane as a potential sporadic ALS exacerbating environmental pollutant.

DARPP-32 distinguishes a subset of adult-born neurons in zebra finch HVC.

Adult male zebra finches (Taeniopygia guttata) continually incorporate adult-born neurons into HVC, a telencephalic brain region necessary for the production of learned song. These neurons express activity-dependent immediate early genes (e.g., zenk and c-fos) following song production, suggesting that these neurons are active during song production. Half of these adult-born HVC neurons (HVC NNs) can be backfilled from the robust nucleus of the arcopallium (RA) and are a part of the vocal motor pathway underlying learned song production, but the other half do not backfill from RA, and they remain to be characterized. Here, we used cell birth-dating, retrograde tract tracing, and immunofluorescence to demonstrate that half of all HVC NNs express the phosphoprotein DARPP-32, a protein associated with dopamine receptor expression. We also demonstrate that DARPP-32+ HVC NNs are contacted by tyrosine hydroxylase immunoreactive fibers, suggesting that they receive catecholaminergic input, have transiently larger nuclei than DARPP-32-neg HVC NNs, and do not backfill from RA. Taken together, these findings help characterize a group of HVC NNs that have no apparent projections to RA and so far have eluded positive identification other than HVC NN status.

Optogenetic Interrogation of ChR2-Expressing GABAergic Interneurons After Transplantation into the Mouse Brain.

This paper describes research methods to investigate the development of synaptic connections between transplanted GABAergic interneurons and endogenous neurons in the adult mouse hippocampus. Our protocol highlights methods for retroviral labeling adult-born GCs, one of the few cell types in the adult brain to be continuously renewed throughout life. By precise targeting of the retrovirus, labeling of adult-born GCs can be combined with optogenetic stimulation of the transplanted cells and electrophysiology in brain slices, to test whether the GABAergic interneurons integrate and establish inhibitory synaptic connections with host brain neurons. Modifications to adult neurogenesis are an important contributing factor in the development and severity of TLE and seizures. When combined with retroviral labeling, the approaches we describe in this chapter can be used to determine whether transplantation modifies the process of adult neurogenesis or other properties of the hippocampus. These approaches are helping to define parameters for potential cell replacement therapies to be used in patients with intractable seizure disorders.

Development of electrophysiological and morphological properties of human embryonic stem cell-derived GABAergic interneurons at different times after transplantation into the mouse hippocampus.

Transplantation of human embryonic stem cell (hESC)-derived neural progenitors is a potential treatment for neurological disorders, but relatively little is known about the time course for human neuron maturation after transplantation and the emergence of morphological and electrophysiological properties. To address this gap, we transplanted hESC-derived human GABAergic interneuron progenitors into the mouse hippocampus, and then characterized their electrophysiological properties and dendritic arborizations after transplantation by means of ex vivo whole-cell patch clamp recording, followed by biocytin staining, confocal imaging and neuron reconstruction software. We asked whether particular electrophysiological and morphological properties showed maturation-dependent changes after transplantation. We also investigated whether the emergence of particular electrophysiological properties were linked to increased complexity of the dendritic arbors. Human neurons were classified into five distinct neuronal types (Type I-V), ranging from immature to mature fast-spiking interneurons. Hierarchical clustering of the dendritic morphology and Sholl analyses suggested four morphologically distinct classes (Class A-D), ranging from simple/immature to highly complex. Incorporating all of our data regardless of neuronal classification, we investigated whether any electrophysiological and morphological features correlated with time post-transplantation. This analysis demonstrated that both dendritic arbors and electrophysiological properties matured after transplantation.

Restrained Dendritic Growth of Adult-Born Granule Cells Innervated by Transplanted Fetal GABAergic Interneurons in Mice with Temporal Lobe Epilepsy.

The dentate gyrus (DG) is a region of the adult rodent brain that undergoes continuous neurogenesis. Seizures and loss or dysfunction of GABAergic synapses onto adult-born dentate granule cells (GCs) alter their dendritic growth and migration, resulting in dysmorphic and hyperexcitable GCs. Additionally, transplants of fetal GABAergic interneurons in the DG of mice with temporal lobe epilepsy (TLE) result in seizure suppression, but it is unknown whether increasing interneurons with these transplants restores GABAergic innervation to adult-born GCs. Here, we address this question by birth-dating GCs with retrovirus at different times up to 12 weeks after pilocarpine-induced TLE in adult mice. Channelrhodopsin 2 (ChR2)-enhanced yellow fluorescent protein (EYFP)-expressing medial-ganglionic eminence (MGE)-derived GABAergic interneurons from embryonic day (E)13.5 mouse embryos were transplanted into the DG of the TLE mice and GCs with transplant-derived inhibitory post-synaptic currents (IPSCs) were identified by patch-clamp electrophysiology and optogenetic interrogation. Putative synaptic sites between GCs and GABAergic transplants were also confirmed by intracellular biocytin staining, immunohistochemistry, and confocal imaging. 3D reconstructions of dendritic arbors and quantitative morphometric analyses were carried out in >150 adult-born GCs. GABAergic inputs from transplanted interneurons correlated with markedly shorter GC dendrites, compared to GCs that were not innervated by the transplants. Moreover, these effects were confined to distal dendritic branches and a short time window of six to eight weeks. The effects were independent of seizures as they were also observed in naïve mice with MGE transplants. These findings are consistent with the hypothesis that increased inhibitory currents over a smaller dendritic arbor in adult-born GCs may reduce their excitability and lead to seizure suppression.

Pluripotent stem cell-derived interneuron progenitors mature and restore memory deficits but do not suppress seizures in the epileptic mouse brain.

GABAergic interneuron dysfunction has been implicated in temporal lobe epilepsy (TLE), autism, and schizophrenia. Inhibitory interneuron progenitors transplanted into the hippocampus of rodents with TLE provide varying degrees of seizure suppression. We investigated whether human embryonic stem cell (hESC)-derived interneuron progenitors (hESNPs) could differentiate, correct hippocampal-dependent spatial memory deficits, and suppress seizures in a pilocarpine-induced TLE mouse model. We found that transplanted ventralized hESNPs differentiated into mature GABAergic interneurons and became electrophysiologically active with mature firing patterns. Some mice developed hESNP-derived tumor-like NSC clusters. Mice with transplants showed significant improvement in the Morris water maze test, but transplants did not suppress seizures. The limited effects of the human GABAergic interneuron progenitor grafts may be due to cell type heterogeneity within the transplants.

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.

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.

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.

STEP regulation of seizure thresholds in the hippocampus.

PURPOSE: To investigate whether striatal enriched protein tyrosine phosphatase (STEP) influences ictogenesis. METHODS: STEP knockout mice were compared to wild-type (WT) mice in pilocarpine-induced seizures. Hippocampal slices were also prepared from these two mouse populations, allowing the examination of ictal-like stimulation in these slices using calcium imaging and electrophysiologic recordings. KEY FINDINGS: To examine seizure thresholds, increasing doses of pilocarpine were administered to adult mice and seizures were scored behaviorally. Significantly fewer STEP knockout mice developed seizures that progressed to the stage of status epilepticus compared to WT mice. To examine potential differences in neural circuits that might account for this finding, seizure-like activity was induced in hippocampal slices. Electrical stimulation of the hippocampal-entorhinal cortex pathway in STEP knockout mice resulted in less activation of the dentate gyrus granule cell layer (GCL), but greater activation of the hilus in STEP knockouts, compared with heterozygous slices. SIGNIFICANCE: STEP deficiency is associated with higher seizure thresholds. The locus of these effects appears to include the dentate gyrus granule cell layer and hilus.

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

Complex 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.

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.

Reverse optical probing (ROPING) of neocortical circuits.

We describe an optical technique to detect circuits of synaptically connected neurons. By combining calcium imaging of the spontaneous activity of neuronal populations with intracellular recordings from a given neuron, we perform a type of reverse correlation analysis to detect neurons that generate action potentials time-locked to the synaptic currents of the recorded cell. This technique can quickly reveal monosynaptically connected neurons.

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.

Synfire chains and cortical songs: temporal modules of cortical activity.

How can neural activity propagate through cortical networks built with weak, stochastic synapses? We find precise repetitions of spontaneous patterns of synaptic inputs in neocortical neurons in vivo and in vitro. These patterns repeat after minutes, maintaining millisecond accuracy. Calcium imaging of slices reveals reactivation of sequences of cells during the occurrence of repeated intracellular synaptic patterns. The spontaneous activity drifts with time, engaging different cells. Sequences of active neurons have distinct spatial structures and are repeated in the same order over tens of seconds, revealing modular temporal dynamics. Higher order sequences are replayed with compressed timing.