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

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

Synaptic characteristics and short-term synaptic plasticity of hippocampal CA3 inhibitory circuits

Inhibitory interneurons of the hippocampus are a diverse population that play vital roles in regulating the activities of the more numerous and relatively homogenous groups of excitatory principal cells. In order to better understand the functions of different groups of interneurons, we used dual whole-cell patch-clamp recordings of interneurons from either stratum lucidum (SL) or stratum oriens (SO) and nearby CA3 pyramidal cells to investigate the morphologies and synaptic properties of IPSCs formed by these two groups of interneurons. SL interneurons have been implicated as mediators of feedforward inhibition between the dentate gyrus and CA3 pyramidal cells, while SO intemeurons mediate both feedback and feedforward inhibition within the highly interconnected CA3 pyramidal cell network. All recordings were performed in organotypic roller-tube cultures. Axodendritic synapses were formed by both interneuron groups on pyramidal cells. The kinetics of IPSCs were similar between the two groups, but the reliability of synaptic transmission of SL IPSCs was significantly lower than the virtually 100% reliability (non-existent failure rates) of SO IPSCs. In addition, SL intemeurons were less likely than SO intemeurons to innervate or to be innervated by nearby CA3 pyramidal cells. Biocytin staining revealed different morphologies between these interneuron groups, both being very similar to those found previously in acute slices. Paired-pulse stimulation at 100 ms interstimulus intervals produced similar paired-pulse depression in both interneuron synapses. However, during repetitive, high frequency stimulation (\u3e10 Hz for 500 ms) the two different synapses exhibited distinctly different forms of short-term plasticity: all SL interneurons displayed significant short-term facilitation, while, by contrast, SO interneuron synapses never displayed facilitation and often demonstrated significant depression. These results indicate that the pattern of innervation and synaptic properties of interneurons are different for interneurons in different hippocampal circuits. These results are relevant to the study of the role of hippocampal CA3 in memory functions in that these two interneuron populations may provide two different strategies for focusing mossy fiber inputs into activating specific networks within the C/A CA3 network. In addition, the short-term plasticities exhibited by these interneurons have implications for the development and propagation of seizures in the hippocampus

Synaptic characteristics and short-term synaptic plasticity of hippocampal CA3 inhibitory circuits

Inhibitory interneurons of the hippocampus are a diverse population that play vital roles in regulating the activities of the more numerous and relatively homogenous groups of excitatory principal cells. In order to better understand the functions of different groups of interneurons, we used dual whole-cell patch-clamp recordings of interneurons from either stratum lucidum (SL) or stratum oriens (SO) and nearby CA3 pyramidal cells to investigate the morphologies and synaptic properties of IPSCs formed by these two groups of interneurons. SL interneurons have been implicated as mediators of feedforward inhibition between the dentate gyrus and CA3 pyramidal cells, while SO intemeurons mediate both feedback and feedforward inhibition within the highly interconnected CA3 pyramidal cell network. All recordings were performed in organotypic roller-tube cultures. Axodendritic synapses were formed by both interneuron groups on pyramidal cells. The kinetics of IPSCs were similar between the two groups, but the reliability of synaptic transmission of SL IPSCs was significantly lower than the virtually 100% reliability (non-existent failure rates) of SO IPSCs. In addition, SL intemeurons were less likely than SO intemeurons to innervate or to be innervated by nearby CA3 pyramidal cells. Biocytin staining revealed different morphologies between these interneuron groups, both being very similar to those found previously in acute slices. Paired-pulse stimulation at 100 ms interstimulus intervals produced similar paired-pulse depression in both interneuron synapses. However, during repetitive, high frequency stimulation (\u3e10 Hz for 500 ms) the two different synapses exhibited distinctly different forms of short-term plasticity: all SL interneurons displayed significant short-term facilitation, while, by contrast, SO interneuron synapses never displayed facilitation and often demonstrated significant depression. These results indicate that the pattern of innervation and synaptic properties of interneurons are different for interneurons in different hippocampal circuits. These results are relevant to the study of the role of hippocampal CA3 in memory functions in that these two interneuron populations may provide two different strategies for focusing mossy fiber inputs into activating specific networks within the C/A CA3 network. In addition, the short-term plasticities exhibited by these interneurons have implications for the development and propagation of seizures in the hippocampus

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

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

Comparison of epileptiform events (EEs).

<p>BIC and 0Mg models are compared. The numbers represent the mean ± SE (n) for each measurement. For all means, only slices with greater than 10 EEs are included (22 out of 27 slices for 0Mg, 17/17 slices for BIC). For the BIC IHL measurements and Proportion of Unilateral EEs, only slices 3 and 4 are included, as no slice 2 was measured in 0Mg.</p>a<p>p<0.01, 0Mg greater than BIC (rank sum test).</p>b<p>p<0.01, 0Mg less than BIC (rank sum test).</p>c<p>p<0.04, 0Mg greater than BIC (rank sum test).</p

Three examples of repeats found using the PHRI detector from a 190 second long cat <i>in vivo</i> current clamp recording.

<p>Each motif-repeat example is labeled with its respective PHRI and its length. The PHRI values are a subset of those that make up the full set of PHRI values for this recording that are displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003983#pone-0003983-g008" target="_blank">Fig. 8c</a>.</p

Comparisons of different seizure models in the callosal slice preparation using dual recordings in two hemispheres simultaneously.

<p>Representative examples of bilateral EEs recorded in BIC (<b>Ai</b> and <b>Aii</b>) versus 0Mg (<b>Bi</b> and <b>Bii</b>) are shown. In each bilateral EE example, there are two simultaneous recordings, where each recording is gathered from opposite hemispheres in a single slice preparation. <b><i>Ai</i></b> and <b><i>Bi</i></b> show events that resemble inter-ictal events (5 sec. or less). These were the predominant kind of events recorded overall, and the only kind of event recorded in the BIC recordings. <b><i>Aii</i></b> and <b><i>Bii</i></b> show the longest event recorded for BIC (approx. 5 sec.), and a typical ictal-like event for 0Mg (approx. 30 sec.), respectively (note differerent time scales in the <b><i>i</i></b> vs. <b><i>ii</i></b> panels). For all panels, 0.4 mV is the y-scale for BIC, while 0.1 mV represents the y-scale for 0Mg, reflecting the significantly larger amplitudes found for these EEs in BIC. (<b>Aiii</b>) Significant differences in the number of EEs arise between the BIC and BIC+MK801 groups during the course of recordings (Wilcoxon rank sum test). (<b>Aiv</b>) The proportion of EEs that are unilateral decreases more in the BIC group than in the BIC+MK801 group (Chi square test of proportions). Data from both (<b>Aiii</b>) and (<b>Aiv</b>) show mean ± SE. n = 17 paired recordings from 17 slices from 9 mice in the BIC group, while n = 17 paired recordings from 17 slices from 5 mice in the BIC+MK801 group. (<b>Biii</b>) The number of EEs increase during long recordings in 0Mg. Data shown are only from slices that demonstrated any EEs during recordings (n = 27 from a total of 46 slices). (<b>Biv</b>) In contrast to BIC data, there is not a significant decrease in the proportion of unilateral events during long recordings. Data shown are only from slices that demonstrated at least one bilateral EE (n = 13 slices).</p