Spiking Signatures of Spontaneous Activity Bursts in Hippocampal Cultures

Dense dissociated hippocampal cultures are known to generate spontaneous bursting electrical activity which can be recorded by multielectrode arrays. We have analyzed spatio-temporal profiles of the distribution of spikes in the bursts recorded after 2 weeks in vitro. We have found a statistically significant similarity between the spiking patterns in sequential bursting events, we refer to these spiking patterns as spiking signatures. Such spiking signatures may appear in different parts of the bursts, including the activation patterns – the first spike times in the bursts, and deactivation patterns – the last spike times in the bursts. Moreover, these patterns may display apparent time scaling, e.g., they may be replayed in the subsequent bursts at different speeds, while preserving the spiking order. We discuss how such properties of the bursts may be associated with the formation of repeatable signaling pathways in cultured networks in vitro

GABAergic Inhibition of Brainstem Neurons Involved in Breathing Regulation is Disrupted in Rett Syndrome

GABA is the prominent inhibitory neurotransmitter in the brain and defects in the GABA system are attributable to several genetic diseases including Rett Syndrome (RTT). People with RTT show characteristic breathing disorders, suggesting a link with defects in the GABA system. Noradrenergic neurons in the locus coeruleus (LC) are CO2 chemosensitive and play a role in breathing regulation. We conducted experiments to find out if activation of a novel group of dorsomedial LC GABAergic neurons causes instantaneous firing rate changes in LC neurons. Spike train recordings with cross-correlation analysis suggests that the dmLC neurons made monosynaptic and oligosynaptic connections with the LC neurons. The GABAergic inhibition appeared to have major effects on medullary respiratory neurons, as our results showed that THIP, the extrasynaptic GABA receptor agonist, suppressed excitations in these cells. The results from these experiments provide new evidence for the GABAergic inhibition of brainstem neurons involved in breathing control

Application of the AAV-Syn-BDNF-EGFP virus vector as a neuroprotective agent in modeling hypoxia in vitro

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Simple model of complex dynamics of activity patterns in developing networks of neuronal cultures

Living neuronal networks in dissociated neuronal cultures are widely known for their ability to generate highly robust spatiotemporal activity patterns in various experimental conditions. These include neuronal avalanches satisfying the power scaling law and thereby exemplifying self-organized criticality in living systems. A crucial question is how these patterns can be explained and modeled in a way that is biologically meaningful, mathematically tractable and yet broad enough to account for neuronal heterogeneity and complexity. Here we propose a simple model which may offer an answer to this question. Our derivations are based on just few phenomenological observations concerning input-output behavior of an isolated neuron. A distinctive feature of the model is that at the simplest level of description it comprises of only two variables, a network activity variable and an exogenous variable corresponding to energy needed to sustain the activity and modulate the efficacy of signal transmission. Strikingly, this simple model is already capable of explaining emergence of network spikes and bursts in developing neuronal cultures. The model behavior and predictions are supported by empirical observations and published experimental evidence on cultured neurons behavior exposed to oxygen and energy deprivation. At the larger, network scale, introduction of the energy-dependent regulatory mechanism enables the network to balance on the edge of the network percolation transition. Network activity in this state shows population bursts satisfying the scaling avalanche conditions. This network state is self-sustainable and represents a balance between global network-wide processes and spontaneous activity of individual elements

Physiological role of PRRT2 and its involvement in the pathogenesis of paroxysmal disorders

Mutations in the PRoline-Rich Transmembrane protein 2 gene (PRRT2) underlie a heterogeneous group of paroxysmal disorders encompassing infantile epilepsy, paroxysmal kinesigenic dyskinesia, a combination of these phenotypes and migraine. For the majority of the pathogenic PRRT2 variants, the mutant proteins are not expressed or not correctly targeted to the plasma membrane, resulting in a loss-of function mechanism for PRRT2-related diseases. PRRT2 is a neuron-specific, type II transmembrane protein of 340 amino acids with an important functional role in synapse formation and maintenance, as well as in the regulation of fast neurotransmitter release at both glutamatergic and GABAergic terminals. The PRRT2 knock-out (PRRT2-KO) mouse, in which PRRT2 has been constitutively inactivated, displays alterations in brain structure and a sharp paroxysmal phenotype, reminiscent of the most common clinical manifestations of the human PRRT2-linked diseases. To gain further insights on the pathogenic role of PRRT2 deficiency, I used Multi-Electrode Arrays (MEAs) to characterize neuronal activity generated by primary hippocampal cultures obtained from the PRRT2-KO mouse embryos and to assess the epileptic propensity of cortico-hippocampal slices obtained from the same animal model. This experimental approach revealed a state of heightened spontaneous activity, hyper-synchronization in population bursts of action potentials (APs) and enhanced responsiveness to external stimuli in mutant networks. A complex interplay between (i) a synaptic phenotype, with weakened spontaneous transmission and increased short-term facilitation, and (ii) a marked increase in intrinsic excitability of excitatory neurons as assessed by single-cell electrophysiology, upholds this network phenotype. Furthermore, our group has generated cortical neurons from induced pluripotent stem cells (iPSCs) derived from heterozygous and homozygous siblings carrying the most common C.649dupC mutation. Patch-clamp recordings in neurons from homozygous patients showed an increased Na+ current that was fully rescued by expression of exogenous wild-type PRRT2. A strikingly similar electrophysiological phenotype was observed in excitatory primary cortical neurons from the PRRT2-KO mouse, which was accompanied by an increased length of the axon initial segment (AIS). At the network level, mutant cortical neurons grown on MEAs also displayed a state of spontaneous and evoked hyper-excitability and elevated propensity to synchronize their activity in network bursting events

Effects of different forms of docosahexaenoic acid supplementations on human neuronal cells

My study showed that Docosahexaenoic Acid (DHA), a major dietary omega-3 polyunsaturated fatty acid, inhibited cell death and promoted electro-physiological activity in cultured neuronal cells. The free fatty acid form was more effective than DHA-phospholipids and DHA-nanoliposomes. This study provides insights into the beneficial effects of dietary omega-3 fatty acids

Reducing the Societal Costs of Traumatic Brain Injury: Astrocyte-Based Therapeutics and Functional Injury Tolerance of the Living Brain

Approximately 1.7 million traumatic brain injuries (TBI) occur annually in the United States, with an annual estimated societal cost of at least $76.5 billion. Addressing the growing TBI epidemic will require a multi pronged approach: developing novel treatment strategies and enhancing existing preventative measures. The specific aims of this thesis are: (1) to modulate astrocyte activation as a potential therapeutic strategy post TBI, (2) to determine the relationship between tissue deformation and alterations in electrophysiological function in the living brain, and (3) to investigate underlying mechanisms of functional changes post TBI by utilizing stretchable microelectrode arrays (SMEAs). In response to disease or injury, astrocytes become activated in a process called reactive astrogliosis. Activated astrocytes generate harmful radicals that exacerbate brain damage and can hinder regeneration of damaged neural circuits by secreting neuro developmental inhibitors and glycosaminoglycans (GAGs). Since mechanically-activated astrocytes upregulate GAG production, delivery of GFP-TAT, a mock therapeutic protein conjugated to the cell-penetrating peptide TAT, increased significantly after activation. A TAT-conjugated peptide JNK inhibitor was delivered to activated astrocytes and significantly reduced activation. These results suggest a potentially new, targeted therapeutic utilizing TAT for preventing astrocyte activation with the possibility of limiting off-target, negative side effects. While modulating astrocyte activation is a promising treatment strategy for TBI, effective therapeutic treatments are still lacking. Preventing TBI, by developing more effective safety systems, remains crucial. We determined functional tolerance criteria for the hippocampus and cortex based on alterations in electrophysiological function in response to controlled mechanical stimuli. Organotypic hippocampal and cortical slice cultures were mechanically injured at tissue strains and strain rates relevant to TBI, and changes in electrophysiological function were quantified. Most changes in electrophysiological function were dependent on strain and strain rate in a complex, nonlinear manner. Our results provide functional data that can be incorporated into finite element (FE) models to improve their biofidelity of accident and collision reconstructions. TBI causes alterations in macroscopic function and behavior, which can be characterized by alterations in electrophysiological function in vitro. We utilized a novel in vitro platform for TBI research, the SMEA, to investigate the effects of TBI on pharmacologically induced, long lasting network synchronization in the hippocampus. Mechanical stimulation of organotypic hippocampal slice cultures significantly disrupted this network synchronization 24 hours after injury. Our results suggest that the ability of the hippocampal neuronal network to develop and sustain network synchronization was disrupted after mechanical injury, while also demonstrating the utility of the SMEA for TBI research. Herein, we identified a novel therapeutic strategy for treating the deleterious effects of astrocyte activation post-TBI. We also developed tolerance criteria relating mechanical injury parameters to electrophysiological function, an important step in developing more accurate computational simulations of TBI. Equipping FE models with new information on the functional response of the living brain will enhance their biofidelity, potentially leading to improved safety systems while reducing development costs. Finally, we utilized a novel in vitro TBI research platform, the SMEA, to investigate the effects of TBI on long-lasting network synchronization in the hippocampus. Compared to more labor intensive in vivo approaches, the ability of the SMEA to efficiently test TBI hypotheses within a single organotypic slice culture over extended durations could increase the speed of drug discovery through high-content screening. This multi-pronged approach is necessary to address the growing public health concern of TBI