Kevin Joseph

Neocortical GABA release at high intracellular sodium and low extracellular calcium: an anti-seizure mechanism

In epilepsy, the GABA and glutamate balance may be disrupted and a transient decrease in extracellular calcium occurs before and during a seizure. Flow Cytometry based fluorescence activated particle sorting experiments quantified synaptosomes from human neocortical tissue, from both epileptic and non‐epileptic patients (27.7% vs. 36.9% GABAergic synaptosomes, respectively). Transporter‐mediated release of GABA in human and rat neocortical synaptosomes was measured using the superfusion technique for the measurement of endogenous GABA. GABA release was evoked by either a sodium channel activator or a sodium/potassium‐ATPase inhibitor when exocytosis was possible or prevented, and when the sodium/calcium exchanger was active or inhibited. The transporter‐mediated release of GABA is because of elevated intracellular sodium. A reduction in the extracellular calcium increased this release (in both non‐epileptic and epileptic, except Rasmussen encephalitis, synaptosomes). The inverse was seen during calcium doubling. In humans, GABA release was not affected by exocytosis inhibition, that is, it was solely transporter‐mediated. However, in rat synaptosomes, an increase in GABA release at zero calcium was only exhibited when the exocytosis was prevented. The absence of calcium amplified the sodium/calcium exchanger activity, leading to elevated intracellular sodium, which, together with the stimulation‐evoked intracellular sodium increment, enhanced GABA transporter reversal. Sodium/calcium exchange inhibitors diminished GABA release. Thus, an important seizure‐induced extracellular calcium reduction might trigger a transporter‐ and sodium/calcium exchanger‐related anti‐seizure mechanism by augmenting transporter‐mediated GABA release, a mechanism absent in rats. Uniquely, the additional increase in GABA release because of calcium‐withdrawal dwindled during the course of illness in Rasmussen encephalitis.

Microfluidic drive for flexible brain implants

Abstract Flexible polyimide probes, used for neuronal signal acquisition, are thought to reduce signal deteriorating gliosis, improving the quality of recordings in brain machine interfacing applications. These probes suffer from the disadvantage that they cannot penetrate brain tissue on their own, owing to their limited stiffness and low buckling forces. A microfluidic device as an external micro-drive which aids in the insertion of flexible polyimide neural probes in 0.6% Agarose gel is presented here.

Modulation of Extracellular Levels of 5-HT in the Caudate Putamen of Freely Moving Rats by High Frequency Stimulation of the Subthalamic Nucleus

Electrical high frequency stimulation (HFS) in the subthalamic nucleus (STN) has been shown to have a thera- peutic effect in several movement disorders. But, debilitating psychiatric effects like depression and suicidality are occa- sionally seen and might be caused by the changes in the serotoninergic activity. Previous studies could show that HFS of the STN results in inhibition of the serotonergic neurons originating in the dorsal raphe nucleus. The aim of this study was to characterize the effect of HFS (124 Hz, 0.5 mA) in the STN, on the extracellular levels of serotonin, dopamine and their metabolites HIAA, DOPAC and HVA in the caudate-putamen (CPu) in conscious and freely moving rats. Extracellular levels of the neurotransmitters and their metabolites were quantified using high performance liquid chromatography with electrochemical detection. Under HFS conditions, a significant reduction in the extracellular levels of serotonin was ob- served. Cessation of HFS showed a recovery back to basal levels. Dopamine levels were not affected, although significant increase of its metabolites DOPAC and HVA were measured. In the case of low frequency stimulation (LFS), levels of se- rotonin and its metabolite HIAA remained unchanged, while the levels of dopamine metabolites, DOPAC and HVA, showed a significant decline. These results demonstrate evidence for a strong linkage between HFS in the STN and reduc- tion of the levels of serotonin in the caudate-putamen, which is likely responsible for psychiatric side effects seen in Park- insonian patients who are treated with STN stimulation.

When the Ostrich-Algorithm Fails: Blanking Method Affects Spike Train Statistics

Modern electroceuticals are bound to employ the usage of electrical high frequency (130–180 Hz) stimulation carried out under closed loop control, most prominent in the case of movement disorders. However, particular challenges are faced when electrical recordings of neuronal tissue are carried out during high frequency electrical stimulation, both in-vivo and in-vitro. This stimulation produces undesired artifacts and can render the recorded signal only partially useful. The extent of these artifacts is often reduced by temporarily grounding the recording input during stimulation pulses. In the following study, we quantify the effects of this method, “blanking,” on the spike count and spike train statistics. Starting from a theoretical standpoint, we calculate a loss in the absolute number of action potentials, depending on: width of the blanking window, frequency of stimulation, and intrinsic neuronal activity. These calculations were then corroborated by actual high signal to noise ratio (SNR) single cell recordings. We state that, for clinically relevant frequencies of 130 Hz (used for movement disorders) and realistic blanking windows of 2 ms, up to 27% of actual existing spikes are lost. We strongly advice cautioned use of the blanking method when spike rate quantification is attempted. Impact statement Blanking (artifact removal by temporarily grounding input), depending on recording parameters, can lead to significant spike loss. Very careful use of blanking circuits is advised.

When a rose is not a rose: Pyramidal neurons respond differently in healthy and epileptic human neocortex

Epilepsy affects a huge number of patients by severe disruption of brain functions and is characterised by recurrent seizures, sometimes hard to be treated by medications. Seizure induced cellular consequences in ionic gradient and homeostasis are expected to result in electrophysiological differences between epileptic and non-epileptic neurons. In the following work we demonstrate these differences in layer III cortical pyramidal neurons sourced from epileptic and non-epileptic human patient tissue. Although visually indistinguishable and featuring similar membrane potentials and latency to first spikes upon whole cell patch stimulation, epileptic pyramidal neurons display a larger rheobase and a smaller membrane resistance, responding less efficiently to electrical stimulation than their peri-tumorous equivalents. This decreased excitability contradicts results in comparable animal models of epilepsy and was further corroborated by detailed analysis of spiking characteristics and phase plot analysis of these events. Both point to an overexpression of K+ channels trying to compensate for the hyperexcited, epileptic network state. A computational model of a pyramidal neuron was utilized to give an estimate of the needful relative changes in K+ and Na+ conductances.

Setup of a white light selective plane microscope to investigate microprobe insertion in a brain model

Little can be seen during the actual, dynamic implantation of microprobes into the bulk of brain tissue, mainly due to the high absorption and scattering properties of the neuropil. Fluorescent selective plane microscopy has revolutionized biology by producing optical 3D stacks of whole tissue, without slicing. The following describes a simple adaptation of white-light selective plane microscopy to visually monitor the insertion of tungsten rods with different velocities into micro-bead charged agarose gels, a good model for brain mechanics. We report on a surprising, speed dependent penetration mechanism resembling bow wave accumulation of gel.

Deep brain stimulation: increasing efficiency by alternative waveforms

Abstract Deep brain stimulation (DBS) is based on the effect of high frequency stimulation (HFS) in neuronal tissue. As a therapy option for patients suffering from e.g. Parkinson’s disease, DBS has been used for decades. Despite the widespread use, the effect of HFS on neurons is not fully investigated. Improving the stimulation efficiency und specificity could increase the efficiency of the INS (internal neuronal stimulator) as well as potentially reduce unwanted side effects. The effect of HFS on the GABAergic system was quantified using whole cell patch clamp electrophysiology during HFS stimulation in cortical human brain slices in vitro. Rectangular, sine, sawtooth and triangular waveforms were applied extracellularly. Since HFS has been hypothesized to increase the activity of the axons of GABAergic interneurons, a decrease in activity can be observed in the pyramidal cells that the interneurons project to. By isolating the incoming non- GABAergic events, we can filter out only the GABAA currents which can be verified using a GABAA antagonist. The results show that all the waveforms effectively increase the GABAA currents. The triangle waveform causes the highest significant increase in the activity which further increases over time after the stimulation was turned off.

Transmitter self-regulation by extracellular glutamate in fresh human cortical slices.

Glutamate is thought to be the most important excitatory neurotransmitter in the CNS, while glutamine predominantly serves as a precursor and metabolite in the glutamate–glutamine cycle. To verify the interaction between intrinsic extracellular glutamate, y-aminobutyric acid (GABA) levels and glial glutamine outflow in human tissue, fresh brain slices from human frontal cortex were incubated in superfusion chambers in vitro. Human neocortical tissue was obtained during surgical treatment of subcortical brain tumors. For superfusion experiments, the white matter was separated and discarded from the gray matter, which finally contained all six neocortical layers. Outflows of endogenous glutamate, GABA and glutamine were established after a 40-min washout period and amounts were simultaneously quantified after two-phase derivatization by high-performance liquid chromatography with electrochemical detection. Under basal conditions, amounts of glutamate could be found 20-fold in comparison to the inhibitory neurotransmitter GABA, whereas this excitatory predominance markedly declined after veratridine-induced activation. The basal glutamate:glutamine ratio of extracellular levels was approximately 1:2. Blockade or activation of the voltage-gated sodium channel by tetrodotoxin or veratridine significantly modulated glutamate levels, but the glutamate:glutamine ratio was nearly constant with 1:2. When the EAAT blocker TBOA was employed, glutamine remained nearly unchanged whereas glutamate significantly enhanced. These results led us to suggest that glutamine release through glial SN1 is related to EAAT activity that can be modulated by intrinsic extracellular glutamate in human cortical slices.