Person:

Rotenberg, Alexander

Neonatal seizures can be refractory to conventional anticonvulsants, and this may in part be due to a developmental increase in expression of the neuronal Na+-K+-2 Cl− cotransporter, NKCC1, and consequent paradoxical excitatory actions of GABAA receptors in the perinatal period. The most common cause of neonatal seizures is hypoxic encephalopathy, and here we show in an established model of neonatal hypoxia-induced seizures that the NKCC1 inhibitor, bumetanide, in combination with phenobarbital is significantly more effective than phenobarbital alone. A sensitive mass spectrometry assay revealed that bumetanide concentrations in serum and brain were dose-dependent, and the expression of NKCC1 protein transiently increased in cortex and hippocampus after hypoxic seizures. Importantly, the low doses of phenobarbital and bumetanide used in the study did not increase constitutive apoptosis, alone or in combination. Perforated patch clamp recordings from ex vivo hippocampal slices removed following seizures revealed that phenobarbital and bumetanide largely reversed seizure-induced changes in EGABA. Taken together, these data provide preclinical support for clinical trials of bumetanide in human neonates at risk for hypoxic encephalopathy and seizures.

Repetitive transcranial magnetic stimulation (rTMS) is a widely-used method for modulating cortical excitability in humans, by mechanisms thought to involve use-dependent synaptic plasticity. For example, when low frequency rTMS (LF rTMS) is applied over the motor cortex, in humans, it predictably leads to a suppression of the motor evoked potential (MEP), presumably reflecting long-term depression (LTD) – like mechanisms. Yet how closely such rTMS effects actually match LTD is unknown. We therefore sought to (1) reproduce cortico-spinal depression by LF rTMS in rats, (2) establish a reliable animal model for rTMS effects that may enable mechanistic studies, and (3) test whether LTD-like properties are evident in the rat LF rTMS setup. Lateralized MEPs were obtained from anesthetized Long-Evans rats. To test frequency-dependence of LF rTMS, rats underwent rTMS at one of three frequencies, 0.25, 0.5, or 1 Hz. We next tested the dependence of rTMS effects on N-methyl-D-aspartate glutamate receptor (NMDAR), by application of two NMDAR antagonists. We find that 1 Hz rTMS preferentially depresses unilateral MEP in rats, and that this LTD-like effect is blocked by NMDAR antagonists. These are the first electrophysiological data showing depression of cortical excitability following LF rTMS in rats, and the first to demonstrate dependence of this form of cortical plasticity on the NMDAR. We also note that our report is the first to show that the capacity for LTD-type cortical suppression by rTMS is present under barbiturate anesthesia, suggesting that future neuromodulatory rTMS applications under anesthesia may be considered.

Objective: Traumatic brain injury (TBI) is a major brain injury type commonly caused by traffic accidents, falls, violence, or sports injuries. To obtain mechanistic insights about TBI, experimental animal models such as weight-drop-induced TBI in rats have been developed to mimic closed-head injury in humans. However, the relationship between the mechanical impact level and neurological severity following weight-drop-induced TBI remains uncertain. In this study, we comprehensively investigated the relationship between physical impact and graded severity at various weight-drop heights. Approach The acceleration, impact force, and displacement during the impact were accurately measured using an accelerometer, a pressure sensor, and a high-speed camera, respectively. In addition, the longitudinal changes in neurological deficits and balance function were investigated at 1, 4, and 7 days post TBI lesion. The inflammatory expression markers tested by Western blot analysis, including glial fibrillary acidic protein, beta-amyloid precursor protein, and bone marrow tyrosine kinase gene in chromosome X, in the frontal cortex, hippocampus, and corpus callosum were investigated at 1 and 7 days post-lesion. Results: Gradations in impact pressure produced progressive degrees of injury severity in the neurological score and balance function. Western blot analysis demonstrated that all inflammatory expression markers were increased at 1 and 7 days post-impact injury when compared to the sham control rats. The severity of neurologic dysfunction and induction in inflammatory markers strongly correlated with the graded mechanical impact levels. Conclusions: We conclude that the weight-drop-induced TBI model can produce graded brain injury and induction of neurobehavioral deficits and may have translational relevance to developing therapeutic strategies for TBI.

Zebrafish epilepsy models are emerging tools in experimental epilepsy. Zebrafish larvae, in particular, are advantageous because they can be easily genetically altered and used for developmental and drug studies since agents applied to the bath penetrate the organism easily. Methods for electrophysiological recordings in zebrafish are new and evolving. We present a novel multi-electrode array method to non-invasively record electrical activity from up to 61 locations of an intact larval zebrafish head. This method enables transcranial noninvasive recording of extracellular field potentials (which include multi-unit activity and EEG) to identify epileptic seizures. To record from the brains of zebrafish larvae, the dorsum of the head of an intact larva was secured onto a multi-electrode array. We recorded from individual electrodes for at least three hours and quantified neuronal firing frequency, spike patterns (continuous or bursting), and synchrony of neuronal firing. Following 15 mM potassium chloride- or pentylenetetrazole-infusion into the bath, spike and burst rate increased significantly. Additionally, synchrony of neuronal firing across channels, a hallmark of epileptic seizures, also increased. Notably, the fish survived the experiment. This non-invasive method complements present invasive zebrafish neurophysiological techniques: it affords the advantages of high spatial and temporal resolution, a capacity to measure multiregional activity and neuronal synchrony in seizures, and fish survival for future experiments, such as studies of epileptogenesis and development.