Katherine L. Perkins

Cell-attached voltage-clamp and current-clamp recording and stimulation techniques in brain slices

Cell-attached recording provides a way to record the activity of - and to stimulate - neurons in brain slices without rupturing the cell membrane. This review uses theory and experimental data to address the proper application of this technique and the correct interpretation of the data. Voltage-clamp mode is best-suited for recording cell firing activity, and current-clamp mode is best-suited for recording resting membrane potential and synaptic potentials. The magnitude of the seal resistance determines what types of experiments can be accomplished with a cell-attached recording: a loose seal is adequate for recording action potential currents, and a tight seal is required for evoking action potentials in the attached cell and for recording resting and synaptic potentials. When recording action potential currents, if the researcher does not want to change the firing activity of the cell, then it is important that no current passes from the amplifier through the patch resistance. In order to accomplish this condition, the recording pipette should be held at the potential that gives a holding current of 0. An advantage of cell-attached current-clamp over whole-cell recording is that it accurately depicts whether a synaptic potential is hyperpolarizing or depolarizing without the risk of changing its polarity.

Hyaluronan Deficiency Due to Has3 Knock-Out Causes Altered Neuronal Activity and Seizures via Reduction in Brain Extracellular Space

Hyaluronan (HA), a large anionic polysaccharide (glycosaminoglycan), is a major constituent of the extracellular matrix of the adult brain. To address its function, we examined the neurophysiology of knock-out mice deficient in hyaluronan synthase (Has) genes. Here we report that these Has mutant mice are prone to epileptic seizures, and that in Has3−/− mice, this phenotype is likely derived from a reduction in the size of the brain extracellular space (ECS). Among the three Has knock-out models, namely Has3−/−, Has1−/−, and Has2CKO, the seizures were most prevalent in Has3−/− mice, which also showed the greatest HA reduction in the hippocampus. Electrophysiology in Has3−/− brain slices demonstrated spontaneous epileptiform activity in CA1 pyramidal neurons, while histological analysis revealed an increase in cell packing in the CA1 stratum pyramidale. Imaging of the diffusion of a fluorescent marker revealed that the transit of molecules through the ECS of this layer was reduced. Quantitative analysis of ECS by the real-time iontophoretic method demonstrated that ECS volume was selectively reduced in the stratum pyramidale by ∼40% in Has3−/− mice. Finally, osmotic manipulation experiments in brain slices from Has3−/− and wild-type mice provided evidence for a causal link between ECS volume and epileptiform activity. Our results provide the first direct evidence for the physiological role of HA in the regulation of ECS volume, and suggest that HA-based preservation of ECS volume may offer a novel avenue for development of antiepileptogenic treatments.

Effects of subtype-selective group I mGluR antagonists on synchronous activity induced by 4-aminopyridine/CGP 55845 in adult guinea pig hippocampal slices

Co-application of the convulsant 4-aminopyridine (4-AP) and the GABA(B) receptor antagonist CGP 55845 to adult guinea pig hippocampal slices elicits giant GABA-mediated postsynaptic potentials (GPSPs) and epileptiform discharges. Here we tested the effects of the group I metabotropic glutamate receptor (mGluR) subtype-selective antagonists LY 367385 (mGlu1, 100 microM), MPEP (mGlu5, 10 microM), and MTEP (mGlu5, 500 nM) on this synchronous activity. Electrophysiological field recordings were performed in the CA3 region of hippocampal slices from adult guinea pigs. The mGlu5 receptor antagonists increased GPSP rate, but the mGlu1 receptor antagonist did not. This ability of mGlu5 receptor antagonists to increase the rate of GPSPs indicates that enough endogenous glutamate is released under these conditions to activate group I mGluR; nevertheless, co-application of a mGlu1 receptor antagonist (LY 367385 or JNJ 16259685) and MPEP did not decrease pre-existing epileptiform activity. Furthermore, co-application of LY 367,385 and MPEP did not prevent the emergence of epileptiform activity. When ionotropic glutamate receptor (iGluR) antagonists were present, neither MPEP nor the group I mGluR agonist DHPG changed GPSP rate, suggesting that pyramidal cell-to-interneuron iGluR-mediated synaptic connections are involved in the rate change mechanism. In contrast to the lack of effect of group I mGluR antagonists on epileptiform activity in the 4-AP/CGP 55845 model, group I mGluR antagonists blocked the emergence of longer epileptiform events and decreased the overall amount of synchronous activity in the GABA(A) antagonist/4-AP model. In conclusion, in the 4-AP/CGP 55845 model, enough glutamate was released to activate group I mGluRs and affect GPSP rate via mGlu5 receptors; however, this group I mGluR activation was not required for the generation of the epileptiform activity.

Brain extracellular space, hyaluronan, and the prevention of epileptic seizures

Abstract Mutant mice deficient in hyaluronan (HA) have an epileptic phenotype. HA is one of the major constituents of the brain extracellular matrix. HA has a remarkable hydration capacity, and a lack of HA causes reduced extracellular space (ECS) volume in the brain. Reducing ECS volume can initiate or exacerbate epileptiform activity in many in vitro models of epilepsy. There is both in vitro and in vivo evidence of a positive feedback loop between reduced ECS volume and synchronous neuronal activity. Reduced ECS volume promotes epileptiform activity primarily via enhanced ephaptic interactions and increased extracellular potassium concentration; however, the epileptiform activity in many models, including the brain slices from HA synthase-3 knockout mice, may still require glutamate-mediated synaptic activity. In brain slice epilepsy models, hyperosmotic solution can effectively shrink cells and thus increase ECS volume and block epileptiform activity. However, in vivo, the intravenous administration of hyperosmotic solution shrinks both brain cells and brain ECS volume. Instead, manipulations that increase the synthesis of high-molecular-weight HA or decrease its breakdown may be used in the future to increase brain ECS volume and prevent seizures in patients with epilepsy. The prevention of epileptogenesis is also a future target of HA manipulation. Head trauma, ischemic stroke, and other brain insults that initiate epileptogenesis are known to be associated with an early decrease in high-molecular-weight HA, and preventing that decrease in HA may prevent the epileptogenesis.