Enhancing GABAergic Tonic Inhibition Reduces Seizure-Like Activity in the Neonatal Mouse Hippocampus and Neocortex.

Approximately one-third of neonatal seizures do not respond to first-line anticonvulsants, including phenobarbital, which enhances phasic inhibition. Whether enhancing tonic inhibition decreases seizure-like activity in the neonate when GABA is mainly depolarizing at this age is unknown. We evaluated if increasing tonic inhibition using THIP [4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol, gaboxadol], a δ-subunit-selective GABAA receptor agonist, decreases seizure-like activity in neonatal C57BL/6J mice (postnatal day P5-8, both sexes) using acute brain slices. Whole-cell patch-clamp recordings showed that THIP enhanced GABAergic tonic inhibitory conductances in layer V neocortical and CA1 pyramidal neurons and increased their rheobase without altering sEPSC characteristics. Two-photon calcium imaging demonstrated that enhancing the activity of extrasynaptic GABAARs decreased neuronal firing in both brain regions. In the 4-aminopyridine and the low-Mg2+ model of pharmacoresistant seizures, THIP reduced epileptiform activity in the neocortex and CA1 hippocampal region of neonatal and adult brain slices in a dose-dependent manner. We conclude that neocortical layer V and CA1 pyramidal neurons have tonic inhibitory conductances, and when enhanced, they reduce neuronal firing and decrease seizure-like activity. Therefore, augmenting tonic inhibition could be a viable approach for treating neonatal seizures.

Brief and diverse excitotoxic insults cause an increase in neuronal nuclear membrane permeability in the neonatal brain.

Neuronal swelling after excitotoxic insults is implicated in neuronal injury and death in the developing brain, yet mitigating brain edema with osmotic and surgical interventions yields poor clinical outcomes. Importantly, neuronal swelling and its downstream consequences during early brain development remain poorly investigated. Using multiphoton Ca2+ imaging in vivo (P12-17) and in acute brain slices (P8-12), we explored Ca2+-dependent downstream effects after neuronal cytotoxic edema. We observed the translocation of cytosolic GCaMP6s into the nucleus of a subpopulation of neurons minutes after various excitotoxic insults. We used automated morphology-detection algorithms for neuronal segmentation and quantified the nuclear translocation of GCaMP6s as the ratio of nuclear and cytosolic intensity (N/C ratio). Elevated neuronal N/C ratios were correlated to higher Ca2+ loads and could occur independently of neuronal swelling. Electron microscopy revealed that the nuclear translocation was associated with increased nuclear pore size. Inhibiting calpains prevented elevated N/C ratios and neuronal swelling. Thus, our results indicate altered nuclear transport in a subpopulation of neurons shortly after injury in the developing brain, which can be used as an early biomarker of acute neuronal injury.

Mannitol decreases neocortical epileptiform activity during early brain development via cotransport of chloride and water.

Seizures and brain injury lead to water and Cl- accumulation in neurons. The increase in intraneuronal Cl- concentration ([Cl-]i) depolarizes the GABAA reversal potential (EGABA) and worsens seizure activity. Neocortical neuronal membranes have a low water permeability due to the lack of aquaporins necessary to move free water. Instead, neurons use cotransport of ions including Cl- to move water. Thus, increasing the extracellular osmolarity during seizures should result in an outward movement of water and salt, reducing [Cl-]i and improving GABAA receptor-mediated inhibition. We tested the effects of hyperosmotic therapy with a clinically relevant dose of mannitol (20 mM) on epileptiform activity, spontaneous multiunit activity, spontaneous inhibitory post-synaptic currents (sIPSCs), [Cl-]i, and neuronal volume in layer IV/V of the developing neocortex of C57BL/6 and Clomeleon mice. Using electrophysiological techniques and multiphoton imaging in acute brain slices (post-natal day 7-12) and organotypic neocortical slice cultures (post-natal day 14), we observed that mannitol: 1) decreased epileptiform activity, 2) decreased neuronal volume and [Cl-]i through CCCs, 3) decreased spontaneous multi-unit activity frequency but not amplitude, and 4) restored the anticonvulsant efficacy of the GABAA receptor modulator diazepam. Increasing extracellular osmolarity by 20 mOsm with hypertonic saline did not decrease epileptiform activity. We conclude that an increase in extracellular osmolarity by mannitol mediates the efflux of [Cl-]i and water through CCCs, which results in a decrease in epileptiform activity and enhances benzodiazepine actions in the developing neocortex in vitro. Novel treatments aimed to decrease neuronal volume may concomitantly decrease [Cl-]i and improve seizure control.

Response to comments on "Local impermeant anions establish the neuronal chloride concentration".

We appreciate the interest in our paper and the opportunity to clarify theoretical and technical aspects describing the influence of Donnan equilibria on neuronal chloride ion (Cl(-)) distributions.

Local impermeant anions establish the neuronal chloride concentration.

Neuronal intracellular chloride concentration [Cl(-)](i) is an important determinant of γ-aminobutyric acid type A (GABA(A)) receptor (GABA(A)R)-mediated inhibition and cytoplasmic volume regulation. Equilibrative cation-chloride cotransporters (CCCs) move Cl(-) across the membrane, but accumulating evidence suggests factors other than the bulk concentrations of transported ions determine [Cl(-)](i). Measurement of [Cl(-)](i) in murine brain slice preparations expressing the transgenic fluorophore Clomeleon demonstrated that cytoplasmic impermeant anions ([A](i)) and polyanionic extracellular matrix glycoproteins ([A](o)) constrain the local [Cl(-)]. CCC inhibition had modest effects on [Cl(-)](i) and neuronal volume, but substantial changes were produced by alterations of the balance between [A](i) and [A](o). Therefore, CCCs are important elements of Cl(-) homeostasis, but local impermeant anions determine the homeostatic set point for [Cl(-)], and hence, neuronal volume and the polarity of local GABA(A)R signaling.

Characterization of tryptophan high affinity transport system in pinealocytes of the rat. Day-night modulation.

Tryptophan is required in the pineal gland for the formation of serotonin, precursor of melatonin biosynthesis. The level of this amino acid in the serum and in the pineal gland of the rat undergoes a circadian rhythm, and reduced plasma tryptophan concentration decreases secretion of melatonin in humans. Tryptophan is transported into the cells by the long chain neutral amine acid system T and by the aromatic amino acid system T. The high affinity component of [(3)H]tryptophan uptake was studied in pinealocytes of the rat. Inhibition was observed in the presence of phenylalanine or tyrosine, but not in the presence of neutral amino acids, alanine, glycine, serine, lysine or by 2-aminobicyclo[2,2,1]-heptane-2-carboxylic acid, a substrate specific for system L. The transport of tryptophan was temperature-dependent and trans-stimulated by phenylalanine and tyrosine, but was energy-, sodium-, chloride-, and pH-independent. In addition, the sulphydryl agent N-ethylmaleimide did not modify the high affinity transport of tryptophan in pinealocytes. The kinetic parameters were not significantly different at 12:00 as compared to 24:00 h. The treatment with the inhibitor of tryptophan hydroxylase, p-chlorophenylalanine, produced an increase in the maximal velocity of the uptake and a reduction in the affinity at 12:00, but not at 24:00 h, probably indicating that during the day, the formation of serotonin in the pineal gland is favoured by elevating the uptake of tryptophan, whereas at 24:00 h other mechanisms, such as induction of enzymes are taking place. High affinity tryptophan uptake in the rat pineal gland occurs through system T and is upregulated during the day when the availability of serotonin is reduced.

Inflammation induced increase of fluoride resistant acid phosphatase (FRAP) activity in the spinal dorsal horn in rats.

Fluoride-resistant acid phosphatase (FRAP) has been suggested as an enzymatic marker for nociceptive primary afferent terminals in the spinal dorsal horn, however there has not been demonstrated a direct functional relation between FRAP activity and an increased nociceptive transmission. For this purpose, we quantitated FRAP activity in the spinal dorsal horn of the rat in a heat-induced cutaneous inflammatory model. Male Sprague-Dawley rats anaesthetised with thiopental were separated in two groups where the left hindpaw was submerged during 60 s either in water at room temperature (control group) or in water at 60 degrees C (inflammation group) which induce in this group a progressive hindpaw inflammation. After 8 h, the lumbar enlargement of the spinal cord was extracted, cut in slices and 1 mm micropunch fragments were obtained from the right and left dorsal horn. The activity of FRAP was determined using the Gomori colorimetric method and corrected by the protein concentrations. FRAP activity in the left dorsal horn was statistically higher than right dorsal horn in the inflammation group (3.05+/-0.54 versus 1.91+/-0.23 u/g per l; P<0.05). Also, FRAP activity from the left dorsal horn of the control and inflammation groups show a significant increase in the last group (3.05+/-0.54 versus 2.17+/-0.23 u/g per l; P<0.05). This results demonstrate that FRAP is not only an enzymatic marker for neuronal and fibre integrity of nociceptive primary afferents but also it is associated to the nociceptive afferent activation.