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.

Optogenetic dissection of ictogenesis: in search of a targeted anti-epileptic therapy.

For over a century, epileptic seizures have been characterized as a state of pathological, hypersynchronous brain activity. Anti-epileptic therapies have been developed largely based on the dogma that the altered brain rhythms result from an overabundance of glutamatergic activity or insufficient GABAergic inhibition. The most effective drugs in use today act to globally decrease excitation, increase inhibition, or decrease all activity. Unfortunately, such broad alterations to brain activity often lead to impactful side effects such as drowsiness, cognitive impairment, and sleep disruption. Recent advances in optical imaging, optogenetics, and chemogenetics have made it feasible to record and alter neuronal activity with single neuron resolution and genetically directed targeting. The goal of this review it to summarize the usage of these research tools in the study of ictogenesis (seizure generation) and propose a translational pathway by which these studies could result in novel clinical therapies. This manuscript is not intended to serve as an exhaustive list of optogenetic tools nor as a summary of all optogenetic manipulations in epilepsy research. Rather, we will focus on the tools and research aimed at dissecting the basic neuron-level interactions underlying ictogenesis.

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.

Service users as collaborators in mental health research: less stick, more carrot.

Involving service users in research improves its quality and relevance. Many research organizations funding and supporting research now ask researchers about involvement as part of their application process. Some researchers are facing challenges in taking forward involvement as the research infrastructure is not always facilitative. Researchers need greater reward and recognition for carrying out good quality involvement to encourage more effective processes.

Interictal spikes precede ictal discharges in an organotypic hippocampal slice culture model of epileptogenesis.

In organotypic hippocampal slice cultures, principal neurons form aberrant excitatory connections with other principal cells in response to slicing induced deafferentation, similar to mechanisms underlying epileptogenesis in posttraumatic epilepsy. To investigate the consequences of this synaptogenesis, the authors recorded field-potential activity from area CA3 during perfusion with the complete growth medium used during incubation. At 7 days in vitro, slice cultures only displayed multiunit activity. At 14 days in vitro, the majority displayed population bursts reminiscent of interictal-like spikes, but sustained synchronous activity was rare. Band-pass filtering of interictal discharges revealed fast ripple-like complexes, similar to in vivo recordings. Spontaneous ictal-like activity became progressively more prevalent with age: at 21 days in vitro, 50% of organotypic hippocampal slice cultures displayed long-lasting, ictal-like discharges that could be suppressed by phenytoin, whereas interictal activity was not suppressed. The fraction of cultures displaying ictal events continually increased with incubation time. Quantification of population spike activity throughout epileptogenesis using automatic detection and clustering algorithms confirmed the appearance of interictal-like activity before ictal-like discharges and also revealed high-frequency pathologic multiunit activity in slice cultures at 14 to 17 days in vitro. These experiments indicate that interictal-like spikes precede the appearance of ictal-like activity in a reduced in vitro preparation. Epileptiform activity in cultures resembled in vivo epilepsy, including sensitivity to anticonvulsants and steadily increasing seizure incidence over time, although seizure frequency and rate of epileptogenesis were higher in vitro. Organotypic hippocampal slice cultures comprise a useful model system for investigating mechanisms of epileptogenesis as well as developing antiepileptic and antiepileptogenic drugs.

Statistical model relating CA3 burst probability to recovery from burst-induced depression at recurrent collateral synapses.

When neuronal excitability is increased in area CA3 of the hippocampus in vitro, the pyramidal cells generate periodic bursts of action potentials that are synchronized across the network. We have previously provided evidence that synaptic depression at the excitatory recurrent collateral synapses in the CA3 network terminates each population burst so that the next burst cannot begin until these synapses have recovered. These findings raise the possibility that burst timing can be described in terms of the probability of recovery of this population of synapses. Here we demonstrate that when neuronal excitability is changed in the CA3 network, the mean and variance of the interburst interval change in a manner that is consistent with a timing mechanism comprised of a pool of exponentially relaxing pacemakers. The relaxation time constant of these pacemakers is the same as the time constant describing the recovery from activity-dependent depression of recurrent collateral synapses. Recovery was estimated from the rate of spontaneous transmitter release versus time elapsed since the last CA3 burst. Pharmacological and long-term alterations of synaptic strength and network excitability affected CA3 burst timing as predicted by the cumulative binomial distribution if the burst pace-maker consists of a pool of recovering recurrent synapses. These findings indicate that the recovery of a pool of synapses from burst-induced depression is a sufficient explanation for burst timing in the in vitro CA3 neuronal network. These findings also demonstrate how information regarding the nature of a pacemaker can be derived from the temporal pattern of synchronous network activity. This information could also be extracted from less accessible networks such as those generating interictal epileptiform discharges in vivo.

Pediatric injuries resulting from use of all-terrain vehicles.

Annually, 20,000 children are injured while operating all-terrain vehicles (ATVs). The purpose of this paper was to review child-ATV injuries in Arkansas and identify any areas in need of further investigation. An analysis of emergency-medical-service transports was done for children 0-19 years who had ATV-related injuries in Arkansas from 1998 to 1999. Prehospital-reported child-ATV emergencies were identified, separated by county, and emergency encounter rates were calculated. Our results indicate that emergency medical services (EMS) transported 319 children in Arkansas from 1998 to 1999. ATV injury information is limited in Arkansas, but available data indicate high injury rates existed for many rural counties.

Characterization of promoter function and cell-type-specific expression from viral vectors in the nervous system.

Viral vectors have become important tools to effectively transfer genes into terminally differentiated cells, including neurons. However, the rational for selection of the promoter for use in viral vectors remains poorly understood. Comparison of promoters has been complicated by the use of different viral backgrounds, transgenes, and target tissues. Adenoviral vectors were constructed in the same vector background to directly compare three viral promoters, the human cytomegalovirus (CMV) immediate-early promoter, the Rous sarcoma virus (RSV) long terminal repeat, and the adenoviral E1A promoter, driving expression of the Escherichia coli lacZ gene or the gene for the enhanced green fluorescent protein. The temporal patterns, levels of expression, and cytotoxicity from the vectors were analyzed. In sensory neuronal cultures, the CMV promoter produced the highest levels of expression, the RSV promoter produced lower levels, and the E1A promoter produced limited expression. There was no evidence of cytotoxicity produced by the viral vectors. In vivo analyses following stereotaxic injection of the vector into the rat hippocampus demonstrated differences in the cell-type-specific expression from the CMV promoter versus the RSV promoter. In acutely prepared hippocampal brain slices, marked differences in the cell type specificity of expression from the promoters were confirmed. The CMV promoter produced expression in hilar regions and pyramidal neurons, with minimal expression in the dentate gyrus. The RSV promoter produced expression in dentate gyrus neurons. These results demonstrate that the selection of the promoter is critical for the success of the viral vector to express a transgene in specific cell types.

Modulation of mammalian dendritic GABA(A) receptor function by the kinetics of Cl- and HCO3- transport.

1. During prolonged activation of dendritic GABAA receptors, the postsynaptic membrane response changes from hyperpolarization to depolarization. One explanation for the change in direction of the response is that opposing HCO3- and Cl- fluxes through the GABAA ionophore diminish the electrochemical gradient driving the hyperpolarizing Cl- flux, so that the depolarizing HCO3- flux dominates. Here we demonstrate that the necessary conditions for this mechanism are present in rat hippocampal CA1 pyramidal cell dendrites. 2. Prolonged GABAA receptor activation in low-HCO3- media decreased the driving force for dendritic but not somatic Cl- currents. Prolonged GABAA receptor activation in low-Cl- media containing physiological HCO3- concentrations did not degrade the driving force for dendritic or somatic HCO3- gradients. 3. Dendritic Cl- transport was measured in three ways: from the rate of recovery of GABAA receptor-mediated currents between paired dendritic GABA applications, from the rate of recovery between paired synaptic GABAA receptor-mediated currents, and from the predicted vs. actual increase in synaptic GABAA receptor-mediated currents at progressively more positive test potentials. These experiments yielded estimates of the maximum transport rate (vmax) for Cl- transport of 5 to 7 mmol l-1 s-1, and indicated that vmax could be exceeded by GABAA receptor-mediated Cl- influx. 4. The affinity of the Cl- transporter was calculated in experiments in which the reversal potential for Cl- (ECl) was measured from the GABAA reversal potential in low-HCO3- media during Cl- loading from the recording electrode solution. The calculated KD was 15 mM. 5. Using a standard model of membrane potential, these conditions are demonstrated to be sufficient to produce the experimentally observed, activity-dependent GABA(A) depolarizing response in pyramidal cell dendrites.

Reciprocal interactions between CA3 network activity and strength of recurrent collateral synapses.

In hippocampal slices, synchronous CA3 network activity induced persistent strengthening of active positive-feedback synapses. This altered network operation by increasing probability of future synchronous network activation. Long-term depression of synaptic strength induced by partial blockade of NMDA receptors during synchronous network activity reversed changes in probability of spontaneous network activation. These results suggest that specific network activity patterns selectively alter strength of active synapses. Stable, reversible alterations in network activity can also be effected by corresponding alterations in synaptic strength. These findings confirm the Hebb memory model at the neural-network level and suggest new therapies for pathological patterns of network activity in epilepsy.

Developmental expression of C1C-2 in the rat nervous system.

Regulation of expression of the voltage-gated chloride channel, C1C-2, was investigated during development and adult life in rat brain. RNase protection assays demonstrated a marked increase in levels of expression of C1C-2 in brain during early postnatal development which was also detected in adult brain. In situ hybridization of E15 and E18 rat brains demonstrated C1C-2 expression in deep brain nuclei and scattered cells within the neuroepithelial layers, but not in the regions of subventricular zone that primarily give rise to glial populations. By E18 all neurons within the emerging cortical plate and its equivalent in other areas of the CNS were heavily labeled. During the first postnatal week, C1C-2 was highly expressed in most neurons. By P7 a pattern of differential expression emerged with evidence of decreased expression of C1C-2 mRNA in many neuronal populations. In adult rat brain, C1C-2 was expressed at highest levels in large neurons as found within layer V of cortex, Ammon's Horn of hippocampus, or mitral cells of the olfactory bulb and Purkinje cells within the cerebellum. Many smaller neurons within the diencephalon maintained significant levels of expression. A functional conductance was readily detected in hippocampal neurons during the first postnatal week, which had the same characteristic properties as the conductance observed in adult neurons. The observed expression and functional presence of C1C-2 suggest a widespread role in neuronal chloride homeostasis in early postnatal life, and demonstrated that cell specific shut-down resulted in the adult pattern of expression.

Presynaptic modulation of CA3 network activity.

The simultaneous discharge of hippocampal CA3 pyramidal cells is a widely studied in vitro model of physiological and pathological network synchronization. This network is rapidly activated because of extensive positive feedback mediated by recurrent axon collaterals. Here we show that population-burst duration is limited by depletion of the releasable glutamate pool at these recurrent synapses. Postsynaptic inhibitory conductances further limit burst duration but are not necessary for burst termination. The interval between bursts in vitro depends on the rate of replenishment of releasable glutamate vesicles and the probability of release of those vesicles at recurrent synapses. Therefore presynaptic factors controlling glutamate release at recurrent synapses regulate the probability and duration of synchronous discharges of the CA3 network.

Expression of LIM protein genes Lmo1, Lmo2, and Lmo3 in adult mouse hippocampus and other forebrain regions: differential regulation by seizure activity.

The LIM domain is a zinc-binding amino acid motif that characterizes various proteins which function in protein-protein interactions and transcriptional regulation. Expression patterns of several LIM protein genes are compatible with roles in vertebrate CNS development, but little is known about the expression, regulation, or function of LIM proteins in the mature CNS. Lmo1, Lmo2, and Lmo3 are LIM-only genes originally identified as putative oncogenes that have been implicated in the control of cell differentiation and are active during CNS development. Using in situ hybridization for mRNA and immunohistochemical detection of reporter protein expression in transgenic mice, we found that Lmo1, Lmo2, and Lmo3 show individually unique but partially overlapping patterns of expression in several regions of the adult mouse forebrain, including hippocampus, caudate putamen, medial habenula, thalamus, amygdala, olfactory bulb, hypothalamus, and cerebral cortex. In the hippocampal formation, Lmo1, Lmo2, and Lmo3 show different combinatorial patterns of expression levels in CA pyramidal and dentate granule neurons, and Lmo1 is present in topographically restricted subpopulations of astrocytes. Kainic acid-induced limbic seizures differentially regulated Lmo1, Lmo2, and Lmo3 mRNA levels in hippocampal pyramidal and granule neurons, such that Lmo1 mRNA increased, whereas Lmo2 and Lmo3 mRNAs decreased significantly, with maximal changes at 6 hr after seizure onset and return to baseline by 24 hr. These findings show that Lmo1, Lmo2, and Lmo3 continue to be expressed in the adult mammalian CNS in a cell type-specific manner, are differentially regulated by neuronal activity, and may thus be involved in cell phenotype-specific regulatory functions.

Apoptotic DNA fragmentation is detected by a semi-quantitative ligation-mediated PCR of blunt DNA ends.

Apoptosis is a form of programmed cell death (PCD) characterized by morphological changes and stereotypical DNA degradation described as a nucleosomal ;ladder'. However, nucleosomal ladders have only been clearly demonstrated in vertebrate tissues when large numbers of cells die in synchrony. Their absence may be explained by asynchronous death under physiological conditions, or by distinct molecular mechanisms. In this study, nucleosomal ladders were revealed by a ligation-mediated polymerase chain reaction (LMPCR), that amplifies DNA fragments with blunt, 5' phosphorylated ends. Numerous tissues from different organisms were examined which demonstrated that nucleosomal ladders (a) accompany physiological cell death in mammalian tissues where previously DNA fragmentation has not been detected; (b) are produced during invertebrate cell death; (c) are invariably generated via the production of blunt, 5' phosphorylated double strand breaks. These results suggest that PCD in multicellular organisms consistently involves apoptotic mechanisms and that the endonuclease activity is evolutionarily conserved.

Alteration of GABAA receptor function following gene transfer of the CLC-2 chloride channel.

The effect of GABAA receptor activation varies from inhibition to excitation depending on the state of the transmembrane anionic concentration gradient (delta anion). delta anion was genetically altered in cultured dorsal root ganglion neurons via adenoviral vector-mediated expression of ClC-2, a Cl- channel postulated to regulate the Cl- concentration in neurons in which GABAA receptor activation is predominantly inhibitory. ClC-2 expression was verified by the presence of the appropriate mRNA, protein, and membrane conductance. CIC-2 expression resulted in a large negative shift in the Cl- equilibrium potential (ECl) that attenuated the GABA-mediated membrane depolarization and prevented GABAA receptor-mediated action potentials. These results establish that gene transfer of transmembrane ion channels to neurons can be used to demonstrate their physiological function, and that delta anion can be genetically manipulated to alter the function of neuronal GABAA receptors in situ.

Widespread programmed cell death in proliferative and postmitotic regions of the fetal cerebral cortex.

A key event in the development of the mammalian cerebral cortex is the generation of neuronal populations during embryonic life. Previous studies have revealed many details of cortical neuron development including cell birthdates, migration patterns and lineage relationships. Programmed cell death is a potentially important mechanism that could alter the numbers and types of developing cortical cells during these early embryonic phases. While programmed cell death has been documented in other parts of the embryonic central nervous system, its operation has not been previously reported in the embryonic cortex because of the lack of cell death markers and the difficulty in following the entire population of cortical cells. Here, we have investigated the spatial and temporal distribution of dying cells in the embryonic cortex using an in situ endlabelling technique called 'ISEL+' that identifies fragmented nuclear DNA in dying cells with increased sensitivity. The period encompassing murine cerebral cortical neurogenesis was examined, from embryonic days 10 through 18. Dying cells were rare at embryonic day 10, but by embryonic day 14, 70% of cortical cells were found to be dying. This number declined to 50% by embryonic day 18, and few dying cells were observed in the adult cerebral cortex. Surprisingly, while dying cells were observed throughout the cerebral cortical wall, the majority were found within zones of cell proliferation rather than in regions of postmitotic neurons. These observations suggest that multiple mechanisms may regulate programmed cell death in the developing cortex. Moreover, embryonic cell death could be an important factor enabling the selection of appropriate cortical cells before they complete their differentiation in postnatal life.

Ionic mechanisms of neuronal excitation by inhibitory GABAA receptors.

Gamma-aminobutyric acid A (GABAA) receptors are the principal mediators of synaptic inhibition, and yet when intensely activated, dendritic GABAA receptors excite rather than inhibit neurons. The membrane depolarization mediated by GABAA receptors is a result of the differential, activity-dependent collapse of the opposing concentration gradients of chloride and bicarbonate, the anions that permeate the GABAA ionophore. Because this depolarization diminishes the voltage-dependent block of the N-methyl-D-aspartate (NMDA) receptor by magnesium, the activity-dependent depolarization mediated by GABA is sufficient to account for frequency modulation of synaptic NMDA receptor activation. Anionic gradient shifts may represent a mechanism whereby the rate and coherence of synaptic activity determine whether dendritic GABAA receptor activation is excitatory or inhibitory.