In central neurons, a tonic conductance is activated by ambient levels of the inhibitory transmitter GABA. Here, we show that in dentate gyrus granule cells, where tonic inhibition is mediated by ␦ subunit-containing GABA A receptors, this conductance is augmented by low concentrations (30 mM) of ethanol. In contrast, the tonic inhibition mediated by ␣5 subunit-containing receptors of CA1 pyramidal cells is not affected. The effect of ethanol on tonic inhibition specifically reduces the excitability of the dentate gyrus and identifies the ␦ subunit-dependent tonic inhibition as a likely site of ethanol action in the brain.

The development of genetically encoded fluorescent voltage probes is essential to image electrical activity from neuronal populations. Previous green fluorescent protein (GFP)-based probes have had limited success in recording electrical activity of neurons because of their low sensitivity and poor temporal resolution. Here we describe a hybrid approach that combines a genetically encoded fluorescent probe (membrane-anchored enhanced GFP) with dipicrylamine, a synthetic voltage-sensing molecule that partitions into the plasma membrane. The movement of the synthetic voltage sensor is translated via fluorescence resonance energy transfer (FRET) into a large fluorescence signal (up to 34% change per 100 mV) with a fast response and recovery time (0.5 ms). Using this two-component approach, we were able to optically record action potentials from neuronal cell lines and trains of action potentials from primary cultured neurons. This hybrid approach may form the basis for a new generation of protein-based voltage probes.

Faria LC, Prince DA. Presynaptic inhibitory terminals are functionally abnormal in a rat model of posttraumatic epilepsy. J Neurophysiol 104: 280-290, 2010. First published May 19, 2010 doi:10.1152/jn.00351.2010. Partially isolated "undercut" neocortex with intact pial circulation is a well-established model of posttraumatic epileptogenesis. Results of previous experiments showed a decreased frequency of miniature inhibitory postsynaptic currents (mIPSCs) in layer V pyramidal (Pyr) neurons of undercuts. We further examined possible functional abnormalities in GABAergic inhibition in rat epileptogenic neocortical slices in vitro by recording whole cell monosynaptic IPSCs in layer V Pyr cells and fast-spiking (FS) GABAergic interneurons using a paired pulse paradigm. Compared with controls, IPSCs in Pyr neurons of injured slices showed increased threshold and decreased peak amplitude at threshold, decreased input/output slopes, increased failure rates, and a shift from paired pulse depression toward paired pulse facilitation (increased paired pulse ratio or PPR). Increasing [Ca 2ϩ ] o from 2 to 4 mM partially reversed these abnormalities in Pyr cells of the epileptogenic tissue. IPSCs onto FS cells also had an increased PPR and failures. Blockade of GABA B receptors did not affect the paired results. These findings suggest that there are functional alterations in GABAergic presynaptic terminals onto both Pyr and FS cells in this model of posttraumatic epileptogenesis. I N T R O D U C T I O NPartially isolated "undercut" (UC) or "injured" neocortex is a well established in vivo model of posttraumatic epileptogenesis in cats and monkeys (Echlin and Battista 1963;Echlin and McDonald 1954;Grafstein and Sastry 1957;Sharpless 1969; reviewed in Halpern 1972). Spontaneous (s) and evoked (e) epileptiform activity persists in rodent neocortical slices cut through the UC neocortex and maintained in vitro (Hoffman et al. 1994;Prince and Tseng 1993;Salin et al. 1995; reviewed in Graber and Prince 2006). Alterations in glutamatergic excitatory synaptic activity/connectivity are present in this model and likely contribute to the development of hyperexcitability (Jin et al. 2006;Li and Prince 2002;Li et al. 2005;Salin et al. 1995).Results of anatomical and electrophysiological experiments in UC cortex indicate that decreases in GABAergic inhibitory activity/connectivity may also contribute to epileptogenesis. Biocytin-filled axons of fast-spiking (FS) GABAergic interneurons in the chronically epileptogenic cortex are structurally abnormal with decreased bouton size and marked reduction in axonal lengths ). Such alterations in presynaptic inhibitory terminals might be associated with functional abnormalities such as increased failure rate, decreased amplitude of inhibitory postsynaptic currents (IPSCs), and decreased probability of GABA release (Pr) that would make inhibitory transmission less effective (Harris and Sultan 1995;Pierce and Lewin 1994). There is also a decrease in the frequency of miniature inhibitory post synapt...

Decreased release probability (Pr) and increased failure rate for monosynaptic inhibitory postsynaptic currents (IPSCs) indicate abnormalities in presynaptic inhibitory terminals on pyramidal (Pyr) neurons of the undercut (UC) model of posttraumatic epileptogenesis. These indices of inhibition are normalized in high [Ca++] ACSF, suggesting dysfunction of Ca2+ channels in GABAergic terminals. We tested this hypothesis using selective blockers of P/Q and N-type Ca2+ channels whose activation underlies transmitter release in cortical inhibitory terminals. Pharmacologically isolated monosynaptic IPSCs were evoked in layer V Pyr cells by extracellular stimuli in adult rat sensorimotor cortical slices. Local perfusion of 0.2/1 μM ω-agatoxin IVa and/or 1 μM ω-conotoxin GVIA was used to block P/Q and N-type calcium channels, respectively. In control layer V Pyr cells, peak amplitude of eIPSCs was decreased by ~50% after treatment with either 1 μM ω-conotoxin GVIA or 1 μM ω-agatoxin IVa. In contrast, there was a lack of sensitivity to 1 μM ω-conotoxin GVIA in UCs. Immunocytochemical results confirmed decreased perisomatic density of N-channels on Pyr cells in UCs. We suggest that decreased calcium influx via N-type channels in presynaptic GABAergic terminals is a mechanism contributing to decreased inhibitory input onto layer V Pyr cells in this model of cortical posttraumatic epileptogenesis.

Faria, Leonardo Coutinho and Istvan Mody. Protective effect of ifenprodil against spreading depression in the mouse entorhinal cortex. J Neurophysiol 92: 2610 -2614, 2004. First published June 16, 2004 10.1152/jn.00466.2004. In the brain, spreading depression (SD) is characterized by a large extracellular DC shift, a massive failure of ion homeostasis and a transient cessation of neuronal function. Clinically, SD is believed to be involved in various neurological disorders including migraine and cerebrovascular diseases. The propagation of cortical SD requires the release of glutamate, and N-methyl-D-aspartate (NMDA) receptors play a crucial role in this process. Here, we have isolated the NMDA receptor-mediated component of extracellularly recorded field excitatory postsynaptic potentials (fEPSPs) in layers 2-3 of the entorhinal cortex of murine brain slices. In the absence of GABA A and AMPA receptor-mediated synaptic transmission, stimulation of layer 6 afferents every 15-90 s elicited spontaneous SD on average within 18.5 min after the start of the stimulation. In the presence of ifenprodil, an NR2B receptor subunit-selective NMDA receptor antagonist, the occurrence of SD was nearly abolished. Our results are consistent with an important role of NR2B subunits in triggering SD in the entorhinal cortex.

Prophylaxis of posttraumatic epilepsy will require a detailed knowledge of the epileptogenic pathophysiological processes that follow brain injury. Results from studies of experimental models and human epilepsy highlight alterations in GABAergic interneurons and formation of excessive new excitatory synaptic connectivity as prominent targets for prophylactic therapies. Promising laboratory results suggest that it will be possible to experimentally modify these aberrant processes and interfere with epileptogenesis. However, a number of key issues must be addressed before these results can be used to frame clinical antiepileptogenic therapy.

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