Chronic loss of inhibition in piriform cortex following brief, daily optogenetic stimulation.

It is well established that seizures beget seizures, yet the cellular processes that underlie progressive epileptogenesis remain unclear. Here, we use optogenetics to briefly activate targeted populations of mouse piriform cortex (PCx) principal neurons in vivo. After just 3 or 4 days of stimulation, previously subconvulsive stimuli trigger massive, generalized seizures. Highly recurrent allocortices are especially prone to "optokindling." Optokindling upsets the balance of recurrent excitation and feedback inhibition. To understand how this balance is disrupted, we then selectively reactivate the same neurons in vitro. Surprisingly, we find no evidence of heterosynaptic potentiation; instead, we observe a marked, pathway-specific decrease in feedback inhibition. We find no loss of inhibitory interneurons; rather, decreased GABA synthesis in feedback inhibitory neurons appears to underlie weakened inhibition. Optokindling will allow precise identification of the molecular processes by which brain activity patterns can progressively and pathologically disrupt the balance of cortical excitation and inhibition.

Parvalbumin and GABA Microcircuits in the Mouse Superior Colliculus.

The mammalian superior colliculus (SC) is a sensorimotor midbrain structure responsible for orienting behaviors. Although many SC features are known, details of its intrinsic microcircuits are lacking. We used transgenic mice expressing reporter genes in parvalbumin-positive (PV+) and gamma aminobutyric acid-positive (GABA+) neurons to test the hypothesis that PV+ neurons co-localize GABA and form inhibitory circuits within the SC. We found more PV+ neurons in the superficial compared to the intermediate SC, although a larger percentage of PV+ neurons co-expressed GABA in the latter. Unlike PV+ neurons, PV+/GABA+ neurons showed predominantly rapidly inactivating spiking patterns. Optogenetic activation of PV+ neurons revealed direct and feedforward GABAergic inhibitory synaptic responses, as well as excitatory glutamatergic synapses. We propose that PV+ neurons in the SC may be specialized for a variety of circuit functions within the SC rather than forming a homogeneous, GABAergic neuronal subtype as they appear to in other regions of the brain.

A circuit model for saccadic suppression in the superior colliculus.

Attenuation of visual activity in the superficial layers (SLs), stratum griseum superficiale and stratum opticum, of the superior colliculus during saccades may contribute to reducing perceptual blur during saccades and also may help prevent subsequent unwanted saccades. GABAergic neurons in the intermediate, premotor, layer (SGI), stratum griseum intermedium, send an inhibitory input to SL. This pathway provided the basis for a model proposing that the SGI premotor cells that project to brainstem gaze centers and discharge before saccades also activate neighboring GABAergic neurons that suppress saccade-induced visual activity in SL. The in vitro method allowed us to test this model. We made whole-cell patch-clamp recordings in collicular slices from either rats or GAD67-GFP knock-in mice, in which GABAergic neurons could be identified by their expression of green fluorescence protein (GFP). Antidromic electrical stimulation of SGI premotor cells was produced by applying pulse currents in which their axons congregate after exiting the superior colliculus. The stimulation evoked monosynaptic EPSCs in SGI GABAergic neurons that project to SL, as would be predicted if these neurons receive excitatory input from the premotor cells. Second, IPSCs were evoked in SL neurons, some of which project to the visual thalamus. These IPSCs were polysynaptically mediated by the GABAergic neurons that were excited by the antidromically activated SGI neurons. These results support the hypothesis that collaterals of premotor neuron axons excite GABAergic neurons that inhibit SL visuosensory cells.

Identity of a pathway for saccadic suppression.

Neurons in the superficial gray layer (SGS) of the superior colliculus receive visual input and excite intermediate layer (SGI) neurons that play a critical role in initiating rapid orienting movements of the eyes, called saccades. In the present study, two types of experiments demonstrate that a population of SGI neurons gives rise to a reciprocal pathway that inhibits neurons in SGS. First, in GAD67-GFP knockin mice, GABAergic SGI neurons that expressed GFP fluorescence were injected with the tracer biocytin to reveal their axonal projections. Axons arising from GFP-positive neurons in SGI terminated densely in SGS. Next, SGI neurons in rats and mice were stimulated by using the photolysis of caged glutamate, and in vitro whole-cell patch-clamp recordings were used to measure the responses evoked in SGS cells. Large, synaptically mediated outward currents were evoked in SGS neurons. These currents were blocked by gabazine, confirming that they were GABA(A) receptor-mediated inhibitory postsynaptic currents. This inhibitory pathway from SGI transiently suppresses visual activity in SGS, which in turn could have multiple effects. These effects could include reduction of perceptual blurring during saccades as well as prevention of eye movements that might be spuriously triggered by the sweep of the visual field across the retina.

An in vitro study of horizontal connections in the intermediate layer of the superior colliculus.

Some models propose that the spatial and temporal distributions of premotor activity in the intermediate layer of the superior colliculus are shaped by neuronal ensembles that give rise to local excitatory and distant inhibitory connections. One function proposed for these connections is to mediate a "winner-take-all" network; the short-range excitatory connections build up the activity of neighboring cells that command orienting movements in one direction, whereas the wide-ranging inhibitory projections attenuate the activity of remote cells that command incompatible movements. We used in vitro photostimulation and whole-cell patch-clamp recording to test these models by measuring the spatial extent of synaptic interactions within the rat intermediate layer. Uncaging glutamate over whole-cell patch-clamped cells in the intermediate layer elicited long-lasting inward currents, resulting from direct activation of glutamate receptors expressed by the cells, and brief synaptic currents evoked by activation of presynaptic neurons. The synaptic responses comprised clusters of excitatory and inhibitory currents. The size of these responses depended on the location of the stimulus with respect to the clamped cell. Large responses were commonly evoked by stimuli within 200 microm of the soma in the intermediate layer; smaller responses could occasionally be evoked from sites as distant as 500 microm. Responses evoked by stimulation beyond this distance were rare. Although the results demonstrated powerful local excitatory and inhibitory connections, they did not support the pattern of short-range excitation and widespread inhibition predicted by the winner-take-all hypothesis.

Neurogranin expression in stably transfected N2A cell line affects cytosolic calcium level by nitric oxide stimulation.

To test a cellular effect of rodent neurogranin (Ng) oxidation as compared to Ng phosphorylation, we develop a cell model capable of stable expression of Ng using the Tet-On system, and determine whether Ng oxidation regulates intracellular calcium level. Our results show that Ng oxidation by nitric oxide donor induces an increase of [Ca(2+)](i) in Ng-expressed cells as compared to the control cells without expressing Ng. These results suggest that Ng oxidation plays a significant role in intracellular Ca(2+) homeostasis, essential for the activated signaling networks in learning and memory.