Two-photon scanning microscopy of in vivo sensory responses of cortical neurons genetically encoded with a fluorescent voltage sensor in rat.

A fluorescent voltage sensor protein "Flare" was created from a Kv1.4 potassium channel with YFP situated to report voltage-induced conformational changes in vivo. The RNA virus Sindbis introduced Flare into neurons in the binocular region of visual cortex in rat. Injection sites were selected based on intrinsic optical imaging. Expression of Flare occurred in the cell bodies and dendritic processes. Neurons imaged in vivo using two-photon scanning microscopy typically revealed the soma best, discernable against the background labeling of the neuropil. Somatic fluorescence changes were correlated with flashed visual stimuli; however, averaging was essential to observe these changes. This study demonstrates that the genetic modification of single neurons to express a fluorescent voltage sensor can be used to assess neuronal activity in vivo.

Visual tuning properties of genetically identified layer 2/3 neuronal types in the primary visual cortex of cre-transgenic mice.

The putative excitatory and inhibitory cell classes within the mouse primary visual cortex V1 have different functional properties as studied using recording microelectrode. Excitatory neurons show high selectivity for the orientation angle of moving gratings while the putative inhibitory neurons show poor selectivity. However, the study of selectivity of the genetically identified interneurons and their subtypes remain controversial. Here we use novel Cre-driver and reporter mice to identify genetic subpopulations in vivo for two-photon calcium dye imaging: Wfs1(+)/Gad1(-) mice that labels layer 2/3 excitatory cell population and Pvalb(+)/Gad1(+) mice that labels a genetic subpopulation of inhibitory neurons. The cells in both mice were identically labeled with a tdTomato protein, visible in vivo, using a Cre-reporter line. We found that the Wfs1(+) cells exhibited visual tuning properties comparable to the excitatory population, i.e., high selectivity and tuning to the angle, direction, and spatial frequency of oriented moving gratings. The functional tuning of Pvalb(+) neurons was consistent with previously reported narrow-spiking interneurons in microelectrode studies, exhibiting poorer selectivity than the excitatory neurons. This study demonstrates the utility of Cre-transgenic mouse technology in selective targeting of subpopulations of neurons and makes them amenable to structural, functional, and connectivity studies.

Current flow in vibrissa motor cortex can phase-lock with exploratory rhythmic whisking in rat.

Rats explore their environment with rhythmic sweeps of their mystacial vibrissae in the range of 5-15 Hz. We tested if vibrissa primary motor (M1) cortex produces electrical activity that locks to this behavioral output. Rats were trained to whisk in air in search of a food reward. The EMG of the mystacial pad served as a surrogate of vibrissa position, while chronically implanted, 16-channel Si-based probes provided a record of field potentials throughout the depth of vibrissa M1 cortex as well as vibrissa primary somatosensory (S1) cortex. The measured potentials were used to estimate the current source density along the radial axis. We observed that current flow throughout the depth of M1 cortex is coherent with the mystacial EMG, i.e., the two signals co-vary with a defined phase relation. This coherence persists after transection of the infraorbital branch (IoN) of the trigeminal nerve, which provides the sole sensory input from the vibrissae. Furthermore, current flow in vibrissa S1 cortex that is coherent with the mystacial EMG also persists after transection of the IoN, consistent with anatomical pathways between M1 and S1. In combination with a previous observation that rhythmic, intracortical microstimulation of vibrissa M1 cortex can drive normal whisking motion, the present data support the hypothesis that, in principle, M1 cortex can initiate motion of the vibrissae on a cycle-by-cycle basis.

Spectral mixing of rhythmic neuronal signals in sensory cortex.

The ability to compute the difference between two frequencies depends on a nonlinear operation that mixes two periodic signals. Behavioral and psychophysical evidence suggest that such mixing is likely to occur in the mammalian nervous system as a means to compare two rhythmic sensory signals, such as occurs in human audition, and as a means to lock an intrinsic rhythm to a sensory input. However, a neurological substrate for mixing has not been identified. Here we address the issue of nonlinear mixing of neuronal activity in the vibrissa primary sensory cortex of rat, a region that receives intrinsic as well as sensory-driven rhythmic input during natural whisking. In our preparation, the intrinsic signal originates from cortical oscillations that were induced by anesthetics, and the extrinsic input is introduced by periodic stimulation of vibrissae. We observed that the local extracellular current in vibrissa primary sensory cortex contained oscillatory components at the sum and difference of the intrinsic and extrinsic frequencies. In complementary experiments, we observed that the simultaneous stimulation of contralateral and ipsilateral vibrissae at different frequencies also led to current flow at the sum and difference frequencies. We show theoretically that the relative amplitudes of the observed mixture terms can be accounted for by a threshold nonlinearity in the input-output relation of the underlying neurons. In general, our results provide a neurological substrate for the modulation and demodulation of rhythmic neuronal signals for sensory coding and feedback stabilization of motor output.