Optogenetic Mapping of Intracortical Circuits Originating from Semilunar Cells in the Piriform Cortex.

Despite its comparatively simple trilaminar architecture, the primary olfactory (piriform) cortex of mammals is capable of performing sophisticated sensory processing, an ability that is thought to depend critically on its extensive associational (intracortical) excitatory circuits. Here, we used a novel transgenic mouse model and optogenetics to measure the connectivity of associational circuits that originate in semilunar (SL) cells in layer 2a of the anterior piriform cortex (aPC). We generated a mouse line (48L) in which channelrhodopsin-2 (ChR) could be selectively expressed in a subset of SL cells. Light-evoked excitatory postsynaptic currents (EPSCs) could be evoked in superficial pyramidal cells (17.4% of n = 86 neurons) and deep pyramidal cells (33.3%, n = 9) in the aPC, but never in ChR- SL cells (0%, n = 34). Thus, SL cells monosynaptically excite pyramidal cells, but not other SL cells. Light-evoked EPSCs were also selectively elicited in 3 classes of GABAergic interneurons in layer 3 of the aPC. Our results show that SL cells are specialized for providing feedforward excitation of specific classes of neurons in the aPC, confirming that SL cells comprise a functionally distinctive input layer.

Noradrenaline Increases mEPSC Frequency in Pyramidal Cells in Layer II of Rat Barrel Cortex via Calcium Release From Presynaptic Stores.

Somatosensory cortex is innervated by afferents originating from the locus coeruleus which typically release noradrenaline. We tested if activation of presynaptic α1-adrenoceptors (AR) coupled to a Gq-mediated signaling cascade resulted in calcium (Ca2+) release from stores and thereby increased spontaneous transmitter release in rat barrel cortex. Adding 1-100 µM noradrenaline (NA) or 5 µM cirazoline (CO), a α1-AR specific agonist, to the standard artificial cerebrospinal fluid increased the frequency of miniature excitatory postsynaptic currents (mEPSC) by 64 ± 7% in 51% of pyramidal cells in layer II (responders) with no effect on the amplitude. In 42 responders, the mEPSC frequency during control was significantly smaller (39 ± 2 vs. 53 ± 4 Hz) and upon NA exposure, the input resistance (Rin) decreased (9 ± 7%) compared to non-responders. Experiments using CO and the antagonist prazosin revealed that NA acted via binding to α1-ARs, which was further corroborated by simultaneously blocking ß- and α2-ARs with propranolol and yohimbine, which did not prevent the increase in mEPSC frequency. To verify elements in the signaling cascade, both the phospholipase C inhibitor edelfosine and the membrane permeable IP3 receptor blocker 2-APB averted the increase in mEPSC frequency. Likewise, emptying Ca2+ stores with cyclopiazonic acid or the chelation of intracellular Ca2+ with BAPTA-AM prevented the frequency increase, suggesting that the frequency increase was caused by presynaptic store release. When group I metabotropic glutamate receptors were activated with DHPG, co-application of NA occluded a further frequency increase suggesting that the two receptor activations may not signal independently of each other. The increased mEPSC frequency in a subset of pyramidal cells results in enhanced synaptic noise, which, together with the reduction in Rin, will affect computation in the network.

TRPA1 expression and its functional activation in rodent cortex.

TRPA1 is a non-selective cation channel involved in pain sensation and neurogenic inflammation. Although TRPA1 is well established in a number of organs including the nervous system, its presence and function in the mammalian cortex remains unclear. Here, we demonstrate the expression of TRPA1 in rodent somatosensory cortex through immunostaining and investigate its functional activation by whole-cell electrophysiology, Ca2+ imaging and two-photon photoswitching. Application of TRPA1 agonist (AITC) and antagonist (HC-030031) produced significant modulation of activity in layer 5 (L5) pyramidal neurons in both rats and mice; AITC increased intracellular Ca2+ concentrations and depolarized neurons, and both effects were blocked by HC-030031. These modulations were absent in the TRPA1 knockout mice. Next, we used optovin, a reversible photoactive molecule, to activate TRPA1 in individual L5 neurons of rat cortex. Optical control of activity was established by applying a tightly focused femtosecond-pulsed laser to optovin-loaded neurons. Light application depolarized neurons (n = 17) with the maximal effect observed at λ = 720 nm. Involvement of TRPA1 was further confirmed by repeating the experiment in the presence of HC-030031, which diminished the light modulation. These results demonstrate the presence of TRPA1 in L5 pyramidal neurons and introduce a highly specific approach to further understand its functional significance.

Improving Focal Photostimulation of Cortical Neurons with Pre-derived Wavefront Correction.

Recent progress in neuroscience to image and investigate brain function has been made possible by impressive developments in optogenetic and opto-molecular tools. Such research requires advances in optical techniques for the delivery of light through brain tissue with high spatial resolution. The tissue causes distortions to the wavefront of the incoming light which broadens the focus and consequently reduces the intensity and degrades the resolution. Such effects are detrimental in techniques requiring focal stimulation. Adaptive wavefront correction has been demonstrated to compensate for these distortions. However, iterative derivation of the corrective wavefront introduces time constraints that limit its applicability to probe living cells. Here, we demonstrate that we can pre-determine and generalize a small set of Zernike modes to correct for aberrations of the light propagating through specific brain regions. A priori identification of a corrective wavefront is a direct and fast technique that improves the quality of the focus without the need for iterative adaptive wavefront correction. We verify our technique by measuring the efficiency of two-photon photolysis of caged neurotransmitters along the dendrites of a whole-cell patched neuron. Our results show that encoding the selected Zernike modes on the excitation light can improve light propagation through brain slices of rats as observed by the neuron's evoked excitatory post-synaptic potential in response to localized focal uncaging at the spines of the neuron's dendrites.

Improved two-photon imaging of living neurons in brain tissue through temporal gating.

We optimize two-photon imaging of living neurons in brain tissue by temporally gating an incident laser to reduce the photon flux while optimizing the maximum fluorescence signal from the acquired images. Temporal gating produces a bunch of ~10 femtosecond pulses and the fluorescence signal is improved by increasing the bunch-pulse energy. Gating is achieved using an acousto-optic modulator with a variable gating frequency determined as integral multiples of the imaging sampling frequency. We hypothesize that reducing the photon flux minimizes the photo-damage to the cells. Our results, however, show that despite producing a high fluorescence signal, cell viability is compromised when the gating and sampling frequencies are equal (or effectively one bunch-pulse per pixel). We found an optimum gating frequency range that maintains the viability of the cells while preserving a pre-set fluorescence signal of the acquired two-photon images. The neurons are imaged while under whole-cell patch, and the cell viability is monitored as a change in the membrane's input resistance.