Stimulus Feature-Specific Control of Layer 2/3 Subthreshold Whisker Responses by Layer 4 in the Mouse Primary Somatosensory Cortex.

In the barrel field of the rodent primary somatosensory cortex (S1bf), excitatory cells in layer 2/3 (L2/3) display sparse firing but reliable subthreshold response during whisker stimulation. Subthreshold responses encode specific features of the sensory stimulus, for example, the direction of whisker deflection. According to the canonical model for the flow of sensory information across cortical layers, activity in L2/3 is driven by layer 4 (L4). However, L2/3 cells receive excitatory inputs from other regions, raising the possibility that L4 partially drives L2/3 during whisker stimulation. To test this hypothesis, we combined patch-clamp recordings from L2/3 pyramidal neurons in S1bf with selective optogenetic inhibition of L4 during passive whisker stimulation in both anesthetized and awake head-restrained mice. We found that L4 optogenetic inhibition did not abolish the subthreshold whisker-evoked response nor it affected spontaneous membrane potential fluctuations of L2/3 neurons. However, L4 optogenetic inhibition decreased L2/3 subthreshold responses to whisker deflections in the preferred direction, and it increased L2/3 responses to stimuli in the nonpreferred direction, leading to a change in the direction tuning. Our results contribute to reveal the circuit mechanisms underlying the processing of sensory information in the rodent S1bf.

Unaltered Network Activity and Interneuronal Firing During Spontaneous Cortical Dynamics In Vivo in a Mouse Model of Severe Myoclonic Epilepsy of Infancy.

Severe myoclonic epilepsy of infancy (SMEI) is associated with loss of function of the SCN1A gene encoding the NaV1.1 sodium channel isoform. Previous studies in Scn1a(-/+) mice during the pre-epileptic period reported selective reduction in interneuron excitability and proposed this as the main pathological mechanism underlying SMEI. Yet, the functional consequences of this interneuronal dysfunction at the circuit level in vivo are unknown. Here, we investigated whether Scn1a(-/+) mice showed alterations in cortical network function. We found that various forms of spontaneous network activity were similar in Scn1a(-/+) during the pre-epileptic period compared with wild-type (WT) in vivo. Importantly, in brain slices from Scn1a(-/+) mice, the excitability of parvalbumin (PV) and somatostatin (SST) interneurons was reduced, epileptiform activity propagated more rapidly, and complex synaptic changes were observed. However, in vivo, optogenetic reduction of firing in PV or SST cells in WT mice modified ongoing network activities, and juxtasomal recordings from identified PV and SST interneurons showed unaffected interneuronal firing during spontaneous cortical dynamics in Scn1a(-/+) compared with WT. These results demonstrate that interneuronal hypoexcitability is not observed in Scn1a(-/+) mice during spontaneous activities in vivo and suggest that additional mechanisms may contribute to homeostatic rearrangements and the pathogenesis of SMEI.

Mechanism underlying unaltered cortical inhibitory synaptic transmission in contrast with enhanced excitatory transmission in CaV2.1 knockin migraine mice.

Familial hemiplegic migraine type 1 (FHM1), a monogenic subtype of migraine with aura, is caused by gain-of-function mutations in CaV2.1 (P/Q-type) calcium channels. In FHM1 knockin mice, excitatory neurotransmission at cortical pyramidal cell synapses is enhanced, but inhibitory neurotransmission at connected pairs of fast-spiking (FS) interneurons and pyramidal cells is unaltered, despite being initiated by CaV2.1 channels. The mechanism underlying the unaltered GABA release at cortical FS interneuron synapses remains unknown. Here, we show that the FHM1 R192Q mutation does not affect inhibitory transmission at autapses of cortical FS and other types of multipolar interneurons in microculture from R192Q knockin mice, and investigate the underlying mechanism. Lowering the extracellular [Ca(2+)] did not reveal gain-of-function of evoked transmission neither in control nor after prolongation of the action potential (AP) with tetraethylammonium, indicating unaltered AP-evoked presynaptic calcium influx at inhibitory autapses in FHM1 KI mice. Neither saturation of the presynaptic calcium sensor nor short duration of the AP can explain the unaltered inhibitory transmission in the mutant mice. Recordings of the P/Q-type calcium current in multipolar interneurons in microculture revealed that the current density and the gating properties of the CaV2.1 channels expressed in these interneurons are barely affected by the FHM1 mutation, in contrast with the enhanced current density and left-shifted activation gating of mutant CaV2.1 channels in cortical pyramidal cells. Our findings suggest that expression of specific CaV2.1 channels differentially sensitive to modulation by FHM1 mutations in inhibitory and excitatory cortical neurons underlies the gain-of-function of excitatory but unaltered inhibitory synaptic transmission and the likely consequent dysregulation of the cortical excitatory-inhibitory balance in FHM1.

Cortico-cortical transfer of socially derived information gates emotion recognition.

Emotion recognition and the resulting responses are important for survival and social functioning. However, how socially derived information is processed for reliable emotion recognition is incompletely understood. Here, we reveal an evolutionarily conserved long-range inhibitory/excitatory brain network mediating these socio-cognitive processes. Anatomical tracing in mice revealed the existence of a subpopulation of somatostatin (SOM) GABAergic neurons projecting from the medial prefrontal cortex (mPFC) to the retrosplenial cortex (RSC). Through optogenetic manipulations and Ca2+ imaging fiber photometry in mice and functional imaging in humans, we demonstrate the specific participation of these long-range SOM projections from the mPFC to the RSC, and an excitatory feedback loop from the RSC to the mPFC, in emotion recognition. Notably, we show that mPFC-to-RSC SOM projections are dysfunctional in mouse models relevant to psychiatric vulnerability and can be targeted to rescue emotion recognition deficits in these mice. Our findings demonstrate a cortico-cortical circuit underlying emotion recognition.

A deep-learning approach for online cell identification and trace extraction in functional two-photon calcium imaging.

In vivo two-photon calcium imaging is a powerful approach in neuroscience. However, processing two-photon calcium imaging data is computationally intensive and time-consuming, making online frame-by-frame analysis challenging. This is especially true for large field-of-view (FOV) imaging. Here, we present CITE-On (Cell Identification and Trace Extraction Online), a convolutional neural network-based algorithm for fast automatic cell identification, segmentation, identity tracking, and trace extraction in two-photon calcium imaging data. CITE-On processes thousands of cells online, including during mesoscopic two-photon imaging, and extracts functional measurements from most neurons in the FOV. Applied to publicly available datasets, the offline version of CITE-On achieves performance similar to that of state-of-the-art methods for offline analysis. Moreover, CITE-On generalizes across calcium indicators, brain regions, and acquisition parameters in anesthetized and awake head-fixed mice. CITE-On represents a powerful tool to speed up image analysis and facilitate closed-loop approaches, for example in combined all-optical imaging and manipulation experiments.

High-Accuracy Detection of Neuronal Ensemble Activity in Two-Photon Functional Microscopy Using Smart Line Scanning.

Two-photon functional imaging using genetically encoded calcium indicators (GECIs) is one prominent tool to map neural activity. Under optimized experimental conditions, GECIs detect single action potentials in individual cells with high accuracy. However, using current approaches, these optimized conditions are never met when imaging large ensembles of neurons. Here, we developed a method that substantially increases the signal-to-noise ratio (SNR) of population imaging of GECIs by using galvanometric mirrors and fast smart line scan (SLS) trajectories. We validated our approach in anesthetized and awake mice on deep and dense GCaMP6 staining in the mouse barrel cortex during spontaneous and sensory-evoked activity. Compared to raster population imaging, SLS led to increased SNR, higher probability of detecting calcium events, and more precise identification of functional neuronal ensembles. SLS provides a cheap and easily implementable tool for high-accuracy population imaging of neural GCaMP6 signals by using galvanometric-based two-photon microscopes.

Extended field-of-view ultrathin microendoscopes for high-resolution two-photon imaging with minimal invasiveness.

Imaging neuronal activity with high and homogeneous spatial resolution across the field-of-view (FOV) and limited invasiveness in deep brain regions is fundamental for the progress of neuroscience, yet is a major technical challenge. We achieved this goal by correcting optical aberrations in gradient index lens-based ultrathin (≤500 µm) microendoscopes using aspheric microlenses generated through 3D-microprinting. Corrected microendoscopes had extended FOV (eFOV) with homogeneous spatial resolution for two-photon fluorescence imaging and required no modification of the optical set-up. Synthetic calcium imaging data showed that, compared to uncorrected endoscopes, eFOV-microendoscopes led to improved signal-to-noise ratio and more precise evaluation of correlated neuronal activity. We experimentally validated these predictions in awake head-fixed mice. Moreover, using eFOV-microendoscopes we demonstrated cell-specific encoding of behavioral state-dependent information in distributed functional subnetworks in a primary somatosensory thalamic nucleus. eFOV-microendoscopes are, therefore, small-cross-section ready-to-use tools for deep two-photon functional imaging with unprecedentedly high and homogeneous spatial resolution.

Temporal Sharpening of Sensory Responses by Layer V in the Mouse Primary Somatosensory Cortex.

The timing of stimulus-evoked spikes encodes information about sensory stimuli. Here we studied the neural circuits controlling this process in the mouse primary somatosensory cortex. We found that brief optogenetic activation of layer V pyramidal cells just after whisker deflection modulated the membrane potential of neurons and interrupted their long-latency whisker responses, increasing their accuracy in encoding whisker deflection time. In contrast, optogenetic inhibition of layer V during either passive whisker deflection or active whisking decreased accuracy in encoding stimulus or touch time, respectively. Suppression of layer V pyramidal cells increased reaction times in a texture discrimination task. Moreover, two-color optogenetic experiments revealed that cortical inhibition was efficiently recruited by layer V stimulation and that it mainly involved activation of parvalbumin-positive rather than somatostatin-positive interneurons. Layer V thus performs behaviorally relevant temporal sharpening of sensory responses through circuit-specific recruitment of cortical inhibition.

Two-Photon Bidirectional Control and Imaging of Neuronal Excitability with High Spatial Resolution In Vivo.

Sensory information is encoded within the brain in distributed spatiotemporal patterns of neuronal activity. Understanding how these patterns influence behavior requires a method to measure and to bidirectionally perturb with high spatial resolution the activity of the multiple neuronal cell types engaged in sensory processing. Here, we combined two-photon holography to stimulate neurons expressing blue light-sensitive opsins (ChR2 and GtACR2) with two-photon imaging of the red-shifted indicator jRCaMP1a in the mouse neocortex in vivo. We demonstrate efficient control of neural excitability across cell types and layers with holographic stimulation and improved spatial resolution by opsin somatic targeting. Moreover, we performed simultaneous two-photon imaging of jRCaMP1a and bidirectional two-photon manipulation of cellular activity with negligible effect of the imaging beam on opsin excitation. This all-optical approach represents a powerful tool to causally dissect how activity patterns in specified ensembles of neurons determine brain function and animal behavior.

An inhibitory gate for state transition in cortex.

Large scale transitions between active (up) and silent (down) states during quiet wakefulness or NREM sleep regulate fundamental cortical functions and are known to involve both excitatory and inhibitory cells. However, if and how inhibition regulates these activity transitions is unclear. Using fluorescence-targeted electrophysiological recording and cell-specific optogenetic manipulation in both anesthetized and non-anesthetized mice, we found that two major classes of interneurons, the parvalbumin and the somatostatin positive cells, tightly control both up-to-down and down-to-up state transitions. Inhibitory regulation of state transition was observed under both natural and optogenetically-evoked conditions. Moreover, perturbative optogenetic experiments revealed that the inhibitory control of state transition was interneuron-type specific. Finally, local manipulation of small ensembles of interneurons affected cortical populations millimetres away from the modulated region. Together, these results demonstrate that inhibition potently gates transitions between cortical activity states, and reveal the cellular mechanisms by which local inhibitory microcircuits regulate state transitions at the mesoscale.

Abnormal cortical synaptic transmission in CaV2.1 knockin mice with the S218L missense mutation which causes a severe familial hemiplegic migraine syndrome in humans.

Familial hemiplegic migraine type 1 (FHM1) is caused by gain-of-function mutations in CaV2.1 (P/Q-type) Ca(2+) channels. Knockin (KI) mice carrying the FHM1 R192Q missense mutation show enhanced cortical excitatory synaptic transmission at pyramidal cell synapses but unaltered cortical inhibitory neurotransmission at fast-spiking interneuron synapses. Enhanced cortical glutamate release was shown to cause the facilitation of cortical spreading depression (CSD) in R192Q KI mice. It, however, remains unknown how other FHM1 mutations affect cortical synaptic transmission. Here, we studied neurotransmission in cortical neurons in microculture from KI mice carrying the S218L mutation, which causes a severe FHM syndrome in humans and an allele-dosage dependent facilitation of experimental CSD in KI mice, which is larger than that caused by the R192Q mutation. We show gain-of-function of excitatory neurotransmission, due to increased action-potential evoked Ca(2+) influx and increased probability of glutamate release at pyramidal cell synapses, but unaltered inhibitory neurotransmission at multipolar interneuron synapses in S218L KI mice. In contrast with the larger gain-of-function of neuronal CaV2.1 current in homozygous than heterozygous S218L KI mice, the gain-of-function of evoked glutamate release, the paired-pulse ratio and the Ca(2+) dependence of the excitatory postsynaptic current were similar in homozygous and heterozygous S218L KI mice, suggesting compensatory changes in the homozygous mice. Furthermore, we reveal a unique feature of S218L KI cortical synapses which is the presence of a fraction of mutant CaV2.1 channels being open at resting potential. Our data suggest that, while the gain-of-function of evoked glutamate release may explain the facilitation of CSD in heterozygous S218L KI mice, the further facilitation of CSD in homozygous S218L KI mice is due to other CaV2.1-dependent mechanisms, that likely include Ca(2+) influx at voltages sub-threshold for action potential generation.

Migraine: a disorder of brain excitatory-inhibitory balance?

Migraine is a common disabling brain disorder whose key manifestations are recurrent attacks of unilateral headache and interictal hypersensitivity to sensory stimuli. Migraine arises from a primary brain dysfunction that leads to episodic activation and sensitization of the trigeminovascular pain pathway and as a consequence to headache. Major open issues concern the molecular and cellular mechanisms of the primary brain dysfunction(s) and of migraine pain. We review here our current understanding of these mechanisms, focusing on recent advances regarding migraine genetics, headache mechanisms, and the primary brain dysfunction(s) underlying migraine onset and susceptibility to cortical spreading depression, the neurophysiological correlate of migraine aura. We also discuss insights obtained from the functional analysis of familial hemiplegic migraine mouse models.

Enhanced excitatory transmission at cortical synapses as the basis for facilitated spreading depression in Ca(v)2.1 knockin migraine mice.

Migraine is a common disabling brain disorder. A subtype of migraine with aura (familial hemiplegic migraine type 1: FHM1) is caused by mutations in Ca(V)2.1 (P/Q-type) Ca(2+) channels. Knockin mice carrying a FHM1 mutation show increased neuronal P/Q-type current and facilitation of induction and propagation of cortical spreading depression (CSD), the phenomenon that underlies migraine aura and may activate migraine headache mechanisms. We studied cortical neurotransmission in neuronal microcultures and brain slices of FHM1 mice. We show gain of function of excitatory neurotransmission due to increased action-potential-evoked Ca(2+) influx and increased probability of glutamate release at pyramidal cell synapses but unaltered inhibitory neurotransmission at fast-spiking interneuron synapses. Using an in vitro model of CSD, we show a causative link between enhanced glutamate release and CSD facilitation. The synapse-specific effect of FHM1 mutations points to disruption of excitation-inhibition balance and neuronal hyperactivity as the basis for episodic vulnerability to CSD ignition in migraine.