Luc J. Gentet

Rapid eye movement (REM) sleep, also referred to as paradoxical sleep for the striking resemblance of its electroencephalogram (EEG) to the one observed in wakefulness, is characterized by the occurrence of transient events such as limb twitches or facial and rapid eye movements. Here, we investigated the local activity of the primary somatosensory or barrel cortex (S1) in naturally sleeping head-fixed male mice during REM. Through local field potential recordings, we uncovered local appearances of spindle waves in the barrel cortex during REM concomitant with strong delta power, challenging the view of a wakefulness-like activity in REM. We further performed extra- and intracellular recordings of thalamic cells in head-fixed mice. Our data show high-frequency thalamic bursts of spikes and subthreshold spindle oscillations in approximately half of the neurons of the ventral posterior medial nucleus which further confirmed the thalamic origin of local cortical spindles in S1 in REM. Cortical spindle oscillations were suppressed, while thalamus spike firing increased, associated with rapid mouse whisker movements and S1 cortical activity transitioned to an activated state. During REM, the sensory thalamus and barrel cortex therefore alternate between high (wake-like) and low (non-REM sleep-like) activation states, potentially providing a neuronal substrate for mnemonic processes occurring during this paradoxical sleep stage.

GABAergic inhibitory neurons, through their molecular, anatomic, and physiological diversity, provide a substrate for the modulation of ongoing cortical circuit activity throughout the sleep/wake cycle. Here, we investigated neuronal activity dynamics of parvalbumin (PV), vasoactive intestinal polypeptide (VIP), and somatostatin (SST) neurons in naturally sleeping head-restrained mice at the level of layer 2/3 of the primary somatosensory barrel cortex of mice. Through calcium imaging and targeted single-unit loose-patch or whole-cell recordings, we found that PV action potential firing activity was largest during both rapid eye movement (REM) and nonrapid eye movement (NREM) sleep stages, that VIP neurons were most active during REM sleep, and that the overall activity of SST neurons remained stable throughout the sleep/wake cycle. Analysis of neuronal activity dynamics uncovered rapid decreases in PV cell firing at wake onset followed by a progressive recovery during wake. Simultaneous local field potential (LFP) recordings further revealed that except for SST neurons, a large proportion of neurons were modulated by ongoing delta and theta oscillations. During NREM sleep spindles, PV and SST activity increased and decreased, respectively. Finally, we uncovered the presence of whisking behavior in mice during REM sleep and show that the activity of VIP and SST is differentially modulated during awake and sleeping whisking bouts, which may provide a neuronal substrate for internal brain representations occurring during sleep. SIGNIFICANCE STATEMENT In the sensory cortex, the balance between excitation and inhibition is believed to be highly dynamic throughout the sleep/wake cycle, shaping the response of cortical circuits to external stimuli while allowing the formation of newly encoded memory. Using in vivo two-photon calcium imaging or targeted single-unit recordings combined with LFP recordings, we describe the vigilance state and whisking-behavior-dependent activity of excitatory pyramidal and inhibitory GABAergic neurons in the supragranular layers of mouse somatosensory cortex. Interneuronal activity was found to be differentially modulated by ongoing delta and theta waves, sleep spindles, and a novel type of whisking observed during REM sleep, potentially providing a neuronal substrate for internal brain representations occurring during sleep.

GABAergic inhibitory neurons, through their molecular, anatomic and physiological diversity, provide a substrate for the modulation of ongoing cortical circuit activity throughout the sleep-wake cycle. Here, we investigated neuronal activity dynamics of parvalbumin (PV), vasoactive intestinal polypeptide (VIP) and somatostatin (SST) neurons in naturally-sleeping head-restrained mice at the level of layer 2/3 of the primary somatosensory barrel cortex of mice. Through calcium-imaging and targeted single-unit loose-patch or whole-cell recordings, we found that PV action potential (AP) firing activity was largest during both NREM (non-rapid eye movement) and REM sleep stages, that VIP neurons were activated during REM sleep and that the overall activity of SST neurons remained stable throughout the sleep/wake cycle. Analysis of neuronal activity dynamics uncovered rapid decreases in PV cell firing at wake onset followed by a progressive recovery during wake. Simultaneous local field potential (LFP) recordings further revealed that, except for SST neurons, a large proportion of neurons were modulated by ongoing delta and theta waves. During NREM sleep spindles, PV and SST activity increased and decreased, respectively. Finally, we uncovered the presence of whisking behavior in mice during REM sleep and show that the activity of VIP and SST is differentially modulated during awake and sleeping whisking bouts, which may provide a neuronal substrate for internal brain representations occurring during sleep.

Neocortical interneurons provide inhibition responsible for organizing neuronal activity into brain oscillations that subserve cognitive functions such as memory, attention or prediction. However, little is known about the interneuronal contribution to the entrainment of neocortical oscillations within and across different cortical layers. Here, using layer-specific optogenetic stimulations with micro-Light-Emitting Diode (µLED) arrays, directed toward parvalbumin-expressing (PV) interneurons in non-anesthetized awake mice, we found that supragranular layer stimulations of PV neurons were most efficient at entraining supragranular local field potential (LFP) oscillations at gamma frequencies (γ: 25 - 80 Hz), whereas infragranular layer stimulation of PV neurons better entrained the LFP at delta (δ: 2 - 5 Hz) and theta (θ: 6 - 10 Hz) frequencies. At the level of neuronal action potential activity, we observed that supragranular neurons better followed the imposed PV stimulation rhythm than their infragranular counterparts at most frequencies when the stimulation was delivered in their respective layer. Moreover, the neuronal entrainment evoked by local stimulation could propagate across layers, though with a lesser impact when the stimulation occurs in deep layers, suggesting an orientation-selective propagation. These results establish a layer-based framework for oscillation to entrain the primary somatosensory cortex in awake conditions.

Quantifying the message communicated by neurons in the cortex by averaging action potentials over repeated trials of a given stimulus can reveal neuronal tuning features. For example, simple cells in the visual cortex have been characterized by reverse correlation based on the detailed structure of their oriented receptive fields. This structure, in turn, has been modeled using large libraries of such receptive fields to allow the simultaneous coding of visual stimuli with small numbers of appropriate combinations of cells selected from the library. This strategy, known as sparse coding , has been shown to produce excellent approximations for natural visual inputs. In concert with this mathematical development has been the discovery of cells’ use of oscillations in the gamma frequency range for general coding tasks, such as a mechanism for synchronizing distal networks of neurons. More recently, spikes timed with oscillations have been shown to exhibit local phase delays within a single gamma cycle, but such delays have resisted a behavioral functional interpretation. We show here that a specific coordinate system for the gamma cycle allows resultant phase delays to be interpreted quantitatively in classical terms. Specifically, extracted phase delays from mice viewing oriented sinusoidal grating images are shown to have the same distributions as those from a computer sparse coding model using natural images, suggesting for the first time a direct link between experimentally measured phase delays and model receptive fields. Significance Statement: Networks of pyramidal cells in the cortex exhibit action potentials (spikes) that are characterized by randomness and low firing rates. Spike averaging methods have been ordinarily useful in dealing with these features to reveal behavioral task structure, but the randomness and slowness so far prevented the specification of a satisfactory generative spike model. We show that a spike can be analyzed using the context of a specific phase of the gamma component of its membrane potential. The result is each spike can be can be assigned a scalar, which makes it immediately useful for network models.

This article reports on the development, i.e., the design, fabrication, and validation of an implantable optical neural probes designed for in vivo experiments relying on optogenetics. The probes comprise an array of ten bare light-emitting diode (LED) chips emitting at a wavelength of 460 nm and integrated along a flexible polyimide-based substrate stiffened using a micromachined ladder-like silicon structure. The resulting mechanical stiffness of the slender, 250-μm-wide, 65-μm-thick, and 5- and 8-mm-long probe shank facilitates its implantation into neural tissue. The LEDs are encapsulated by a fluropolymer coating protecting the implant against the physiological conditions in the brain. The electrical interface to the external control unit is provided by 10-μm-thick, highly flexible polyimide cables making the probes suitable for both acute and chronic in vivo experiments. Optical and electrical properties of the probes are reported, as well as their in vivo validation in acute optogenetic studies in transgenic mice. The depth-dependent optical stimulation of both excitatory and inhibitory neurons is demonstrated by altering the brain activity in the cortex and the thalamus. Local network responses elicited by 20-ms-long light pulses of different optical power (20 μW and 1 mW), as well as local modulation of single unit neuronal activity to 1-s-long light pulses with low optical intensity (17 μW) are presented. The ability to modulate neural activity makes these devices suitable for a broad variety of optogenetic experiments.

Cortical gamma activity (30–80 Hz) is believed to play important functions in neural computation and arises from the interplay of parvalbumin-expressing interneurons (PV) and pyramidal cells (PYRs). However, the subthreshold dynamics underlying its emergence in the cortex of awake animals remain unclear. Here, we characterized the intracellular dynamics of PVs and PYRs during spontaneous and visually evoked gamma activity in layers 2/3 of V1 of awake mice using targeted patch-clamp recordings and synchronous local field potentials (LFPs). Strong gamma activity patterned in short bouts (one to three cycles), occurred when PVs and PYRs were depolarizing and entrained their membrane potential dynamics regardless of the presence of visual stimulation. PV firing phase locked unconditionally to gamma activity. However, PYRs only phase locked to visually evoked gamma bouts. Taken together, our results indicate that gamma activity corresponds to short pulses of correlated background synaptic activity synchronizing the output of cortical neurons depending on external sensory drive. The neocortex is the main substrate of cognitive activity of the mammalian brain. During active wakefulness, it exhibits an oscillatory activity in the gamma range (30–80Hz), which is believed to play an important functional role and is altered in schizophrenic patients. Experimental studies have shown that gamma activity arises from the interaction of excitatory pyramidal neurons, the main neuronal type of the cortex, and local inhibitory neurons expressing the protein parvalbumin (PV). However, how these neuronal types behave during gamma activity remains largely unknown. Here, we recorded the intracellular activity of pyramidal and PV-expressing neurons in the visual cortex of awake mice while acquiring Local Field Potentials (LFPs)—extracellular voltage fluctuations within a small volume of the cortex—to monitor gamma activity. We found that gamma activity arises when PV-expressing neurons synchronize their output in response to a correlated input, reflecting the general activation of the local cortical network. This happens even in the absence of visual input. On the other hand, the output of pyramidal neurons only becomes entrained to gamma activity when the mice are exposed to visual stimulation. Thus, our results suggest that gamma activity synchronizes pyramidal neurons specifically when the cortex is engaged in processing external inputs.

Beta and gamma rhythms have been hypothesized to be involved in global and local coordination of neuronal activity, respectively. Here, we investigated how cells in rodent area S1BF are entrained by rhythmic fluctuations at various frequencies within the local area and in connected areas, and how this depends on behavioral state and cell type. We performed simultaneous extracellular field and unit recordings in four connected areas of the freely moving rat (S1BF, V1M, perirhinal cortex, CA1). S1BF spiking activity was strongly entrained by both beta and gamma S1BF oscillations, which were associated with deactivations and activations, respectively. We identified multiple classes of fast spiking and excitatory cells in S1BF, which showed prominent differences in rhythmic entrainment and in the extent to which phase locking was modulated by behavioral state. Using an additional dataset acquired by whole-cell recordings in head-fixed mice, these cell classes could be compared with identified phenotypes showing gamma rhythmicity in their membrane potential. We next examined how S1BF cells were entrained by rhythmic fluctuations in connected brain areas. Gamma-synchronization was detected in all four areas, however we did not detect significant gamma coherence among these areas. Instead, we only found long-range coherence in the theta-beta range among these areas. In contrast to local S1BF synchronization, we found long-range S1BF-spike to CA1-LFP synchronization to be homogeneous across inhibitory and excitatory cell types. These findings suggest distinct, cell-type contributions of low and high-frequency synchronization to intra- and inter-areal neuronal interactions.