Localized chemogenetic silencing of inhibitory neurons: a novel mouse model of focal cortical epileptic activity.

Focal cortical epilepsies are frequently refractory to available anticonvulsant drug therapies. One key factor contributing to this state is the limited availability of animal models that allow to reliably study focal cortical seizures and how they recruit surrounding brain areas in vivo. In this study, we selectively expressed the inhibitory chemogenetic receptor, hM4D, in GABAergic neurons in focal cortical areas using viral gene transfer. GABAergic silencing using Clozapine-N-Oxide (CNO) demonstrated reliable induction of local epileptiform events in the electroencephalogram signal of awake freely moving mice. Anesthetized mice experiments showed consistent induction of focal epileptiform-events in both the barrel cortex (BC) and the medial prefrontal cortex (mPFC), accompanied by high-frequency oscillations, a known characteristic of human seizures. Epileptiform-events showed propagation indication with favored propagation pathways: from the BC on 1 hemisphere to its counterpart and from the BC to the mPFC, but not vice-versa. Lastly, sensory whisker-pad stimulation evoked BC epileptiform events post-CNO, highlighting the potential use of this model in studying sensory-evoked seizures. Combined, our results show that targeted chemogenetic inhibition of GABAergic neurons using hM4D can serve as a novel, versatile, and reliable model of focal cortical epileptic activity suitable for systematically studying cortical ictogenesis in different cortical areas.

A novel theoretical framework for simultaneous measurement of excitatory and inhibitory conductances.

The firing of neurons throughout the brain is determined by the precise relations between excitatory and inhibitory inputs, and disruption of their balance underlies many psychiatric diseases. Whether or not these inputs covary over time or between repeated stimuli remains unclear due to the lack of experimental methods for measuring both inputs simultaneously. We developed a new analytical framework for instantaneous and simultaneous measurements of both the excitatory and inhibitory neuronal inputs during a single trial under current clamp recording. This can be achieved by injecting a current composed of two high frequency sinusoidal components followed by analytical extraction of the conductances. We demonstrate the ability of this method to measure both inputs in a single trial under realistic recording constraints and from morphologically realistic CA1 pyramidal model cells. Future experimental implementation of our new method will facilitate the understanding of fundamental questions about the health and disease of the nervous system.

Reduction of corpus callosum activity during whisking leads to interhemispheric decorrelation.

Interhemispheric correlation between homotopic areas is a major hallmark of cortical physiology and is believed to emerge through the corpus callosum. However, how interhemispheric correlations and corpus callosum activity are affected by behavioral states remains unknown. We performed laminar extracellular and intracellular recordings simultaneously from both barrel cortices in awake mice. We find robust interhemispheric correlations of both spiking and synaptic activities that are reduced during whisking compared to quiet wakefulness. Accordingly, optogenetic inactivation of one hemisphere reveals that interhemispheric coupling occurs only during quiet wakefulness, and chemogenetic inactivation of callosal terminals reduces interhemispheric correlation especially during quiet wakefulness. Moreover, in contrast to the generally elevated firing rate observed during whisking epochs, we find a marked decrease in the activity of imaged callosal fibers. Our results indicate that the reduction in interhemispheric coupling and correlations during active behavior reflects the specific reduction in the activity of callosal neurons.

Cross-Whisker Adaptation of Neurons in Layer 2/3 of the Rat Barrel Cortex.

Neurons in the barrel cortex respond preferentially to stimulation of one principal whisker and weakly to several adjacent whiskers. Such integration exists already in layer 4, the pivotal recipient layer of thalamic inputs. Previous studies show that cortical neurons gradually adapt to repeated whisker stimulations and that layer 4 neurons exhibit whisker specific adaptation and no apparent interactions with other whiskers. This study aimed to study the specificity of adaptation of layer 2/3 cortical cells. Towards this aim, we compared the synaptic response of neurons to either repetitive stimulation of one of two responsive whiskers or when repetitive stimulation of the two whiskers was interleaved. We found that in most layer 2/3 cells adaptation is whisker-specific. These findings indicate that despite the multi-whisker receptive fields in the cortex, the adaptation process for each whisker-pathway is mostly independent of other whiskers. A mechanism allowing high responsiveness in complex environments.

Multiple Timescales Account for Adaptive Responses across Sensory Cortices.

Sensory systems encounter remarkably diverse stimuli in the external environment. Natural stimuli exhibit timescales and amplitudes of variation that span a wide range. Mechanisms of adaptation, a ubiquitous feature of sensory systems, allow for the accommodation of this range of scales. Are there common rules of adaptation across different sensory modalities? We measured the membrane potential responses of individual neurons in the visual, somatosensory, and auditory cortices of male and female mice to discrete, punctate stimuli delivered at a wide range of fixed and nonfixed frequencies. We find that the adaptive profile of the response is largely preserved across these three areas, exhibiting attenuation and responses to the cessation of stimulation, which are signatures of response to changes in stimulus statistics. We demonstrate that these adaptive responses can emerge from a simple model based on the integration of fixed filters operating over multiple time scales.SIGNIFICANCE STATEMENT Our recent sensations affect our current expectations and perceptions of the environment. Neural correlates of this process exist throughout the brain and are loosely termed adaptation. Adaptive processes have been described across sensory cortices, but direct comparisons of these processes have not been possible because paradigms have been tailored specifically for each modality. We developed a common stimulus set that was used to characterize adaptation in somatosensory, visual, and auditory cortex. We describe here the similarities and differences in adaptation across these cortical areas and demonstrate that adaptive responses may emerge from a set of static filters that operate over a broad range of timescales.

In-vivo optogenetics and pharmacology in deep intracellular recordings.

Single cell intracellular recordings in-vivo at deep brain structures are seldomly accompanied by nearby optogenetics or drug application. The use of such tools is limited as both light and drugs cannot penetrate deep inside brain tissue. Hence, the optical fiber or drug delivery pipette needs to be placed within the brain close to the recording pipette. So far, however, this has required highly accurate hardware to achieve. These complications have now been solved by new approaches enabling intracellular recordings both for optogenetics and pharmacological application by the use of a single manipulator. In this manuscript we review these technologies - their pros, cons and implications.

Stereotactic system for accurately targeting deep brain structures in awake head-fixed mice.

Deep brain nuclei, such as the amygdala, nucleus basalis, and locus coeruleus, play a crucial role in cognition and behavior. Nonetheless, acutely recording electrical activity from these structures in head-fixed awake rodents has been very challenging due to the fact that head-fixed preparations are not designed for stereotactic accuracy. We overcome this issue by designing the DeepTarget, a system for stereotactic head fixation and recording, which allows for accurately directing recording electrodes or other probes into any desired location in the brain. We then validated it by performing intracellular recordings from optogenetically tagged amygdalar neurons followed by histological reconstruction, which revealed that it is accurate and precise to within ~100 µm. Moreover, in another group of mice we were able to target both the mammillothalamic tract and subthalamic nucleus. This approach can be adapted to any type of extracellular electrode, fiber optic, or other probe in cases where high accuracy is needed in awake, head-fixed rodents.NEW & NOTEWORTHY Accurate targeting of recording electrodes in awake head-restrained rodents is currently beyond our reach. We developed a device for stereotactic implantation of a custom head bar and a recording system that together allow the accurate and precise targeting of any brain structure, including deep and small nuclei. We demonstrated this by performing histology and intracellular recordings in the amygdala of awake mice. The system enables the targeting of any probe to any location in the awake brain.

Membrane Potential Correlates of Network Decorrelation and Improved SNR by Cholinergic Activation in the Somatosensory Cortex.

The nucleus basalis (NB) projects cholinergic axons to the cortex, where they play a major role in arousal, attention, and learning. Cholinergic inputs shift cortical dynamics from synchronous to asynchronous and improve the signal-to-noise ratio (SNR) of sensory responses. However, the underlying mechanisms of these changes remain unclear. Using simultaneous extracellular and whole-cell patch recordings in layer 4 of the mouse barrel cortex, we show that electrical or optogenetic activation of the cholinergic system has a differential effect on ongoing and sensory evoked activities. Cholinergic activation profoundly reduced the large spontaneous fluctuations in membrane potential and decorrelated ongoing activity. However, NB stimulation had no effect on the response to whisker stimulation or on signal correlations. These effects of cholinergic activation provide a unified explanation for the increased SNR of sensory response and for the reduction in noise correlations and explain the shift into the desynchronized cortical state, which are the hallmarks of arousal and attention.SIGNIFICANCE STATEMENT Attention increases the signal-to-noise ratio (SNR) of cortical sensory response, which may reflect either reduction in background firing rate or increased sensory response. Extracellular recordings showed that attention also reduces the correlation in network activity. These effects are partially mediated by cholinergic axons from the nucleus basalis projecting to the entire cortex. To reveal the cellular and synaptic correlates of these cholinergic effects, we performed simultaneous intracellular and LFP recordings in the somatosensory cortex. Global or local cholinergic activation increased the SNR of sensory response mainly by reducing the rate and amplitude of background synaptic activity and also reduced network correlations. Therefore, coding of sensory information is enhanced by the cholinergic system mainly due to a reduction in spontaneous activity.

Cortical balance of excitation and inhibition is regulated by the rate of synaptic activity.

Cortical activity is determined by the balance between excitation and inhibition. To examine how shifts in brain activity affect this balance, we recorded spontaneous excitatory and inhibitory synaptic inputs into layer 4 neurons from rat somatosensory cortex while altering the depth of anesthesia. The rate of excitatory and inhibitory events was reduced by ∼50% when anesthesia was deepened. However, whereas both the amplitude and width of inhibitory synaptic events profoundly increased under deep anesthesia, those of excitatory events were unaffected. These effects were found using three different types of anesthetics, suggesting that they are caused by the network state and not by local specific action of the anesthetics. To test our hypothesis that the size of inhibitory events increased because of the decreased rate of synaptic activity under deep anesthesia, we blocked cortical excitation and replayed the slow and fast patterns of inhibitory inputs using intracortical electrical stimulation. Evoked inhibition was larger under low-frequency stimulation, and, importantly, this change occurred regardless of the depth of anesthesia. Hence, shifts in the balance between excitation and inhibition across distinct states of cortical activity can be explained by the rate of inhibitory inputs combined with their short-term plasticity properties, regardless of the actual global brain activity.

Opposite adaptive processing of stimulus intensity in two major nuclei of the somatosensory brainstem.

Tactile information ascends from the brainstem to the somatosensory cortex via two major parallel pathways, lemniscal and paralemniscal. In both pathways, and throughout all processing stations, adaptation effects are evident. Although parallel processing of sensory information is not unique to this system, the distinct information carried by these adaptive pathways remains unclear. Using in vivo intracellular recordings at their divergence point (brainstem trigeminal complex) in rats, we found opposite adaptation effects in the corresponding nuclei of these two pathways. Increasing the intensity of vibrissa stimulation entailed more adaption in paralemniscal neurons, whereas it caused less adaptation in lemniscal cells. Furthermore, increasing the intensity sharpens lemniscal receptive field profile as adaptation progresses. We hypothesize that these pathways evolved to operate optimally at different dynamic ranges of sustained sensory stimulation. Accordingly, the two pathways are likely to serve different functional roles in the transmission of weak and strong inputs. Hence, our results suggest that due to the disparity in the adaptation properties of two major parallel pathways in this system, high and reliable throughput of information can be achieved at a wider range of stimulation intensities than by each pathway alone.

Imbalance between excitation and inhibition in the somatosensory cortex produces postadaptation facilitation.

Adaptation is typically associated with attenuation of the neuronal response during sustained or repetitive sensory stimulation, followed by a gradual recovery of the response to its baseline level thereafter. Here, we examined the process of recovery from sensory adaptation in layer IV cells of the rat barrel cortex using in vivo intracellular recordings. Surprisingly, in approximately one-third of the cells, the response to a test stimulus delivered a few hundred milliseconds after the adapting stimulation was significantly facilitated. Recordings under different holding potentials revealed that the enhanced response was the result of an imbalance between excitation and inhibition, where a faster recovery of excitation compared with inhibition facilitated the response. Hence, our data provide the first mechanistic explanation of sensory facilitation after adaptation and suggest that adaptation increases the sensitivity of cortical neurons to sensory stimulation by altering the balance between excitation and inhibition.

Optopatcher--an electrode holder for simultaneous intracellular patch-clamp recording and optical manipulation.

Optogenetics has rapidly become a standard method in neuroscience research. Although significant progress has been made in the development of molecular tools, refined techniques for combined light delivery and recording in vivo are still lacking. For example, simultaneous intracellular recording and light stimulation have only been possible by using two separate positioning systems. To overcome this limitation, we have developed a glass pipette holder which contains an additional port for the insertion of an optical fiber into the pipette. This device, which we called "optopatcher" allows whole cell patch-clamp recording simultaneously with direct projection of light from the recording pipette. The holder spares the use of an additional manipulator and, importantly, enables accurate, stable and reproducible illumination. In addition, replacement of standard pipettes is done as easily as with the available commercial holders. Here we used the optopatcher in vivo to record the membrane potential of neurons from different cortical layers in the motor cortex of transgenic mice expressing channelrhodopsin-2 under the Thy1 promoter. We demonstrate the utility of the optopatcher by recording LFP and intracellular responses to light stimulation.

Trial-to-trial correlation between thalamic sensory response and global EEG activity.

Thalamic gating of sensory inputs to the cortex varies with behavioral conditions, such as sleep-wake cycles, or with different stages of anesthesia. Behavioral conditions in turn are accompanied by stereotypic spectral content of the EEG. In the rodent somatosensory system, the receptive field size of the ventral posteromedial thalamic nucleus (VPM) shrinks when anesthesia is deepened. Here we examined whether evoked thalamic responses are correlated with global EEG activity on a fine time scale of a few seconds. Trial-by-trial analysis of responses of VPM cells to whisker stimulation in lightly anesthetized rats indicated that increased EEG power in the delta band (1-4 Hz) was accompanied by a small, but highly significant, reduction in spontaneous and evoked thalamic firing. The opposite effect was found for the gamma EEG band (30-50 Hz). These significant correlations were not accompanied by an apparent change in the size of the receptive fields and were not EEG phase-related. The correlation between EEG and firing rate was observed only in neurons that responded to multiple whiskers and was higher for the non-principal whiskers. Importantly, the contributions of the two EEG bands to the modulation of VPM responses were to a large extent independent of each other. Our findings suggest that information conveyed by different whiskers can be rapidly modulated according to the global brain activity.

Intensity-dependent adaptation of cortical and thalamic neurons is controlled by brainstem circuits of the sensory pathway.

Current views of sensory adaptation in the rat somatosensory system suggest that it results mainly from short-term synaptic depression. Experimental and theoretical studies predict that increasing the intensity of sensory stimulation, followed by an increase in firing probability at early sensory stages, is expected to attenuate the response at later stages disproportionately more than weaker stimuli, due to greater depletion of synaptic resources and the relatively slow recovery process. This may lead to coding ambiguity of stimulus intensity during adaptation. In contrast, we found that increasing the intensity of repetitive whisker stimulation entails less adaptation in cortical neurons. In a series of recordings, from the trigeminal ganglion to the thalamus, we pinpointed the source of the unexpected pattern of adaptation to the brainstem trigeminal complex. We suggest that low-level sensory processing counterbalances later effects of short-term synaptic depression by increasing the throughput of high-intensity sensory inputs.

Shift in the balance between excitation and inhibition during sensory adaptation of S1 neurons.

Sustained stimulation of sensory organs results in adaptation of the neuronal response along the sensory pathway. Whether or not cortical adaptation affects equally excitation and inhibition is poorly understood. We examined this question using patch recordings of neurons in the barrel cortex of anesthetized rats while repetitively stimulating the principal whisker. We found that inhibition adapts more than excitation, causing the balance between them to shift toward excitation. A comparison of the latency of thalamic firing and evoked excitation and inhibition in the cortex strongly suggests that adaptation of inhibition results mostly from depression of inhibitory synapses rather than adaptation in the firing of inhibitory cells. The differential adaptation of the evoked conductances that shifts the balance toward excitation may act as a gain mechanism which enhances the subthreshold response during sustained stimulation, despite a large reduction in excitation.

Stochastic emergence of repeating cortical motifs in spontaneous membrane potential fluctuations in vivo.

It was recently discovered that subthreshold membrane potential fluctuations of cortical neurons can precisely repeat during spontaneous activity, seconds to minutes apart, both in brain slices and in anesthetized animals. These repeats, also called cortical motifs, were suggested to reflect a replay of sequential neuronal firing patterns. We searched for motifs in spontaneous activity, recorded from the rat barrel cortex and from the cat striate cortex of anesthetized animals, and found numerous repeating patterns of high similarity and repetition rates. To test their significance, various statistics were compared between physiological data and three different types of stochastic surrogate data that preserve dynamical characteristics of the recorded data. We found no evidence for the existence of deterministically generated cortical motifs. Rather, the stochastic properties of cortical motifs suggest that they appear by chance, as a result of the constraints imposed by the coarse dynamics of subthreshold ongoing activity.

Cross-whisker adaptation of neurons in the rat barrel cortex.

Neurons in the barrel cortex and the thalamus respond preferentially to stimulation of one whisker (the principal whisker) and weakly to several adjacent whiskers. Cortical neurons, unlike thalamic cells, gradually adapt to repeated whisker stimulations. Whether cortical adaptation is specific to the stimulated whisker is not known. The aim of this intracellular study was to determine whether the response of a cortical cell to stimulation of an adjacent whisker would be affected by previous adaptation induced by stimulation of the principal whisker and vice versa. Using a high-frequency stimulation that causes substantial adaptation in the cortex and much less adaptation in the thalamus, we show that cortical adaptation evoked by a train of stimuli applied to one whisker does not affect the synaptic response to subsequent stimulation of a neighboring whisker. Our data indicate that intrinsic mechanisms are not involved in cortical adaptation. Thalamic recordings obtained under the same conditions demonstrated that an adjacent whisker response was not generated in the thalamus, indicating that the observed whisker-specific adaptation results from diverging thalamic inputs or from cortical integration.