Multiple two-photon targeted whole-cell patch-clamp recordings from monosynaptically connected neurons in vivo

Although we know a great deal about monosynaptic connectivity, transmission and integration in the mammalian nervous system from in vitro studies, very little is known in vivo. This is partly because it is technically difficult to evoke action potentials and simultaneously record small amplitude subthreshold responses in closely (<150 µm) located pairs of neurons. To address this, we have developed in vivo two-photon targeted multiple (2–4) whole-cell patch clamp recordings of nearby neurons in superficial cortical layers 1–3. Here, we describe a step-by-step guide to this approach in the anesthetized mouse primary somatosensory cortex, including: the design of the setup, surgery, preparation of pipettes, targeting and acquisition of multiple whole-cell recordings, as well as in vivo and post hoc histology. The procedure takes ∼4 h from start of surgery to end of recording and allows examinations both into the electrophysiological features of unitary excitatory and inhibitory monosynaptic inputs during different brain states as well as the synaptic mechanisms of correlated neuronal activity

Corollary discharge inhibition of ascending auditory neurons in the stridulating cricket

Acoustically communicating animals are able to process external acoustic stimuli despite generating intense sounds during vocalization. We have examined how the crickets' ascending auditory pathway copes with self-generated, intense auditory signals (chirps) during singing (stridulation). We made intracellular recordings from two identified ascending auditory interneurons, ascending neuron 1 (AN1) and ascending neuron 2 (AN2), during pharmacologically elicited sonorous (two-winged), silent (one-winged), and fictive (isolated CNS) stridulation. During sonorous chirps, AN1 responded with bursts of spikes, whereas AN2 was inhibited and rarely spiked. Low-amplitude hyperpolarizing potentials were recorded in AN1 and AN2 during silent chirps. The potentials were also present during fictive chirps. Therefore, they were the result of a centrally generated corollary discharge from the stridulatory motor network. The spiking response of AN1 and AN2 to acoustic stimuli was inhibited during silent and fictive chirps. The maximum period of inhibition occurred in phase with the maximum spiking response to self-generated sound in a sonorously stridulating cricket. In some experiments (30%) depolarizing potentials were recorded during silent chirps. Reafferent feedback elicited by wing movement was probably responsible for the depolarizing potentials. In addition, two other sources of inhibition were present in AN1: (1) IPSPs were elicited by stimulation with 12.5 kHz stimuli and (2) a long-lasting hyperpolarization followed spiking responses to 4.5 kHz stimuli. The hyperpolarization desensitized the response of AN1 to subsequent quieter stimuli. Therefore, the corollary discharge will reduce desensitization by suppressing the response of AN1 to self-generated sounds

The cortical states of wakefulness

Cortical neurons process information on a background of spontaneous, ongoing activity with distinct spatiotemporal profiles defining different cortical states. During wakefulness, cortical states alter constantly in relation to behavioral context, attentional level or general motor activity. In this review article, we will discuss our current understanding of cortical states in awake rodents, how they are controlled, their impact on sensory processing, and highlight areas for future research. A common observation in awake rodents is the rapid change in spontaneous cortical activity from high-amplitude, low-frequency (LF) fluctuations, when animals are quiet, to faster and smaller fluctuations when animals are active. This transition is typically thought of as a change in global brain state but recent work has shown variation in cortical states across regions, indicating the presence of a fine spatial scale control system. In sensory areas, the cortical state change is mediated by at least two convergent inputs, one from the thalamus and the other from cholinergic inputs in the basal forebrain. Cortical states have a major impact on the balance of activity between specific subtypes of neurons, on the synchronization between nearby neurons, as well as the functional coupling between distant cortical areas. This reorganization of the activity of cortical networks strongly affects sensory processing. Thus cortical states provide a dynamic control system for the moment-by-moment regulation of cortical processing

Single synaptic inputs drive high-precision action potentials in parvalbumin expressing GABA-ergic cortical neurons in vivo

A defining feature of cortical layer 2/3 excitatory neurons is their sparse activity, often firing in singlets of action potentials. Local inhibitory neurons are thought to play a major role in regulating sparseness, but which cell types are recruited by single excitatory synaptic inputs is unknown. Using multiple, targeted, in vivo whole-cell recordings, we show that single EPSPs have little effect on the firing rates of excitatory neurons and somatostatin-expressing GABA-ergic inhibitory neurons but evoke precisely timed action potentials in parvalbumin-expressing inhibitory neurons. Despite a EPSP decay time of 7.8 ms, the evoked action potentials were almost completely restricted to the EPSP rising phase (~0.5 ms). Evoked parvalbumin-expressing neuron action potentials go on to inhibit the local excitatory network, thus providing a pathway for single spike evoked disynaptic inhibition which may enforce sparse and precisely timed cortical signaling

Matching cell type to function in cortical circuits

In this issue of Neuron, Pinto and Dan (2015) performed single-cell calcium imaging in the mouse dorsomedial prefrontal cortex to reveal correlated, cell-type-specific responses in three major GABA-ergic interneuron subtypes during a goal-directed sensory discrimination task

The neural circuits of thermal perception

Thermal information about skin surface temperature is a key sense for the perception of object identity and valence. The identification of ion channels involved in the transduction of thermal changes has provided a genetic access point to the thermal system. However, from sensory specific 'labeled-lines' to multimodal interactive pathways, the functional organization and identity of the neural circuits mediating innocuous thermal perception have been debated for over 100 years. Here we highlight points in the system that require further attention and review recent advances using in vivo electrophysiology, cellular resolution calcium imaging, optogenetics and thermal perceptual tasks in behaving mice that have begun to uncover the anatomical principles and neural processing mechanisms underlying innocuous thermal perception

The cellular coding of temperature in the mammalian cortex

Temperature is a fundamental sensory modality separate from touch, with dedicated receptor channels and primary afferent neurons for cool and warm. Unlike for other modalities, however, the cortical encoding of temperature remains unknown, with very few cortical neurons reported that respond to non-painful temperature, and the presence of a 'thermal cortex' is debated. Here, using widefield and two-photon calcium imaging in the mouse forepaw system, we identify cortical neurons that respond to cooling and/or warming with distinct spatial and temporal response properties. We observed a representation of cool, but not warm, in the primary somatosensory cortex, but cool and warm in the posterior insular cortex (pIC). The representation of thermal information in pIC is robust and somatotopically arranged, and reversible manipulations show a profound impact on thermal perception. Despite being positioned along the same one-dimensional sensory axis, the encoding of cool and that of warm are distinct, both in highly and broadly tuned neurons. Together, our results show that pIC contains the primary cortical representation of skin temperature and may help explain how the thermal system generates sensations of cool and warm

Brain-wide connectivity map of mouse thermosensory cortices

In the thermal system, skin cooling is represented in the primary somatosensory cortex (S1) and the posterior insular cortex (pIC). Whether S1 and pIC are nodes in anatomically separate or overlapping thermal sensorimotor pathways is unclear, as the brain-wide connectivity of the thermal system has not been mapped. We address this using functionally targeted, dual injections of anterograde viruses or retrograde tracers into the forelimb representation of S1 (fS1) and pIC (fpIC). Our data show that inputs to fS1 and fpIC originate from separate neuronal populations, supporting the existence of parallel input pathways. Outputs from fS1 and fpIC are more widespread than their inputs, sharing a number of cortical and subcortical targets. While, axonal projections were separable, they were more overlapping than the clusters of input cells. In both fS1 and fpIC circuits, there was a high degree of reciprocal connectivity with thalamic and cortical regions, but unidirectional output to the midbrain and hindbrain. Notably, fpIC showed connectivity with regions associated with thermal processing. Together, these data indicate that cutaneous thermal information is routed to the cortex via parallel circuits and is forwarded to overlapping downstream regions for the binding of somatosensory percepts and integration with ongoing behavior

Functional diversity of subicular principal cells during hippocampal ripples

Cortical and hippocampal oscillations play a crucial role in the encoding, consolidation, and retrieval of memory. Sharp-wave associated ripples have been shown to be necessary for the consolidation of memory. During consolidation, information is transferred from the hippocampus to the neocortex. One of the structures at the interface between hippocampus and neocortex is the subiculum. It is therefore well suited to mediate the transfer and distribution of information from the hippocampus to other areas. By juxtacellular and whole-cell-recordings in awake mice, we show here that in the subiculum a subset of pyramidal cells is activated, whereas another subset is inhibited during ripples. We demonstrate that these functionally different subgroups are predetermined by their cell subtype. Bursting cells are selectively used to transmit information during ripples, whereas the firing probability in regular firing cells is reduced. With multiple patch-clamp recordings in vitro, we show that the cell subtype-specific differences extend into the local network topology. This is reflected in an asymmetric wiring scheme where bursting cells and regular firing cells are recurrently connected among themselves but connections between subtypes exclusively exist from regular to bursting cells. Furthermore, inhibitory connections are more numerous onto regular firing cells than onto bursting cells. We conclude that the network topology contributes to the observed functional diversity of subicular pyramidal cells during sharp-wave associated ripples

A somatosensory circuit for cooling perception in mice

The temperature of an object provides important somatosensory information for animals performing tactile tasks. Humans can perceive skin cooling of less than one degree, but the sensory afferents and central circuits that they engage to enable the perception of surface temperature are poorly understood. To address these questions, we examined the perception of glabrous skin cooling in mice. We found that mice were also capable of perceiving small amplitude skin cooling and that primary somatosensory (S1) cortical neurons were required for cooling perception. Moreover, the absence of the menthol-gated transient receptor potential melastatin 8 ion channel in sensory afferent fibers eliminated the ability to perceive cold and the corresponding activation of S1 neurons. Our results identify parts of a neural circuit underlying cold perception in mice and provide a new model system for the analysis of thermal processing and perception and multimodal integration