ABSTRACT The ears of stridulating crickets are exposed to loud self-generated sounds that might desensitise the auditory system and reduce its responsiveness to environmental sounds. We examined whether crickets prevent self-induced auditory desensitisation, and measured the responsiveness of the peripheral auditory system of the cricket (acoustic spiracle, tympanic membrane and tympanic nerve) during pharmacologically induced sonorous (two-winged) and silent (one-winged) stridulation. The acoustic spiracles remained open during stridulation, so the self-generated auditory signal had full access to both the external side and the internal side of the tympanic membrane. When the spiracles shut in resting crickets, the responsiveness of the tympanic membrane to acoustic stimuli varied according to the phase of ventilation and was minimal during expiration. The tympanic membrane oscillated in phase with the self-generated sounds during sonorous chirps and did not oscillate during silent chirps. In both sonorously and silently singing crickets, the responses of the tympanic membrane to acoustic stimuli were identical during the chirps and the chirp intervals. Bursts of activity were recorded in the tympanic nerve during sonorous chirps; however, activity was minor during silent chirps. In sonorously and in silently singing crickets, the summed nerve response to acoustic stimuli in the chirp intervals was the same as in resting crickets. The response to stimuli presented during the syllable intervals of sonorous chirps was slightly reduced compared with the response in the chirp intervals as a consequence of receptor habituation. In silently singing crickets, acoustic stimuli elicited the same summed nerve response during chirps and chirp intervals. These data indicate that in the cricket no specific mechanism acts to reduce the responsiveness of the peripheral auditory pathway during stridulation.
Abstract Optogenetics has revolutionized neurobiological research by providing tools for modulating neuronal activity. As these tools utilise light-activated ion fluxes, they afford new opportunities to examine the nature of transmembrane ion gradients. Traditional investigation into the equilibrium potential for chloride (E Cl ) has been limited to studying endogenous chloride-permeable receptors. Here we demonstrate the utility of using a light-activated chloride channel, stGtACR2, to probe somatic E Cl in rodent. This agonist-independent optogenetic strategy is validated in vitro and in vivo , captures differences in E Cl dynamics following manipulations of endogenous chloride fluxes, and reveals distinct resting E Cl across genetically-defined neuronal subpopulations. Using this approach to challenge chloride homeostasis, we uncover cell-specific E Cl dynamics that are supported by the differential expression of endogenous handling mechanisms. Our findings establish an optical method for investigating transmembrane chloride gradients and thereby expand the repertoire of optogenetics.
Optical imaging techniques are widely used in biological research, but their penetration depth is limited by tissue scattering. Wavefront shaping techniques are able to overcome this problem in principle, but are often slow and their performance depends on the sample. This greatly reduces their practicability for biological applications. Here we present a scattering compensation technique based on three-photon (3P) excitation, which converges faster than comparable two-photon (2P) techniques and works reliably even on densely labeled samples, where 2P approaches fail. To demonstrate its usability and advantages for biomedical imaging we apply it to the imaging of dendritic spines on GFP-labeled layer 5 neurons in an anesthetized mouse.
The sensory responses in the barrel cortex of mice aged postnatal day (P)7-P12 evoked by a single whisker deflection are smaller in amplitude and spread over a smaller area than those measured in P13-P21 mice. However, repetitive 10-Hz stimulation or paired pulse whisker stimulation in P7-P12 mice evoked facilitating sensory responses, contrasting with the depressing sensory responses observed in P13-P21 mice. This facilitation occurred during an interval ranging 300-1,000 ms after the first stimulus and was measured using whole cell recordings, voltage-sensitive dye imaging, and calcium-sensitive dye imaging. The facilitated responses were not only larger in amplitude but also propagated over a larger cortical area. The facilitation could be blocked by local application of pharmacological agents reducing cortical excitability. Local cortical microstimulation could substitute for the first whisker stimulus to produce a facilitated sensory response. The enhanced sensory responses evoked by repetitive sensory stimuli in P7-P12 mice may contribute to the activity-dependent specification of the developing cortical circuits. In addition, the facilitating sensory responses allow long integration times for sensory processing compatible with the slow behavior of mice during early postnatal development.
Abstract It has long been known that orofacial movements for feeding can be triggered, coordinated, and often rhythmically organized at the level of the brainstem, without input from higher centers. We uncover two nuclei that can organize the movements for ingesting fluids in mice. These neuronal groups, IRt Phox2b and Peri5 Atoh1 , are marked by expression of the pan-autonomic homeobox gene Phox2b and are located, respectively, in the intermediate reticular formation of the medulla and around the motor nucleus of the trigeminal nerve. They are premotor to all jaw-opening and tongue muscles. Stimulation of either, in awake animals, opens the jaw, while IRt Phox2b alone also protracts the tongue. Moreover, stationary stimulation of IRt Phox2b entrains a rhythmic alternation of tongue protraction and retraction, synchronized with jaw opening and closing, that mimics lapping. Finally, fiber photometric recordings show that IRt Phox2b is active during volitional lapping. Our study identifies one of the subcortical nuclei underpinning a stereotyped feeding behavior.
How do animals discriminate self-generated from external stimuli during behavior and prevent desensitization of their sensory pathways? A fundamental concept in neuroscience states that neural signals, termed corollary discharges or efference copies, are forwarded from motor to sensory areas. Neurons mediating these signals have proved difficult to identify. We show that a single, multisegmental interneuron is responsible for the pre- and postsynaptic inhibition of auditory neurons in singing crickets (Gryllus bimaculatus). Therefore, this neuron represents a corollary discharge interneuron that provides a neuronal basis for the central control of sensory responses.
Summary Humans easily discriminate tiny skin temperature changes that are perceived as warming or cooling. Dedicated thermoreceptors forming distinct thermosensory channels or “labelled lines” are thought to underlie thermal perception. We show that mice have similar perceptual thresholds for forepaw warming to humans (~1 °C change) and do not mistake warming for cooling. Mice perform warm discrimination tasks without dedicated thermoreceptors, but use information carried by unmyelinated polymodal C-fibers. Deletion of the heat-sensitive transduction channels TRPM2 and TRPV1 did not impact warming perception or afferent coding of warm. However, without the cold sensitive TRPM8 channel, afferent coding of cooling was impaired and these mice cannot perceive warming or cooling. Our data is incompatible with the existence of thermospecific labelled lines, but can be reconciled by the existence of central circuits that compare and integrate the input from at least two types of polymodal afferents, hitherto thought to exclusively signal pain.
Abstract The patch-clamp technique has revolutionized neurophysiology by allowing to study single neuronal excitability, synaptic connectivity, morphology, and the transcriptomic profile. However, the throughput in recordings is limited because of the manual replacement of patch-pipettes after each attempt which are often also unsuccessful. This has been overcome by automated cleaning the tips in detergent solutions, allowing to reuse the pipette for further recordings. Here, we developed a novel method of automated cleaning by sonicating the tips within the bath solution wherein the cells are placed, reducing the risk of contaminating the bath solution or internal solution of the recording pipette by any detergent and avoiding the necessity of a separate chamber for cleaning. We showed that the patch-pipettes can be used consecutively at least ten times and that the cleaning process does not negatively impact neither the brain slices nor other patched neurons. This method, combined with automated patch-clamp, highly improves the throughput for single and especially multiple recordings.
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