Light-evoked somatosensory perception of transgenic rats that express channelrhodopsin-2 in dorsal root ganglion cells

In vertebrate somatosensory systems, each mode of touch-pressure, temperature or pain is sensed by sensory endings of different dorsal root ganglion (DRG) neurons, which conducted to the specific cortical loci as nerve impulses. Therefore, direct electrical stimulation of the peripheral nerve endings causes an erroneous sensation to be conducted by the nerve. We have recently generated several transgenic lines of rat in which channelrhodopsin-2 (ChR2) transgene is driven by the Thy-1.2 promoter. In one of them, W-TChR2V4, some neurons were endowed with photosensitivity by the introduction of the ChR2 gene, coding an algal photoreceptor molecule. The DRG neurons expressing ChR2 were immunohistochemically identified using specific antibodies to the markers of mechanoreceptive or nociceptive neurons. Their peripheral nerve endings in the plantar skin as well as the central endings in the spinal cord were also examined. We identified that ChR2 is expressed in a certain population of large neurons in the DRG of W-TChR2V4. On the basis of their morphology and molecular markers, these neurons were classified as mechanoreceptive but not nociceptive. ChR2 was also distributed in their peripheral sensory nerve endings, some of which were closely associated with CK20-positive cells to form Merkel cell-neurite complexes or with S-100-positive cells to form structures like Meissner's corpuscles. These nerve endings are thus suggested to be involved in the sensing of touch. Each W-TChR2V4 rat showed a sensory-evoked behavior in response to blue LED flashes on the plantar skin. It is thus suggested that each rat acquired an unusual sensory modality of sensing blue light through the skin as touch-pressure. This light-evoked somatosensory perception should facilitate study of how the complex tactile sense emerges in the brain.     

Robo2 determines subtype-specific axonal projections of trigeminal sensory neurons

How neurons connect to form functional circuits is central to the understanding of the development and function of the nervous system. In the somatosensory system, perception of sensory stimuli to the head requires specific connections between trigeminal sensory neurons and their many target areas in the central nervous system. Different trigeminal subtypes have specialized functions and downstream circuits, but it has remained unclear how subtype-specific axonal projection patterns are formed. Using zebrafish as a model system, we followed the development of two trigeminal sensory neuron subtypes: one that expresses trpa1b, a nociceptive channel important for sensing environmental chemicals; and a distinct subtype labeled by an islet1 reporter (Isl1SS). We found that Trpa1b and Isl1SS neurons have overall similar axon trajectories but different branching morphologies and distributions of presynaptic sites. Compared with Trpa1b neurons, Isl1SS neurons display reduced branch growth and synaptogenesis at the hindbrain-spinal cord junction. The subtype-specific morphogenesis of Isl1SS neurons depends on the guidance receptor Robo2. robo2 is preferentially expressed in the Isl1SS subset and inhibits branch growth and synaptogenesis. In the absence of Robo2, Isl1SS afferents acquire many of the characteristics of Trpa1b afferents. These results reveal that subtype-specific activity of Robo2 regulates subcircuit morphogenesis in the trigeminal sensory system.     

Ionic and neuromodulatory regulation of burst discharge controls frequency tuning

Sensory neurons encode natural stimuli by changes in firing rate or by generating specific firing patterns, such as bursts. Many neural computations rely on the fact that neurons can be tuned to specific stimulus frequencies. It is thus important to understand the mechanisms underlying frequency tuning. In the electrosensory system of the weakly electric fish, Apteronotus leptorhynchus, the primary processing of behaviourally relevant sensory signals occurs in pyramidal neurons of the electrosensory lateral line lobe (ELL). These cells encode low frequency prey stimuli with bursts of spikes and high frequency communication signals with single spikes. We describe here how bursting in pyramidal neurons can be regulated by intrinsic conductances in a cell subtype specific fashion across the sensory maps found within the ELL, thereby regulating their frequency tuning. Further, the neuromodulatory regulation of such conductances within individual cells and the consequences to frequency tuning are highlighted. Such alterations in the tuning of the pyramidal neurons may allow weakly electric fish to preferentially select for certain stimuli under various behaviourally relevant circumstances.     

Arrays of MicroLEDs and Astrocytes: Biological Amplifiers to Optogenetically Modulate Neuronal Networks Reducing Light Requirement

In the modern view of synaptic transmission, astrocytes are no longer confined to the role of merely supportive cells. Although they do not generate action potentials, they nonetheless exhibit electrical activity and can influence surrounding neurons through gliotransmitter release. In this work, we explored whether optogenetic activation of glial cells could act as an amplification mechanism to optical neural stimulation via gliotransmission to the neural network. We studied the modulation of gliotransmission by selective photo-activation of channelrhodopsin-2 (ChR2) and by means of a matrix of individually addressable super-bright microLEDs (μLEDs) with an excitation peak at 470 nm. We combined Ca²⁺ imaging techniques and concurrent patch-clamp electrophysiology to obtain subsequent glia/neural activity. First, we tested the μLEDs efficacy in stimulating ChR2-transfected astrocyte. ChR2-induced astrocytic current did not desensitize overtime, and was linearly increased and prolonged by increasing μLED irradiance in terms of intensity and surface illumination. Subsequently, ChR2 astrocytic stimulation by broad-field LED illumination with the same spectral profile, increased both glial cells and neuronal calcium transient frequency and sEPSCs suggesting that few ChR2-transfected astrocytes were able to excite surrounding not-ChR2-transfected astrocytes and neurons. Finally, by using the μLEDs array to selectively light stimulate ChR2 positive astrocytes we were able to increase the synaptic activity of single neurons surrounding it. In conclusion, ChR2-transfected astrocytes and μLEDs system were shown to be an amplifier of synaptic activity in mixed corticalneuronal and glial cells culture.     

Changing reference frames during the encoding of tactile events

The mindless act of swatting a mosquito on the hand poses a remarkable challenge for the brain. Given that the primary somatosensory cortex maps skin location independently of arm posture [1, 2], the brain must realign tactile coordinates in order to locate the origin of the stimuli in extrapersonal space. Previous studies have highlighted the behavioral relevance of such an external mapping of touch, which results from combining somatosensory input with proprioceptive and visual cues about body posture [3-7]. However, despite the widely held assumption about the existence of this remapping process from somatotopic to external space and various findings indirectly suggesting its consequences [8-11], a demonstration of its changing time course and nature was lacking. We examined the temporal course of this multisensory interaction and its implications for tactile awareness in humans using a crossmodal cueing paradigm [12, 13]. What we show is that before tactile events are referred to external locations [12-15], a fleeting, unconscious image of the tactile sensation abiding to a somatotopic frame of reference rules performance. We propose that this early somatotopic "glimpse" arises from the initial feed-forward sweep of neural activity to the primary somatosensory cortex, whereas the later externally-based, conscious experience reflects the activity of a somatosensory network involving recurrent connections from association areas.     

In vivo birthdating by BAPTISM reveals that trigeminal sensory neuron diversity depends on early neurogenesis

Among sensory systems, the somatic sense is exceptional in its ability to detect a wide range of chemical, mechanical and thermal stimuli. How this sensory diversity is established during development remains largely elusive. We devised a method (BAPTISM) that uses the photoconvertible fluorescent protein Kaede to simultaneously analyze birthdate and cell fate in live zebrafish embryos. We found that trigeminal sensory ganglia are formed from early-born and late-born neurons. Early-born neurons give rise to multiple classes of sensory neurons that express different ion channels. By contrast, late-born neurons are restricted in their fate and do not form chemosensory neurons expressing the ion channel TrpA1b. Accordingly, larvae lacking early-born neurons do not respond to the TrpA1b agonist allyl isothiocyanate. These results indicate that the multimodal specification and function of trigeminal sensory ganglia depends on the timing of neurogenesis.     

Prefrontal neurons predict choices during an auditory same-different task

The detection of stimuli is critical for an animal's survival [1]. However, it is not adaptive for an animal to respond automatically to every stimulus that is present in the environment [2-5]. Given that the prefrontal cortex (PFC) plays a key role in executive function [6-8], we hypothesized that PFC activity should be involved in context-dependent responses to uncommon stimuli. As a test of this hypothesis, monkeys participated in a same-different task, a variant of an oddball task [2]. During this task, a monkey heard multiple presentations of a "reference" stimulus that were followed by a "test" stimulus and reported whether these stimuli were the same or different. While they participated in this task, we recorded from neurons in the ventrolateral prefrontal cortex (vPFC; a cortical area involved in aspects of nonspatial auditory processing [9, 10]). We found that vPFC activity was correlated with the monkeys' choices. This finding demonstrates a direct link between single neurons and behavioral choices in the PFC on a nonspatial auditory task.     

Repulsive interactions shape the morphologies and functional arrangement of zebrafish peripheral sensory arbors

BACKGROUND: Trigeminal sensory neurons detect thermal and mechanical stimuli in the skin through their elaborately arborized peripheral axons. We investigated the developmental mechanisms that determine the size and shape of individual trigeminal arbors in zebrafish and analyzed how these interactions affect the functional organization of the peripheral sensory system. RESULTS: Time-lapse imaging indicated that direct repulsion between growing axons restricts arbor territories. Removal of one trigeminal ganglion allowed axons of the contralateral ganglion to cross the midline, and removal of both resulted in the expansion of spinal cord sensory neuron arbors. Generation of embryos with single, isolated sensory neurons resulted in axon arbors that possessed a vast capacity for growth and expanded to encompass the entire head. Embryos in which arbors were allowed to aberrantly cross the midline were unable to respond in a spatially appropriate way to mechanical stimuli. CONCLUSIONS: Direct repulsive interactions between developing trigeminal and spinal cord sensory axon arbors determine sensory neuron organization and control the shapes and sizes of individual arbors. This spatial organization is crucial for sensing the location of objects in the environment. Thus, a combination of undirected growth and mutual repulsion results in the formation of a functionally organized system of peripheral sensory arbors.     

Neuropilin 1 and 2 control cranial gangliogenesis and axon guidance through neural crest cells

Neuropilin (NRP) receptors and their class 3 semaphorin (SEMA3) ligands play well-established roles in axon guidance, with loss of NRP1, NRP2, SEMA3A or SEMA3F causing defasciculation and errors in growth cone guidance of peripherally projecting nerves. Here we report that loss of NRP1 or NRP2 also impairs sensory neuron positioning in the mouse head, and that this defect is a consequence of inappropriate cranial neural crest cell migration. Specifically, neural crest cells move into the normally crest-free territory between the trigeminal and hyoid neural crest streams and recruit sensory neurons from the otic placode; these ectopic neurons then extend axons between the trigeminal and facioacoustic ganglia. Moreover, we found that NRP1 and NRP2 cooperate to guide cranial neural crest cells and position sensory neurons; thus, in the absence of SEMA3/NRP signalling, the segmentation of the cranial nervous system is lost. We conclude that neuropilins play multiple roles in the sensory nervous system by directing cranial neural crest cells, positioning sensory neurons and organising their axonal projections.     

New transgenic reporters identify somatosensory neuron subtypes in larval zebrafish

To analyze somatosensory neuron diversity in larval zebrafish, we identified several enhancers from the zebrafish and pufferfish genomes and used them to create five new reporter transgenes. Sequential deletions of three of these enhancers identified small sequence elements sufficient to drive expression in zebrafish trigeminal and Rohon‐Beard (RB) neurons. One of these reporters, using the Fru.p2x3‐2 enhancer, highlighted a somatosensory neuron subtype that expressed both the p2rx3a and pkcα genes. Comparison with a previously described trpA1b reporter revealed that it highlighted the same neurons as the Fru.p2x3‐2 reporter. To determine whether neurons of this subtype possess characteristic peripheral branching morphologies or central axon projection patterns, we analyzed the morphology of single neurons. Surprisingly, although these analyses revealed diversity in peripheral axon branching and central axon projection, PKCα/p2rx3a/trpA1b‐expressing RB cells did not possess obvious characteristic morphological features, suggesting that even within this molecularly defined subtype, individual neurons may possess distinct properties. The new transgenes created in this study will be powerful tools for further characterizing the molecular, morphological, and developmental diversity of larval somatosensory neurons. © 2012 Wiley Periodicals, Inc., 2013     

Zebrafish collapsin response mediator protein (CRMP)-2 is expressed in developing neurons

The collapsin response mediator proteins (CRMPs) are highly expressed in the vertebrate nervous system. CRMP2 has been shown to function in Semaphorin and lysophosphatidic acid induced growth cone collapse. Correspondingly, the highest levels of CRMP2 protein are found in the distal portion of growing axons. To understand the role of CRMP2 during embryonic development we have documented its expression pattern in zebrafish embryos at multiple stages. We find that CRMP2 is expressed in the major neural clusters of the embryonic brain during the primary stages of neurogenesis. From 20 somites through 30 hpf CRMP2 is expressed in the dorsal rostral cluster of the telencephalon, the ventral rostral cluster of the diencephalon, the ventral caudal cluster of the mesencephalon, and the hindbrain clusters. CRMP2 is also expressed in the trigeminal sensory ganglia and the Rohon Beard cells of the neural tube from 15 somites. By 48 hpf, we find expression of CRMP2 throughout the developing brain, trigeminal sensory ganglia, and Rohon Beard cells. CRMP2 is also detected in the retinal ganglion cell layer of the eye, and in the otic vesicle. Finally, we have compared the expression of CRMP2 to PlexinA4, a Semaphorin receptor expressed in sensory neurons, and find that their expression partially overlaps.     

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. Due to their external fertilization, rapid development, and translucency, zebrafish are particularly well suited for optogenetic approaches to investigate neural circuit function. In this approach, light-sensitive ion channels are expressed in specific neurons, enabling the experimenter to activate or inhibit them at will and thus assess their contribution to specific behaviors. Applying these methods in larval zebrafish is conceptually simple but requires the optimization of technical details. Here we demonstrate a procedure for expressing a channelrhodopsin variant in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. By introducing a few modifications to previously established methods, this approach could be used to elicit behavioral responses from single neurons activated up to at least 4 days post-fertilization (dpf). Specifically, we created a transgene using a somatosensory neuron enhancer, CREST3, to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Injecting this transgene into 1-cell stage embryos results in mosaic expression in somatosensory neurons, which can be imaged with confocal microscopy. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicits behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. This technique could be adapted to study behaviors elicited by activating any zebrafish neuron. Combining this approach with genetic or pharmacological perturbations will be a powerful way to investigate circuit formation and function.     

Inducible Prrxl1-CreER(T2) recombination activity in the somatosensory afferent pathway

Prrxl1-CreER(T2) transgenic mice expressing tamoxifen-inducible Cre recombinase were generated by modifying a Prrxl1-containing BAC clone. Cre recombination activity was examined in Prrxl1-CreER(T2); Rosa26 reporter mice at various embryonic and postnatal stages. Pregnant mice were treated with a single dose of tamoxifen at embryonic day (E) 9.5 or E12.5, and X-gal staining was performed 2 days later. Strong X-gal staining was observed in the somatosensory ganglia (e.g., dorsal root and trigeminal ganglia) and the first central sites for processing somatosensory information (e.g., spinal dorsal horn and trigeminal nerve-associated nuclei). When tamoxifen was administered at postnatal day (P) 20 or in adulthood (P120), strong Cre recombination activity was present in the primary somatosensory ganglia, while weak Cre recombination activity was found in the spinal dorsal horn, mesencephalic trigeminal nucleus, principal sensory trigeminal nucleus, and spinal trigeminal nucleus. This mouse line provides a useful tool for exploring genes' functions in the somatosensory system in a time-controlled way.     

Learned odor discrimination in Drosophila without combinatorial odor maps in the antennal lobe

A unifying feature of mammalian and insect olfactory systems is that olfactory sensory neurons (OSNs) expressing the same unique odorant-receptor gene converge onto the same glomeruli in the brain [1-7]. Most odorants activate a combination of receptors and thus distinct patterns of glomeruli, forming a proposed combinatorial spatial code that could support discrimination between a large number of odorants [8-11]. OSNs also exhibit odor-evoked responses with complex temporal dynamics [11], but the contribution of this activity to behavioral odor discrimination has received little attention [12]. Here, we investigated the importance of spatial encoding in the relatively simple Drosophila antennal lobe. We show that Drosophila can learn to discriminate between two odorants with one functional class of Or83b-expressing OSNs. Furthermore, these flies encode one odorant from a mixture and cross-adapt to odorants that activate the relevant OSN class, demonstrating that they discriminate odorants by using the same OSNs. Lastly, flies with a single class of Or83b-expressing OSNs recognize a specific odorant across a range of concentration, indicating that they encode odorant identity. Therefore, flies can distinguish odorants without discrete spatial codes in the antennal lobe, implying an important role for odorant-evoked temporal dynamics in behavioral odorant discrimination.     

Attentional load modulates responses of human primary visual cortex to invisible stimuli

Visual neuroscience has long sought to determine the extent to which stimulus-evoked activity in visual cortex depends on attention and awareness. Some influential theories of consciousness maintain that the allocation of attention is restricted to conscious representations [1, 2]. However, in the load theory of attention [3], competition between task-relevant and task-irrelevant stimuli for limited-capacity attention does not depend on conscious perception of the irrelevant stimuli. The critical test is whether the level of attentional load in a relevant task would determine unconscious neural processing of invisible stimuli. Human participants were scanned with high-field fMRI while they performed a foveal task of low or high attentional load. Irrelevant, invisible monocular stimuli were simultaneously presented peripherally and were continuously suppressed by a flashing mask in the other eye [4]. Attentional load in the foveal task strongly modulated retinotopic activity evoked in primary visual cortex (V1) by the invisible stimuli. Contrary to traditional views [1, 2, 5, 6], we found that availability of attentional capacity determines neural representations related to unconscious processing of continuously suppressed stimuli in human primary visual cortex. Spillover of attention to cortical representations of invisible stimuli (under low load) cannot be a sufficient condition for their awareness.     

Embryonic expression of a P2X(3) receptor encoding gene in zebrafish

From studies performed primarily in mammals, it is thought that the P2X(3) purinoreceptor is involved in mediating sensory and nociceptive signals in adult tissues. However, little is known concerning the expression or function of P2X family genes during early development. Here we describe the expression of a gene (p2x3) encoding a P2X(3) receptor during zebrafish development. We find that zebrafish p2x3 is expressed in the anlage of the trigeminal ganglion from very early stages of development, most likely in neural crest derived trigeminal cells as opposed to placode derived cells. p2x3 is also expressed in the spinal sensory Rohon-Beard cells and in the putative posterior lateral line ganglion.     

Encoding of Naturalistic Stimuli by Local Field Potential Spectra in Networks of Excitatory and Inhibitory Neurons

Recordings of local field potentials (LFPs) reveal that the sensory cortex displays rhythmic activity and fluctuations over a wide range of frequencies and amplitudes. Yet, the role of this kind of activity in encoding sensory information remains largely unknown. To understand the rules of translation between the structure of sensory stimuli and the fluctuations of cortical responses, we simulated a sparsely connected network of excitatory and inhibitory neurons modeling a local cortical population, and we determined how the LFPs generated by the network encode information about input stimuli. We first considered simple static and periodic stimuli and then naturalistic input stimuli based on electrophysiological recordings from the thalamus of anesthetized monkeys watching natural movie scenes. We found that the simulated network produced stimulus-related LFP changes that were in striking agreement with the LFPs obtained from the primary visual cortex. Moreover, our results demonstrate that the network encoded static input spike rates into gamma-range oscillations generated by inhibitory–excitatory neural interactions and encoded slow dynamic features of the input into slow LFP fluctuations mediated by stimulus–neural interactions. The model cortical network processed dynamic stimuli with naturalistic temporal structure by using low and high response frequencies as independent communication channels, again in agreement with recent reports from visual cortex responses to naturalistic movies. One potential function of this frequency decomposition into independent information channels operated by the cortical network may be that of enhancing the capacity of the cortical column to encode our complex sensory environment.