Ch Neurons Research Articles

A bidirectional network for appetite control in larval zebrafish.

Medial and lateral hypothalamic loci are known to suppress and enhance appetite, respectively, but the dynamics and functional significance of their interaction have yet to be explored. Here we report that, in larval zebrafish, primarily serotonergic neurons of the ventromedial caudal hypothalamus (cH) become increasingly active during food deprivation, whereas activity in the lateral hypothalamus (LH) is reduced. Exposure to food sensory and consummatory cues reverses the activity patterns of these two nuclei, consistent with their representation of opposing internal hunger states. Baseline activity is restored as food-deprived animals return to satiety via voracious feeding. The antagonistic relationship and functional importance of cH and LH activity patterns were confirmed by targeted stimulation and ablation of cH neurons. Collectively, the data allow us to propose a model in which these hypothalamic nuclei regulate different phases of hunger and satiety and coordinate energy balance via antagonistic control of distinct behavioral outputs.

The regulation of mechanosensory motile cilium formation

In contrast to the progress in understanding ciliogenesis and cilium function, we know less about the pathways for generating ciliary diversity. Drosophila has a variety of sensory neurons with ciliary dendrites that are structurally and functionally specialised for receiving different sensory modalities. For instance, chordotonal (Ch) neurons have mechanosensory ciliary dendrites and are required for proprioception and hearing. Time-course gene expression profiling of differentiating Ch neurons allowed us to characterise the roles of two transcription factors for ciliogenic gene regulation: the well-known cilia gene regulator, Rfx, and a factor of the Forkhead family (Fd3F), which appears to be a diverged homologue of FOXJ1. Fd3F and Rfx cooperate to regulate a cohort of genes required for ciliary motility - in Drosophila this is a specialisation unique to Ch neuron cilia and is essential for the hearing mechanism. Analysing the target genes of Fd3F has led to the implication of new factors in the assembly of axonemal dynein complexes. Two of these are also mutated in human primary ciliary dyskinesia. Further analysis of these genes in Drosophila and mouse suggests that ZMYND10 may be linked to an emerging chaperone pathway, while HEATR2 appears to have a distinct function related to transport.

High-throughput analysis of stimulus-evoked behaviors in Drosophila larva reveals multiple modality-specific escape strategies.

All organisms react to noxious and mechanical stimuli but we still lack a complete understanding of cellular and molecular mechanisms by which somatosensory information is transformed into appropriate motor outputs. The small number of neurons and excellent genetic tools make Drosophila larva an especially tractable model system in which to address this problem. We developed high throughput assays with which we can simultaneously expose more than 1,000 larvae per man-hour to precisely timed noxious heat, vibration, air current, or optogenetic stimuli. Using this hardware in combination with custom software we characterized larval reactions to somatosensory stimuli in far greater detail than possible previously. Each stimulus evoked a distinctive escape strategy that consisted of multiple actions. The escape strategy was context-dependent. Using our system we confirmed that the nociceptive class IV multidendritic neurons were involved in the reactions to noxious heat. Chordotonal (ch) neurons were necessary for normal modulation of head casting, crawling and hunching, in response to mechanical stimuli. Consistent with this we observed increases in calcium transients in response to vibration in ch neurons. Optogenetic activation of ch neurons was sufficient to evoke head casting and crawling. These studies significantly increase our understanding of the functional roles of larval ch neurons. More generally, our system and the detailed description of wild type reactions to somatosensory stimuli provide a basis for systematic identification of neurons and genes underlying these behaviors.

Dynamic Changes in Heat Transducing Channel TRPV1 Expression Regulate Mechanically Insensitive, Heat Sensitive C-Fiber Recruitment after Axotomy and Regeneration

Peripheral injury leads to a significant increase in the prevalence of mechanically insensitive, heat-sensitive C-fibers (CH) that contain the heat transducing TRPV1 (transient receptor potential vanilloid type I) channel in mice. We have recently shown that this recruitment of CH fibers is associated with increased expression of the receptor for GDNF (glial cell line-derived neurotrophic factor) family neurotrophic factor artemin (GFRα3), and that in vivo inhibition of GFRα3 prevented the increase in TRPV1 expression normally observed following axotomy. Here we have directly tested the hypothesis that the recruitment of functional CH fibers following nerve regeneration requires enhanced TRPV1 levels. We used in vivo siRNA-mediated knockdown to inhibit the injury-induced expression of TRPV1 coupled with ex vivo recording to examine response characteristics and neurochemical phenotypes of different functionally defined cutaneous sensory neurons after regeneration. We confirmed that inhibition of TRPV1 did not affect the axotomy-induced decrease in polymodal C-fiber (CPM) heat threshold, but transiently prevented the recruitment of CH neurons. Moreover, a recovery of TRPV1 protein was observed following resolution of siRNA-mediated inhibition that was correlated with a concomitant rebound in CH neuron recruitment. Thus dynamic changes in TRPV1 expression, not absolute levels, may underlie the functional alterations observed in CH neurons and may contribute to the development of heat hyperalgesia after nerve injury.

The transcriptional regulation of ciliogenesis in differentiating Drosophila sensory neurons

In contrast to the progress in understanding ciliogenesis and cilium function, we know less about the transcriptional regulation of ciliogenesis genes and how this regulatory program is modulated to generate diverse cilia. Drosophila sensory neurons have ciliary dendrites that are structurally and functionally specialised for receiving different sensory modalities. Time-course gene expression profiling of differentiating chordotonal (Ch) mechanosensory neurons allowed us to determine how Atonal, a proneural bHLH factor, regulates events leading to mechanosensory cilium formation and specialisation. Atonal regulates ciliogenesis via activation of two downstream transcription factors: the well-known cilia gene regulator, Rfx, and a novel factor of the Forkhead family (Fd3F). Rfx regulates a variety of ciliogenesis genes in all ciliated sensory neurons. In contrast, Fd3F is unique to Ch neurons, where it regulates a cohort of genes required for ciliary motility – a unique specialisation of Ch cilia in Drosophila and an essential part of the hearing mechanism. Among the targets of Fd3F are genes with human homologues linked to primary ciliary dyskinesia, a congenital condition resulting from defective ciliary motility. We provide evidence that Fd3F is a previously unrecognised orthologue of Foxj1, the so-called master regulator of motile ciliated cells in vertebrates. Interestingly, Fd3F and Rfx cooperate to regulate motility target genes directly via adjacent DNA binding sites, thus providing a mechanism for how Rfx can regulate genes for cilium specialisation in addition to ‘core’ ciliogenesis genes. Our study suggests how Foxj1 and Rfx factors may interact in vertebrates to generate ciliary diversity. http://www.ed.ac.uk/schools-departments/integrative-physiology/staff-profiles/andrew-jarman

Neural mechanism of binding amplitude information of echo sound with its frequency one in echolocating bat

Most species of bats making echolocation use the sound pressure level (SPL) and Doppler-shifted frequency of ultrasonic echo pulse to measure the size and velocity of target. The neural circuits for detecting these target features are specialized for amplitude and frequency analysis of the second harmonic constant frequency (CF2) component of Doppler-shifted echoes. The neuronal circuits involved in detecting these echo features have been well known [1]. In natural situation, large natural objects in environment, like bushes or trees, produce complex stochastic echoes, which can be characterized by the echo roughness. The echo signal reflecting from a target insect is embedded in the complex signal. Even in such an environment, bats can detect accurately the detailed information of flying insect. We consider here two questions: how the neural circuits bind amplitude and frequency information of echo sound, and how bat distinguishes target information from information of background signal. To address these issues, we developed a neural network model for detecting SPL amplitude and Doppler-shifted frequency of echo sound. The model contains two hemispheres, in each of which, the network model consists of cochlea (Ch), inferior colliculus (IC), and Doppler-shifted constant frequency (DSCF) processing area. The Ch network has a frequency map by which sound frequency is encoded. The model for detecting frequency information of echo sound was based on the model previously presented by us [2].The SPL amplitude is encoded in to firing rate of IC neurons. The IC neurons encode SPL amplitude by means of a balance between excitatory connection from contralateral Ch neurons and inhibitory connection from ipsilateral ones, and then combine the amplitude and frequency information of echo sound. The DSCF network has two types of sub-networks detecting AC and DC components of echo sound, which represent the information of target and background signals, respectively. We showed that in IC, the amplitude information of echo sound is encoded by integrating the outputs of ipsi and contralateral Ch neurons and then combined with the Doppler-shifted frequency information encoded by tonotopical map of Ch neurons. The accuracy of the amplitude and frequency information was improved in the DSCF area. The model reproduced well several experimental results observed in IC and DSCF neurons. We also showed that AC and DC components of echo signal are discriminated in the two sub-networks of DSCF. The discrimination ability is due to the difference in time constant between DSCF neurons in the two sub-networks.

A Combinatorial Semaphorin Code Instructs the Initial Steps of Sensory Circuit Assembly in the Drosophila CNS

Longitudinal axon fascicles within the Drosophila embryonic CNS provide connections between body segments and are required for coordinated neural signaling along the anterior-posterior axis. We show here that establishment of select CNS longitudinal tracts and formation of precise mechanosensory afferent innervation to the same CNS region are coordinately regulated by the secreted semaphorins Sema-2a and Sema-2b. Both Sema-2a and Sema-2b utilize the same neuronal receptor, plexin B (PlexB), but serve distinct guidance functions. Localized Sema-2b attraction promotes the initial assembly of a subset of CNS longitudinal projections and subsequent targeting of chordotonal sensory afferent axons to these same longitudinal connectives, whereas broader Sema-2a repulsion serves to prevent aberrant innervation. In the absence of Sema-2b or PlexB, chordotonal afferent connectivity within the CNS is severely disrupted, resulting in specific larval behavioral deficits. These results reveal that distinct semaphorin-mediated guidance functions converge at PlexB and are critical for functional neural circuit assembly.

Enhanced Artemin/GFRα3 Levels Regulate Mechanically Insensitive, Heat-Sensitive C-Fiber Recruitment after Axotomy and Regeneration

We have shown recently that following saphenous nerve transection and successful regeneration, cutaneous polymodal nociceptors (CPMs) lacking transient receptor potential vanilloid 1 (TRPV1) are sensitized to heat stimuli and that mechanically insensitive, heat-sensitive C-fibers (CHs) that contain TRPV1 increase in prevalence. Target-derived neurotrophic factor levels were also enhanced after axotomy and regeneration. In particular, the glial-cell line-derived neurotrophic factor (GDNF) family member artemin was found to be significantly enhanced in the hairy hindpaw skin and its receptor GDNF family receptor α3 (GFRα3) was increased in the L2/L3 dorsal root ganglia (DRGs) following nerve injury. In this study, we assessed the role of enhanced artemin/GFRα3 levels on the changes in mouse cutaneous CH neurons following saphenous nerve regeneration. We used a newly developed siRNA-mediated in vivo knockdown strategy to specifically inhibit the injury-induced expression of GFRα3 and coupled this with an ex vivo recording preparation to examine response characteristics and neurochemical phenotype of different types of functionally defined neurons after injury. We found that inhibition of GFRα3 did not affect the axotomy-induced decrease in CPM threshold, but transiently prevented the recruitment of CH neurons. Western blot and real-time PCR analysis of hairy hindpaw skin and L2/L3 DRGs after saphenous nerve regeneration suggested that inhibition of the potential initial injury-induced increase in enhanced target-derived artemin signaling resulted in dynamic changes in TRPV1 expression after regeneration. These changes in TRPV1 expression may underlie the functional alterations observed in CH neurons after nerve regeneration.

Chronic hypoxia alters anoxia‐induced activation of ATP‐sensitive K+ (KATP) channels in second‐order peripheral chemoreceptor neurons in the NTS

The impact of tissue hypoxia on 2nd‐order NTS neurons receiving chemoreceptor afferent inputs is unknown. We investigated the neuronal response to anoxia (bath solution bubbled in 95% N2/5% CO2 vs control 95% O2/5% CO2) in normoxic rats (NORM) and rats exposed to chronic hypoxia (CH; 7‐10 days at 10% FIO2). Whole‐cell recordings of NTS 2nd‐order neurons identified following DiA labeling of the carotid bodies were obtained in a brainstem slice. In 8 cells from NORM rats, anoxia (3 min) induced outward currents of 13.1±1.0 pA that reversed at −73±2 mV and decreased input resistance (59±2%). This current was completely blocked by the KATP channel blocker tolbutamide (100 μM). In NORM rats the KATP channel opener diazoxide (200 μM, 3 min) evoked an outward current of 27.7±5.5 pA (n=4), which was blocked by tolbutamide. In 10 CH neurons the same anoxia protocol induced a significantly smaller outward current (5.9±1.5 pA, p<0.01) and decreased input resistance (21±2%, p<0.01). In CH, diazoxide (200 μM, 3 min) induced an outward current significantly smaller than in NORM neurons (2.5±0.6 pA, n=4, p<0.01). These results suggest that anoxia activates KATP channels in 2nd‐order NTS neurons receiving chemoreceptor afferent inputs. CH reduces KATP channel function in NTS neurons. This could represent a neuronal adaptation to CH that preserves neuronal excitability in these crucial relay neurons in peripheral chemoreflex pathways.

Golgi analysis of tangential neurons in the lobula plate of Drosophila melanogaster.

The lobula plate (LP), which is the third order optic neuropil of flies, houses wide-field neurons which are exquisitely sensitive to motion. Among Diptera, motion-sensitive neurons of larger flies have been studied at the anatomical and physiological levels. However, the neurons of Drosophila lobula plate are relatively less explored. As Drosophila permits a genetic analysis of neural functions, we have analysed the organization of lobula plate of Drosophila melanogaster. Neurons belonging to eight anatomical classes have been observed in the present study. Three neurons of the horizontal system (HS) have been visualized. The HS north (HSN) neuron, occupying the dorsal lobula plate is stunted in its geometry compared to that of larger flies. Associated with the HS neurons, thinner horizontal elements known as h-cells have also been visualized in the present study. Five of the six known neurons of the vertical system (VS) have been visualized. Three additional neurons in the proximal LP comparable in anatomy to VS system have been stained. We have termed them as additional VS (AVS)-like neurons. Three thinner tangential cells that are comparable to VS neurons, which are elements of twin vertical system (tvs); and two cells with wide dendritic fields comparable to CH neurons of Diptera have been also observed. Neurons comparable to VS cells but with 'tufted' dendrites have been stained. The HSN and VS1-VS2 neurons are dorsally stunted. This is possibly due to the shape of the compound eye of Drosophila which is reduced in the fronto-dorsal region as compared to larger flies.

The Role of the cut Gene in the Specification of Central Projections by Sensory Axons in Drosophila

Mutations in the cut gene transform sense organs in Drosophila embryos from external sensory (es) receptors to chordotonal (ch) organs. We have investigated whether their central axonal projections are also transformed. Following Lucifer yellow injection of the sensory neuron, wild-type es and ch organs show characteristic, different projection patterns in the CNS. Transformed es neurons in cut embryos are variable in their projection patterns: some resemble wild-type es neurons, others ch neurons, while yet others are unlike either of these. We conclude that the cut gene influences axonal projections, although its action as a simple modality switch is open to question. Additional genes could be involved in the specification of the central axonal projection the transformed neurons.

Morphological differentiation of the embryonic peripheral neurons in Drosophila.

The stereotyped segmental and dorso-ventral organization of the peripheral nervous system (PNS) of Drosophila embryos allows the identification of all the neurons in the body wall. Distinct classes of neurons are distinguishable according to their location, the targets they innervate, the particular shape of their dendrites and their cell size. Those neurons innervating external sensory structures (es) and chordotonal organs (ch) have single dendrites and have been previously described (Ghysen et al. 1986; Dambly-Chaudiere and Ghysen 1986; Campos-Ortega and Hartenstein 1985). We describe here the identity and morphological features of three other classes of neurons in the body segments which have multiple dendrites (md neurons): 1) neurons that give rise to elaborate dendritic arborisations (da neurons); 2) neurons that have bipolar dendrites (bd neurons); 3) neurons that arborize around particular tracheal branches (td neurons). The thoracic hemisegment (T2 and T3) contains 13 da, one bd, one td, 21 es and four ch neurons; the abdominal hemisegment (A1 to A7) contains 14 da, three bd, three td, 15 es and eight ch neurons. The arrangement of the segmented peripheral neurons is highly invariant and provides a favorable assay system for the genetic analysis of neurodevelopment.