Expression of the cold thermoreceptor TRPM8 in rodent brain thermoregulatory circuits

The cold‐ and menthol‐activated ion channel transient receptor potential channel subfamily M member 8 (TRPM8) is the principal detector of environmental cold in mammalian sensory nerve endings. Although it is mainly expressed in a subpopulation of peripheral sensory neurons, it has also been identified in non‐neuronal tissues. Here, we show, by in situ hybridization (ISH) and by the analysis of transgenic reporter expression in two different reporter mouse strains, that TRPM8 is also expressed in the central nervous system. Although it is present at much lower levels than in peripheral sensory neurons, we found cells expressing TRPM8 in restricted areas of the brain, especially in the hypothalamus, septum, thalamic reticular nucleus, certain cortices and other limbic structures, as well as in some specific nuclei in the brainstem. Interestingly, positive fibers were also found traveling through the major limbic tracts, suggesting a role of TRPM8‐expressing central neurons in multiple aspects of thermal regulation, including autonomic and behavioral thermoregulation. Additional ISH experiments in rat brain demonstrated a conserved pattern of expression of this ion channel between rodent species. We confirmed the functional activity of this channel in the mouse brain using electrophysiological patch‐clamp recordings of septal neurons. These results open a new window in TRPM8 physiology, guiding further efforts to understand potential roles of this molecular sensor within the brain.     

Partial deletion of p75NTR in large-diameter DRG neurons exerts no influence upon the survival of peripheral sensory neurons in vivo

The p75 neurotrophin receptor (p75^NTR) is required for maintaining peripheral sensory neuron survival and function; however, the underlying cellular mechanism remains unclear. The general view is that expression of p75^NTR by the neuron itself is required for maintaining sensory neuron survival and myelination in the peripheral nervous system (PNS). Adopting a neuronal-specific conditional knockout strategy, we demonstrate the partial depletion of p75^NTR in neurons exerts little influence upon maintaining sensory neuron survival and peripheral nerve myelination in health and after demyelinating neuropathy. Our data show that the density and total number of dorsal root ganglion (DRG) neurons in 2-month-old mice is not affected following the deletion of p75^NTR in large-diameter myelinating neurons, as assessed by stereology. Adopting experimental autoimmune neuritis induced in adult male mice, an animal model of demyelinating peripheral neuropathy, we identify that deleting p75^NTR in myelinating neurons exerts no influence upon the disease progression, the total number of DRG neurons, and the extent of myelin damage in the sciatic nerve, indicating that the expression of neuronal p75^NTR is not essential for maintaining peripheral neuron survival and myelination after a demyelinating insult in vivo. Together, results of this study suggest that the survival and myelination of peripheral sensory neurons is independent of p75^NTR expressed by a subtype of neurons in vivo. Thus, our findings provide new insights into the mechanism underpinning p75^NTR-mediated neuronal survival in the PNS.     

Innervation of the syrinx of the zebra finch (Taeniopygia guttata)

In songbirds, the learning and maintenance of song is dependent on auditory feedback, but little is known about the presence or role of other forms of sensory feedback. Here, we studied the innervation of the avian vocal organ, the syrinx, in the zebra finch. Using a combination of immunohistochemistry, immunofluorescence and neural tracing with subunit B of cholera toxin (CTB), we analysed the peripheral and central endings of the branch of the hypoglossal nerve that supplies the syrinx, the tracheosyringeal nerve. In the syringeal muscles, we show the presence of numerous choline acetyl transferase‐like immunoreactive en plaque motor endplates and substance P‐like immunoreactive, thin and varicose free nerve endings. Substance P‐like immunoreactive free nerve endings were also present in the luminal syringeal tissues, especially in the luminal epithelium of the trachea and pessulus. Also, by a combination of immunofluorescence and transganglionic tracing following injections of CTB in the tracheosyringeal nerve, we identified as central targets of the syringeal receptors the caudolateral part of the interpolaris subnucleus of the descending trigeminal tract, a caudolateral region of the nucleus tractus solitarius, and a lateral band of the principal sensory trigeminal nucleus. Further studies are required to determine the sensory modalities of these receptors and the connections of their specific synaptic targets.     

Olfactory ensheathing cells from the nasal mucosa and olfactory bulb have distinct membrane properties

Transplantation of olfactory ensheathing cells (OECs) is a potential therapy for the regeneration of damaged neurons. While they maintain tissue homeostasis in the olfactory mucosa (OM) and olfactory bulb (OB), their regenerative properties also support the normal sense of smell by enabling continual turnover and axonal regrowth of olfactory sensory neurons (OSNs). However, the molecular physiology of OECs is not fully understood, especially that of OECs from the mucosa. Here, we carried out whole-cell patch-clamp recordings from individual OECs cultured from the OM and OB of the adult rat, and from the human OM. A subset of OECs from the rat OM cultured 1–3 days in vitro had large weakly rectifying K⁺ currents, which were sensitive to Ba²⁺ and desipramine, blockers of Kir4-family channels. Kir4.1 immunofluorescence was detectable in cultured OM cells colabeled for the OEC marker S100, and in S100-labeled cells found adjacent to OSN axons in mucosal sections. OECs cultured from rat OB had distinct properties though, displaying strongly rectifying inward currents at hyperpolarized membrane potentials and strongly rectifying outward currents at depolarized potentials. Kir4.1 immunofluorescence was not evident in OECs adjacent to axons of OSNs in the OB. A subset of human OECs cultured from the OM of adults had membrane properties comparable to those of the rat OM that is dominated by Ba²⁺-sensitive weak inwardly rectifying currents. The membrane properties of peripheral OECs are different to those of central OECs, suggesting they may play distinct roles during olfaction.     

A population of descending tyraminergic/octopaminergic projection neurons of the insect deutocerebrum

In this study, we describe a cluster of tyraminergic/octopaminergic neurons in the lateral dorsal deutocerebrum of desert locusts (Schistocerca gregaria) with descending axons to the abdominal ganglia. In the locust, these neurons synthesize octopamine from tyramine stress-dependently. Electrophysiological recordings in locusts reveal that they respond to mechanosensory touch stimuli delivered to various parts of the body including the antennae. A similar cluster of tyraminergic/octopaminergic neurons was also identified in the American cockroach (Periplaneta americana) and the pink winged stick insect (Sipyloidea sipylus). It is suggested that these neurons release octopamine in the ventral nerve cord ganglia and, most likely, convey information on arousal and/or stressful stimuli to neuronal circuits thus contributing to the many actions of octopamine in the central nervous system.     

Morphology,innervation, and peripheral sensory cells of the siphon of aplysia californica

The siphon of Aplysia californica has several functions, including involvement in respiration, excretion, and defensive inking. It also provides sensory input for defensive withdrawals that have been studied extensively to examine mechanisms that underlie learning. To better understand the neuronal bases of these functions, we used immunohistochemistry to catalogue peripheral cell types and innervation of the siphon in stage 12 juveniles (chosen to allow observation of tissues in whole‐mounts). We found that the siphon nerve splits into three major branches, leading ultimately to a two‐part FMRFamide‐immunoreactive plexus and an apparently separate tyrosine hydroxylase–immunoreactive plexus. Putative sensory neurons included four distinct types of tubulin‐immunoreactive bipolar cells (one likely also tyrosine hydroxylase immunoreactive) that bore ciliated dendrites penetrating the epithelium. A fifth bipolar neuron type (tubulin‐ and FMRFamide‐immunoreactive) occurred deeper in the tissue, associated with part of the FMRFamide‐immunoreactive plexus. Our observations emphasize the structural complexity of the peripheral nervous system of the siphon, and the importance of direct tests of the various components to better understand the functioning of the entire organ, including its role in defensive withdrawal responses. J. Comp. Neurol. 523:2409–2425, 2015. © 2015 Wiley Periodicals, Inc.     

Topographic organization and possible function of the posterior optic tubercles in the brain of the desert locust Schistocerca gregaria

Migrating desert locusts, Schistocerca gregaria, are able to use the skylight polarization pattern for navigation. They detect polarized light with a specialized dorsal rim area in their compound eye. After multistage processing, polarization signals are transferred to the central complex, a midline‐spanning brain area involved in locomotor control. Polarization‐sensitive tangential neurons (TB‐neurons) of the protocerebral bridge, a part of the central complex, give rise to a topographic arrangement of preferred polarization angles in the bridge, suggesting that the central complex acts as an internal sky compass. TB‐neurons connect the protocerebral bridge with two adjacent brain areas, the posterior optic tubercles. To analyze the polarotopic organization of the central complex further, we investigated the number and morphologies of TB‐neurons and the presence and colocalization of three neuroactive substances in these neurons. Triple immunostaining with antisera against Diploptera punctata allatostatin (Dip‐AST), Manduca sexta allatotropin (Mas‐AT), and serotonin (5HT) raised in the same host species revealed three spatially distinct TB‐neuron clusters, each consisting of 10 neurons per hemisphere: cluster 1 and 3 showed Dip‐AST/5HT immunostaining, whereas cluster 2 showed Dip‐AST/Mas‐AT immunostaining. Five subtypes of TB‐neuron could be distinguished based on ramification patterns. Corresponding to ramification domains in the protocerebral bridge, the neurons invaded distinct but overlapping layers within the posterior optic tubercle. Similarly, neurons interconnecting the tubercles of the two hemispheres also targeted distinct layers of these neuropils. From these data we propose a neuronal circuit that may be suited to stabilize the internal sky compass in the central complex of the locust. J. Comp. Neurol. 523:1589–1607, 2015. © 2015 Wiley Periodicals, Inc.     

Novel insights into the glia limitans of the olfactory nervous system

Olfactory ensheathing cells (OECs) are often described as being present in both the peripheral and the central nervous systems (PNS and CNS). Furthermore, the olfactory nervous system glia limitans (the glial layer defining the PNS–CNS border) is considered unique as it consists of intermingling OECs and astrocytes. In contrast, the glia limitans of the rest of the nervous system consists solely of astrocytes which create a distinct barrier to Schwann cells (peripheral glia). The ability of OECs to interact with astrocytes is one reason why OECs are believed to be superior to Schwann cells for transplantation therapies to treat CNS injuries. We have used transgenic reporter mice in which glial cells express DsRed fluorescent protein to study the cellular constituents of the glia limitans. We found that the glia limitans layer of the olfactory nervous system is morphologically similar to elsewhere in the nervous system, with a similar low degree of intermingling between peripheral glia and astrocytes. We found that the astrocytic layer of the olfactory bulb is a distinct barrier to bacterial infection, suggesting that this layer constitutes the PNS–CNS immunological barrier. We also found that OECs interact with astrocytes in a similar fashion as Schwann cells in vitro. When cultured in three dimensions, however, there were subtle differences between OECs and Schwann cells in their interactions with astrocytes. We therefore suggest that glial fibrillary acidic protein–reactive astrocyte layer of the olfactory bulb constitutes the glia limitans of the olfactory nervous system and that OECs are primarily “PNS glia.”     

The effects of low intensity focused ultrasonic stimulation on dorsal root ganglion neurons and Schwann cells in vitro

Satisfactory treatment of peripheral nerve injury (PNI) faces difficulties owing to the intrinsic biological barriers in larger injuries and invasive surgical interventions. Injury gaps >3 cm have low chances of full motor and sensory recovery, and the unmet need for PNI repair techniques which increase the likelihood of functional recovery while limiting invasiveness motivate this work. Building upon prior work in ultrasound stimulation (US) of dorsal root ganglion (DRG) neurons, the effects of US on DRG neuron and Schwann cell (SC) cocultures were investigated to uncover the role of SCs in mediating the neuronal response to US in vitro. Acoustic intensity‐dependent alteration in selected neuromorphometrics of DRG neurons in coculture with SCs was observed in total outgrowth, primary neurites, and length compared to previously reported DRG monoculture in a calcium‐independent manner. SC viability and proliferation were not impacted by US. Conditioned medium studies suggest secreted factors from SCs subjected to US impact DRG neuron morphology. These findings advance the current understanding of mechanisms by which these cell types respond to US, which may lead to new noninvasive US therapies for treating PNI.     

Morphological and functional changes in TRPM8‐expressing corneal cold thermoreceptor neurons during aging and their impact on tearing in mice

Morphological and functional alterations of peripheral somatosensory neurons during the aging process lead to a decline of somatosensory perception. Here, we analyze the changes occurring with aging in trigeminal ganglion (TG), TRPM8‐expressing cold thermoreceptor neurons innervating the mouse cornea, which participate in the regulation of basal tearing and blinking and have been implicated in the pathogenesis of dry eye disease (DED). TG cell bodies and axonal branches were examined in a mouse line (TRPM8^BAC‐EYFP) expressing a fluorescent reporter. In 3 months old animals, about 50% of TG cold thermoreceptor neurons were intensely fluorescent, likely providing strongly fluorescent axons and complex corneal nerve terminals with ongoing activity at 34°C and low‐threshold, robust responses to cooling. The remaining TRPM8⁺ corneal axons were weakly fluorescent with nonbeaded axons, sparsely ramified nerve terminals, and exhibited a low‐firing rate at 34°C, responding moderately to cooling pulses as do weakly fluorescent TG neurons. In aged (24 months) mice, the number of weakly fluorescent TG neurons was strikingly high while the morphology of TRPM8⁺ corneal axons changed drastically; 89% were weakly fluorescent, unbranched, and often ending in the basal epithelium. Functionally, 72.5% of aged cold terminals responded as those of young animals, but 27.5% exhibited very low‐background activity and abnormal responsiveness to cooling pulses. These morpho‐functional changes develop in parallel with an enhancement of tear's basal flow and osmolarity, suggesting that the aberrant sensory inflow to the brain from impaired peripheral cold thermoreceptors contributes to age‐induced abnormal tearing and to the high incidence of DED in elderly people.     

An epilepsy‐associated ACTL6B variant captures neuronal hyperexcitability in a human induced pluripotent stem cell model

ACTL6B is a component of the neuronal BRG1/brm‐associated factor (nBAF) complex, which is required for chromatin remodeling in postmitotic neurons. We recently reported biallelic pathogenic variants in ACTL6B in patients diagnosed with early infantile epileptic encephalopathy, subtype 76 (EIEE‐76), presenting with severe, global developmental delay, epileptic encephalopathy, cerebral atrophy, and abnormal central nervous system myelination. However, the pathophysiological mechanisms underlying their phenotype is unknown. Here, we investigate the molecular pathogenesis of ACTL6B p.(Val421_Cys425del) using in silico 3D protein modeling predictions and patient‐specific induced pluripotent stem cell‐derived neurons. We found neurons derived from EIEE‐76 patients showed impaired accumulation of ACTL6B compared to unaffected relatives, caused by reduced protein stability. Furthermore, EIEE‐76 patient‐derived neurons had dysregulated nBAF target gene expression, including genes important for neuronal development and disease. Multielectrode array system analysis unveiled elevated electrophysiological activity of EIEE‐76 patients‐derived neurons, consistent with the patient phenotype. Taken together, our findings validate a new model for EIEE‐76 and reveal how reduced ACTL6B expression affects neuronal function.     

Differential deletion of GDNF in the auditory system leads to altered sound responsiveness

Glial-derived neurotrophic factor (GDNF) has been proposed as a potent neurotrophic factor with the potential to cure neurodegenerative diseases. In the cochlea, GDNF has been detected in auditory neurons and sensory receptor cells and its expression is upregulated upon trauma. Moreover, the application of GDNF in different animal models of deafness has shown its capacity to prevent hearing loss and promoted its future use in therapeutic trials in humans. In the present study we have examined the endogenous requirement of GDNF during auditory development in mice. Using a lacZ knockin allele we have confirmed the expression of GDNF in the cochlea including its sensory regions during development. Global inactivation of GDNF throughout the hearing system using a Foxg1-Cre line causes perinatal lethality but reveals no apparent defects during formation of the cochlea. Using TrkC-Cre and Atoh1-Cre lines, we were able to generate viable mutants lacking GDNF in auditory neurons or both auditory neurons and sensory hair cells. These mutants show normal frequency-dependent auditory thresholds. However, mechanoelectrical response properties of outer hair cells (OHCs) in TrkC-Cre GDNF mutants are altered at low thresholds. Furthermore, auditory brainstem wave analysis shows an abnormal increase of wave I. On the other hand, Atoh1-Cre GDNF mutants show normal OHC function but their auditory brainstem wave pattern is reduced at the levels of wave I, III and IV. These results show that GDNF expression during the development is required to maintain functional hearing at different levels of the auditory system.     

Variability in the number of abdominal leucokinergic neurons in adult Drosophila melanogaster

Developmental plasticity allows individuals with the same genotype to show different phenotypes in response to environmental changes. An example of this is how neuronal diversity is protected at the expense of neuronal number under sustained undernourishment during the development of the Drosophila optic lobe. In the development of the Drosophila central nervous system, neuroblasts go through two phases of neurogenesis separated by a period of mitotic quiescence. Although during embryonic development much evidence indicates that both cell number and the cell fates generated by each neuroblast are very precisely controlled in a cell autonomous manner, after quiescence extrinsic factors control the reactivation of neuroblast proliferation in a fashion that has not yet been elucidated. Moreover, there is very little information about whether environmental changes affect lineage progression during postembryonic neurogenesis. Using as a model system the pattern of abdominal leucokinergic neurons (ABLKs), we have analyzed how changes in a set of environmental factors affect the number of ABLKs generated during postembryonic neurogenesis. We describe the variability in ABLK number between individuals and between hemiganglia of the same individual and, by genetic analysis, we identify the bithorax‐complex genes and the ecdysone hormone as critical factors in these differences. We also explore the possible adaptive roles involved in this process. J. Comp. Neurol. 525:639–660, 2017. © 2016 Wiley Periodicals, Inc.     

The astrocyte network in the ventral nerve cord neuropil of the Drosophila third-instar larva

Understanding neuronal function at the local and circuit level requires understanding astrocyte function. We have provided a detailed analysis of astrocyte morphology and territory in the Drosophila third-instar ventral nerve cord where there already exists considerable understanding of the neuronal network. Astrocyte shape varies more than previously reported; many have bilaterally symmetrical partners, many have a high percentage of their arborization in adjacent segments, and many have branches that follow structural features. Taken together, our data are consistent with, but not fully explained by, a model of a developmental growth process dominated by competitive or repulsive interactions between astrocytes. Our data suggest that the model should also include cell-autonomous aspects, as well as the use of structural features for growth. Variation in location of arborization territory for identified astrocytes was great enough that a standardized scheme of neuropil division among the six astrocytes that populate each hemi-segment is not possible at the third instar. The arborizations of the astrocytes can extend across neuronal functional domains. The ventral astrocyte in particular, whose territory can extend well into the proprioceptive region of the neuropil, has no obvious branching pattern that correlates with domains of particular sensory modalities, suggesting that the astrocyte would respond to neuronal activity in any of the sensory modalities, perhaps integrating across them. This study sets the stage for future studies that will generate a robust, functionally oriented connectome that includes both partners in neuronal circuits—the neurons and the glial cells, providing the foundation necessary for studies to elucidate neuron–glia interactions in this neuropil.     

5HTR3A‐driven GFP labels immature olfactory sensory neurons

The ionotropic serotonin receptor, 5‐HT₃, is expressed by many developing neurons within the central nervous system. Since the olfactory epithelium continues to generate new olfactory sensory neurons (OSNs) throughout life, we investigated the possibility that 5‐HT₃ is expressed in the adult epithelium. Using a transgenic mouse in which the promoter for the 5‐HT_3a subunit drives expression of green fluorescent protein (GFP), we assessed the expression of this marker in the olfactory epithelium of adult mice. Both the native 5‐HT_3a mRNA and GFP are expressed within globose basal cells of the olfactory and vomeronasal epithelium in adult mice. Whereas the 5‐HT_3a mRNA disappears relatively quickly after final cell division, the GFP label persists for about 5 days, thereby labeling immature OSNs in both the main olfactory system and vomeronasal organ. The GFP‐labeled cells include both proliferative globose basal cells as well as immature OSNs exhibiting the hallmarks of ongoing differentiation including GAP43, PGP9.5, but the absence of olfactory marker protein. Some of the GFP‐labeled OSNs show characteristics of more mature yet still developing OSNs including the presence of cilia extending from the apical knob and expression of NaV1.5, a component of the transduction cascade. These findings suggest that 5‐HT_3a is indicative of a proliferative or developmental state, regardless of age, and that the 5‐HT_3AGFP mice may prove useful for future studies of neurogenesis in the olfactory epithelium. J. Comp. Neurol. 525:1743–1755, 2017. © 2016 Wiley Periodicals, Inc.