Corneal innervation and sensitivity to noxious stimuli in trkA knockout mice

Most primary sensory neurones depend on neurotrophins for survival. Mutant mice in which TrkA, the high-affinity receptor for nerve growth factor (NGF), has been inactivated lack nociceptive neurones in sensory ganglia and do not respond to noxious stimuli. The cornea of the eye is innervated by trigeminal neurones that are activated by noxious mechanical, thermal and chemical stimuli. In the human cornea, these stimuli evoke only sensations of pain. We have analysed the innervation pattern and the response to noxious stimulation of the cornea of trkA (–/–) mutant mice. Corneal nerves were stained with the gold chloride impregnation method. Corneal sensitivity to noxious stimuli was assessed by counting blinking movements evoked by von Frey hairs, topical application of saline at different temperatures and application of acetic acid and capsaicin at different concentrations. In the cornea of trkA (–/–) mutant animals, we observed a drastic reduction in the number of nerve trunks and branches in the corneal stroma. Furthermore, quantitative analysis of the number of thin nerve terminals revealed a marked decrease in the corneal epithelium of trkA (–/–) mice when compared to those present in wild type and trkA (+/–) animals. The blinking response of trkA (–/–) mice to mechanical, thermal and chemical noxious stimuli was also significantly reduced. These results indicate that the population of corneal sensory neurones is markedly depleted in trkA (–/–) mutant mice. However, a small portion of corneal sensory neurones survive in these mice suggesting that they may be NGF independent. On the basis of our results, we propose that these surviving cells are polymodal nociceptive neurones, sensitive to mechanical stimulation, noxious heat and acid.     

Immunohistochemical analysis of the mouse celiac ganglion: An integrative relay station of the peripheral nervous system

Celiac ganglia are important sites of signal integration and transduction. Their complex neurochemical anatomy has been studied extensively in guinea pigs but not in mice. The goal of this study was to provide detailed neurochemical characterization of mouse celiac ganglia and noradrenergic nerves in two target tissues, spleen and stomach. A vast majority of mouse celiac neurons express a noradrenergic phenotype, which includes tyrosine hydroxylase (TH), vesicular monoamine transporter 2, and the norepinephrine transporter. Over 80% of these neuron also express neuropeptide Y (NPY), and this coexpression is maintained by dissociated neurons in culture. Likewise, TH and NPY were colocalized in noradrenergic nerves throughout the spleen and in stomach blood vessels. Somatostatin was not detected in principal neurons but did occur in small, TH-negative cells presumed to be interneurons and in a few varicose nerve fibers. Cholinergic nerves provided the most abundant input to the ganglia, and small percentages of these also contained nitric oxide synthase or vasoactive intestinal polypeptide. A low-to-moderate density of nerves also stained separately for the latter markers. Additionally, nerve bundles and varicose nerve fibers containing the sensory neuropeptides, calcitonin gene-related polypeptide, and substance P, occurred at variable density throughout the ganglia. Collectively, these findings demonstrate that principal neurons of mouse celiac ganglia have less neurochemical diversity than reported for guinea pig and other species but receive input from nerves expressing an array of neurochemical markers. This profile suggests celiac neurons integrate input from many sources to influence target tissues by releasing primarily norepinephrine and NPY.     

Local and generalized sensitization of thermally evoked defensive behavior in caterpillars

In addition to camouflage and chemical toxicity, many caterpillars defend themselves against predators with sudden sharp movements. For smaller species, these movements propel the body away from the threat, but in larger caterpillars, such as the tobacco hornworm, Manduca sexta, the movement is a defensive strike targeted to a noxious stimulus on the abdomen. Previously, strikes have been studied using mechanical stimulation like poking or pinching the insect, but such stimuli are hard to control. They also introduce mechanical perturbations that interfere with measurements of the behavior. We have now established that strike behavior can be evoked using infra-red lasers to provide a highly localized and repeatable heat stimulus. The latency from the end of an effective stimulus to the start of head movement decreased with repeated stimuli and this effect generalized to other stimulus locations indicating a centrally mediated component of sensitization. The tendency to strike increased with two successive subthreshold stimuli. When delivered to different locations or to a single site, this split-pulse stimulation revealed an additional site-specific sensitization that has not previously been described in Manduca. Previous work shows that strong stimuli increases the effectiveness of sensory stimulation by activating a long-lasting muscarinic cation current in motoneurons. Injection of muscarinic cholinergic antagonists, scopolamine methyl bromide or quinuclidinyl benzilate, only decreased the strike probability evoked by paired stimuli at two locations and not at a single site. This strongly suggests a role of muscarinic acetylcholine receptors in the generalized sensitization of nociceptive responses in caterpillars.     

Mediolateral and dorsoventral projection patterns of cutaneous afferents within transverse planes of the mouse spinal dorsal horn

The central projection patterns of cutaneous afferents from the forelimb and shoulder of mice were studied in the spinal dorsal horn after intracutaneous injection of AlexaFluor 488-conjugated and/or 594-conjugated cholera toxin subunit B (CTB). Based on their dermatomes, the following eight skin regions are thought to be innervated by spinal nerves from the sixth to eighth cervical spinal nerve roots: the dorsal surface of the shoulder, brachium, proximal forearm, distal forearm, hand, palmar surface of the second and third digits, and palm. The termination areas of afferents from the dorsal surface of the shoulder and forearm were narrow, distributed in a dorsoventral direction, and aligned in order from lateral to medial within the sixth to eighth cervical dorsal horns. By contrast, the termination areas of the palmar surface of the second and third digits largely overlapped. We also injected CTB into the dorsal surface of the hindlimb and pelvic regions. Skin regions there are thought to be innervated by nerves from the third to fifth lumbar spinal nerve roots. The observed projection patterns in the lumbar dorsal horn were similar to the cervical patterns. Injection of a mixture of CTB and wheat-germ agglutinin-conjugated horseradish peroxidase (WGA-HRP), which are thought to label Aβ and Aδ/C fibers, respectively, showed segregated termination areas of CTB- and WGA-HRP-labeled afferents. Moreover, alignment of the termination areas was in the dorsoventral direction. These results suggest there is fine somatotopic (mediolateral axis) and modality-specific (dorsoventral axis) organization within the spinal dorsal horn.     

Perineural application of resiniferatoxin on uninjured L3 and L4 nerves completely alleviates thermal and mechanical hypersensitivity following L5 nerve injury in rats

Fifth lumbar (L5) nerve injury in rats causes neuropathic pain manifested with thermal and mechanical hypersensitivity in the ipsilateral hind paw. This study aimed to determine whether the elimination of unmyelinated primary afferents of the adjacent uninjured nerves (L3 and L4) would alleviate peripheral neuropathic pain. Different concentrations of capsaicin or its analog, resiniferatoxin (RTX), were applied perineurally on either the left L4 or L3 and L4 nerves in Wistar rats whose left L5 nerves were ligated and cut. The application of both capsaicin and RTX on the L4 nerve significantly reduced both thermal and mechanical hypersensitivity. However, only the application of RTX on both L3 and L4 nerves completely alleviated all neuropathic manifestations. Interestingly, responses to thermal and mechanical stimuli were preserved, despite RTX application on uninjured L3, L4, and L5 nerves, which supply the plantar skin in rats. Perineural application of RTX caused downregulation of TRPV1, CGRP, and IB4 binding and upregulation of VIP in the corresponding dorsal root ganglia (DRG) and the dorsal horn of the spinal cord. In comparison, VGLUT1 and NPY immunoreactivities were not altered. RTX application did not cause degenerative or ultrastructural changes in the treated nerves and corresponding DRGs. The results demonstrate that RTX induces neuroplasticity, rather than structural changes in primary afferents, that are responsible for alleviating hypersensitivity and chronic pain. Furthermore, this study suggests that treating uninjured adjacent spinal nerves may be used to manage chronic neuropathic pain following peripheral nerve injury.     

Immunocytochemical organization and sour taste activation in the rostral nucleus of the solitary tract of mice

Sensory inputs from the oropharynx terminate in both the trigeminal brainstem complex and the rostral part of the nucleus of the solitary tract (nTS). Taste information is conveyed via the facial and glossopharyngeal nerves, while general mucosal innervation is carried by the trigeminal and glossopharyngeal nerves. In contrast, the caudal nTS receives general visceral information largely from the vagus nerve. Although the caudal nTS shows clear morphological and molecularly delimited subdivisions, the rostral part does not. Thus, linking taste‐induced patterns of activity to morphological subdivisions in the nTS is challenging. To test whether molecularly defined features of the rostral nTS correlate with patterns of taste‐induced activity, we combined immunohistochemistry for markers of various visceral afferent and efferent systems with c‐Fos–based activity maps generated by stimulation with a sour tastant, 30 mM citric acid. We further dissociated taste‐related activity from activity arising from acid‐sensitive general mucosal innervation by comparing acid‐evoked c‐Fos in wild‐type and “taste blind” P2X₂/P2X₃ double knockout (P2X‐dbl KO) mice. In wild‐type mice, citric acid stimulation evoked significant c‐Fos activation in the central part of the rostral nTS—activity that was largely absent in the P2X‐dbl KO mice. P2X‐dbl KO mice, like wild‐type mice, did exhibit acid‐induced c‐Fos activity in the dorsomedial trigeminal brainstem nucleus situated laterally adjacent to the rostral nTS. This dorsomedial nucleus also showed substantial innervation by trigeminal nerve fibers immunoreactive for calcitonin gene‐related peptide (CGRP), a marker for polymodal nociceptors, suggesting that trigeminal general mucosal innervation carries information about acids in the oral cavity. J. Comp. Neurol. 525:271–290, 2017. © 2016 Wiley Periodicals, Inc.     

Cross-modal modulation of cell activity by sound in first-order visual thalamic nucleus

Cross-modal auditory influence on cell activity in the primary visual cortex emerging at short latencies raises the possibility that the first-order visual thalamic nucleus, which is considered dedicated to unimodal visual processing, could contribute to cross-modal sensory processing, as has been indicated in the auditory and somatosensory systems. To test this hypothesis, the effects of sound stimulation on visual cell activity in the dorsal lateral geniculate nucleus were examined in anesthetized rats, using juxta-cellular recording and labeling techniques. Visual responses evoked by light (white LED) were modulated by sound (noise burst) given simultaneously or 50–400 ms after the light, even though sound stimuli alone did not evoke cell activity. Alterations of visual response were observed in 71% of cells (57/80) with regard to response magnitude, latency, and/or burst spiking. Suppression predominated in response magnitude modulation, but de novo responses were also induced by combined stimulation. Sound affected not only onset responses but also late responses. Late responses were modulated by sound given before or after onset responses. Further, visual responses evoked by the second light stimulation of a double flash with a 150–700 ms interval were also modulated by sound given together with the first light stimulation. In morphological analysis of labeled cells projection cells comparable to X-, Y-, and W-like cells and interneurons were all susceptible to auditory influence. These findings suggest that the first-order visual thalamic nucleus incorporates auditory influence into parallel and complex thalamic visual processing for cross-modal modulation of visual attention and perception.     

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.     

Central neural alterations predominate in an insect model of nociceptive sensitization

Many organisms respond to noxious stimuli with defensive maneuvers. This is noted in the hornworm, Manduca sexta, as a defensive strike response. After tissue damage, organisms typically display sensitized responses to both noxious or normally innocuous stimuli. To further understand this phenomenon, we used novel in situ and in vitro preparations based on paired extracellular nerve recordings and videography to identify central and peripheral nerves responsible for nociception and sensitization of the defensive behavior in M. sexta. In addition, we used the in vivo defensive strike response threshold assayed with von Frey filaments to examine the roles that N‐methyl‐D‐aspartate receptor (NMDAR) and hyperpolarization‐activated, cyclic nucleotide‐gated (HCN) channels play in this nociceptive sensitization using the inhibitors MK‐801 and AP5 (NMDAR), and ivabradine and ZD7288 (HCN). Using our new preparations, we found that afferent activity evoked by noxious pinch in these preparations was conveyed to central ganglia by axons in the anterior‐ and lateral‐dorsal nerve branches, and that sensitization induced by tissue damage was mediated centrally. Furthermore, sensitization was blocked by all inhibitors tested except the inactive isomer L‐AP5, and reversed by ivabradine both in vivo and in vitro. Our findings suggest that M. sexta's sensitization occurs through central signal amplification. Due to the relatively natural sensitization method and conserved molecular actions, we suggest that M. sexta may be a valuable model for studying the electrophysiological properties of nociceptive sensitization and potentially related conditions such as allodynia and hyperalgesia in a comparative setting that offers unique experimental advantages. J. Comp. Neurol. 525:1176–1191, 2017. © 2016 Wiley Periodicals, Inc.     

The reduction of intraepidermal P2X3 nerve fiber density correlates with behavioral hyperalgesia in a rat model of nerve injury‐induced pain

Skin biopsies from patients with neuropathic pain often show changes in epidermal innervation, although it remains to be elucidated to what extent such changes can be linked to a particular subgroup of nerve fibers and how these changes are correlated with pain intensity. Here, we investigated to what extent behavioral signs of hyperalgesia are correlated with immunohistochemical changes of peptidergic and non‐peptidergic epidermal nerve fibers in a rat model of nerve injury‐induced pain. Rats subjected to unilateral partial ligation of the sciatic nerve developed significant mechanical and thermal hyperalgesia as tested by the withdrawal responses of the ipsilateral footpad to von Frey hairs and hotplate stimulation. At day 14, epidermal nerve fiber density and total epidermal nerve fiber length/mm² were significantly and consistently reduced compared to the contralateral side, following testing and re‐testing by two blinded observers. The expression of calcitonin gene‐related peptide, a marker for peptidergic nerve fibers, was not significantly changed on the ipsilateral side. In contrast, the expression of the P2X₃ receptor, a marker for non‐peptidergic nerve fibers, was not only significantly reduced but could also be correlated with behavioral hyperalgesia. When labeling both peptidergic and non‐peptidergic nerve fibers with the pan‐neuronal marker PGP9.5, the expression was significantly reduced, albeit without a significant correlation with behavioral hyperalgesia. In conjunction, our data suggest that the pathology of the P2X₃ epidermal nerve fibers can be selectively linked to neuropathy, highlighting the possibility that it is the degeneration of these fibers that drives hyperalgesia.     

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.     

Surface topography during neural stem cell differentiation regulates cell migration and cell morphology

We sought to determine the contribution of scaffold topography to the migration and morphology of neural stem cells by mimicking anatomical features of scaffolds found in vivo. We mimicked two types of central nervous system scaffolds encountered by neural stem cells during development in vitro by constructing different diameter electrospun polycaprolactone (PCL) fiber mats, a substrate that we have shown to be topographically similar to brain scaffolds. We compared the effects of large fibers (made to mimic blood vessel topography) with those of small‐diameter fibers (made to mimic radial glial process topography) on the migration and differentiation of neural stem cells. Neural stem cells showed differential migratory and morphological reactions with laminin in different topographical contexts. We demonstrate, for the first time, that neural stem cell biological responses to laminin are dependent on topographical context. Large‐fiber topography without laminin prevented cell migration, which was partially reversed by treatment with rock inhibitor. Cell morphology complexity assayed by fractal dimension was inhibited in nocodazole‐ and cytochalasin‐D–treated neural precursor cells in large‐fiber topography, but was not changed in small‐fiber topography with these inhibitors. These data indicate that cell morphology has different requirements on cytoskeletal proteins dependent on the topographical environment encountered by the cell. We propose that the physical structure of distinct scaffolds induces unique signaling cascades that regulate migration and morphology in embryonic neural precursor cells. J. Comp. Neurol. 524:3485–3502, 2016. © 2016 Wiley Periodicals, Inc.     

Developmental organization of central neurons in the adult Drosophila ventral nervous system

We have used MARCM to reveal the adult morphology of the post embryonically produced neurons in the thoracic neuromeres of the Drosophila VNS. The work builds on previous studies of the origins of the adult VNS neurons to describe the clonal organization of the adult VNS. We present data for 58 of 66 postembryonic thoracic lineages, excluding the motor neuron producing lineages (15 and 24) which have been described elsewhere. MARCM labels entire lineages but where both A and B hemilineages survive (e.g., lineages 19, 12, 13, 6, 1, 3, 8, and 11), the two hemilineages can be discriminated and we have described each hemilineage separately. Hemilineage morphology is described in relation to the known functional domains of the VNS neuropil and based on the anatomy we are able to assign broad functional roles for each hemilineage. The data show that in a thoracic hemineuromere, 16 hemilineages are primarily involved in controlling leg movements and walking, 9 are involved in the control of wing movements, and 10 interface between both leg and wing control. The data provide a baseline of understanding of the functional organization of the adult Drosophila VNS. By understanding the morphological organization of these neurons, we can begin to define and test the rules by which neuronal circuits are assembled during development and understand the functional logic and evolution of neuronal networks.     

Nogo‐A does not inhibit retinal axon regeneration in the lizard Gallotia galloti

The myelin‐associated protein Nogo‐A contributes to the failure of axon regeneration in the mammalian central nervous system (CNS). Inhibition of axon growth by Nogo‐A is mediated by the Nogo‐66 receptor (NgR). Nonmammalian vertebrates, however, are capable of spontaneous CNS axon regeneration, and we have shown that retinal ganglion cell (RGC) axons regenerate in the lizard Gallotia galloti. Using immunohistochemistry, we observed spatiotemporal regulation of Nogo‐A and NgR in cell bodies and axons of RGCs during ontogeny. In the adult lizard, expression of Nogo‐A was associated with myelinated axon tracts and upregulated in oligodendrocytes during RGC axon regeneration. NgR became upregulated in RGCs following optic nerve injury. In in vitro studies, Nogo‐A‐Fc failed to inhibit growth of lizard RGC axons. The inhibitor of protein kinase A (pkA) activity KT5720 blocked growth of lizard RGC axons on substrates of Nogo‐A‐Fc, but not laminin. On patterned substrates of Nogo‐A‐Fc, KT5720 caused restriction of axon growth to areas devoid of Nogo‐A‐Fc. Levels of cyclic adenosine monophosphate (cAMP) were elevated over sustained periods in lizard RGCs following optic nerve lesion. We conclude that Nogo‐A and NgR are expressed in a mammalian‐like pattern and are upregulated following optic nerve injury, but the presence of Nogo‐A does not inhibit RGC axon regeneration in the lizard visual pathway. The results of outgrowth assays suggest that outgrowth‐promoting substrates and activation of the cAMP/pkA signaling pathway play a key role in spontaneous lizard retinal axon regeneration in the presence of Nogo‐A. Restriction of axon growth by patterned Nogo‐A‐Fc substrates suggests that Nogo‐A may contribute to axon guidance in the lizard visual system. J. Comp. Neurol. 525:936–954, 2017. © 2016 Wiley Periodicals, Inc.     

Neurochemistry of neurons in the ventrolateral medulla activated by hypotension: Are the same neurons activated by glucoprivation?

Previous studies have demonstrated that a range of stimuli activate neurons, including catecholaminergic neurons, in the ventrolateral medulla. Not all catecholaminergic neurons are activated and other neurochemical content is largely unknown hence whether stimulus specific populations exist is unclear. Here we determine the neurochemistry (using in situ hybridization) of catecholaminergic and noncatecholaminergic neurons which express c‐Fos immunoreactivity throughout the rostrocaudal extent of the ventrolateral medulla, in Sprague Dawley rats treated with hydralazine or saline. Distinct neuronal populations containing PPCART, PPPACAP, and PPNPY mRNAs, which were largely catecholaminergic, were activated by hydralazine but not saline. Both catecholaminergic and noncatecholaminergic neurons containing preprotachykinin and prepro‐enkephalin (PPE) mRNAs were also activated, with the noncatecholaminergic population located in the rostral C1 region. Few GlyT2 neurons were activated. A subset of these data was then used to compare the neuronal populations activated by 2‐deoxyglucose evoked glucoprivation (Brain Structure and Function (2015) 220:117). Hydralazine activated more neurons than 2‐deoxyglucose but similar numbers of catecholaminergic neurons. Commonly activated populations expressing PPNPY and PPE mRNAs were defined. These likely include PPNPY expressing catecholaminergic neurons projecting to vasopressinergic and corticotrophin releasing factor neurons in the paraventricular nucleus, which when activated result in elevated plasma vasopressin and corticosterone. Stimulus specific neurons included noncatecholaminergic neurons and a few PPE positive catecholaminergic neuron but neurochemical codes were largely unidentified. Reasons for the lack of identification of stimulus specific neurons, readily detectable using electrophysiology in anaesthetized preparations and for which neural circuits can be defined, are discussed.     

Extensive branching of radially-migrating neurons in the mammalian cerebral cortex

Excitatory neurons of the cerebral cortex migrate radially from their place of birth to their final position in the cortical plate during development. Radially-migrating neurons display a single leading process that establishes the direction of movement. This leading process has been described as being unbranched, and the occurrence of branches proposed to impair radial migration. Here we have analyzed the detailed morphology of leading process in radially-migrating pyramidal neurons and its impact on radial migration. We have compared ferret and mouse to identify differences between cortices that undergo folding or not. In mouse, we find that half of radially-migrating neurons exhibit a branched leading process, this being even more frequent in ferret. Branched leading processes are less parallel to radial glia fibers than those unbranched, suggesting some independence from radial glia fibers. Two-photon videomicroscopy revealed that a vast majority of neurons branch their leading process at some point during radial migration, but this does not reduce their migration speed. We have tested the functional impact of exuberant leading process branching by expressing a dominant negative Cdk5. We confirm that loss of Cdk5 function significantly impairs radial migration, but this is independent from increased branching of the leading process. We propose that excitatory neurons may branch their leading process as an evolutionary mechanism to allow cells changing their trajectory of migration to disperse laterally, such that increased branching in gyrencephalic species favors the tangential dispersion of radially-migrating neurons, and cortical folding.