The Characterization of AT<Subscript>1</Subscript> Expression in the Dorsal Root Ganglia After Chronic Constriction Injury

To clarify the role of Angiotensin II in the regulation of sensory signaling, we characterized the AT₁ expression in neuronal subpopulation of lower lumbar dorsal root ganglia under normal conditions and its alteration in neuropathic pain model. The characterization of AT₁ expression was done under control and after the chronic constriction injury induced by four loose ligatures of the sciatic nerve representing the model of posttraumatic painful peripheral neuropathy. Major Angiotensin II receptor type was expressed in approximately 43 % of small-sized and 62 % of large-sized neurons in control. The AT₁ overexpression after sciatic nerve ligation lasting 7 days was detected predominantly in small-sized AT₁ immunoreactive neurons (about 38 % increase). Chronic constriction injury caused a statistically marked increase in number of the small-sized peptidergic (CGRP immunoreactive) neuronal subpopulation expressing AT₁ (about 64 %). The subpopulations of AT₁-immunoreactive and nonpeptide-containing primary sensory neurons revealed by IB4 binding, tyrosine hydroxylase- and parvalbumin-immunoreactive neurons were not markedly changed. Our results indicate that: (1) the AT₁ overexpression after the chronic constriction injury is an important factor in Angiotensin II-potentiated pain perception; (2) Angiotensin II is involved in pathological mechanisms of neuropathic pain and this effect can be mediated perhaps in combination with other neuropeptides synthesized in the primary sensory neurons.     

c-fos Expression in Gracilothalamic Tract Neurons after Electrical Stimulation of the Injured Sciatic Nerve in the Adult Rat

The number of c-fos protein-like immunoreactive (Fos-LI) cells in the gracile nucleus was determined after electrical stimulation at Aα/Aβ-fiber strength of the normal and of the previously injured sciatic nerve in adult rats. No Fos-LI cells were seen after electrical stimulation of the noninjured sciatic nerve, or after sciatic nerve injury without electrical stimulation. However, stimulation 21 days after sciatic nerve transection resulted in numerous Fos-LI cells in the ipsilateral gracile nucleus. Combined Fos immunocytochemistry and retrograde labeling from the thalamus showed that the majority (76%; range = 70–80%) of the cells in the gracile nucleus that expressed Fos-LI after nerve injury projected to the thalamus. The results indicate that morphological, biochemical, and physiological alterations in primary sensory central endings and second-order neurons, which have earlier been demonstrated in the dorsal column nuclei after peripheral nerve injury, are accompanied by changes in the c-fos gene activation pattern after stimulation of the injured sciatic nerve. A substantial number of the c-fos-expressing neurons project to the thalamus.     

Changes in lectin binding of lumbar dorsal root ganglia neurons and peripheral axons after sciatic and spinal nerve injury in the rat

Summary The effects of chronic lesions of rat lumbar spinal or sciatic nerves on the binding of Glycine max (soybean) agglutinin to galacto-conjugates, in small-and medium-size primary sensory neurons of the L₄ and L₅ dorsal root ganglia, were examined over a 580-day period. Spinal nerve section resulted in a marked decrease in the population of stained neurons within 7 days. However, despite some retrograde morphological changes triggered by axonal injury, the proportion of stained nerve cells was normalized 180 days postoperatively. This temporary decrease in perikaryal lectin reactivity was initially associated with a marked accumulation of stained material in the nerve, proximal and distal to the site of section, with similar accumulations also being noticeable at each level of injury in sciatic nerves subjected to double ligature. This may reflect the presence of glycocompounds linked to the autolysis of nerve fibers during the phase of retrograde dying-back and Wallerian degeneration. At later stages, stained deposits could be seen scattered along central and peripheral axonal processes of the dorsal root ganglion neurons in the vicinity of the cell body. They may indicate a disturbance in the peripheral turnover of glycoproteins in chronically-transected nerves, with piling up of neuronal products. Sciatic nerve injury caused similar but less severe effects which, except for the L₄ ganglion cells, were rapidly reversible.     

The Plasticity of the DRG Neurons Belonging to Different Subpopulations After Dorsal Rhizotomy

SUMMARY 1. The plasticity of sensory neurons following the injury to their axons is very important for prognosis of recovery of afferent fibers with different modality. It is evident that the response of dorsal root ganglion (DRG) neurons after peripheral axotomy is different depending on the deficiency in neurotrophic factors from peripheral region. The loss of cells appears earlier and is more severe in B-cells (small, dark cells with unmyelinated axons) than in A-cells (large, light cells with myelinated axons).2. We studied using immunohistochemical methods the response of DRG neurons to dorsal rhizotomy and combined injury of central and peripheral neuronal processes. A quantitative analysis of DRG neurons tagged by the selective markers isolectin B4 (IB4) and the heavy molecular component of the neurofilament triplet (NF200) antibody, selective for subpopulations of small and large/medium DRG neurons, respectively, was performed after dorsal rhizotomy, peripheral axotomy, and their combination.3. The number of NF200⁺-neurons is reduced substantially after both dorsal rhizotomy and peripheral axotomy, while the decrease of IB4⁺-neurons is observed only in combined injury, i.e., dorsal rhizotomy accompanied with sciatic nerve injury.4. Our results show that distinct subpopulations of DRG neurons respond differently to the injury of their central processes. The number of NF200⁺-neurons decreases to greater degree following dorsal rhizotomy in comparison to IB4⁺-neurons.     

Rapid axoplasmic transport of insulin-like growth factor I in the sciatic nerve of adult rats

Summary Somatomedin C (Sm-C; insulin-like growth factor I; IGF-I) is a polypeptide (M_r 7649), often dependent on growth hormone (GH), with trophic effects on several different tissues. Monospecific IGF-I antisera were used to investigate its localization in the sciatic nerve and corresponding nerve cells, as well as its possible axoplasmic transport in the adult rat. IGF-I-like immunoreactivity was demonstrated in anterior horn motor nerve cells in the spinal cord and in spinal- and autonomic ganglion nerve cells. Faint IGF-I immunoreactivity was under normal conditions observed in axons of the sciatic nerve and in the Schwann cells. Using crush technique, accumulation of IGF-I immunoreactivity was seen in dilated axons within 2 h, both proximal and distal to the crush. However, only a small fraction of the anterogradely transported IGF-I immunoreactive material could be demonstrated to be transported in retrograde direction. Colchicine injected proximal to a crush prevented accumulation of IGF-I immunoreactivity proximal to the crush, but not distal to it.IGF-I-immunoreactive material is synthesized in the cell bodies of peripheral sensory and motor nerve cells. It is transported at rapid rates in the axoplasm of the sciatic nerve of adult rats both in anterograde and retrograde directions. We propose that axonally transported IGF-I may be released and exert trophic influence on innervated cells, tissues and organs.     

Primary sensory neurons of the rat showing calcitonin gene-related peptide immunoreactivity and their relation to substance P-, somatostatin-, galanin-, vasoactive intestinal polypeptide- and cholecystokinin-immunoreactive ganglion cells

Summary By use of the indirect immunofluorescence technique the distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) has been analyzed in cervical and lumbar dorsal root ganglia of untreated and colchicine-treated rats. In addition, lumbar ganglia were examined 2 weeks after transection of the sciatic nerve. The occurrence of CGRP-positive cells in relation to ganglion cells containing substance P-, somatostatin-, galanin-, cholecystokinin (CCK)-, and vasoactive intestinal polypeptide (VIP)/peptide histidine isoleucin (PHI)-LI has been evaluated on consecutive sections as well as using elution-restaining and double-staining techniques.CGRP-LI was observed in many ganglion cells of all sizes ranging in diameter from 15 m to 65 m. Thus, this peptide occurs also in the large primary sensory neurons. In contrast to the sensory peptides described to date, CGRP-positive cells constituted up to 50% of all and 70% of the medium-sized neurons, thus being the most frequently occurring peptide in sensory neurons so far encountered. Subpulations of CGRP-positive neurons were shown to contain substance P-, somatostatin-, or galanin-LI and some CGRP-positive neurons contained both substance P- and galanin-LI. In fact, most substance P-, somatostatin- and galanin-positive cell bodies were CGRP-immunoreactive. The coexistence analysis further revealed that galanin and substance P often coexisted and that some cells contained both substance P- and somatostatin-LI, whereas no coexistence between galanin and somatostatin has as yet been seen. VIP/PHI-LI was only shown in a few cells in untreated or colchicine-treated rats. However, after transcetion of the sciatic nerve numerous VIP/PHI-positive cells were observed, some of which also contained CGRP-LI.The present results indicate that a CGRP-like peptide is present in a wide range of primary sensory neurons probably not related to specific sensory modalities. Often this peptide coexists with other biologically active peptides. Taken together these findings suggest that CGRP may have a generalized function.     

Enhanced axon outgrowth and improved long‐distance axon regeneration in sprouty2 deficient mice

Sprouty (Spry) proteins are negative feedback inhibitors of receptor tyrosine kinase signaling. Downregulation of Spry2 has been demonstrated to promote elongative axon growth of cultured peripheral and central neurons. Here, we analyzed Spry2 global knockout mice with respect to axon outgrowth in vitro and peripheral axon regeneration in vivo. Neurons dissociated from adult Spry2 deficient sensory ganglia revealed stronger extracellular signal‐regulated kinase activation and enhanced axon outgrowth. Prominent axon elongation was observed in heterozygous Spry2^+/? neuron cultures, whereas homozygous Spry2^?/? neurons predominantly exhibited a branching phenotype. Following sciatic nerve crush, Spry2^+/? mice recovered faster in motor but not sensory testing paradigms (Spry2^?/? mice did not tolerate anesthesia required for nerve surgery). We attribute the improvement in the rotarod test to higher numbers of myelinated fibers in the regenerating sciatic nerve, higher densities of motor endplates in hind limb muscles and increased levels of GAP‐43 mRNA, a downstream target of extracellular regulated kinase signaling. Conversely, homozygous Spry2^?/? mice revealed enhanced mechanosensory function (von Frey's test) that was accompanied by an increased innervation of the epidermis, elevated numbers of nonmyelinated axons and more IB4‐positive neurons in dorsal root ganglia. The present results corroborate the functional significance of receptor tyrosine kinase signaling inhibitors for axon outgrowth during development and nerve regeneration and propose Spry2 as a novel potential target for pharmacological inhibition to accelerate long‐distance axon regeneration in injured peripheral nerves. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 217–231, 2015     

Choline acetyltransferase immunoreactivity of putative intrinsic primary afferent neurons in the rat ileum

The colocalisation of choline acetyltransferase (ChAT) with markers of putative intrinsic primary afferent neurons was determined in whole-mount preparations of the myenteric and submucosal plexuses of the rat ileum. In the myenteric plexus, prepared for the simultaneous localisation of ChAT and nitric oxide synthase (NOS), all nerve cells were immunoreactive (IR) for ChAT or NOS, but seldom for both; only 1.6 +/- 1.8% of ChAT-IR neurons displayed NOS-IR and, conversely, 2.8 +/- 3.3% of NOS-IR neurons were ChAT-IR. In preparations double labelled for NOS-IR and the general nerve cell marker, neuron-specific enolase, 24% of all nerve cells were immunoreactive for NOS, indicating that about 75% of all nerve cells have ChAT-IR. All putative intrinsic primary afferent neurons in the myenteric plexus, identified by immunoreactivity for the neurokinin 1 (NK1) receptor and the neurokinin 3 (NK3) receptor, were ChAT-IR. Conversely, of the ChAT-IR nerve cells, about 45% were putative intrinsic primary afferent neurons (this represents 34% of all nerve cells). The cell bodies of putative intrinsic primary afferent neurons had Dogiel type II morphology and were also immunoreactive for calbindin. All, or nearly all, nerve cells in the submucosal plexus were immunoreactive for ChAT. About 46% of all submucosal nerve cells were immunoreactive for both neuropeptide Y (NPY) and calbindin; 91.8 +/- 10.5% of NPY/calbindin cells were also ChAT-IR and 99.1 +/- 0.7% were NK3 receptor-IR. Of the nerve cells with immunoreactivity for ChAT, 44.3 +/- 3.8% were NPY-IR, indicating that about 55% of submucosal nerve cells had ChAT but not NPY-IR. Only small proportions of the ChAT-IR, non-NPY, nerve cells had NK3 receptor or calbindin-IR. It is concluded that about 45% of submucosal nerve cells are ChAT/calbindin/NPY/VIP/NK3 receptor-IR and are likely to be secretomotor neurons. Most of the remaining submucosal nerve cells are immunoreactive for ChAT, but their functions were not deduced. They may include the cell bodies of intrinsic primary afferent neurons.     

Phenotype of distinct primary sensory afferent subpopulations and caspase-3 expression following axotomy

Specific sensory neuronal subpopulations show contrasting responses to peripheral nerve injury, as shown by the axotomy-induced death of many cutaneous sensory neurons whilst muscular sensory afferents survive an identical insult. We used a novel combination of retrograde neuronal tracing with immunohistochemistry and laser microdissection techniques, in order to describe the neurochemistry of medial gastrocnemius (muscular sensory afferents) and sural (cutaneous sensory afferents) branches of the rat sciatic nerve and relate this to the pro-apoptotic caspase-3 gene expression following nerve transection. Our results demonstrated distinctions in medial gastrocnemius and sural neuron populations with the most striking difference in the respective proportions of isolectin B4 (IB4) staining neurons (3.7 V 32.8%). The mean neuronal area of the medial gastrocnemius (MG) neurons was larger than that of the sural (SUR) neurons (1,070.8 V 646.2 μm2) and each phenotypic group was significantly smaller in sural neurons than in MG neurons. At 1 week post-axotomy, MG neurons markedly downregulated caspase-3, whilst SUR neurons upregulated caspase-3 gene expression; this may be attributable to the differing IB4-positive composition of the subpopulations. These findings provide further clarification in the understanding of two distinct neuronal populations used increasingly in nerve injury models.     

Intrathecal NGF Administration Reduces Reactive Astrocytosis and Changes Neurotrophin Receptors Expression Pattern in a Rat Model of Neuropathic Pain

Nerve growth factor (NGF), an essential peptide for sensory neurons, seems to have opposite effects when administered peripherally or directly to the central nervous system. We investigated the effects of 7-days intrathecal (i.t.) infusion of NGF on neuronal and glial spinal markers relevant to neuropathic behavior induced by chronic constriction injury (CCI) of the sciatic nerve. Allodynic and hyperalgesic behaviors were investigated by Von Frey and thermal Plantar tests, respectively. NGF-treated animals showed reduced allodynia and thermal hyperalgesia, compared to control animals. We evaluated on lumbar spinal cord the expression of microglial (ED-1), astrocytic (GFAP and S-100β), and C- and Aδ-fibers (SubP, IB-4 and Cb) markers. I.t. NGF treatment reduced reactive astrocytosis and the density of SubP, IB4 and Cb positive fibers in the dorsal horn of injured animals. Morphometric parameters of proximal sciatic nerve stump fibers and cells in DRG were also analyzed in CCI rats: myelin thickness was reduced and DRG neurons and satellite cells appeared hypertrophic. I.t. NGF treatment showed a beneficial effect in reversing these molecular and morphological alterations. Finally, we analyzed by immunohistochemistry the expression pattern of neurotrophin receptors TrkA, pTrkA, TrkB and p75^NTR. Substantial alterations in neurotrophin receptors expression were observed in the spinal cord of CCI and NGF-treated animals. Our results indicate that i.t. NGF administration reverses the neuro-glial morphomolecular changes occurring in neuropathic animals paralleled by alterations in neurotrophin receptors ratio, and suggest that NGF is effective in restoring homeostatic conditions in the spinal cord and maintaining analgesia in neuropathic pain.     

Small cardioactive peptide-like immunoreactivity and its colocalization with FMRFamide-like immunoreactivity in the central nervous system of the leech Hirudo medicinalis

Summary The distributions of small cardioactive peptide (SCP)- and FMRFamide-like immunoreactivities in the central nervous system of the medicinal leech Hirudo medicinalis were studied. A subset of neurons in the segmental ganglia and brains was immunoreactive to an antibody directed against SCP_B. Immunoreactive cell bodies were regionally distributed throughout the nerve cord, and occurred both as bilaterally paired and unpaired neurons. The majority of the unpaired cells displayed a tendency to alternate from side to side in adjacent ganglia. A small number of neurons were immunoreactive only in a minority of nerve cords investigated. Intracellular injections of Lucifer yellow dye and subsequent processing for immunocytochemistry revealed SCP-like immunoreactivity in heart modulatory neurons but not in heart motor neurons. FMRFamide-like immunoreactivity was also detected in cell bodies throughout the central nervous system. A subset of neurons contained both SCP- and FMRFamide-like immunoreactivities; others stained for only one or the other antigen. These data suggest that an antigen distinct from FMRFamide is responsible for at least part of the SCP-like immunoreactivity. This antigen likely bears some homology to the carboxyl terminal of SCP_A and SCP_B.     

Nestin-Expressing Stem Cells Promote Nerve Growth in Long-Term 3-Dimensional Gelfoam?-Supported Histoculture

We have previously reported that hair follicles contain multipotent stem cells which express nestin. The nestin-expressing cells form the hair follicle sensory nerve. In vitro, the nestin-expressing hair follicle cells can differentiate into neurons, Schwann cells, and other cell types. In the present study, the sciatic nerve was excised from transgenic mice in which the nestin promoter drives green fluorescent protein (ND-GFP mice). The ND-GFP cells of the sciatic nerve were also found to be multipotent as the ND-GFP cells in the hair follicle. When the ND-GFP cells in the mouse sciatic nerve cultured on Gelfoam® and were imaged by confocal microscopy, they were observed forming fibers extending the nerve. The fibers consisted of ND-GFP-expressing spindle cells, which co-expressed the neuron marker β-III tubulin, the immature Schwann-cell marker p75^NTR and TrkB which is associated with neurons. The fibers also contain nestin-negative spherical cells expressing GFAP, a Schwann-cell marker. The β-III tubulin-positive fibers had growth cones on their tips expressing F-actin, indicating they are growing axons. When the sciatic nerve from mice ubiquitously expressing red fluorescent protein (RFP) was co-cultured on Gelfoam® with the sciatic nerve from ND-GFP transgenic mice, the interaction of nerves was observed. Proliferating nestin-expressing cells in the injured sciatic nerve were also observed in vivo. Nestin-expressing cells were also observed in posterior nerves but not in the spinal cord itself, when placed in 3-D Gelfoam® culture. The results of the present report suggest a critical function of nestin-expressing cells in peripheral nerve growth and regeneration.     

Immunoreactive endozepine-like peptides in the brain and pituitary of the Atlantic hagfish, Myxine glutinosa

Endozepines are a family of peptides capable of displacing benzodiazepines from their specific binding sites, to which belong the diazepam-binding inhibitor and the octadecaneuropeptide (ODN). This paper reports the distribution of ODN-related peptides, investigated for the first time by immunocytochemistry, in different brain and pituitary regions of the Atlantic hagfish, Myxine glutinosa. Immunoreactive ODN-like material was found in the telencephalon at the level of bundles of different olfactory nerve fibres. Moreover, at the level of the pallium, immunoreactive multipolar neurons were observed in the pars parvocellularis of the stratum griseum superficialis. Similar immunopositive nerve cell bodies were found in the nucleus medialis of the central prosencephalic complex. In the mesencephalon, few immunoreactive neurons lining and contacting the mesencephalic ventricle were detected; such nerve cells could be involved in the regulation of cerebrospinal fluid homeostasis. Dorsally in the mesencephalon, numerous ODN-containing cell bodies were present in the area praetectalis. The rhomboencephalon was immunostained only in the octavolateral area and in the nucleus motorius magnocellularis of the trigeminal nerve. Furthermore, ODN immunoreactivity was also present in the nerve cells of ganglia of the ophthalmic division of the trigeminal nerve complex. The immunocytochemical patterns described here in the brain of M. glutinosa suggest an involvement of ODN-like peptides as neuromodulators in sensory pathways, such as olfactory and visual. Finally, ODN-like substances were localized in discrete populations of adenohypophysial cells and in tanycytes lining the neurohypophyseal walls, suggesting for endozepines a paracrine and/or endocrine control of pituitary hormones release and a neurohormone role respectively. These results could give new insights into the chemioarchitecture of the brain of myxinoids.     

Distribution and functional significance of Met-enkephalin-Arg6-Phe7-and Met-enkephalin-Arg6-Gly7-Leu8-like peptides in the blowflyCalliphora vomitoria

Summary Neuronal pathways in the retrocerebral complex and thoracico-abdominal ganglionic mass of the blowflyCalliphora vomitoria have been identified immunocytochemically with antisera against the extended-enkephalins, Met-enkephalin-Arg⁶-Phe⁷ (Met-7) and Met-enkephalin-Arg⁶-Gly⁷-Leu⁸ (Met-8). Neurons of the hypocerebral ganglion, immunoreactive to Met-8, have axons in the crop duct nerve and terminals in muscles of the crop and its duct. Certain neurons of the hypocerebral ganglion are also immunoreactive to Met-7, and axons from these cells innervate the heart. Met-8 immunoreactive nerve terminals invest the cells of the corpus allatum. The source of this material is believed to ve a single pair of lateral neurosecretory cells in the brain. There is no Met-7 immunoreactive material in the corpus allatum. In the corpus cardiacum neither Met-7 nor Met-8 immunoreactivity is present in the cells. However, in the neuropil of the gland certain fibres, with their origins elsewhere, do contain Met-8 immunoreactivity. The most prominent neurons in the thoracic ganglion are the Met-7 immunoreactive ventral thoracic neurosecretory cells, axons from which project to neurohaemal areas in the dorsal neural sheath and also, via the ventral connective, to the brain. Co-localisation studies show that the perikarya of these cells are immunoreactive to antisera raised against several vertebrate-type peptides, such as Met-7, gastrin/cholecystokinin and pancreatic polypeptide. However, their axons and terminals show varying amounts of the peptides, suggesting differential transport and utilisation. Only a few cells in the thoracic ganglion are immunoreactive to Met-8 antisera. These lie close to the nerve bundles suppling the legs. In the abdominal ganglion, Met-8 immunoreactive neurons project to the muscles of the hindgut. This study suggests that the extended enkephalin-like peptides ofCalliphora may have a variety of different roles: as neurotransmitter or neuromodulator substances; in the direct innervation of effector organs; and as neurohormones.     

Identification of the populations of enteric neurons that have NK1 tachykinin receptors in the guinea-pig small intestine

Simultaneous immunofluorescence labelling was used to investigate the patterns of colocalisation of the NK1 tachykinin receptor with other neuronal markers, and hence determine the functional classes of neuron that bear the NK1 receptor in the guinea-pig ileum. In the myenteric plexus, 85% of NK1 receptor-immunoreactive (NK1r-IR) nerve cells had nitric oxide synthase (NOS) immunoreactivity and the remaining 15% were immunoreactive for choline acetyltransferase (ChAT). Of the latter group, about 50% were immunoreactive for both neuropeptide Y (NPY) and somatostatin (SOM), and had the morphologies of secretomotor neurons. Many of the remaining ChAT neurons were immunoreactive for calbindin or tachykinins (TK), but not both. These calbindin immunoreactive neurons had Dogiel type II morphology. No NK1r-IR nerve cells in the myenteric plexus had serotonin or calretinin immunoreactivity. In the submucosal ganglia, 84% of NK1r-IR nerve cells had neuropeptide Y immunoreactivity and 16% were immunoreactive for TK. It is concluded that NK1r-IR occurs in five classes of neuron; namely, in the majority of NOS-immunoreactive inhibitory motor neurons, in ChAT/TK-immunoreactive excitatory neurons to the circular muscle, in all ChAT/NPY/SOM-immunoreactive secretomotor neurons, in a small proportion of ChAT/calbindin myenteric neurons, and in about 50% of ChAT/TK submucosal neurons.     

Histamine-like immunoreactivity in the visual system and brain of Drosophila melanogaster

Summary In this study, immunohistochemistry on cryostat sections is used to demonstrate anti-histamine immunoreactivity in the Drosophila brain. The results support earlier findings that histamine is probably a transmitter of insect photoreceptors. It is further shown that, in Drosophila, all imaginal photoreceptors including receptor type R7 are anti-histamine immunoreactive, whereas the larval photoreceptors do not seem to contain histamine. In addition to the photoreceptors, fibres in the antennal nerve and approximately 12 neurons in each brain hemisphere show strong histamine-like immunoreactivity. These cells arborize extensively in large parts of the central brain.     

Ultrastructural and immunocytochemical observations of the nervous systems of three macrodasyidan gastrotrichs

The nervous systems of three macrodasyidan gastrotrichs, Dactylopodola baltica, Macrodasys caudatus and Dolichodasys elongatus, were investigated using immunocytochemistry and electron microscopy. Labelling of neural structures against serotonin revealed the presence of two pairs of cerebral cells, a dorsal cerebral connective, and paired ventral nerve cords in D. baltica. In M. caudatus and D. elongatus serotonin immunoreactivity was present in a single pair of dorsal cerebral cells and the ventral nerve cords; the dorsal connective of D. elongatus was also immunoreactive to serotonin and acetylated α‐tubulin. The presence of paired, serotonin‐like immunoreactive cells in D. baltica and other species may represent the plesiomorphic condition in Macrodasyida. The fine structure of the photoreceptors in D. baltica was also investigated to explore the potential ground pattern for eyes in the Macrodasyida. The pigmented photoreceptors of D. baltica contain a unicellular pigment cup, sheath cell and sensory receptor. The pigment cup contains numerous osmiophilic granules that presumably function to shield the eyes from downwelling light in the red part of the spectrum. Projecting into the pigment cup and sheath cell are numerous microvilli from a bipolar sensory cell. A single sensory cell may represent the plesiomorphic condition in Macrodasyida, with multiplication of sensory cells representative of more derived taxa.     

Pluripotent Hair Follicle Neural Crest Stem-Cell-Derived Neurons and Schwann Cells Functionally Repair Sciatic Nerves in Rats

In this paper, we constructed a novel acellular nerve xenograft (ANX) seeded with neurons and Schwann cells to bridge long-distance gaps in rat sciatic nerves. The neurons and Schwann cells were induced from Sprague Dawley (SD) rat hair follicle neural crest stem cells with sonic hedgehog/retinoic acid and neuregulin 1, respectively. Fifty male SD rats were randomly divided into two groups (n = 25): ANX + cells group and ANX group. A 4-cm-long sciatic nerve defect was created on the right hind limb and bridged with cell-seeded ANX in ANX + cells group or ANX alone in ANX group. We found that the implanted neurons and Schwann cells could survive by 4 weeks and as far as 52 weeks posttransplantation. In implanted grafts, chemical synaptic structures were also found under transmission electron microscope and confirmed with immunostaining of synapsin 1, a synaptic marker. The number of regenerated axons in ANX + cells group was higher than that in ANX group (P < 0.01). This novel implantation of neurons and Schwann cells via acellular nerve graft may provide an alternative way for repairing peripheral nerve defect.     

Cupular organs in two species of Corella (Tunicata: Ascidiacea)

Abstract. Simple cupular organs similar to those described in Ciona intestinalis were observed in Corella eumyota. They consist of a macula containing the cell bodies of 20–30 primary sensory neurons whose cilia project into a dome‐ or finger‐shaped structure, the cupula. Rather than being found in the mantle lining as in C. intestinalis, the organs were located on the atrial surface of the branchial sac. The sensory innervation was examined in whole‐mount preparations using anti‐tubulin immunohistochemistry. Sensory neurons in C. eumyota showed no immunoreactivity with antisera raised against gonadotropin‐releasing hormone (GnRH). A novel, elongated sense organ termed the cupular strand was found in Corella inflata. It has the same basic components as the simple type of cupular organ but consists of a single, long structure containing ～1500 sensory cells. Located on the atrial surface of the branchial sac, it extends along the midline of the dorsal fold, from the gonoduct openings almost as far as the brain. Preparations were examined using optical and electron microscopy. Nerves and cilia were visualized by anti‐tubulin immunofluorescence microscopy. It was possible to follow the sensory axons from the macula of the cupular strand to points where they joined branches of the visceral nerve, which enters a nerve root at the back of the brain. In C. inflata the sensory cell bodies and their axons were immunoreactive not only with anti‐tubulin but also with an antiserum raised against Tunicate I GnRH. There was no immunoreactivity, however, with Chicken II and catfish GnRH antisera. All three GnRH antisera labeled the dorsal strand plexus, a structure associated with production of GnRH in its role as a reproductive hormone. We concluded that the GnRH‐like molecule labeled in sensory neurons differs from the form of GnRH found in the dorsal strand plexus, and may have a different function, perhaps in the neural control of ciliary activity. The function of the cupular organs in species of Corella has not yet been investigated physiologically, but by analogy with such structures in other metazoans, cupular organs are probably hydrodynamic sensors registering local disturbances or changes in water flow through the atrial cavity.