Localization of a Passively Transferred Human Recombinant Monoclonal Antibody to Herpes Simplex Virus Glycoprotein D to Infected Nerve Fibers and Sensory Neurons In Vivo

A human recombinant monoclonal antibody to herpes simplex virus (HSV) glycoprotein D labeled with the fluorescent dye Cy5 was administered to mice infected in the cornea with HSV type 1 (HSV-1). The distribution of such antibody in the corneas and trigeminal ganglia of the mice was then investigated by confocal microscopy. The antibody was detected on HSV-infected nerve fibers in the cornea--identified by colocalization with HSV antigens and the neuritic markers neurofilament, GAP-43, synapsin-1, and CNPase--and on the perikarya of sensory neurons in the HSV-1-infected neurons in ipsilateral trigeminal ganglia. Antibodies have been shown to be effective against many neurotropic viruses, often in the absence of obvious cell damage. Observations from experimental HSV infections suggest that antibodies could act in part by interfering with virus expression in the ganglia and/or with axonal spread. The present results provide morphological evidence of the localization of antiviral antibodies at anatomical sites relevant to such putative antibody-mediated protective actions and suggest that viral glycoproteins are accessible to antibodies on infected nerve fibers and sensory neurons.     

Enhancement of CGRP sensory afferent innervation in the gut during the development of food allergy in an experimental murine model

Recent advances in neuroscience and immunology have revealed a bidirectional interaction between the nervous and immune systems. Therefore, the gastrointestinal tract may be modulated by neuro–immune interactions, but little information about this interaction is available. Intrinsic and extrinsic primary afferent neurons play an important role in this interaction because of their abilities to sense, process and transmit various information in the intestinal microenvironment. Calcitonin gene-related peptide (CGRP) is exclusively contained in intrinsic and extrinsic primary afferent neurons in the mouse intestine. Therefore, we investigated CGRP-immunoreactive nerve fibers in the colonic mucosa of mice induced to develop food allergy. CGRP-immunoreactive nerve fibers were specifically increased with the development of food allergy, and the fibers were juxtaposed to mucosal mast cells in the colonic mucosa of food allergy mice. Denervation of the extrinsic afferent neurons using neonatal capsaicin treatment did not affect the development of food allergy or the density and distribution of CGRP-immunoreactive nerve fibers in the colonic mucosa of food allergy mice. Furthermore, the mRNA and plasma level of CGRP was increased in food allergy mice. These results suggest that the activation of intrinsic primary afferent neurons in the intestine contributes to the development and pathology of food allergy.     

Impaired pain sensation in mice lacking Aquaporin-1 water channels

Aquaporin-1 (AQP1), a membrane water channel, is expressed in choroid plexus where it contributes to cerebrospinal fluid production. Here, we show that AQP1 is also expressed in the dorsal horn of the spinal cord and the trigeminal nucleus caudalis, regions that process pain information. Within the dorsal root and trigeminal sensory ganglia, AQP1 is concentrated in small diameter cell bodies, most of which give rise to unmyelinated C-fibers. To study the role of AQP1 in pain signaling, we compared acute pain responses in wild-type mice and in mice lacking AQP1. AQP1^−/− mice had reduced responsiveness to thermal and capsaicin chemical stimuli, but not to mechanical stimuli or formalin. These results provide evidence for AQP1 expression in nociceptive neurons and suggest that AQP1 may play a role in pain signal transduction.     

Expression of aquaporin-4 water channels in the digestive tract of the guinea pig

Expression of the aquaporin-4 (AQP4) water channel was systematically studied in the digestive tract of the guinea pig using Western blot and immunofluorescence techniques. The results showed that AQP4 was expressed widely in different segments of the guinea pig digestive tract. AQP4-immunoreactivity was confined to parietal cells in the stomach, and absorptive and glandular epithelial cells of small and large intestine. AQP4 protein was also expressed by enteric glial cells of submucosal and myenteric ganglia and primary nerve trunks. AQP4 was expressed by both type I and type II enteric gliocytes, but not by type III or type IV enteric gliocytes, indicating that enteric gliocytes have a heterogeneous distribution in the gut wall. In addition, different patterns of AQP4 expression in the enteric nervous system of human, guinea pig, rat and mouse colon mucosa were identified: in rat and mouse AQP4 was localised to a small subpopulation of neurons; in the guinea pig AQP4 was localised to enteric glial cells; and in the human colon mucosa, AQP4 was also detected mainly in the glial cells. It has been speculated that AQP4 may be involved in water transport in the gastrointestinal tract. Its role in enteric neurons and glia is unknown, but, by analogy with the brain, AQP4 may be involved in the formation and resolution of edema.     

Mechanical sensitivity of the facial nerve fibers innervating the anterior palate of the puffer,Fugu pardalis,and their central projection to the primary taste center

Mechanical and chemical sensitivity of the palatine nerve, ramus palatinus facialis, innervating the anterior palate of the puffer, Fugu pardalis, and their central projection to the primary taste center were investigated. Application of horseradish peroxidase (HRP) to the central cut end of the palatine nerve resulted in retrogradely labeled neurons in the geniculate ganglion but no such neurons in the trigeminal ganglion, suggesting that the palatine nerve is represented only by the facial component. Tracing of the facial sensory root in serial histological sections of the brain stem suggested that the facial sensory nerve fibers project only to the visceral sensory column of the medulla. Peripheral recordings from the palatine nerve bundle showed that both mechanical and chemical stimuli caused marked responses. Mechanosensitive fibers were rather uniformly distributed in the nerve bundle. Intra-cranial recordings from the trigeminal and facial nerves at their respective roots revealed that tactile information produced in the anterior palate was carried by the facial nerve fibers. Elimination of the sea water current over the receptive field also caused a marked response in the palatine nerve bundle or facial nerve root while this did not cause any detectable responses in the trigeminal nerve root. Single fiber analyses of the mechanical responsiveness of the palatine nerve were performed by recording unit responses of 106 single fibers to mechanical stimuli (water flow), HCl (0.005 M), uridine-5'-monophosphate (UMP, 0.001 M), proline (0.01 M), CaCl2 (0.5 M), and NaSCN (0.5 M). All these fibers responded well to one of the above stimuli; however, most taste fibers did not respond well to the inorganic salts. The palatine fibers (n = 36), identified as mechanosensitive, never responded to any of the chemical stimuli, whereas chemosensitive fibers (n = 70) did not respond to mechanical stimuli at all. The chemosensitive units showed a high specificity to the above stimuli: they tended to respond selectively to hydrochloric acid, UMP, or proline. The responses of the mechanosensitive units consisted of phasic and tonic impulse trains and the sensitivity of the units varied considerably. The results reveal that the facial nerve fibers innervating the anterior palate of the puffer contain two kinds of afferent fibers, chemosensory and mechanosensory respectively, and suggest that the convergence of the tactile and gustatory information first occurs in the neurons of the primary gustatory center in the medulla.     

Motor unit abnormalities in Dystonia musculorum mice

Dystonia musculorum (dt) is a mouse inherited sensory neuropathy caused by mutations in the dystonin gene. While the primary pathology lies in the sensory neurons of dt mice, the overt movement disorder suggests motor neurons may also be affected. Here, we report on the contribution of motor neurons to the pathology in dt(27J) mice. Phenotypic dt(27J) mice display reduced alpha motor neuron cell number and eccentric alpha motor nuclei in the ventral horn of the lumbar L1 spinal cord region. A dramatic reduction in the total number of motor axons in the ventral root of postnatal day 15 dt(27J) mice was also evident. Moreover, analysis of the trigeminal nerve of the brainstem showed a 2.4 fold increase in number of degenerating neurons coupled with a decrease in motor neuron number relative to wild type. Aberrant phosphorylation of neurofilaments in the perikaryon region and axonal swellings within the pre-synaptic terminal region of motor neurons were observed. Furthermore, neuromuscular junction staining of dt(27J) mouse extensor digitorum longus and tibialis anterior muscle fibers showed immature endplates and a significant decrease in axon branching compared to wild type littermates. Muscle atrophy was also observed in dt(27J) muscle. Ultrastructure analysis revealed amyelinated motor axons in the ventral root of the spinal nerve, suggesting a possible defect in Schwann cells. Finally, behavioral analysis identified defective motor function in dt(27J) mice. This study reveals neuromuscular defects that likely contribute to the dt(27J) pathology and identifies a critical role for dystonin outside of sensory neurons.     

Intravenous neuromyelitis optica autoantibody in mice targets aquaporin-4 in peripheral organs and area postrema

The pathogenesis of neuromyelitis optica (NMO) involves binding of IgG autoantibodies (NMO-IgG) to aquaporin-4 (AQP4) on astrocytes in the central nervous system (CNS). We studied the in vivo processing in mice of a recombinant monoclonal human NMO-IgG that binds strongly to mouse AQP4. Following intravenous administration, serum [NMO-IgG] decreased with t_1/2 ∼18 hours in wildtype mice and ∼41 hours in AQP4 knockout mice. NMO-IgG was localized to AQP4-expressing cell membranes in kidney (collecting duct), skeletal muscle, trachea (epithelial cells) and stomach (parietal cells). NMO-IgG was seen on astrocytes in the area postrema in brain, but not elsewhere in brain, spinal cord, optic nerve or retina. Intravenously administered NMO-IgG was also seen in brain following mechanical disruption of the blood-brain barrier. Selective cellular localization was not found for control (non-NMO) IgG, or for NMO-IgG in AQP4 knockout mice. NMO-IgG injected directly into brain parenchyma diffused over an area of ∼5 mm² over 24 hours and targeted astrocyte foot-processes. Our data establish NMO-IgG pharmacokinetics and tissue distribution in mice. The rapid access of serum NMO-IgG to AQP4 in peripheral organs but not the CNS indicates that restricted antibody access cannot account for the absence of NMO pathology in peripheral organs.     

Chemosensory Information Processing between Keratinocytes and Trigeminal Neurons

Trigeminal fibers terminate within the facial mucosa and skin and transmit tactile, proprioceptive, chemical, and nociceptive sensations. Trigeminal sensations can arise from the direct stimulation of intraepithelial free nerve endings or indirectly through information transmission from adjacent cells at the peripheral innervation area. For mechanical and thermal cues, communication processes between skin cells and somatosensory neurons have already been suggested. High concentrations of most odors typically provoke trigeminal sensations in vivo but surprisingly fail to activate trigeminal neuron monocultures. This fact favors the hypothesis that epithelial cells may participate in chemodetection and subsequently transmit signals to neighboring trigeminal fibers. Keratinocytes, the major cell type of the epidermis, express various receptors that enable reactions to multiple environmental stimuli. Here, using a co-culture approach, we show for the first time that exposure to the odorant chemicals induces a chemical communication between human HaCaT keratinocytes and mouse trigeminal neurons. Moreover, a supernatant analysis of stimulated keratinocytes and subsequent blocking experiments with pyrodoxalphosphate-6-azophenyl-2′,4′-disulfonate revealed that ATP serves as the mediating transmitter molecule released from skin cells after odor stimulation. We show that the ATP release resulting from Javanol® stimulation of keratinocytes was mediated by pannexins. Consequently, keratinocytes act as chemosensors linking the environment and the trigeminal system via ATP signaling.     

人十二指肠血管活性肠肽能P物质能神经分布的研究

本文采用免疫组织化学ＡＢＣ法研究血管活性肠肽（ＶＩＰ） 能神经和Ｐ物质（ＳＰ） 能神经在人十二指肠壁内的分布。结果显示： ＶＩＰ能和ＳＰ能神经纤维和神经元均呈棕褐色; ＶＩＰ能神经纤维遍布肠壁各层,ＳＰ能神经纤维主要分布于肌层和神经丛; ＶＩＰ能和ＳＰ能神经元见于肌间和粘膜下神经, 尤以后者为多, 但形态特点不同; 在肌间神经丛, ＳＰ能神经元比ＶＩＰ能神经元多。粘膜内可见ＶＩＰ能和ＳＰ能神经元, 多单个分布在粘膜肌层内。结果表明： １ＶＩＰ能和ＳＰ能神经在人十二指肠壁内分布有差异。２粘膜内存在ＶＩＰ能和ＳＰ能神经元     

小鼠眼神经注入Dil后三叉神经节的形态学研究

目的：经眼神经注入DiI研究小鼠三叉神经节的形态学结构。方法：小鼠10只,体重25—30克,雌雄不拘,进行灌注固定后,在外科显微镜下开颅并确认三叉神经节和眼神经,分别于双侧眼神经植入DiI染色晶体。37℃恒温箱放置3个月,待DiI染色晶体扩散后,取出植入DiI染色晶体的眼神经和三叉神经节,再根据神经走向切片,通过荧光显微镜观察DiI染色晶体在三又神经节内的分布。结果：眼神经离三叉神经节约1cm处植入DiI染色晶体后,应用荧光显微镜明视野观察,均可见到高密度标记的眼神经纤维,行向后内,穿经眶上裂入颅。逐步靠近三叉神经节外上方,并进入三叉神经节内,眼神经标记的神经元位于三叉神经节的前内侧。在三叉神经节内可见到DiI标记的神经节细胞及神经纤维。神经纤维平行致密排列,并被神经节细胞神经纤维分隔成群或簇。神经节细胞呈圆形和卵圆形,大小不一,部分节细胞呈蜂窝状排列。亦可见神经元的突起,有的呈螺旋状连于胞体,有的呈线状连于胞体,并可见到双极神经元。结论：小鼠经眼神经注入DiI后,三叉神经节细胞和神经纤维的排列循序跟其他动物基本一致。     

Immunohistochemical mapping of galanin-like neurons in the rat central nervous system

Using an antiserum generated in rabbits against synthetic galanin (GA) and the indirect immunofluorescence method, the distribution of GA-like immunoreactive cell bodies and nerve fibers was studied in the rat central nervous system (CNS) and a detailed stereotaxic atlas of GA-like neurons was prepared. GA-like immunoreactivity was widely distributed in the rat CNS. Appreciable numbers of GA-positive cell bodies were observed in the rostral cingulate and medial prefrontal cortex, the nucleus interstitialis striae terminalis, the caudate, medial preoptic, preoptic periventricular, and preoptic suprachiasmatic nuclei, the medial forebrain bundle, the supraoptic, the hypothalamic periventricular, the paraventricular, the arcuate, dorsomedial, perifornical, thalamic periventricular, anterior dorsal and lateral thalamic nuclei, medial and central amygdaloid nuclei, dorsal and ventral premamillary nuclei, at the base of the hypothalamus, in the central gray matter, the hippocampus, the dorsal and caudoventral raphe nuclei, the interpeduncular nucleus, the locus coeruleus, ventral parabrachial, solitarii and commissuralis nuclei, in the A1, C1 and A4 catechaolamine areas, the posterior area postrema and the trigeminal and dorsal root ganglia. Fibers were generally seen where cell bodies were observed. Very dense fiber bundles were noted in the septohypothalamic tract, the preoptic area, in the hypothalamus, the habenula and the thalamic periventricular nucleus, in the ventral hippocampus, parts of the reticular formation, in the locus coeruleus, the dorsal parabrachial area, the nucleus and tract of the spinal trigeminal area and the substantia gelatinosa, the superficial layers of the spinal cord and the posterior lobe of the pituitary. The localization of the GA-like immunoreactivity in the locus coeruleus suggests a partial coexistence with catecholaminergic neurons as well as a possible involvement of the GA-like peptide in a neuroregulatory role.     

SCN2B in the Rat Trigeminal Ganglion and Trigeminal Sensory Nuclei

The beta-2 subunit of the mammalian brain voltage-gated sodium channel (SCN2B) was examined in the rat trigeminal ganglion (TG) and trigeminal sensory nuclei. In the TG, 42.6 % of sensory neurons were immunoreactive (IR) for SCN2B. These neurons had various cell body sizes. In facial skins and oral mucosae, corpuscular nerve endings contained SCN2B-immunoreactivity. SCN2B-IR nerve fibers formed nerve plexuses beneath taste buds in the tongue and incisive papilla. However, SCN2B-IR free nerve endings were rare in cutaneous and mucosal epithelia. Tooth pulps, muscle spindles and major salivary glands were also innervated by SCN2B-IR nerve fibers. A double immunofluorescence method revealed that about 40 % of SCN2B-IR neurons exhibited calcitonin gene-related peptide (CGRP)-immunoreactivity. However, distributions of SCN2B- and CGRP-IR nerve fibers were mostly different in facial, oral and cranial structures. By retrograde tracing method, 60.4 and 85.3 % of TG neurons innervating the facial skin and tooth pulp, respectively, showed SCN2B-immunoreactivity. CGRP-immunoreactivity was co-localized by about 40 % of SCN2B-IR cutaneous and tooth pulp TG neurons. In trigeminal sensory nuclei of the brainstem, SCN2B-IR neuronal cell bodies were common in deep laminae of the subnucleus caudalis, and the subnuclei interpolaris and oralis. In the mesencephalic trigeminal tract nucleus, primary sensory neurons also exhibited SCN2B-immunoreactivity. In other regions of trigeminal sensory nuclei, SCN2B-IR cells were very infrequent. SCN2B-IR neuropil was detected in deep laminae of the subnucleus caudalis as well as in the subnuclei interpolaris, oralis and principalis. These findings suggest that SCN2B is expressed by various types of sensory neurons in the TG. There appears to be SCN2B-containing pathway in the TG and trigeminal sensory nuclei.     

Association of substance-P-immunoreactive nerves with the murine olfactory mucosa

Location and distribution of nerve fibers immunoreactive to substance P were studied in the mouse olfactory mucosa. A moderately dense plexus of fibers is present at the interface of the olfactory epithelium and the connective tissue of the lamina propria. In addition, many immunoreactive nerve fibers are noted in close association with Bowman's glands and blood vessels in the lamina propria. However, such fibers were not observed in olfactory epithelium proper nor in the fila olfactoria. Substance-P-immunoreactivity is almost totally abolished by treatment of animals with capsaicin, an agent known to deplete substance P from primary sensory neurons. It is suggested that the substance-P-immunoreactive fibers are of sensory origin, with their perikarya most likely located in the trigeminal ganglia. Functionally, they might influence local blood flow and/or the secretion of Bowman's glands.     

Central Projections of Identified Trigeminal Primary Afferents after Molar Pulp Deafferentation in Adult Rats

It is known that removal of the tooth pulp from mandibular molar teeth in adult rats alters the mechanoreceptive field properties of many low-threshold mechanoreceptive neurons in the trigeminal brainstem nuclear complex. The present study investigates one possible way that such deafferentation-induced receptive field changes could occur: altered central projections of uninjured trigeminal low-threshold mechanoreceptive primary afferent fibers. Intra-axonal injection of horseradish peroxidase (n = 22) or neurobiotin (n = 44) into characterized fibers was performed ipsilateral to, and 10–32 days after, removal of the coronal pulp from the left mandibular molars in adult rats. Collaterals were reconstructed, quantified, and compared by means of multivariate analyses of variance to equivalent fibers stained in normal adult rats.

Stained mechanosensitive fibers from experimental animals were rapidly conducting and responded to light mechanical stimulation of one vibrissa, one tooth, oral mucosa, facial hairy skin, or guard hairs. Their central projections were indistinguishable from those of control axons in all four trigeminal subnuclei. The numbers of collaterals, areas subtended by collateral arbors, numbers of boutons per collateral, and arbor circularity did not differ from those of control afferents. Collateral somatotopy was also unaffected.

These data suggest that following pulpotomy, the central collaterals of uninjured trigeminal afferents display normal morphologies and maintain normal somatotopy. Changes in the morphology of low-threshold primary afferents cannot account for the changes that occur in the receptive field properties of trigeminal brainstem neurons after pulp deafferentation.     

Distribution of serotonin-immunoreactive nerve cells and fibers in the rat gastrointestinal tract

 The distribution of serotonin-immunoreactive (5HT-IR) nerve cells and fibers was thoroughly investigated immunohistochemically in the rat stomach, duodenum, jejunum, ileum, and colon. The immunoreactivity of the 5HT neurons was compared between non-treated controls and animals treated with colchicine, colchicine plus 5-hydroxytryptophan (5HTP), colchicine plus pargyline, and reserpine. The intensity of immunoreactivity in nerve fibers as well as nerve cell bodies was enhanced mostly in colchicine plus pargyline treated animals, therefore these animals were used for an observation of precise localization of 5HT in the rat gastrointestinal (GI) tract. Immunoreactivity in the nerve cell bodies and fibers was completely abolished in the GI tract of reserpine treated animals. The pattern of localization and projection of 5HT-IR neurons was similar in all segments of the rat GI tract. 5HT-IR nerve cell bodies were located in the myenteric plexus and showed the distinctive features of Dogiel type I neurons. Prominent bundles of varicose fibers traversed the myenteric ganglia and some of them surrounded the cell bodies of immunopositive and immunonegative neurons. 5HT-IR nerve fibers were located in the submucous plexus, densely entwined about the submucosal blood vessels. Most characteristically, 5HT-IR nerve fibers invaded the lamina propria of mucosa where they underlay the crypt epithelium. In conclusion, the present study showed that 5HT-IR neurons located in the myenteric plexus projected fibers widely in the rat GI tract. The localization of fibers in the lamina propria of mucosa implies that this neuron may exert an important role in the epithelial function of the GI tract. Accepted: 8 October 1996     

两种软体动物神经系统一氧化氮合酶的组织化学定位

运用一氧化氮合酶(NOS)组织化学方法研究了软体动物门双壳纲种类中国蛤蜊和腹足纲种类嫁Qi神经系统中NOS阳性细胞以及阳性纤维的分布。结果表明：在蛤蜊脑神经节腹内侧,每侧约有10-15个细胞呈强NOS阳性反应,其突起也呈强阳性反应,并经脑足神经节进入足神经节的中央纤维网中;足神经节内只有2个细胞呈弱阳性反应,其突起较短,进入足神经节中央纤维网中,但足神经节中,来自脑神经节阳性细胞和外周神经系统的纤维大多呈NOS阳性反应;脏神经节的前内侧部和后外侧部各有一个阳性细胞团,其突起分别进入后闭壳肌水管后外套膜神经和脑脏神经索。脏神经节背侧小细胞层以及联系两侧小细胞层的纤维也呈NOS阳性反应。嫁Qi中枢神经系统各神经节中没有发现NOS阳性胞体存在;脑神经节、足神经节、侧神经节以及脑—侧、脑—足、侧—脏连索中均有反应程度不同的NOS阳性纤维,这些纤维均源于外周神经。与已研究的软体动物比较,嫁Qi和前鳃亚纲其它种类一样,神经系统中NO作为信息分子可能主要存在于感觉神经。而中国蛤蜊的神经系统中一氧化氮作为信息分子则可能参与更广泛的神经调节过程。     

Sensory innervation of the ear drum and middle-ear mucosa: Retrograde tracing and immunocytochemistry

Summary The distribution and origin of nerve fibers of presumed sensory nature in the ear drum and middle-ear mucosa of the rat were studied by a retrograde tracing technique in combination with immunocytochemistry.Application of True Blue (TB) on the ear drum or on the middle-ear mucosa labeled nerve cell bodies in the jugular, trigeminal, geniculate and cervical dorsal root ganglia (C₂–C₄). Judging from the number of TB-labeled nerve cell bodies the jugular and trigeminal ganglia contributed the major component to the sensory innervation of the ear drum and the middle-ear mucosa, while the contribution from the geniculate and cervical dorsal root ganglia was relatively minor.The majority of the TB-labeled nerve cell bodies contained calcitonin gene-related peptide (CGRP), whereas minor populations stored substance P (SP) and neurokinin A (NKA). Nerve fibers containing SP, NKA and CGRP were moderate in number in the middle-ear mucosa and few in the ear drum. Double immunostaining revealed that SP invariably coexisted with NKA in nerve cell bodies in the ganglia examined. The SP/NKA-containing nerve cell bodies constituted a subpopulation of those storing CGRP.The findings indicate that several ganglia project to the ear drum and middle-ear mucosa and that many neuropeptides are involved in the mediation of middle-ear sensitivity.