Carbonic anhydrase enzyme histochemistry of cranial nerve primary sensory afferent neurons in the rat

Summary Hansson's enzyme histochemical method for the demonstration of carbonic anhydrase has been used to examine primary sensory neurons of cranial nerves in the rat (cochlear ganglion cells excluded). Numerous carbonic anhydrase positive neurons were present in the trigeminal and geniculate ganglia as well as in the mecencephalic trigeminal nucleus. A few carbonic anhydrase positive ganglion cells were found in the nodose ganglion, but none in the petrosal and vestibular ganglia. However, in the latter ganglia, satellite cells surrounding the neurons frequently showed staining for carbonic anhydrase.     

Trigeminal ganglion cells cocultured with gut express vasoactive intestinal peptide

The plasticity of neural crest cells for the expression of adrenergic and cholinergic transmitter phenotypes has been well studied. The object of this study was to determine if cells of a sensory ganglion are capable of neuropeptide transmitter plasticity. We studied whether cells of the trigeminal ganglion, which do not express the neuropeptide vasoactive intestinal peptide (VIP) in vivo, would express this peptide when grown with a tissue the gut, that contains large numbers of VIP neurons. Embryonic aneural chick rectum was explanted with the embryonic quail trigeminal ganglion on the chorioallantoic membrane of chick hosts for 7-8 days. The explants were fixed, sectioned, and stained for VIP immunoreactivity (IR), for neurofilament protein immunoreactivity, and for the quail nucleolar marker. In sections of the explants we observed two populations of quail neurons: small (10-13 microns) VIP-IR cells and large (25-32 microns) cells lacking VIP-IR and resembling native trigeminal neurons. Trigeminal ganglia explanted with embryonic heart or trigeminal ganglia explanted alone lacked small VIP-IR cells but contained large VIP-negative neurons. These results show that cells of the trigeminal ganglion grown with the gut can express a neuropeptide they do not express in the absence of the gut or in vivo. Thus the embryonic trigeminal ganglion contains cells that are plastic with respect to neuropeptide expression.     

Cadherin‐7 mediates proper neural crest cell–placodal neuron interactions during trigeminal ganglion assembly

The cranial trigeminal ganglia play a vital role in the peripheral nervous system through their relay of sensory information from the vertebrate head to the brain. These ganglia are generated from the intermixing and coalescence of two distinct cell populations: cranial neural crest cells and placodal neurons. Trigeminal ganglion assembly requires the formation of cadherin‐based adherens junctions within the neural crest cell and placodal neuron populations; however, the molecular composition of these adherens junctions is still unknown. Herein, we aimed to define the spatio‐temporal expression pattern and function of Cadherin‐7 during early chick trigeminal ganglion formation. Our data reveal that Cadherin‐7 is expressed exclusively in migratory cranial neural crest cells and is absent from trigeminal neurons. Using molecular perturbation experiments, we demonstrate that modulation of Cadherin‐7 in neural crest cells influences trigeminal ganglion assembly, including the organization of neural crest cells and placodal neurons within the ganglionic anlage. Moreover, alterations in Cadherin‐7 levels lead to changes in the morphology of trigeminal neurons. Taken together, these findings provide additional insight into the role of cadherin‐based adhesion in trigeminal ganglion formation, and, more broadly, the molecular mechanisms that orchestrate the cellular interactions essential for cranial gangliogenesis.     

Contributions of placodal and neural crest cells to avian cranial peripheral ganglia

The method of embryonic tissue transplantation was used to confirm the dual origin of avian cranial sensory ganglia, to map precise locations of the anlagen of these sensory neurons, and to identify placodal and neural crest-derived neurons within ganglia. Segments of neural crest or strips of presumptive placodal ectoderm were excised from chick embryos and replaced with homologous tissues from quail embryos, whose cells contain a heterochromatin marker. Placode-derived neurons associated with cranial nerves V, VII, IX, and X are located distal to crest-derived neurons. The generally larger, embryonic placodal neurons are found in the distal portions of both lobes of the trigeminal ganglion, and in the geniculate, petrosal and nodose ganglia. Crest-derived neurons are found in the proximal trigeminal ganglion and in the combined proximal ganglion of cranial nerves IX and X. Neurons in the vestibular and acoustic ganglia of cranial nerve VIII derive from placodal ectoderm with the exception of a few neural crest-derived neurons localized to regions within the vestibular ganglion. Schwann sheath cells and satellite cells associated with all these ganglia originate from neural crest. The ganglionic anlagen are arranged in cranial to caudal sequence from the level of the mesencephalon through the third somite. Presumptive placodal ectoderm for the VIIIth, the Vth, and the VIIth, IXth, and Xth ganglia are located in a medial to lateral fashion during early stages of development reflecting, respectively, the dorsolateral, intermediate, and epibranchial positions of these neurogenic placodes.     

The control of avian cephalic neural crest cytodifferentiation. II. Neural tissues.

A series of neural crest transplantations has been performed to (1) analyze whether avian premigratory cranial neural crest cells are pluripotential or restricted to specific developmental pathways and (2) examine the ability of trunk neural crest cells to develop in an environment usually occupied by cranial crest cells. Quail embryos, the cells of which have a unique nuclear marker, were used as donors and chick embryos as hosts. Hindbrain crest cells grafted in the place of diencephalic crest cells failed to form neurons in all but one case, in which a small ectopic ganglion was found. In the reciprocal transplants, neural crest cells emigrating from a segment of forebrain crest tissue grafted in the place of metencephalic crest cells produced trigeminal and ciliary ganglia which were completely normal. Thus, crest cells which normally never form ganglionic neurons will do so if placed in a suitable neurogenic environment. These results prove that premigratory avian cranial crest cells are not restricted to specific developmental pathways, but are initially pluripotential. Trunk crest cells grafted in the place of metencephalic crest cells form neuronal ganglia along the proximal trigeminal motor roots but do not form normal trigeminal ganglia. These root ganglia do not display normal peripheral projections, and placode cells, a normal component of the trigeminal ganglion, form ganglia in ectopic locations. Thus, while trunk crest cells respond to the metencephalic environment and form neurons, their response is different from that of cranial crest cells in the same location. Whether this is due to differences in developmental potential or in initial population size is not known.     

Pax3-expressing trigeminal placode cells can localize to trunk neural crest sites but are committed to a cutaneous sensory neuron fate

The cutaneous sensory neurons of the ophthalmic lobe of the trigeminal ganglion are derived from two embryonic cell populations, the neural crest and the paired ophthalmic trigeminal (opV) placodes. Pax3 is the earliest known marker of opV placode ectoderm in the chick. Pax3 is also expressed transiently by neural crest cells as they emigrate from the neural tube, and it is reexpressed in neural crest cells as they condense to form dorsal root ganglia and certain cranial ganglia, including the trigeminal ganglion. Here, we examined whether Pax3+ opV placode-derived cells behave like Pax3+ neural crest cells when they are grafted into the trunk. Pax3+ quail opV ectoderm cells associate with host neural crest migratory streams and form Pax3+ neurons that populate the dorsal root and sympathetic ganglia and several ectopic sites, including the ventral root. Pax3 expression is subsequently downregulated, and at E8, all opV ectoderm-derived neurons in all locations are large in diameter, and virtually all express TrkB. At least some of these neurons project to the lateral region of the dorsal horn, and peripheral quail neurites are seen in the dermis, suggesting that they are cutaneous sensory neurons. Hence, although they are able to incorporate into neural crest-derived ganglia in the trunk, Pax3+ opV ectoderm cells are committed to forming cutaneous sensory neurons, their normal fate in the trigeminal ganglion. In contrast, Pax3 is not expressed in neural crest-derived neurons in the dorsal root and trigeminal ganglia at any stage, suggesting either that Pax3 is expressed in glial cells or that it is completely downregulated before neuronal differentiation. Since Pax3 is maintained in opV placode-derived neurons for some considerable time after neuronal differentiation, these data suggest that Pax3 may play different roles in opV placode cells and neural crest cells.     

The cranial sensory ganglia in culture: Differences in the response of placode-derived and neural crest-derived neurons to nerve growth factor

Explants of cranial sensory ganglia and dorsal root ganglia from embryonic chicks of 4 to 16 days incubation (E4 to E16) were grown for 24 hr in collagen gels with and without nerve growth factor (NGF) in the culture medium. NGF elicited marked neurite outgrowth from neural crest-derived explants, i.e., dorsal root ganglia, the dorsomedial part of the trigeminal ganglion, and the jugular ganglion. This response was first observed in ganglia taken from E6 embryos, reached a maximum between E8 and E11, and gradually declined through E16. Explants in which the neurons were of placodal origin varied in their response to NGF. There was negligible neurite outgrowth from explants of the ventrolateral part of the trigeminal ganglion and the vestibular ganglion grown in the presence of NGF. The geniculate, petrosal, and nodose ganglia exhibited an early moderate response to NGF. This was first evident in ganglia taken from E5 embryos, reached a maximum by E6, and declined through later ages, becoming negligible by E13. Dissociated neuron-enriched cultures of vestibular, petrosal, jugular, and dorsal root ganglia were established from embryos taken at E6 and E9. At both ages NGF elicited neurite outgrowth from a substantial proportion of neural crest-derived neurons (jugular and dorsal root ganglia) but did not promote the growth of placode-derived neurons (vestibular and petrosal ganglia). Our findings demonstrate a marked difference in the response of neural crest and placode-derived sensory neurones to NGF. The data from dissociated neuron-enriched cultures suggest that NGF promotes survival and growth of sensory ganglionic neurons of neural crest origin but not of placodal origin. The data from explant cultures suggest that NGF promotes neurite outgrowth from placodal neurons of the geniculate, petrosal, and nodose ganglia early in their ontogeny. However, we argue that this fibre outgrowth emanates not from the placodal neurons but from neural crest-derived cells which normally give rise only to satellite cells of these ganglia.     

Assembly of trigeminal sensory ganglia by chemokine signaling

Sensory neurons with related functions form ganglia, but how these precisely positioned clusters are assembled has been unclear. Here, we use the zebrafish trigeminal sensory ganglion as a model to address this question. We find that some trigeminal sensory neurons are born at the position where the ganglion is assembled, whereas others are born at a distance and have to migrate against opposing morphogenetic movements to reach the site of ganglion assembly. Loss of Cxcr4b-mediated chemokine signaling results in the formation of mispositioned ganglia. Conversely, ectopic sources of the chemokine SDF1a can attract sensory neurons. Transplantation experiments reveal that neuron-neuron interaction and the adhesion molecules E- and N-Cadherin also contribute to ganglion assembly. These results indicate that ganglion formation depends on the interplay of birthplace, chemokine attraction, cell-cell interaction, and cadherin-mediated adhesion.     

Evidence for Glutamate as a Neuroglial Transmitter within Sensory Ganglia

This study examines key elements of glutamatergic transmission within sensory ganglia of the rat. We show that the soma of primary sensory neurons release glutamate when depolarized. Using acute dissociated mixed neuronal/glia cultures of dorsal root ganglia (DRG) or trigeminal ganglia and a colorimetric assay, we show that when glutamate uptake by satellite glial cells (SGCs) is inhibited, KCl stimulation leads to simultaneous increase of glutamate in the culture medium. With calcium imaging we see that the soma of primary sensory neurons and SGCs respond to AMPA, NMDA, kainate and mGluR agonists, and selective antagonists block this response. Using whole cell patch-clamp technique, inward currents were recorded from small diameter (<30 µm) DRG neurons from intact DRGs (ex-vivo whole ganglion preparation) in response to local application of the above glutamate receptor agonists. Following a chronic constriction injury (CCI) of either the inferior orbital nerve or the sciatic nerve, glutamate expression increases in the trigeminal ganglia and DRG respectively. This increase occurs in neurons of all diameters and is present in the somata of neurons with injured axons as well as in somata of neighboring uninjured neurons. These data provides additional evidence that glutamate can be released within the sensory ganglion, and that the somata of primary sensory neurons as well as SGCs express functional glutamate receptors at their surface. These findings, together with our previous gene knockdown data, suggest that glutamatergic transmission within the ganglion could impact nociceptive threshold.     

Differentiation of postmitotic neuroblasts into substance P-immunoreactive sensory neurons in dissociated cultures of chick dorsal root ganglion

Counts performed on dissociated cell cultures of E10 chick embryo dorsal root ganglia (DRG) showed after 4-6 days of culture a pronounced decline of the neuronal population in neuron-enriched cultures and a net gain in the number of ganglion cells in mixed DRG cell cultures (containing both neurons and nonneuronal cells). In the latter case, the increase in the number of neurons was found to depend on NGF and to average 119% in defined medium or 129% in horse serum-supplemented medium after 6 days of culture. The lack of [3H]thymidine incorporation into the neuronal population indicated that the newly formed ganglion cells were not generated by proliferation. On the contrary, the differentiation of postmitotic neuroblasts present in the nonneuronal cell compartment was supported by sequential microphotographs of selected fields taken every hour for 48-55 hr after 3 days of culture. Apparently nonneuronal flat dark cells exhibited morphological changes and gradually evolved into neuronal ovoid and refringent cell bodies with expanding neurites. The ultrastructural organization of these evolving cells corresponded to that of primitive or intermediate neuroblasts. The neuronal nature of these rounding up cell bodies was indeed confirmed by the progressive expression of various neuronal cell markers (150 and 200-kDa neurofilament triplets, neuron specific enolase, and D2/N-CAM). Besides a constant lack of immunoreactivity for tyrosine hydroxylase, somatostatin, parvalbumin, and calbindin-D 28K and a lack of cytoenzymatic activity for carbonic anhydrase, all the newly produced neurons expressed three main phenotypic characteristics: a small cell body, a strong immunoreactivity to MAG, and substance P. Hence, ganglion cells newly differentiated in culture would meet characteristics ascribed to small B sensory neurons and more specifically to a subpopulation of ganglion cells containing substance P-immunoreactive material.     

Intrinsic choroidal neurons in the chicken eye: chemical coding and synaptic input

Intrinsic choroidal neurons (ICNs) exist in some primates and bird species. They may act on both vascular and non-vascular smooth muscle cells, potentially influencing choroidal blood flow. Here, we report on the chemical coding of ICNs and eye-related cranial ganglia in the chicken, an important model in myopia research, and further to determine synaptic input onto ICN. Chicken choroid, ciliary, superior cervical, pterygopalatine, and trigeminal ganglia were prepared for double or triple immunohistochemistry of calcitonin gene-related peptide (CGRP), choline acetyltransferase (ChAT), dopamine-β-hydroxylase, galanin (GAL), neuronal nitric oxide synthase (nNOS), somatostatin (SOM), tyrosine hydroxylase (TH), vasoactive intestinal polypeptide (VIP), vesicular monoamine-transporter 2 (VMAT2), and α-smooth muscle actin. For documentation, light, fluorescence, and confocal laser scanning microscopy were used. Chicken ICNs express nNOS/VIP/GAL and do not express ChAT and SOM. ICNs are approached by TH/VMAT2-, CGRP-, and ChAT-positive nerve fibers. About 50% of the pterygopalatine ganglion neurons and about 9% of the superior cervical ganglion neurons share the same chemical code as ICN. SOM-positive neurons in the ciliary ganglion are GAL/NOS negative. CGRP-positive neurons in the trigeminal ganglion lack GAL/SOM. The neurochemical phenotype and synaptic input of ICNs in chicken resemble that of other bird and primate species. Because ICNs lack cholinergic markers, they cannot be readily incorporated into current concepts of the autonomic nervous system. The data obtained provide the basis for the interpretation of future functional experiments to clarify the role of these cells in achieving ocular homeostasis.     

Wise promotes coalescence of cells of neural crest and placode origins in the trigeminal region during head development

While most cranial ganglia contain neurons of either neural crest or placodal origin, neurons of the trigeminal ganglion derive from both populations. The Wnt signaling pathway is known to be required for the development of neural crest cells and for trigeminal ganglion formation, however, migrating neural crest cells do not express any known Wnt ligands. Here we demonstrate that Wise, a Wnt modulator expressed in the surface ectoderm overlying the trigeminal ganglion, play a role in promoting the assembly of placodal and neural crest cells. When overexpressed in chick, Wise causes delamination of ectodermal cells and attracts migrating neural crest cells. Overexpression of Wise is thus sufficient to ectopically induce ganglion-like structures consisting of both origins. The function of Wise is likely synergized with Wnt6, expressed in an overlapping manner with Wise in the surface ectoderm. Electroporation of morpholino antisense oligonucleotides against Wise and Wnt6 causes decrease in the contact of neural crest cells with the delaminated placode-derived cells. In addition, targeted deletion of Wise in mouse causes phenotypes that can be explained by a decrease in the contribution of neural crest cells to the ophthalmic lobe of the trigeminal ganglion. These data suggest that Wise is able to function cell non-autonomously on neural crest cells and promote trigeminal ganglion formation.     

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。     

Immunohistochemical localization of carbonic anhydrase isoenzymes I and II in human bone,cartilage and giant cell tumor

Summary Carbonic anhydrase isoenzymes I and II have been localized in human bone and cartilage. Osteoclasts are strongly positive for carbonic anhydrase II but very little if any reaction is observed for carbonic anhydrase I. In tendon giant cell tumor osteoclastlike-giant cells contained high amounts of carbonic anhydrase II suggesting the close relation of these cells to normal osteoclasts. In growth plate cartilage strong staining was obtained in late proliferative and hypertrophic chondroxytes as well as in extracellular matrix of hypertrophic zone also only with anti-human carbonic anhydrase II.     

Carbonic Anhydrase Immunostaining in Astrocytes in the Rat Cerebral Cortex

Carbonic anhydrase is known to occur in the choroid plexus, oligodendrocytes, and myelin, and to be virtually absent from neurons, in the mammalian CNS; however, there is significant controversy whether it is also present in astrocytes. When brain sections from adult rats were stained for simultaneous immunofluorescence of carbonic anhydrase and the astrocyte marker glutamine synthetase, both antigens were detected in the same glial cells in the cortical gray matter, whereas the oligodendrocytes and myelinated fibers in and adjacent to the white matter showed immunofluorescence only for carbonic anhydrase. Some glial cells in the gray matter also showed double immunofluorescence for carbonic anhydrase and glial fibrillary acidic protein. These results indicate that there is carbonic anhydrase in some astrocytes in the mammalian CNS.