METABOLISM OF PUTATIVE TRANSMITTERS IN INDIVIDUAL NEURONS OF APLYSIA CALIFORNICA

The activities of acetylcholinesterase (acetylcholine acetyl-hydrolase; EC 3.1.1.7) and catechol-O-methyl transferase (S-adenosylmethionine: catechol-O-methyl transferase; EC 2.1.1 .a) were measured in the various ganglia of the nervous system of Aplysia californica and in some of the individually identifiable neurons in these ganglia. All of the neurons studied had measurable levels of activity for both enzymes. Since different individual neurons exhibited approximately the same level of activity we concluded that neither of these enzymes could be used to classify neurons as ‘cholinergic’ vs. ‘aminergic’ or ‘cholinoceptive’ vs. ‘aminoceptive'. The ubiquitous distribution of either or both of these enzymes in different single neurons may be related to glial contamination.     

RESPIRATION BY CULTIVATED ASTROCYTES AND NEURONS FROM THE CEREBRAL HEMISPHERES

-Rates of oxygen uptake were measured in chick and/or rat astrocytes and neuronal cells cultivated for 2–4 weeks in Falcon flasks or Rose chambers. All the preparations were found to have respiratory rates between 0.4 and 0.8 × 10^?5μl/h O₂ per cell. Based upon measurements of cell diameters these values were recalculated to about 570 μmol/g wet wt. for the neuronal cells and 130 μmol/g wet wt. for the glial cells. The results are compared with previous data of oxygen uptake by neurons and glial cells separated by other procedures.     

THE CHARACTERIZATION OF HISTIDINE DECARBOXYLASE AND ITS DISTRIBUTION IN NERVES, GANGLIA AND IN SINGLE NEURONAL CELL BODIES FROM THE CNS OF APLYSIA CALIFORNICA

Histamine (HA) is present in substantial quantities in all ganglia of Aplysia californica. Within the cerebral ganglia this amine is known to be concentrated in at least two identified neurons designated C-2 neurons. In this study a combination of chemical and enzymatic analyses was employed to provide evidence for the existence of a biochemical pathway for HA synthesis in ganglia and individual neurons of this marine mollusk. Examination of extracts of individual neurons dissected from ganglia organ-cultured in the presence of [³H]histidine showed that every neuron accumulated labelled histidine, but only the HA-containing C-2 neurons synthesized and stored labelled HA suggesting that the formation of HA in Aplysia could be catalyzed by the enzyme histidine decarboxylase (HDC). HDC activity was studied with a new microradiometric assay. Many of the properties of the molluscan HDC studied were found to correspond to the vertebrate enzyme. Enzyme activity was inhibited by α-hydrazino-histidine but unaffected by concentrations of α-methyldopa or by 5-(3,4-dihydroxycinnamoyl) salicylic acid which produced nearly complete inhibition of aromatic amino acid decarboxylase activity. HDC was measurable in nervous but not other Aplysia tissues assayed. All 5 major ganglia contained HDC activity which spanned a 15-fold range between the least and most active ganglia. Only 4 of the 13 nerve trunks assayed yielded measurable enzymic activity; these active nerves were associated with the cerebral ganglia which has the highest HDC activity of all measured ganglia. Of the numerous individual neurons assayed for HDC, only the C-2 cells showed measurable enzyme activity, about 25 pmol/cell/h or 70 μmol/g protein/h. Since the activity of HDC in the HA-containing neurons was at least three orders of magnitude larger than all other neurons assayed in the cerebral and other ganglia, these data appear to provide a direct metabolic basis for the selective presence of HA in these cells, and they indicate that the cellular presence of HDC provides a useful biochemical marker for the location of HA-rich neurons in Aplysia.     

CELLS ISOLATED FROM TRYPSIN-TREATED BRAIN CONTAIN TRYPSIN

—Neuronal and oligodendroglial cell fractions isolated from trypsinized brain contain trypsin. Neuronal cell bodies contained approximately 5 μg of trypsin/mg of cell protein; oligodendroglial cells had approx 1 μg of enzyme activity. Studies with fluorescein-conjugated trypsin suggested that least a portion of the fluorescent enzyme was internalized in neurons.     

PREPARATION OF PLASMA MEMBRANE FROM ISOLATED NEURONS

A bulk fraction enriched with respect to neuronal cell bodies was used as starting material for the isolation of neuronal plasma membrane The cells were gently homogenized in isotonic sucrose and a crude membrane containing fraction sedimented at 3000 g. Subsequently, the membrane fraction was purified on a discontinuous sucrose density gradient between 35% and 25 5% sucrose (w/w). Enzymatic analyses showed a 4–5-fold enrichment in plasma membrane markers, and a 10–15% contamination of mitochondrial and microsomal material. Electron micrographs of the membrane fraction confirmed the enzymatic data Fragmented membranes were found, mainly in vesicular form No ribosomes, but a few mitochondria and some multilamellar membranes were seen     

Neural crest and placode roles in formation and patterning of cranial sensory ganglia in lamprey

Vertebrates possess paired cranial sensory ganglia derived from two embryonic cell populations, neural crest and placodes. Cranial sensory ganglia arose prior to the divergence of jawed and jawless vertebrates, but the developmental mechanisms that facilitated their evolution are unknown. Using gene expression and cell lineage tracing experiments in embryos of the sea lamprey, Petromyzon marinus, we find that in the cranial ganglia we targeted, development consists of placode‐derived neuron clusters in the core of ganglia, with neural crest cells mostly surrounding these neuronal clusters. To dissect functional roles of neural crest and placode cell associations in these developing cranial ganglia, we used CRISPR/Cas9 gene editing experiments to target genes critical for the development of each population. Genetic ablation of SoxE2 and FoxD‐A in neural crest cells resulted in differentiated cranial sensory neurons with abnormal morphologies, whereas deletion of DlxB in cranial placodes resulted in near‐total loss of cranial sensory neurons. Taken together, our cell‐lineage, gene expression, and gene editing results suggest that cranial neural crest cells may not be required for cranial ganglia specification but are essential for shaping the morphology of these sensory structures. We propose that the association of neural crest and placodes in the head of early vertebrates was a key step in the organization of neurons and glia into paired sensory ganglia.     

Demonstration of insulin-related substances in the central nervous systems of pulmonates and Aplysia californica

Summary the occurrence of insulin-related substances in the central nervous system of pulmonates and Aplysia californica was investigated by means of immunocytochemistry and in situ hybridization. Previous experiments have shown that, in Lymnaea stagnalis, the growth hormone-producing neurons in the cerebral ganglia (the so-called light green cells) express at least 5 genes that are related to the vertebrate insulin genes, i.e., they encode prohormones that are composed of a B- and A-chain and a connecting C peptide. These insulin related molecules also have the amino acids essential for their tertiary structure (viz. cysteines) at identical positions to those of the vertebrate insulins. In the investigated basommatophoran and stylommatophoran snails and slugs, neurons reacted with an antiserum raised against the C peptide of one of the molluscan insulin-related peptides. These neurons can be considered to be, based on morphological and endocrinological criteria, homologous to the light green cells of L. stagnalis. In A. californica, all central ganglia contain immunoreactive neurons. The highest number (about 50) was observed in the abdominal ganglion. The present results indicate that insulin-related substances are generally occurring neuropeptides in the central nervous system of molluscs.     

PHYLOGENETIC ANALYSIS OF LEARNING-RELATED NEUROMODULATION IN MOLLUSCAN MECHANOSENSORY NEURONS

In spite of significant advances in our understanding of mechanisms of learning and memory in a variety of organisms, little is known about how such mechanisms evolve. Even mechanisms of simple forms of learning, such as habituation and sensitization, have not been studied phylogenetically. Here we begin an evolutionary analysis of learning-related neuromodulation in species related to the well-studied opisthobranch gastropod, Aplysia californica. In Aplysia, increased spike duration and excitability in mechanosensory neurons contribute to several forms of learning-related changes to defensive withdrawal reflexes. The modulatory transmitter serotonin (5-hydroxytryptamine, or 5-HT), is thought to play a critical role in producing these firing property changes. In the present study, we tested mechanosensory homologs of the tail-withdrawal reflex in species related to Aplysia for 5-HT-mediated increases in spike duration and excitability. Criteria used to identify homologous tail-sensory neurons included position, relative size, resting electrical properties, expression of a sensory neuron-specific protein, neuroanatomy, and receptive field. The four ingroup species studied (Aplysia californica, Dolabella auricularia, Bursatella leachii, and Dolabrifera dolabrifera) belong to two clades (two species each) within the family Aplysiidae. In the first clade (Aplysia/Dolabella), we found that the tail-sensory neurons of A. californica and tail-sensory homologs of a closely related species, D. auricularia, responded to bath-applied serotonin in essentially similar fashion: significant increases in spike duration as well as excitability. In the other clade (Dolabrifera/Bursatella), more distantly related to Aplysia, one species (B. leachii) showed spike broadening and increased excitability. However, the other species (D. dolabrifera) showed neither spike broadening nor increased excitability. The firing properties of tail-sensory homologs of D. dolabrifera were insensitive to 5-HT over a wide range of concentrations. We also performed experiments on two outgroup species (Akera bullata and Bulla gouldiana) and found that spike duration was unaffected by 5-HT, whereas excitability was increased. This study suggests that 5-HT-induced spike broadening arose more recently in opisthobranch evolution, whereas 5-HT-induced excitability increase is a more ancestral trait that may have been expressed in the earliest opisthobranchs. Both traits are absent in the aplysiid species D. dolabrifera, demonstrating that a lineage can lose learning-related mechanisms. The phylogenetic variation observed in the present study presents the opportunity to test general models about learning mechanisms and their evolution in unique ways.     

P物质对大鼠分离的DRG细胞GABA激活电流的抑制作用

本文就用全细胞膜片箝技术,在新鲜分离的大鼠ＤＲＧ细胞上证明,在部分细胞Ｐ物质（１０＾－７－１０＾－５ｍｏｌ／Ｌ）可引起浓度依赖性的内向流（４／２６）;在多数细胞虽未检测到ＳＰ引起的膜电流,但却能对ＧＡＢＡＡ受体激活介导的膜内向流产生抑制效应（１８／２２）,并有加速去敏感的作用。本文就有关ＳＰ以ＧＡＢＡ激活电流抑制效应的可能意义进行了讨论。     

Neuronal localization of dopamine and 5-Hydroxytryptamine in some mollusca

Summary The localization of biogenic monoamines in ganglionic tissues from Anodonta piscinalis, Helix pomatia, and Buccinum undatum has been studied by means of the histochemical fluorescence method of Falck and Hillarp.In cerebral, visceral, and pedal ganglia (besides nonfluorescent nerve cells) neurons emitting a green or yellow fluorescence were found. No other cell systems exhibiting a specific fluorescence were observed. An abundance of monoaminergic terminals were found in the central parts of these ganglia. Spectrophotofluorimetric determinations showed that there are large quantities of dopamine and 5-hydroxytryptamine in the tissues investigated. The amounts of dopamine and 5-hydroxytryptamine agree well with the distribution of green and yellow fluorescence, respectively, in the ganglia.There are many similarities between the vertebrate and the molluscan monoaminergic neurons. The morphology of the neurons is the same, the intraneuronal distribution of the monoamines is identical, depletion experiments with reserpine and denervation experiments give the same results, and the synaptic arrangement of monoaminergic fibres on non-adrenergic neurons has the same appearance. Apparently, however, dopamine and 5-hydroxytryptamine are the only monoamines acting as neuronal transmitters in the species investigated.The research reported in this document has been sponsored by the Air Force Office of Scientific Research under Grant AF EOAR 64-5 through the European Office of Aerospace Research (OAR), United States Air Force and by the Swedish Natural Science Research Council.     

Evidence for homologous peptidergic neurons in the buccal ganglia of diverse nudibranch mollusks

The buccal ganglia of seven nudibranches (Aeolidia papillosa, Armina californica, Dirona albolineata, D. picta, Hermissenda crassicornis, Melibe leonina, and Tritonia diomedea) were examined to explore possible homologies between large cells that reacted with antibodies directed against small cardioactive peptide B (SCP_B). The buccal ganglion of each species possessed a pair of large, dorsal–lateral, whitish neurons that contained an SCP_B-like peptide. We refer to these neurons as the SLB (SCP_B-immunoreactive Large Buccal) cells. In all species examined, the SLB cells project out the gastroesophageal nerves and appear to innervate the esophagus. In each species, an apparent rhythmic feeding motor program (FMP) was observed by intracellular recording from both SLB neurons and other neurons in isolated preparations of the buccal ganglia. SLB cells often fire at a high frequency, and usually burst in a specific phase relation to the FMP activity. Stimulation of SLB cells enhances expression of the feeding motor program, either by potentiating existing activity or eliciting the FMP in quiescent preparations. Finally, perfusion of isolated buccal ganglia with SCP_B excites the SLB cells and activates FMPs. Thus, both the immunohistochemical and electrophysiological data suggest that the SLB cells within three suborders of the opistobranchia (Dendronotacea, Arminacea, and Aeolidacea) are homologous. A comparison of our data with previously published studies indicates that SLB cell homologs may exist in other gastropods as well.     

Pigment‐dispersing factor in the locust abdominal ganglia may have roles as circulating neurohormone and central neuromodulator

Pigment‐dispersing factor (PDF) is a neuropeptide that has been indicated as a likely output signal from the circadian clock neurons in the brain of Drosophila. In addition to these brain neurons, there are PDF‐immunoreactive (PDFI) neurons in the abdominal ganglia of Drosophila and other insects; the function of these neurons is not known. We have analyzed PDFI neurons in the abdominal ganglia of the locust Locusta migratoria. These PDFI neurons can first be detected at about 45% embryonic development and have an adult appearance at about 80%. In each of the abdominal ganglia (A3–A7) there is one pair of lateral PDFI neurons and in each of the A5–A7 ganglia there is additionally a pair of median neurons. The lateral neurons supply varicose branches to neurohemal areas of the lateral heart nerves and perisympathetic organs, whereas the median cells form processes in the terminal abdominal ganglion and supply terminals on the hindgut. Because PDF does not influence hindgut contractility, it is possible that also these median neurons release PDF into the circulation. Release from one or both the PDFI neuron types was confirmed by measurements of PDF‐immunoreactivity in hemolymph by enzyme immunoassay. PDF applied to the terminal abdominal ganglion triggers firing of action potentials in motoneurons with axons in the genital nerves of males and the 8th ventral nerve of females. Because this action is blocked in calcium‐free saline, it is likely that PDF acts via interneurons. Thus, PDF seems to have a modulatory role in central neuronal circuits of the terminal abdominal ganglion that control muscles of genital organs. © 2001 John Wiley & Sons, Inc. J Neurobiol 48: 19–41, 2001     

Distribution of enteric nerve cells projecting to the superior and inferior mesenteric ganglia of the guinea-pig

Retrograde tracing, using Fast Blue dye, was employed to determine the distribution of enteric nerve cells that project to the superior mesenteric and inferior mesenteric ganglia of the guinea-pig. Retrogradely labelled neurons were found in the myenteric but not submucous ganglia. When the superior mesenteric ganglion was injected, labelled neurons were found in low frequencies (less than 5 nerve cell bodies/cm²) in the duodenum, jejunum, ileum, caecum and proximal colon. The distal colon was analysed in five segments of equal length (1–5; oral to anal). Segment 1 had about 4 labelled nerve cells/cm², whereas segments 2 to 5 displayed an average of about 25 nerve cells/cm². The rectum contained about 36 labelled neurons/cm². After injection of the inferior mesenteric ganglia with Fast Blue, no labelled neurons were found in the duodenum, jejunum, ileum or caecum. No labelled cells were observed in the gallbladder. A small number of labelled cells occurred in the proximal colon and in segment 1 of the distal colon. The frequency of labelled cells increased markedly in the more anal regions of the distal colon, and reached a peak in the rectum (138 cells/cm²). Both nerve lesions and immersion of the cut nerve in Fast Blue solution showed that the superior mesenteric nerve carries the axons of neurons located in the middle distal colon to the superior mesenteric ganglion. Almost half of the neurons in the rectum that project to the inferior mesenteric ganglia do so via the hypogastric nerves. Of neurons that projected to the inferior or superior mesenteric ganglia from the colon or rectum, similar proportions (about 75–80%) showed immunoreactivity for calbindin or VIP. For each of the prevertebral ganglia (coeliac, superior mesenteric and inferior mesenteric) the great majority of peripheral inputs arise from the large intestine.     

THE UPTAKE OF BIOGENIC MONOAMINES BY THE ISOLATED AURICLE OF THE SNAIL (HELIX POMATIA)

—The uptake of [³H]5HT, [³H]dopamine, [³H]noradrenaline and [³H]octopamine into the auricle of Helix pomatia was studied. When tissues were incubated at 25°C in media containing radioactive amines, tissue:medium ratios of about 49:1, 14:1 and 5:1 for 5-HT, dopamine, noradrenaline, and octopamine respectively were obtained after a 20–30 min incubation time. Tissues incubated at 25°C in media containing radioactive amines for 20–30 mins showed that almost all (96%) the radioactivity was present as unchanged [³H]5-HT, [³H]dopamine, [³H]octopamine or [³H]noradrenaline. The high tissue:medium ratios for 5-HT and dopamine, but not for noradrenaline and octopamine, showed saturation kinetics which were dependent upon temperature and sodium ions. From the Lineweaver–Burk plots, two uptake mechanisms for 5-HT at 25°C were resolved; the high affinity uptake process having a K_m1 value of 6.0 ± 10^?8m and a V_m1 value of 0.115 nmol/g/min while the lower affinity process had a K_m2 value of 1.04 ± 10^?6m and a V_m2 value of 0.66nmol/g/min. At 0°C a single uptake mechanism for 5-HT occurred which gave a K_m value of 5.02 ± 10^?8m and a V_m value of 0.0165 nmol/g/min. In the case of dopamine, the Lineweaver–Burk plot at 25°C showed a single uptake process with values for K_m and V_m of 1.55 ± 10^?7m and 0.086 nmol/g/min respectively. This process did not function at 0°C. The effect of various agents and ions upon the accumulation processes for all amines was also studied, and the data indicate that the same neurons probably accumulate more than one amine type. It is concluded that 5-HT and dopamine uptake in the auricle is a mechanism for inactivating these substances at 25°C and that an uptake mechanism for 5-HT also functions at 0°C. The results are discussed from the point of view of 5-HT's being the cardioexcitatory substance in the snail heart.     

Phenotypes of neural-crest-derived cells in vagal and sacral pathways

Enteric neurons arise from vagal and sacral level neural crest cells. To examine the phenotype of neural-crest-derived cells in vagal and sacral pathways, we used antisera to Sox10, p75, Phox2b, and Hu, and transgenic mice in which the expression of green fluorescent protein was under the control of the Ret promoter. Sox10 was expressed prior to the emigration of vagal cells, whereas p75 was expressed shortly after their emigration. Most crest-derived cells that emigrated adjacent to somites 1–4 migrated along a pathway that was later followed by the vagus nerve. A sub-population of these vagal cells coalesced to form vagal ganglia, whereas others continued their migration towards the heart and gut. Cells that coalesced into vagal ganglia showed a different phenotype from cells in the migratory streams proximal and distal to the ganglia. Only a sub-population of the vagal cells that first entered the foregut expressed Phox2b or Ret. Sacral neural crest cells gave rise to pelvic ganglia and some neurons in the hindgut. The pathways of sacral neural crest cells were examined by using DβH-nlacZ mice. Sacral cells appeared to enter the distal hindgut around embryonic day 14.5. Very few of the previously demonstrated, but rare, neurons that were present in the large intestine of Ret null mutants and that presumably arose from the sacral neural crest expressed nitric oxide synthase, unlike their counterparts in Ret heterozygous mice. This study was supported by the National Health and Medical Research Council of Australia (project grants nos. 145628 and 350311, C.J. Martin Fellowship no. 007144, and Senior Research Fellowship no. 170224).     

Transneuronal uptake of horseradish peroxidase in the central nervous system of dipterous insects

Summary Horseradish peroxidase (HRP) applied to lesioned neurons in the retina and thoracic ganglia of the flies Musca, Calliphora and Drosophila labeled axon terminals, dendrites and perikarya of the severed neurons after anterograde or retrograde passage. In addition, HRP reaction product secondarily labeled intact neurons that are contiguous with injured nerve cells. In many cases labeling of optic lobe neurons remote from primarily filled ones was also seen (here called tertiary labeling). HRP labeling was extensive and both primarily and transneuronally filled neurons could be resolved in almost as much detail as Golgi-impregnated or cobalt-silver-labeled cells. Electron microscopy showed that in both primarily and secondarily filled neurons, reaction product was distributed diffusely in the cytoplasm.Transneuronal uptake of HRP was specific to certain types of neurons in the brain and thus displayed certain pathways. The pathways resolved by transneuronal labeling with HRP extend from the optic lobes to the thoracic ganglia and include visual neurons previously identified electrophysiologically and anatomically.Transneuronal HRP uptake, although believed to occur in vivo, could not be shown to be dependent on synaptic activity. Three other heme peptides tested were taken up by injured neurons, but showed no transneuronal labeling: lactoperoxidase, cytochrome c, and microperoxidase.     

DNA CONTENT OF PURIFIED PREPARATIONS OF MOUSE PURKINJE NEURONS ISOLATED BY A VELOCITY SEDIMENTATION TECHNIQUE

Abstract— The DNA content of mouse Purkinje neurons was investigated employing a biochemical approach. Material for the biochemical assay was provided by means of a sedimentation velocity separation technique which yields bulk quantities of well-preserved Purkinje perikarya in a high degree of purity. The same amount of DNA/cell was recorded for mixed cerebellar cell somata (7·6 ± 0±2 pg/cell), as for the Purkinje perikarya enriched fractions (7±2 & 0·2 pg/cell). No evidence could be found for the existence of a tetraploid DNA complement in mouse Purkinje neurons despite indications to the contrary from a parallel cytophotometric study.     

Mapping of serotonin-immunoreactive neurons in the larval nervous system of the flies Calliphora erythrocephala and Sarcophaga bullata

Summary Serotonin-immunoreactive (5-HTi) neurons were mapped in the larval central nervous system (CNS) of the dipterous flies Calliphora erythrocephala and Sarcophaga bullata. Immunocytochemistry was performed on cryostat sections, paraffin sections, and on the entire CNS (whole mounts).The CNS of larvae displays 96–98 5-HTi cell bodies. The location of the cell bodies within the segmental cerebral and ventral ganglia is consistent among individuals. The pattern of immunoreactive fibers in tracts and within neuropil regions of the CNS was resolved in detail. Some 5-HTi neurons in the CNS possess axons that run through peripheral nerves (antenno-labro-frontal nerves).The suboesophagealand thoracico-abdominal ganglia of the adult blowflies were studied for a comparison with the larval ventral ganglia. In the thoracico-abdominal ganglia of adults the same number of 5-HTi cell bodies was found as in the larvae except in the metathoracic ganglion, which in the adult contains two cell bodies less than in the larva. The immunoreactive processes within the neuropil of the adult thoracico-abdominal ganglia form more elaborate patterns than those of the larvae, but the basic organization of major fiber tracts was similar in larval and adult ganglia. Some aspects of postembryonic development are discussed in relation to the transformation of the distribution of 5-HTi neurons and their processes into the adult pattern.     

大鼠心内神经元至颈上神经节的分支投射研究

应用CB－HRP逆行追踪法研究了大鼠心内神经元至颈上神经节的分支投射。将CB－HRP注入大鼠颈上神经节内,在心脏壁内神经节见到CB－HRP标记细胞,这些细胞为中小型梭形或园形,多位于心房后壁。结果表明心内神经元有分支投射到颈上神经节,并对此进行了讨论。     

Patterns of serotonin-immunoreactive neurons in the central nervous system of the earthworm Lumbricus terrestris L.

Summary The distribution patterns of serotonin-immunoreactive somata in the cerebral and subpharyngeal ganglion, and in the head and tail ganglia of the nerve cord of Lumbricus terrestris are described from whole-mount preparations. A small number of serotonin-immunoreactive neurons occurs in the cerebral ganglion, in contrast to the large population of serotonin-immunoreactive neurons that exists in all parts of the ventral nerve cord. From the arrangement of serotonin-immunoreactive somata in the subpharyngeal ganglion, we suggest that this ganglion arises from the fusion of two primordial ganglia. In head and tail ganglia, the distribution of serotonin-immunoreactive somata resembles that in midbody segments. Segmental variations in the pattern and number of serotonin-immunoreactive somata in the different body regions are discussed on the background of known developmental mechanisms that result in metameric neuronal populations in annelids and arthropods.Abbreviations CG1, CG2 cerebral soma group 1, 2 - CNS central nervous system - GINs giant interneurons - 5-HT 5-hydroxytryptamine, serotonin - 5-HTi 5-HT-immunoreactive - N side nerve - SG19 subpharyngeal soma group 1–9 - SN segmental nerve