Neonatal Dorsal Root Ganglion Research Articles

Glycosylation Alters Steady-State Inactivation of Sodium Channel Nav1.9/NaN in Dorsal Root Ganglion Neurons and Is Developmentally Regulated

Na channel NaN (Na(v)1.9) produces a persistent TTX-resistant (TTX-R) current in small-diameter neurons of dorsal root ganglia (DRG) and trigeminal ganglia. Na(v)1.9-specific antibodies react in immunoblot assays with a 210 kDa protein from the membrane fractions of adult DRG and trigeminal ganglia. The size of the immunoreactive protein is in close agreement with the predicted Na(v)1.9 theoretical molecular weight of 201 kDa, suggesting limited glycosylation of this channel in adult tissues. Neonatal rat DRG membrane fractions, however, contain an additional higher molecular weight immunoreactive protein. Reverse transcription-PCR analysis did not show additional longer transcripts that could encode the larger protein. Enzymatic deglycosylation of the membrane preparations converted both immunoreactive proteins into a single faster migrating band, consistent with two states of glycosylation of Na(v)1.9. The developmental change in the glycosylation state of Na(v)1.9 is paralleled by a developmental change in the gating of the persistent TTX-R Na(+) current attributable to Na(v)1.9 in native DRG neurons. Whole-cell patch-clamp analysis demonstrates that the midpoint of steady-state inactivation is shifted 7 mV in a hyperpolarized direction in neonatal (postnatal days 0-3) compared with adult DRG neurons, although there is no significant difference in activation. Pretreatment of neonatal DRG neurons with neuraminidase causes an 8 mV depolarizing shift in the midpoint of steady-state inactivation of Na(v)1.9, making it indistinguishable from that of adult DRG neurons. Our data show that extensive glycosylation of rat Na(v)1.9 is developmentally regulated and changes a critical property of this channel in native neurons.

Influence of Peripheral Inflammation on the Postnatal Maturation of Primary Sensory Neuron Phenotype in Rats

The influence of early peripheral inflammation upon the postnatal development of rat primary sensory neuron subtypes was investigated. Lumbar dorsal root ganglia (DRG) were immunostained for calcitonin gene-related peptide (CGRP), neurofilament (NF200), and isolectin B4 (IB4) binding. Proportions of each subpopulation were measured at postnatal day (P) 0, P3, P7, and P21 in normal pups and in those that had received a unilateral hindpaw carrageenan injection at P1. The effects were compared with those following a similar injury in adults. Both the IB4 (positive [+ve]) and NF200+ve cell populations increased postnatally (IB4+ve: 23 ± 1.6% to 32.6 ± 1.3%; NF200+ve: 33.8 ± 1.2% to 43.3 ± 1.9%), whereas the population of CGRP+ve cells stayed the same. After neonatal inflammation, the rise in IB4+ve binding occurred earlier but was the same as that in controls by P21. The CGRP+ve population increased at 2 and 6 days after carrageenan in neonates, because of an increase in both small CGRP/IB4 and larger CGRP/NF200 double-labeled cells, but was normal by 3 weeks. Carrageenan in adults caused an increase in CGRP/IB4 cells only. The effects of peripheral inflammation differ in neonatal and adult DRG. Neonatal inflammation causes CGRP upregulation in both small and large cells and accelerates the postnatal increase in IB4 binding. These effects might influence subsequent central development.

Actions of cannabinoid receptor ligands on rat cultured sensory neurones: implications for antinociception

Cannabinoids modulate nociceptive processing in models of acute, inflammatory and neuropathic pain. We have investigated the location and function of cannabinoid receptors on cultured neonatal dorsal root ganglion (DRG) neurones and F-11 cells, a dorsal root ganglion×neuroblastoma hybridoma which displays several of the features of authentic DRG neurones. CB 1 receptor immunolabelling was observed on the cell bodies and as fine puncta on processes of both cultured DRG neurones and F-11 cells. Additionally, fluorescence-activated cell sorting (FACS) analysis provided evidence that both CB 1 and CB 2 receptors are expressed on populations of cells within the cultured DRG and F-11 cells. The cannabinoid receptor agonist (+)-WIN55212 (10 and 100 nM) inhibited the mean voltage-activated Ca 2+ current in DRG neurones by 21% and 30%, respectively. The isomer, (−)-WIN55212 (10 and 100 nM) produced significantly less inhibition of 6% and 10% respectively. The CB 1 selective receptor antagonist SR141716A (100 nM) enhanced the peak high voltage-activated Ca 2+ current by 24% and simultaneous application of SR141716A (100 nM) and (+)-WIN55212 (100 nM) resulted in a significant attenuation of the inhibition obtained with (+)-WIN55212 alone. These data give functional evidence for the hypothesis that the analgesic actions of cannabinoids may be mediated by presynaptic inhibition of transmitter release in sensory neurones.

Transport of mitochondria during axonogenesis.

The cellular mechanisms involved in axonogenesis are still unclear. In the present work we found that formation of neurites in cultured neonatal dorsal root ganglion neurons co-incided with the redistribution of highly charged mitochondria. Radially distributed in subplasmalemmal space 3 h after plating, highly charged mitochondria formed clusters in the hillocks of predominant neurites during the next 2448 h and then redistributed into the axons. These results provide evidence that accumulation of a critical mass of charged mitochondria at the site of the future axonal hillock may represent the slow initiation stage of axonogenesis, followed by a fast growth phase.

Metabotropic glutamate receptor activation and intracellular cyclic ADP-ribose release Ca2+from the same store in cultured DRG neurones

The whole cell patch clamp technique has been used to record Ca2+-activated cation and chloride conductances evoked by release of Ca2+from intracellular stores of cultured neonatal dorsal root ganglion neurones. The aim of this study was to investigate metabotropic glutamate receptor (mGluR) mechanisms and evaluate a possible role for cyclic ADP-ribose as an intracellular signalling molecule. Glutamate and the metabotropic glutamate receptor agonist (1S,3R)-ACPD-evoked transient depolarizations, Ca2+-activated inward currents and rises in intracellular Ca2+. The (1S,3R)-ACPD-activated currents were insensitive to InsP3signalling inhibitors, heparin and pentosan polysulphate. Intracellular application of ryanodine alone activated currents in this study and proved a difficult tool to use as a potential inhibitor of cyclic ADP-ribose-mediated responses. However, intracellular dantrolene did attenuate both (1S,3R)-ACPD and cyclic ADP-ribose responses. Intracellular photorelease of cGMP and cyclic ADP-ribose mimicked the responses to mGluR receptor activation. Intracellular application of nicotinamide and W7 inhibited the responses to photoreleased cGMP but did not prevent responses to mGluR activation. The cyclic ADP-ribose receptor antagonist 8-amino cyclic ADP-ribose attenuated responses to (1S,3R)-ACPD, cGMP and cyclic ADP-ribose, but some Ca2+-activated inward currents were still observed in the presence of this antagonist. In conclusion, mGluR receptor activation, cGMP and cyclic ADP-ribose release Ca2+from intracellular stores. Some evidence suggests that pharmacologically related pathways are involved.

DSD-1-Proteoglycan Is the Mouse Homolog of Phosphacan and Displays Opposing Effects on Neurite Outgrowth Dependent on Neuronal Lineage

DSD-1-PG is a chondroitin sulfate proteoglycan (CSPG) expressed by glial cells that can promote neurite outgrowth from rat embryonic mesencephalic (E14) and hippocampal (E18) neurons, an activity that is associated with the CS glycosaminoglycans (GAGs). Further characterization of DSD-1-PG has included sequencing of peptides from the core protein and the cloning of the corresponding cDNA using polyclonal antisera against DSD-1-PG to screen phage expression libraries. On the basis of these studies we have identified DSD-1-PG as the mouse homolog of phosphacan, a neural rat CSPG. Monoclonal antibodies 3H1 and 3F8 against carbohydrate residues on rat phosphacan recognize these epitopes on DSD-1-PG. The epitopes of the antibodies, L2/HNK-1 and L5/Lewis-X, which have been implicated in functional interactions, are also found on DSD-1-PG. Although DSD-1-PG has previously been shown to promote neurite outgrowth, its upregulation after stab wounding of the CNS and its localization in regions that are considered boundaries to axonal extension suggested that it may also have inhibitory functions. Neonatal dorsal root ganglion (DRG) explants grown on a rich supportive substrate (laminin) with and without DSD-1-PG were strikingly inhibited by the proteoglycan. The inhibitory effects of DSD-1-PG on the DRG explants were not relieved by removal of the CS GAGs, indicating that this activity is associated with the core glycoprotein. The neurite outgrowth from embryonic hippocampal neurons on laminin was not affected by the addition of DSD-1-PG. This indicates that DSD-1-PG/mouse phosphacan can have opposing effects on the process of neurite outgrowth dependent on neuronal lineage.

TUNEL, Hoechst and immunohistochemistry triple-labelling: an improved method for detection of apoptosis in tissue sections—an update

We describe an updated protocol which allows the combination of the previously described TUNEL and Hoechst 33342 double-labelling (G. Whiteside, N. Cougnon, S.P. Hunt, R. Munglani, An improved method for the detection of apoptosis in tissue sections and cell culture, using the TUNEL technique combined with Hoechst stain, Brain Res. Protocols, 2, 1998, 160–164 [3]), with immunohistochemistry. The inclusion of the immunohistochemistry step allows identification of apoptosis in distinct cell types by using cell specific markers. It also permits investigation into the proteins expressed in cells undergoing apoptotic cell death. We have evaluated the triple staining in sections of post-axotomy neonatal dorsal root ganglia. Themes: Development and regeneration Topics: Neuronal death

An improved method for detection of apoptosis in tissue sections and cell culture, using the TUNEL technique combined with Hoechst stain

We describe a novel procedure for combining a fluorescent variant of the TUNEL technique with Hoechst 33342 stain ( bis-benzimide) to identify apoptosis in tissue sections and cell culture. The biochemical hallmark of apoptosis is internucleosomal DNA cleavage, which gives rise to oligonucleosome-sized fragments (multiples of approximately 180 bp) that can be directly visualised by labelling with biotinylated or digoxygenin-conjugated nucleosides in a reaction that employs terminal deoxynucleotide transferase (TUNEL). TUNEL and Hoechst 33342 have been used separately to identify apoptosis. TUNEL specifically labels dying cells, yet a low background makes comparison of labelled cells with surrounding normal cells difficult and causes disorientation in tissue sections. Hoechst 33342 binds all DNA therefore staining all nuclear material, allowing identification of apoptotic nuclei, but the analysis is laborious. Combining the two fluorescent labels allows the initial recognition of apoptotic cells using the TUNEL technique then, by simply changing the filter, the TUNEL positive nuclei can be compared to surrounding normal nuclei to assess changes in morphology and size. Hoechst 33342 acts as a counterstain, allowing identification of anatomical structures, and permits quantitative comparison between TUNEL positive versus normal cells. We have evaluated the technique using sections of rat embryo, post-axotomy neonatal dorsal root ganglia and adult dorsal root ganglia cell culture.

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor for sensory neurons: Comparison with the effects of the neurotrophins

We compared the effects of glial cell line-derived neurotrophic factor (GDNF) on dorsal root ganglion (DRG) sensory neurons to that of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3). All of these factors were retrogradely transported to subpopulations of sensory neuron cell bodies in the L4/ L5 DRG of neonatal rats. The size distribution of 125I-GDNF-labeled neurons was variable and consisted of both small and large DRG neurons (mean of 506.60 microns2). 125I-NGF was preferentially taken up by small neurons with a mean cross-sectional area of 383.03 microns2. Iodinated BDNF and NT-3 were transported by medium to large neurons with mean sizes of 501.48 and 529.27 microns2, respectively. A neonatal, sciatic nerve axotomy-induced cell death model was used to determine whether any of these factors could influence DRG neuron survival in vivo. GDNF and NGF rescued nearly 100% of the sensory neurons. BDNF and NT-3 did not promote any detectable level of neuronal survival despite the fact that they underwent retrograde transport. We examined the in vitro survival-promoting ability of these factors on neonatal DRG neuronal cultures derived from neonatal rats. GDNF, NGF, and NT-3 were effective in vitro, while BDNF was not. The range of effects seen in the models described here underscores the importance of testing neuronal responsiveness in more than one model. The biological responsiveness of DRG neurons to GDNF in multiple models suggests that this factor may play a role in the development and maintenance of sensory neurons.

Functional involvement of sciatic nerve-derived versican- and decorin-like molecules and other chondroitin sulphate proteoglycans in ECM-mediated cell adhesion and neurite outgrowth.

We have previously described two proteoglycans from human sciatic nerve which are immunochemically related to the chondroitin sulphate proteoglycans versican and decorin. The chondroitin sulphate of the versican-like molecule and the core protein of the decorin-like molecule have been found previously to be up-regulated after lesioning the adult mouse sciatic nerve. To investigate if the versican- and decorin-like molecules are involved in cell-extracellular matrix interactions, we studied the effect of both molecules on cell adhesion. The versican- and decorin-like molecules, substrate-coated in a mixture with fibronectin, but not with laminin or collagen types I or IV, inhibited the adhesion of several cell lines, neonatal dorsal root ganglion neurons and Schwann cells. The inhibitory activity was concentration-dependent and mediated by the chondroitin sulphate. Furthermore, when different proteoglycans were incubated with fibronectin, only the versican- and decorin-like molecules and the chondroitin sulphate proteoglycan aggrecan, but not the heparan sulphate proteoglycan perlecan, were able to inhibit fibronectin-mediated cell adhesion. The versican- and decorin-like molecules, substrate-coated alone or in a mixture with fibronectin or laminin, were at most slightly inhibitory to neurite outgrowth from PC12 phaeochromocytoma cells and neonatal dorsal root ganglion neurons. In a solid-phase ligand-binding assay the versican- and decorin-like molecules interacted with fibronectin, but not with laminin or collagen types I and IV. Binding of the versican-like molecule to fibronectin and inhibition of cell adhesion by this molecule was mediated via the heparin and cell-binding domains of fibronectin. These observations suggest that binding of the two proteoglycans to fibronectin is involved in the modulation of adhesion of cells to fibronectin.

Modulation of Ca2+‐dependent currents in metabolically stressed cultured sensory neurones by intracellular photorelease of ATP

1. The whole cell recording technique was used to study high voltage-activated Ca2+ currents and Ca(2+)-activated Cl- tail currents from cultured neonatal dorsal root ganglion neurones of the rat which were metabolically stressed. The neurones were metabolically stressed with 2-deoxy-D-glucose (5 mM) for 30 min to 3 h. The aim of the project was to examine the actions of intracellular photorelease of ATP on the properties of Ca(2+)-dependent currents and determine if the effects of metabolic stress could be reversed. 2. The mean duration of Ca(2+)-activated Cl- tail currents was significantly increased by metabolic stress and this effect was reversed by intracellular photorelease of approximately 300 microM ATP. Intracellular photolysis of 'caged' photolabile compounds was achieved with a xenon flash lamp. 3. Intracellular photorelease of ATP and adenosine 3':5'-cyclic monophosphate (cyclic AMP) (about 40 microM) also accelerated the inactivation of high voltage-activated Ca2+ currents evoked by 500 ms depolarizing step commands from -90 mV to 0 mV. This effect was prevented by intracellular application of the calcineurin (protein phosphatase-2B) inhibitor cyclosporin A (14 nM) and cyclophilin A (50 nM) either applied together or individually. In contrast the protein phosphatase 1 and 2A inhibitor, calyculin A, increased voltage-activated Ca2+ currents, but failed to prevent enhanced inactivation induced by intracellular photorelease of ATP. Intracellular photorelease of ATP had no effect on Ca2+ currents recorded from control neurones which were not metabolically stressed and supplied with glucose and ATP in the extracellular and patch pipette solutions respectively. 4. In conclusion, intracellular photorelease of ATP increases the decay of Ca2+-activated Cl- tail currents in metabolically stressed neurones suggesting that the efficiency of intracellular Ca2+ buffering was improved. Additionally, an ATP/cyclic AMP-dependent component of high voltage-activated Ca2+current inactivation which is mediated by calcineurin is revealed following photolysis of 'caged' ATP or cyclic AMP in metabolically stressed neurones.

Molecular cloning of a resiniferatoxin-binding protein

Capsaicin and resiniferatoxin are neurotoxins which act on a sensory neuron membrane-associated receptor. In order to identify sensory neuron capsaicin binding proteins, expressed fusion proteins encoded by a directionally-cloned rat neonatal dorsal root ganglion library in λ Zap-II were photoaffinity-labelled with the potent resiniferatoxin and capsaicin-like agonist resiniferanol-9,13,14-orthophenylacetate-20-(3-azido,4-methoxyphenyl) acetate. Four clones encoding possible binding proteins were detected with rabbit anti-resiniferanotoxin antiserum and sequenced. Two clones were homologous and hybridised on Northern blots with a 1.6 kb transcript enriched in dorsal root ganglia, but also present in other non-neuronal tissues. The full-length sequence corresponding to this transcript (RTX-42) was verified using primer extension and found to encode a putative 235 amino acid protein of molecular weight 26,000 which we named RBP-26. In vitro translation of transcribed cRNA resulted in the synthesis of radiolabelled protein of the predicted molecular weight. In situ hybridisation showed that the mRNA encoding this protein was present in sensory neuron cell bodies. Both expressed bacterial fusion proteins and cytoplasmic fractions from COS cells transfected with an expression vector encoding RTX-42 showed [ 3H]resiniferatoxin binding activity (IC 50 ∼ 10 nM). RBP-26 is expressed in non-neuronal and capsaicin-insensitive neuronal tissues, and shows distinct binding characteristics from the resiniferatoxin binding site defined on DRG membranes. The functional role of RBP-26 thus remains to be established.

Effects of metabolic blockers on Ca2+‐dependent currents in cultured sensory neurones from neonatal rats

1. The whole cell variant of the patch clamp technique was used to record high voltage-activated Ca2+ currents and Ca(2+)-activated Cl- tail currents from cultured neonatal rat dorsal root ganglion neurones. The aim of the project was to use these currents as physiological indices of intracellular Ca2+ regulation under control conditions and in the presence of metabolic inhibitors. 2. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (5 microM) and sodium cyanide (1 microM) inhibited Ca2+ currents within 20 s, even when ATP was present in the patch pipette solution, suggesting a direct action on Ca2+ channels. These metabolic inhibitors did not affect Ca2+ current 'run down' or inactivation kinetics. 3. Cultured neonatal dorsal root ganglion neurones of the rat were relatively insensitive to the removal of glucose and ATP from the recording solutions for up to 3 h. These data suggest that the Ca2+ homeostatic mechanisms in these cells are highly resistant to metabolic insult. 4. However 2-deoxy-D-glucose (5 mM) in the extracellular recording medium with no ATP or glucose present did prolong the deactivation time of Ca(2+)-activated Cl- tail currents and increase the total charge flow following activation of a 500 ms voltage-activated Ca2+ current. This effect was prevented by inclusion of D-fructose 1,6-diphosphate (500 microM) in the patch pipette solution. 5. We conclude that some agents used to induce chemical hypoxia, such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone and sodium cyanide, may interact directly with voltage-activated Ca2+ channels and are therefore not appropriate for use in studying disturbed neuronal Ca2+ homeostasis. However, the use of 2-deoxy-D-glucose in the absence of glucose and ATP does represent a model of disturbed Ca2+ homeostasis in cultured dorsal root ganglion neurones. In this study we have combined the whole cell recording technique with cultured neurones under conditions which produce a degree of metabolic stress as reflected by prolonged Ca(2+)-activated Cl- tail currents. The reduced efficiency of handling of intracellular Ca2+ loads may be an important factor contributing to the onset of neuronal damage during hypoxia and ischaemia.

Embryonic optic nerve tissue fails to support neurite outgrowth by central and peripheral neurons in vitro.

The failure of axon regeneration in the injured mammalian central nervous system has been ascribed, in part, to the inhibitory effects of myelin proteins. To investigate the influence of myelination on neurite growth and regeneration by both central nervous system and peripheral nervous system neurons, isolated rat neonatal retinal ganglion cells and adult and neonatal dorsal root ganglion neurons were cultured on cryostat sections of both immature unmyelinated and mature fully myelinated adult rat optic nerve. In agreement with earlier studies using neonatal peripheral neurons, the adult optic nerve failed to support neurite outgrowth from any of the neurons tested. A new finding was that tissue sections from unmyelinated optic nerve (aged embryonic days 18 and 20, and postnatal days 1-3), also failed to support the growth of neurites from neonatal retinal ganglion cells and both neonatal and adult dorsal root ganglion neurons. Neonatal retinal ganglion cells also failed to extend neurites on sections of pre-degenerated sciatic nerve, a tissue shown in our previous work to be a good substratum for supporting neurite growth for both neonatal and adult DRG neurons. These results suggest that cells in the immature optic nerve either express widely acting axon growth inhibitory molecules unrelated to previously described myelin proteins, or do not synthesize appropriate axon growth promoting molecules. They also reveal that, for axon regeneration, central nervous system and peripheral sensory neurons require distinct substratum interactions.

Nitric oxide may act as a messenger between dorsal root ganglion neurones and their satellite cells

The distribution of NADPH-diaphorase and cyclic GMP in neonatal dorsal root ganglia in vitro has been investigated under control conditions and in response to incubation with either sodium nitroprusside or N- methyl- d-aspartate . NADPH-diaphorase activity which reveals the distribution of nitric oxide (NO) synthase in neurones was found to be intense in some dorsal root ganglion neurones and present at a lower level in the majority. Basal levels of cGMP were found to be low but when stimulated by sodium nitroprusside were found to be selectively increased in satellite cells. The results suggest that NO may function as a signalling system between neurones and satellite cells in sensory ganglia.

NF‐L and peripherin immunoreactivities define distinct classes of rat sensory ganglion cells

Double immunofluorescence studies using antibodies against NF-L and peripherin revealed three distinct subpopulations of neurons in rat dorsal root ganglia (DRG). In the adult rat, 46% of the DRG neurons were small and peripherin-positive (NF-L-negative), and 48% were large and NF-L-positive (peripherin-negative). About 6% were both peripherin- and NF-L-positive. All of the DRG neurons reacted with antibodies to NF-M and nonphosphorylation-dependent or phosphorylation-independent antibodies to NF-H. The neuropeptides were predominantly found in the peripherin-positive small cell population. Eighty-seven percent of the peripherin-positive small cell population contained substance P immunoreactivity, while 43% of this cell population contained CGRP. In contrast, only 18-24% of the NF-L-positive large-cell population contained neuropeptides, and these were primarily in a smaller sized subpopulation. Similar patterns of antigen representation were observed in neonatal (PN2) DRG cell populations. Tissue cultures of sensory ganglion cells from PN2 DRG, in serum-free medium, stably maintained exclusively peripherin-positive neurons, with about 5% of these containing coexistent NF-L immunoreactivity. Very high levels of neuropeptide gene expression were exhibited by these postnatal neurons in culture.

Neuron‐Enriched cultures of adult rat dorsal root ganglia: Establishment, characterization, survival, and neuropeptide expression in response to trophic factors

It is unknown whether adult dorsal root ganglion (DRG) neurons require trophic factors for their survival and maintenance of neuropeptide phenotypes. We have established and characterized neuron-enriched cultures of adult rat DRGs and investigated their responses to nerve growth factor (NGF), ciliary neuronotrophic factor (CNTF), pig brain extract (PBE, crude fraction of brain-derived neuronotrophic factor, BDNF), and laminin (LN). DRGs were dissected from levels C1 through L6 and dissociated and freed from myelin fragments and most satellite (S-100-immunoreactive) cells by centrifugation on Percoll and preplating. The enriched neurons, characterized by their morphology and immunoreactivity for neuron-specific enolase, constituted a population representative of the in vivo situation with regard to expression of substance P (SP), somatostatin (SOM), and cholecystokinin-8 (CCK) immunoreactivities. In the absence of trophic factors and using polyornithine (PORN) as a substratum, 60-70% of the neurons present initially (0.5 days) had died after 7 days. LN as a substratum did not prevent a 30% loss of neurons up to day 4.5, but it subsequently maintained DRG neurons at a plateau. This behavior might reflect a cotrophic effect of LN and factors provided by non-neuronal cells, whose proliferation between 4.5 and 7 days could not be prevented by addition of mitotic inhibitors of gamma-irradiation. CNTF, but not NGF, slightly enhanced survival at 7 days on either PORN or LN. No neuronal losses were found in non-enriched cultures or when enriched neurons were supplemented with PBE, indicating that non-neuronal cells and PBE provide factor(s) essential for adult DRG neuron survival. Proportions of SP-, SOM-, and CCK-immunoreactive cells were unaltered under any experimental condition, with the exception of a numerical decline in SP cells in 7-day cultures with LN, but not PORN, as the substratum. Our data, considered in the context of recent in vivo and vitro studies, suggest that a combination of trophic factors or an unidentified factor, rather than the established molecules NGF, CNTF, and BDNF, which address embryonic and neonatal DRG neurons, are required for the in vitro maintenance of adult DRG neurons.

Serotonin decreases the duration of action potentials recorded from tetraethylammonium-treated bullfrog dorsal root ganglion cells

Neurotransmitter effects on calcium currents activated by sensory neuron action potentials have been previously studied in embryonic or neonatal dorsal root ganglion (DRG) cells in culture. In the present study we examined the effects of serotonin (5-HT) on the shape of action potentials recorded from fully differentiated primary afferent neurons in isolated DRG of adult bullfrogs. Intracellular recordings were obtained from cell bodies of type A and C neurons. Concentrations of 5-HT that had no effect on membrane potential or input resistance had little or no effect on action potential shape. Treatment with 5-20 mM tetraethylammonium ion (TEA) led to the appearance of a plateau phase on the falling limb of the spike. This plateau phase appears to result from calcium influx, as it was dramatically reduced in amplitude and duration by solutions containing low concentrations of calcium or the calcium channel blocker, manganese. In preparations treated with 7.5 mM TEA, low concentrations of 5-HT (10 nM-1 microM) produced a dose-dependent narrowing of the calcium-dependent plateau phase of the mixed sodium/calcium spike. A decrease in spike afterhyperpolarization was also noted. The decrease in spike duration was recorded from 74% of type A neurons and 57% of type C neurons, and was not secondary to a change in resting potential or input resistance. The 5-HT receptor antagonists methysergide and metergoline did not block the response to 5-HT. Instead, they exhibited weak agonist-like actions. Serotonin also reduced the rate of rise and peak amplitude of calcium spikes recorded in the presence of tetrodotoxin and TEA.(ABSTRACT TRUNCATED AT 250 WORDS)

Lactoseries carbohydrates specify subsets of dorsal root ganglion neurons projecting to the superficial dorsal horn of rat spinal cord

Cell surface carbohydrates are thought to play important roles in the development and differentiation of mammalian cells. In previous studies we have found that one population of dorsal root ganglion (DRG) neurons is specified by the expression of complex globoseries oligosaccharides (Dodd, J., D. Solter, and T. M. Jessell (1984) Nature 311: 469-472; Jessell, T. M., and J. Dodd (1985) Philos. Trans. R. Soc. Lond. (Biol.) 308: 271-281). We now report that monoclonal antibodies (MAbs) directed against backbone structures of lactoseries oligosaccharides define antigens present in the cytoplasm of a second, anatomically and functionally distinct subset of DRG neurons. Lactoseries carbohydrate structures identified by MAb A5 are restricted to small- and intermediate-diameter DRG neurons with central projections in the superficial dorsal horn of the rat spinal cord. The distribution of labeled terminals suggests that many of the DRG neurons that express lactoseries carbohydrates are likely to convey nociceptive information. More complex galactose- and fucose-substituted lactoseries structures recognized by MAbs LD2, KH10, TC6, TD10, LA4, and anti-Lewis a are segregated on subsets of DRG neurons that differ in their expression of substance P, somatostatin, and fluoride-resistant acid phosphatase and in their laminar termination in the superficial dorsal horn. The majority of lactoseries carbohydrate antigens identified in the cytoplasm of DRG neurons are also expressed on the surface of subsets of DRG neurons in culture. These studies establish that structurally defined carbohydrate differentiation antigens specify the majority of primary sensory neurons with cutaneous receptive fields. Moreover, lactoseries carbohydrate structures similar or identical to those expressed on neonatal DRG neurons in culture have been implicated in cell-cell interactions at early stages of preimplantation embryonic development. Our observations suggest strategies for testing the hypothesis that carbohydrates present on the surface of subsets of DRG neurons play a role in cell interactions that contribute to the laminar organization of sensory afferents in the developing spinal cord.

Expression of neural cell adhesion molecule L1 during development, in neurological mutants and in the peripheral nervous system

Neural cell adhesion molecule L1 consists of two glycoprotein bands of 140 and 200 kdaltons at all developmental stages studied (from birth to adulthood in murine cerebellum and cerebral hemispheres) and in the 4 neurological mouse mutants reeler, weaver, staggerer and Purkinje cell degeneration. In histological sections L1 antigen is detectable at birth in the Purkinje cell layer and fiber tracts in the prospective white matter, but not in the external granular layer. From postnatal day 4 onwards L1 antigen additionally appears in the inner part of the external granular layer, the zone of postmitotic premigratory granule cell neurons. The outer part of the external granular layer remains L1 antigen-negative until it disappears at approximately day 12. From then onwards, the antigen remains prominent in the nascent molecular layer and is less detectable in white matter and internal granular layer, the location of the cell bodies of postmigratory granule cells. The four neurological mouse mutants show development of L1 antigen expression analogous to the normal situation, despite an abnormal cellular architecture. In contrast to the central nervous system, Western blots of adult sciatic nerve show a more complex pattern of L1 immunoreactive bands. L1 antigen is detectable on most, if not all Schwann cells in histological sections of sciatic nerve from 17-day-old embryos. At postnatal day 2, only some Schwann cells appear L1 antigen-positive. From then onwards L1 seems most prominently associated with non-myelinating Schwann cells. In monolayer cultures of neonatal dorsal root ganglia the antigen is observed on the surface of neurons and of some Schwann cells. The mutant, trembler, shows a more immature staining pattern for L1 antigen in adult sciatic nerve.