Mammalian Dorsal Root Ganglion Neurons Research Articles

SrGAP3 promotes neurite outgrowth of dorsal root ganglion neurons by inactivating RAC1

ObjectiveTo explore effect of srGAP3 promotes neurite outgrowth of dorsal root ganglion neurons. MethodsIn this study, expression of Slit1 was observed predominantly in the glia, while expression of Robo2 and srGAP3 was detected in sensory neurons of postnatal rat cultured dorsal root ganglion (DRG). Furthermore, upregulation of srGAP3 following sciatic nerve transection was detected by immunohistochemistry and Western blotting. ResultsIt was observed that inhibition of neurite outgrowth in cultured adult DRG neurons following treatment with anti-srGAP3 or anti-Robo2 was more effectively (1.5-fold higher) than that following treatment with an anti-BDNF positive control antibody. It demonstrated that srGAP3 interacted with Robo2 and Slit1 protein to decrease Rac1-GTP activity in cultured adult rat DRG neurons and the opposite effect on Rac1-GTP activity was detected by co-immunoprecipitation and immunoblotting analyses following treatment with anti-Robo2 or anti-srGAP3. These data demonstrated a role for srGAP3 in neurite outgrowth of DRG sensory neurons. ConclusionsOur observations suggest that srGAP3 promotes neurite outgrowth and filopodial growth cones by interacting with Robo2 to inactivate Rac1 in mammalian DRG neurons.

A family of excitatory peptide toxins from venomous crassispirine snails: Using Constellation Pharmacology to assess bioactivity

The toxinology of the crassispirine snails, a major group of venomous marine gastropods within the superfamily Conoidea, is largely unknown. Here we define the first venom peptide superfamily, the P-like crassipeptides, and show that the organization of their gene sequences is similar to conotoxin precursors. We provide evidence that one peptide family within the P-like crassipeptide superfamily includes potassium-channel (K-channel) blockers, the κP-crassipeptides. Three of these peptides were chemically synthesized (cce9a, cce9b and iqi9a). Using conventional electrophysiology, cce9b was shown to be an antagonist of both a human Kv1.1 channel isoform (Shaker subfamily of voltage-gated K channels) and a Drosophila K-channel isoform. We assessed the bioactivity of these peptides in native mammalian dorsal root ganglion neurons in culture. We demonstrate that two of these crassipeptides, cce9a and cce9b, elicited an excitatory phenotype in a subset of small-diameter capsaicin-sensitive mouse DRG neurons that were also affected by κJ-conotoxin PlXIVA (pl14a), a blocker of Kv1.6 channels. Given the vast complexity of heteromeric K-channel isoforms, this study demonstrates that the crassispirine venoms are a potentially rich source for discovering novel peptides that can help to identify and characterize the diversity of K-channel subtypes expressed in native neurons and other cell types.

Modulation of Cav2.2 Channels via Activation of Human GABAB Receptors Expressed in HEK293 Cells by Analgesic α-Conotoxins

Numerous G protein-coupled receptors, including the GABAB receptor (GABABR), provide negative feedback to modulate the activity of neuronal (N)-type voltage-gated calcium channels (Cav2.2), which play a critical role in nociception. We have previously shown that analgesic α-conotoxins indirectly inhibit Cav2.2 channels via GABABR activation in mammalian dorsal root ganglion neurons (1). We reconstituted GABABR-mediated modulation of stably expressed CaV2.2 channels (α1, α2δ and β3) in HEK293 cells, co-transfected with cDNAs of cloned human GABABR subunits. GABABR expression was demonstrated using receptors labelled with fluorescent antibodies against the epitope tags of GABAB1 and GABAB2 subunits. Modulation of Cav2.2 by the agonists GABA and baclofen, and the α-conotoxins Vc1.1 and RgIA was studied using the whole-cell recording configuration of the patch clamp technique. Voltage-dependent Ba2+ currents were inhibited by baclofen, GABA and α-conotoxins Vc1.1 and RgIA in HEK293 cells transfected with Cav2.2 and GABABRs but not in cells transfected with Cav2.2 alone. In the presence of the GABABR, the biophysical properties of the Cav2.2 channels, including the current-voltage relationship, and activation and steady-state inactivation curves, were unchanged. The concentration-response relationships obtained for inhibition of Cav2.2 by baclofen, GABA, RgIA, Vc1.1, and cyclized Vc1.1 resulted in half-maximal inhibitory concentrations (IC50) of 3 μM, 68 nM, 130 nM, 120 nM and 10 nM, respectively. The inhibition by baclofen and GABA could be reversed by a depolarizing prepulse to +80 mV, whereas the effect of the α-conotoxins was slower and unaffected by a prepulse, suggesting the involvement of a voltage-independent pathway. Taken together, HEK293 cells provide a suitable expression system to study GABABR modulation of Cav2.2 channels and confirm the role of GABABRs in mediating the effects of analgesic α-conotoxins.1. Callaghan et al. (2008) J. Neurosci. 28:10943-51.

Voltage-clamp and current-clamp recordings from mammalian DRG neurons

We provide here detailed electrophysiological protocols to study voltage-gated sodium channels and to investigate how wild-type and mutant channels influence firing properties of transfected mammalian dorsal root ganglion (DRG) neurons. Whole-cell voltage-clamp recordings permit us to analyze kinetic and voltage-dependence properties of ion channels and to determine the effect and mode of action of pharmaceuticals on specific channel isoforms. They also permit us to analyze the role of individual sodium channels and their mutant derivatives in regulating firing of DRG neurons. Five to ten cells can be recorded daily, depending on the extent of analysis that is required. Because of different internal solutions that are used in voltage-clamp and current-clamp recordings, only limited information can be obtained from recording the same neuron in both modes. These electrophysiological studies help to elucidate the role of specific channels in setting threshold and suprathreshold responses of neurons, under normal and pathological conditions.

Impaired regenerative response of primary sensory neurons in ZPK/DLK gene-trap mice

Rapid and persistent activation of c-JUN is necessary for axonal regeneration after nerve injury, although upstream molecular events leading to c-JUN activation remain largely unknown. ZPK/DLK/MAP3K12 activates the c-Jun N-terminal kinase pathway at an apical level. We investigated axonal regeneration of the dorsal root ganglion (DRG) neurons of homozygous ZPK/DLK gene-trap mice. In vitro neurite extension assays using DRG explants from 14 day-old mice revealed that neurite growth rates of the ZPK/DLK gene-trap DRG explants were reduced compared to those of the wild-type DRG explants. Three ZPK/DLK gene-trap mice which survived into adulthood were subjected to sciatic nerve axotomy. At 24 h after axotomy, phosphorylated c-JUN-positive DRG neurons were significantly less frequent in ZPK/DLK gene-trap mice than in wild-type mice. These results indicate that ZPK/DLK is involved in regenerative responses of mammalian DRG neurons to axonal injury through activation of c-JUN.

A simulation study on the Ca2+-independent but voltage-dependent exocytosis and endocytosis in dorsal root ganglion neurons

In patch-clamped somata of dorsal root ganglion (DRG) neurons, two types of secretion have been proposed: Ca(2+)-dependent secretion and Ca(2+)-independent but voltage-dependent secretion (CIVDS). The Ca(2+)-induced and the depolarization-induced membrane capacitance (C(m)) increases contribute 80 and 20% to the total C(m) increase, respectively (Zhang and Zhou in Nat Neurosci 5:425, 2002). In order to explore the mechanism of the voltage-dependent C(m) change (DeltaC(m)), we constructed a model with sequential states. The simulation with this model closely approximates all the experimental data. The model predicts that the majority of fusion events (approximately 80%) are so-called "kiss-and-run" events, which account for the fast recovery or the rapid retrieval feature of the signals. The remaining 20% are attributed to full fusion events, which account for a slow retrieval feature. On the basis of the model, one mechanism of the activity-dependent endocytosis has revealed a differential distribution of vesicles between the kiss-and-run and full fusion states at different stimulation frequencies. The quantitative model presented in this study may help us to understand the mechanism of the CIVDS and the tightly coupled endocytosis found in mammalian DRG neurons.

Neuropilin-1 Is Expressed on Adult Mammalian Dorsal Root Ganglion Neurons and Mediates Semaphorin3a/Collapsin-1-Induced Growth Cone Collapse by Small Diameter Sensory Afferents

Neuropilin-1 on the growth cones of NGF-dependent embryonic dorsal root ganglion (DRG) neurons mediates the repulsive effects of secreted semaphorin3a, but its role in adult neurons is unknown. Here we show that most adult rat DRG neurons, regardless of cell diameter/afferent phenotype, express neuropilin-1 protein in vitro. However, the response of growth cones belonging to these neurons (induced by recombinant collapsin-1/semaphorin3a and blocked by the anti-neuropilin-1 antibody) was restricted to those of small cell body diameter (<30 μm), corresponding primarily to nociceptive sensory afferents. Neurotrophic factors had a differential effect on neuropilin-1 expression in vitro, with DRG neurons cultured in either NGF or GDNF expressing the highest levels on their neurites. These findings suggest that neuropilin-1-mediated repellent effects of semaphorins may regulate the behavior of nociceptive sensory axons in the adult as well as the embryonic peripheral nervous system.

Selective dependence of mammalian dorsal root ganglion neurons on nerve growth factor during embryonic development

We have investigated the NGF dependence of dorsal root ganglion (DRG) neurons in mammals using a paradigm of multiple in utero injections of a high titer anti-NGF antiserum. We have determined the specificity of our antiserum in relation to other members of the NGF neurotrophin family and found no cross-reactivity with brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3). To identify various classes of DRG neurons, we have stained their characteristic central projections with Dill. We show here that the NGF dependence of DRG neurons is strikingly selective. Although a majority of DRG neurons are lost after NG F deprivation during embryonic life, these are almost exclusively small diameter neurons that project to laminae I and II of the dorsal horn and presumably subserve nociception and thermoreception. Larger neurons that project to more ventral spinal laminae and subserve other sensory modalities do not require NGF for survival. These NGF-independent DRG neurons likely require one of the more recently identified neurotrophins, BDNF or NT-3.

Subtypes of dorsal root ganglion neurons based on different inward currents as measured by whole-cell voltage clamp.

Electrophysiological and pharmacological properties distinguished subtypes of adult mammalian dorsal root ganglion neurons (DRGn) in monolayer dissociated cell culture. By analogy of action potential waveform and duration, neurons with short duration (SDn) and long duration (LDn) action potentials resembled functionally distinct subtypes of DRGn in intact ganglia. Patch clamp and conventional intracellular recording techniques were combined here to elucidate differences in the ionic basis of excitability of subtypes of DRGn in vitro. Both SDn and LDn were quiescent at the resting potential. Action potentials of SDn were brief (less than 2 msec), sensitive to tetrodotoxin (TTX, 5-10 nM), exhibited damped firing during long depolarizations, and did not respond to algesic agents applied by pressure ejection. Action potentials of LDn were 2-6 msec in duration, persisted in 30 microM TTX, and fired repetitively during depolarizing current pulses or exposure to algesic agents (e.g., capsaicin, histamine and bradykinin). Whole-cell recordings from freshly dissociated neurons revealed two inward sodium currents (INa; variable with changes in sodium but not calcium concentration in the superfusate) in various proportions: a rapidly activating and inactivating, TTX-sensitive current; and, a slower, TTX (30 microM)-resistant INa. Large neurons, presumable SDn, had predominantly TTX-sensitive current and little TTX-resistant current. The predominant inward current of small neurons, presumably LDn, was TTX-resistant with a smaller TTX-sensitive component. By analogy to findings from intact ganglia, these results suggest that fundamentally different ionic currents controlling excitability of subtypes of DRGn in vitro may contribute to functional differences between subtypes of neurons in situ.

Nerve growth factor.

Nerve growth factor.