Neonatal Dorsal Root Ganglion Neurons Research Articles

Characterization of dorsal root ganglion neurons cultured on silicon micro-pillar substrates.

Our study focuses on characterization of dorsal root ganglion (DRG) neurons cultured on silicon micro-pillar substrates (MPS) with the ultimate goal of designing micro-electrode arrays (MEAs) for successful electrophysiological recordings of DRG neurons. Adult and neonatal DRG neurons were cultured on MPS and glass coverslips for 7 days in vitro. DRG neuronal distribution and morphometric analysis, including neurite alignment and length, was performed on MPS areas with different pillar width and spacing. We showed that MPS provide an environment for growth of adult and neonatal DRG neurons as permissive as control glass surfaces. Neonatal DRG neurons were present on MPS areas with narrow pillar spacing, while adult neurons preferred wider pillar spacing. Compared to the control glass surfaces the neonatal and adult DRG neurons in regions with narrow pillar spacing range developed a smaller number of longer neurites. In the same area, neurites were preferentially oriented along three directional axes at 30°, 90° and 150°. MPS architecture influenced growth directionality of all main DRG neuronal subtypes. We can conclude that specific micro-pillar substrate topography affects the morphology of DRG neurons. This knowledge can enable development of MEAs with precisely defined physical features for various neuroscience applications.

Axonal Neuropathy-associated TRPV4 Regulates Neurotrophic Factor-derived Axonal Growth

Spinal muscular atrophy and hereditary motor and sensory neuropathies are characterized by muscle weakness and atrophy caused by the degenerations of peripheral motor and sensory nerves. Recent advances in genetics have resulted in the identification of missense mutations in TRPV4 in patients with these hereditary neuropathies. Neurodegeneration caused by Ca(2+) overload due to the gain-of-function mutation of TRPV4 was suggested as the molecular mechanism for the neuropathies. Despite the importance of TRPV4 mutations in causing neuropathies, the precise role of TRPV4 in the sensory/motor neurons is unknown. Here, we report that TRPV4 mediates neurotrophic factor-derived neuritogenesis in developing peripheral neurons. TRPV4 was found to be highly expressed in sensory and spinal motor neurons in early development as well as in the adult, and the overexpression or chemical activation of TRPV4 was found to promote neuritogenesis in sensory neurons as well as PC12 cells, whereas its knockdown and pharmacologic inhibition had the opposite effect. More importantly, nerve growth factor or cAMP treatment up-regulated the expression of phospholipase A(2) and TRPV4. Neurotrophic factor-derived neuritogenesis appears to be regulated by the phospholipase A(2)-mediated TRPV4 pathway. These findings show that TRPV4 mediates neurotrophic factor-induced neuritogenesis in developing peripheral nerves. Because neurotrophic factors are essential for the maintenance of peripheral nerves, these findings suggest that aberrant TRPV4 activity may lead to some types of pathology of sensory and motor nerves.

Differential contribution of SNARE‐dependent exocytosis to inflammatory potentiation of TRPV1 in nociceptors

Potentiation of the pain-integrator ion channel transient receptor potential vanilloid type 1 (TRPV1) underlies thermal hyperalgesia mediated by a variety of proinflammatory factors. Two complementary mechanisms of TRPV1 inflammatory sensitization have been proposed, namely a decrease of its activation threshold and an increment of its surface expression in nociceptors. Here we investigated the involvement of regulated exocytosis to the inflammatory sensitization of TRPV1 in rat neonatal dorsal root ganglion neurons by proalgesic agents. The contribution of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent exocytosis was evaluated using a small peptide patterned after the synaptosomal-associated protein of 25 kDa (SNAP-25) protein that acts as a specific and potent inhibitor of neuronal exocytosis. We found that TRPV1 sensitization mediated by nerve growth factor, ATP, and IGF-I was accompanied by a higher channel expression in the neuronal plasma membrane, which was prevented by blockade of regulated exocytosis. In contrast, TRPV1 sensitization caused by bradykinin, IL-1beta, and artemin was insensitive to inhibition of SNARE-dependent vesicular fusion and was not due to an increase in TRPV1 surface expression. Therefore, it appears that some, but not all, proinflammatory agents sensitize rat nociceptors by promoting the recruitment of TRPV1 channels to the neuronal surface. These findings support the tenet that SNARE complex-mediated exocytosis of TRPV1 may be a valid therapeutic target to treat inflammatory pain.

Estrogen Elicits Dorsal Root Ganglion Axon Sprouting via a Renin-Angiotensin System

Many painful conditions occur more frequently in women, and estrogen is a predisposing factor. Estrogen may contribute to some pain syndromes by enhancing axon outgrowth by sensory dorsal root ganglion (DRG) neurons. The objective of the present study was to define mechanisms by which estrogen elicits axon sprouting. The estrogen receptor-alpha agonist propyl pyrazole triol induced neurite outgrowth from cultured neonatal DRG neurons, whereas the estrogen receptor-beta agonist diarylpropionitrile was ineffective. 17beta-Estradiol (E2) elicited sprouting from peripherin-positive unmyelinated neurons, but not larger NF200-positive myelinated neurons. Microarray analysis showed that E2 up-regulates angiotensin II (ANGII) receptor type 2 (AT2) mRNA in vitro, and studies in adult rats confirmed increased DRG mRNA and protein in vivo. AT2 plays a central role in E2-induced axon sprouting because AT2 blockade by PD123,319 eliminated estrogen-mediated sprouting in vitro. We assessed whether AT2 may be responding to locally synthesized ANGII. DRG from adult rats expressed mRNA for renin, angiotensinogen, and angiotensin converting enzyme (ACE), and protein products were present and occasionally colocalized within neurons and other DRG cells. We determined if locally synthesized ANGII plays a role in estrogen-mediated sprouting by blocking its formation using the ACE inhibitor enalapril. ACE inhibition prevented estrogen-induced neuritogenesis. These findings support the hypothesis that estrogen promotes DRG nociceptor axon sprouting by up-regulating the AT2 receptor, and that locally synthesized ANGII can induce axon formation. Therefore, estrogen may contribute to some pain syndromes by enhancing the pro-neuritogenic effects of AT2 activation by ANGII.

Selective inhibition of voltage‐activated Ca2+ channels by mu‐opioid receptor agonist in the primary sensory neurons devoid of T‐type Ca2+ channels: Mechanisms of action

Opioids have a unique effect on nociception with little effect on other sensory modalities. However, the cellular mechanisms for this preferential effect are not clear. We have found recently that the μ‐opioid agonists inhibited high voltage‐activated Ca2+ channels (HVACC) only when the low voltage‐activated (T‐type) Ca2+ channels are not present in dorsal root ganglion (DRG) neurons (15 – 30μm). In this study, we determined the potential mechanisms for the differential effect of the μ‐opioid agonists on HVACC in DRG neurons with and without T‐type Ca2+ channel currents. Whole‐cell voltage clamp recordings were performed in acutely isolated DRG neurons in adult rats. In DRG neurons with T‐type Ca2+ channel currents, the μ‐opioid agonist DAMGO had no significant effect on both T‐type and HVACC currents. This occurred despite the fact that a large ω‐conotoxin GVIA‐sensitive (N‐type) Ca2+ current was present in the cells with T‐type currents. DAMGO still had no effect on HVACC currents when T‐type Ca2+ currents were blocked by amiloride or Ni2+ in these neurons. However, intracellular dialysis of GTP‐γ‐S to activate G proteins significantly attenuated HVACC currents, and the strong depolarization partially relieved GTP‐γ‐S‐induced inhibition. In the neonatal DRG neurons with T‐type Ca2+ currents, the GABAB receptor agonist baclofen but not DAMGO potently inhibited HVACC. Importantly, single‐cell RT‐PCR analysis showed that none of the DRG neurons with T‐type Ca2+ currents had μ‐opioid receptor mRNA. Thus, this study provides novel information about the heterogenous effects of μ‐opioid receptor agonists on HVACC in different populations of sensory neurons. This finding provides an intrinsic cellular mechanism for the selective effect of μ‐opioids on pain transmission.

Ro5‐4864 promotes neonatal motor neuron survival and nerve regeneration in adult rats

The translocator protein (18 kDa; TSPO), formerly known as the peripheral benzodiazepine receptor, is an outer mitochondrial membrane protein that associates with the mitochondrial permeability transition pore to regulate both steroidogenesis and apoptosis. TSPO expression is induced in adult dorsal root ganglion (DRG) sensory neurons after peripheral nerve injury and a TSPO receptor ligand, Ro5-4864, enhances DRG neurite growth in vitro and axonal regeneration in vivo. We have now found that TSPO is induced in neonatal motor neurons after peripheral nerve injury and have evaluated its involvement in neonatal and adult sensory and motor neuron survival, and in adult motor neuron regeneration. The TSPO ligand Ro5-4864 rescued cultured neonatal DRG neurons from nerve growth factor withdrawal-induced apoptosis and protected neonatal spinal cord motor neurons from death due to sciatic nerve axotomy. However, Ro5-4864 had only a small neuroprotective effect on adult facial motor neurons after axotomy, did not delay onset or prolong survival in SOD1 mutant mice, and failed to protect adult DRG neurons from sciatic nerve injury-induced death. In contrast, Ro5-4864 substantially enhanced adult facial motor neuron nerve regeneration and restoration of function after facial nerve axotomy. These data indicate a selective sensitivity of neonatal sensory and motor neurons to survival in response to Ro5-4864, which highlights that survival in injured immature neurons cannot necessarily predict success in adults. Furthermore, although Ro5-4864 is only a very weak promoter of survival in adult neurons, it significantly enhances regeneration and functional recovery in adults.

Estrogen increases sensory nociceptor neuritogenesis in vitro by a direct, nerve growth factor‐independent mechanism

Estrogen affects many aspects of the nervous system, including pain sensitivity and neural regulation of vascular function. We have shown that estrogen elevation increases sensory nociceptor innervation of arterioles in Sprague-Dawley rat mammary gland, external ear and mesentery, suggesting widespread effects on sensory vasodilatory innervation. However, it is unclear whether estrogen elicits nociceptor hyperinnervation by promoting target release of neurotrophic factors, or by direct effects on sensory neurons. To determine if estrogen may promote axon sprouting by increasing release of target-derived diffusible factors, dorsal root ganglia explants were co-cultured with mesenteric arterioles for 36 h in the absence or presence of 17beta-estradiol (E2). Mesenteric arteriolar target substantially increased neurite outgrowth from explanted ganglia, but estrogen had no effect on outgrowth, suggesting that estrogen does not increase the availability of trophic proteins responsible for target-induced neurite outgrowth. To assess the direct effects of estrogen, dissociated neonatal dorsal root ganglion neurons were cultured for 3 days in the absence or presence of E2 and nerve growth factor (NGF; 1-10 ng/mL), and immunostained for the nociceptor markers peripherin or calcitonin gene-related peptide. NGF increased neuron size, survival and numbers of neurons with neurites, but did not affect neurite area per neuron. Estrogen did not affect neuron survival, size or numbers of neurons with neurites, but did increase neurite area per neuron. The effects of these agents were not synergistic. We conclude that estrogen exerts direct effects on nociceptor neurons to promote axon outgrowth, and this occurs through an NGF-independent mechanism.

Ca2+ permeability of nicotinic acetylcholine receptors from rat dorsal root ganglion neurones

Ca2+ entry through neuronal nicotinic ACh receptors (nAChRs) modulates many biological processes in nervous tissue. In order to study the functional role of nAChRs in peripheral sensory signalling, we measured their Ca2+ permeability in rat dorsal root ganglion (DRG) neurones, and analysed the effects of nAChR-mediated Ca2+ influx on the function of the vanilloid receptor TRPV1. The fractional Ca2+ current (P(f), i.e. the percentage of current carried by Ca2+ ions) flowing through nAChR channels was measured by Ca2+ imaging fluorescence microscopy in combination with the patch-clamp technique. Functional nAChRs were expressed in a subset of adult DRG neurones (about 24% of the cells), typically with small to medium size as measured by their capacitance (40 +/- 3 pF). In most cells, ACh evoked slowly desensitizing currents, insensitive to methyllycaconitine (MLA, 10 nm), a potent antagonist of homomeric nAChRs. Fast decaying currents, probably mediated by alpha7*-nAChRs (i.e. native alpha7-containing nAChRs), were observed in 15% of ACh-responsive cells, in which slowly decaying currents, mediated by heteromeric nAChRs, were simultaneously present. The nAChRs of adult DRG neurones exhibited a P(f) value of 2.2 +/- 0.6% in the presence of MLA and 1.9 +/- 0.6% (P > 0.1) in the absence of MLA, indicating that homomeric MLA-sensitive nAChRs do not contribute to Ca2+ entry into adult DRG neurones. Conversely, 10% of neonatal DRG neurones showed ACh-evoked currents completely blocked by MLA. In these neurones, nAChRs showed a larger P(f) value (9.5 +/- 1.5%), indicating the expression of bona fide alpha7*-nAChRs. Finally, we report that Ca2+ influx through nAChRs in adult DRG neurones negatively modulated the TRPV1-mediated responses, representing a possible mechanism underlying the analgesic properties of nicotinic agonists on sensory neurones.

Either nitric oxide or nerve growth factor is required for dorsal root ganglion neurons to survive during embryonic and neonatal development

During early embryonic (E12) development almost all dorsal root ganglion (DRG) neurons express the neuronal isoform of nitric oxide synthase (nNOS). At this stage, the axons of these neurons are rudimentary and have not made contact with peripheral tissue targets. As their axons establish contact with peripheral targets such as the skin, the number of neurons expressing nNOS decrease that correspond to increased immunoreactivity for nerve growth factor (NGF) in the skin, and its high affinity receptor, tyrosine kinase A ( trkA) in both skin and DRG neurons. During late postnatal development, very few DRG neurons express nNOS; however, axotomy or NGF deprivation of cultured DRG neurons induce nNOS and NOS blockade causes neuronal death. In contrast, NGF-deprived embryonic and neonatal DRG neurons die by apoptosis, while NOS blockade has no effect. Overall, these observations suggest that NGF and nitric oxide (NO) interact during embryonic and postnatal development to facilitate neuronal selection and survival. The roles of NO, NGF and its receptor trkA in DRG neurons during different stages of development are discussed.

Glial-mediated neuroprotection: evidence for the protective role of the NO-cGMP pathway via neuron-glial communication in the peripheral nervous system.

The NO-cGMP pathway has emerged as a neuroprotective signaling system involved in communication between neurons and glia. We have previously shown that axotomy or nerve growth factor (NGF)-deprivation of dorsal root ganglion (DRG) neurons leads to increased production of NO and at the same time an increase in cGMP production in their satellite glia cells. Blockade of NO or its receptor, the cGMP synthesizing enzyme soluble guanylate cyclase (sGC), results in apoptosis of neurons and glia. We now show that co-culture of neonatal DRG neurons with either Schwann cells pre-treated with an NO donor or a membrane-permeant cGMP analogue; or neurons maintained in the medium from Schwann cell cultures treated in the same way, prevents neuronal apoptosis. Both NO donor and cGMP treatment of Schwann cells results in synthesis of NGF and NT3. Furthermore, if the Schwann cells are previously infected with adenoviral vectors expressing a dominant negative sGC mutant transgene, treatment of these Schwann cells with an NO donor now fails to prevent neuronal apoptosis. Schwann cells treated in this way also fail to express neither cGMP nor neurotrophins. These findings suggest NO-sGC-cGMP-mediated NGF and NT3 synthesis by Schwann cells protect neurons.

The functional expression of mu opioid receptors on sensory neurons is developmentally regulated; morphine analgesia is less selective in the neonate

Opioid requirements in neonatal patients are reported to be lower than older infants and this may be a reflection of the developmental regulation of opioid receptors. In this study we have investigated the postnatal regulation of Mu opioid receptor (MOR) function in both rat lumbar dorsal root ganglion (DRG) cultures and behavioural mechanical and thermal reflex tests in rat pups. Immunostaining with MOR and selective neurofilament (NF200) antibodies was combined with calcium imaging of MOR function in cultured neonatal and adult rat dorsal root ganglion cells. Calcium imaging showed that a significantly greater number of neonatal DRG neurons expressed functional MOR compared to adult (56.5±3.4 versus 39.9±1.5%, n=8, mean±SEM, P<0.001). This expression is confined to the large, neurofilament positive sensory neurons, while expression in small, nociceptive, neurofilament negative neurons remains unchanged. Sensory threshold testing in rat pups showed that the analgesic potency of systemic morphine to mechanical stimulation is significantly greater in the neonate and declines with postnatal age. Morphine analgesic potency in thermal nociceptive tests did not change with postnatal age. These experiments show that the MOR expressed on large DRG neurons in neonates are functional and are subject to postnatal developmental regulation. This changing functional receptor profile is consistent with greater morphine potency in mechanical, but not thermal, sensory tests in young animals. These results have important clinical implications for the use of morphine in neonates and provide a possible explanation for the differences in morphine requirements observed in the youngest patients.

A developmental switch in acute sensitization of small dorsal root ganglion (DRG) neurons to capsaicin or noxious heating by NGF.

Using dissociated rat dorsal root ganglion (DRG) neurons, we have explored the ability of nerve growth factor (NGF) to acutely (within minutes) sensitize responses of nociceptors to capsaicin or noxious heat during postnatal development. While robust sensitization of noxious heat or capsaicin responses by NGF is observed in adult DRG neurons, responses to such stimuli in trkA-positive neurons from early postnatal animals are not sensitized by NGF. Neurons acquire sensitivity to the hyperalgesic effects of NGF between postnatal days 4 and 10 (P4-P10). In contrast to NGF, bradykinin sensitizes responses to noxious heat in both adult and neonatal DRG neurons. These observations suggest a developmental switch in signal transduction cascades linking trkA receptors to hyperalgesia during postnatal development and differences in the signaling pathways mediating bradykinin- and NGF-induced sensitization.

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.

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.

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.

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.

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.