Cultured Rat Sensory Neurons Research Articles

Purinergic synapses formed between rat sensory neurons in primary culture

Though there is some evidence to the contrary, dogma claims that primary sensory neurons in the dorsal root ganglion do not interact, that the ganglion serves as a through-station in which no signal processing occurs. Here we use patch clamp and immunocytochemistry to show that sensory neurons in primary culture can form chemical synapses on each other. The resulting neurotransmitter release is calcium dependent and uses synaptotagmin-containing vesicles. On many cells studied, the postsynaptic receptor for the neurotransmitter is a P2X receptor, an ion channel activated by extracellular ATP. This shows that sensory neurons have the machinery to form purinergic synapses on each other and that they do so when placed in short-term tissue culture.

Purinergic synapses formed between rat sensory neurons in primary culture

Though there is some evidence to the contrary, dogma claims that primary sensory neurons in the dorsal root ganglion do not interact, that the ganglion serves as a through-station in which no signal processing occurs. Here we use patch clamp and immunocytochemistry to show that sensory neurons in primary culture can form chemical synapses on each other. The resulting neurotransmitter release is calcium dependent and uses synaptotagmin-containing vesicles. On many cells studied, the postsynaptic receptor for the neurotransmitter is a P2X receptor, an ion channel activated by extracellular ATP. This shows that sensory neurons have the machinery to form purinergic synapses on each other and that they do so when placed in short-term tissue culture.

ATP augments peptide release from rat sensory neurons in culture through activation of P2Y receptors.

ATP has recently emerged as an important proinflammatory mediator that has direct excitatory actions on sensory neurons through activation of ion channel-coupled P2X receptors. The purpose of the current work is to assess whether ATP alters the release of neuropeptides from sensory neurons and the receptors mediating this putative action. Exposing embryonic sensory neurons in culture to concentrations of ATP up to 300 microm did not increase the release of immunoreactive substance P or calcitonin gene-related peptide from sensory neurons. However, pre-exposing sensory neurons to 0.1 to 100 microm ATP prior to and throughout administration of 30 nM capsaicin resulted in a significant augmentation of release evoked by the vanilloid. This sensitizing action of ATP is blocked by suramin but not pyridoxal phosphate-6-azobenzene-2,4-disulfonic acid and is mimicked by the P2Y receptor agonists, 2-2-chloroadenosine triphosphate and UTP, but not by 2-(methylthio)adenosine 5'-triphosphate or alpha,beta-methyleneadenosine 5'-diphosphate. This profile of drug actions suggests that the sensitizing actions of ATP are mediated by P2Y receptors. Pretreating sensory neurons with bisindolylmaleimide I, a selective protein kinase C (PKC) inhibitor, attenuates the augmentation of capsaicin-induced peptide release by ATP, further implicating P2Y receptors in the actions of ATP. Immunoblotting also indicates the presence of P2Y2-like immunoreactive substance in embryonic dorsal root ganglia neurons. Together, these data support the notion that ATP acts at P2Y receptors in sensory neurons in a PKC-dependent manner to augment their sensitivity to other stimuli.

Calcium-activated potassium channel SK1- and IK1-like immunoreactivity in injured human sensory neurones and its regulation by neurotrophic factors.

Calcium-activated potassium ion channels SK and IK (small and intermediate conductance, respectively) may be important in the pathophysiology of pain following nerve injury, as SK channels are known to impose a period of reduced excitability after each action potential by afterhyperpolarization. We studied the presence and changes of human SK1 (hSK1)- and hIK1-like immunoreactivity in control and injured human dorsal root ganglia (DRG) and peripheral nerves and their regulation by key neurotrophic factors in cultured rat sensory neurones. Using specific antibodies, hSK-1 and hIK-1-like immunoreactivity was detected in a majority of large and small/medium-sized cell bodies of human DRG. hSK1 immunoreactivity was decreased significantly in cell bodies of avulsed human DRG (n = 8, surgery delay 8 h to 12 months). There was a decrease in hIK1-like immunoreactivity predominantly in large cells acutely (<3 weeks after injury), but also in small/medium cells of chronic cases. Twenty-three injured peripheral nerves were studied (surgery delay 8 h to 12 months); in five of these, hIK1-like immunoreactivity was detected proximally but not distally to injury, whereas neurofilament staining confirmed the presence of nerve fibres in both regions. These five nerves, unlike the others, had all undergone Wallerian degeneration previously and the loss of hIK1-like immunoreactivity may therefore reflect reduced axonal transport of this ion channel across the injury site in regenerated fibres, as well as decreased expression in the cell body. In vitro studies of neonatal rat DRG neurones showed that nerve growth factor (NGF) significantly increased the percentage of hSK1-positive cells, whereas neurotrophin 3 (NT-3) and glial cell line-derived neurotrophic factor (GDNF) failed to show a significant effect. NT-3 stimulated hIK1 expression, while NGF and GDNF were ineffective. As expected, NGF increased expression of the voltage-gated sodium channel SNS1/PN3 in this system. Decreased retrograde transport of these neurotrophic factors in injured sensory neurones may thus reduce expression of these ion channels and increase excitability. Blockade of IK1-like and other potassium channels by aminopyridines (4-AP and 3,4-DAP) may also explain the paraesthesiae induced by these medications. Selective potassium channel openers are likely to represent novel therapies for pain following nerve injury.

The inhibitory effects of dantrolene on action potential-induced calcium transients in cultured rat dorsal root ganglion neurons

We investigated the actions of dantrolene Ca2+-induced on Ca2+-release (CICR) evoked by action potentials in cultured rat sensory neurons. The effect of dantrolene on action potential after-depolarization and voltage-activated calcium currents was studied in cultured neonatal rat dorsal root ganglion cells (DRG) using the whole-cell patch-clamp technique. Depolarizing current injection evoked action potentials and depolarizing after-potentials, which are activated as a result of CICR following a single action potential in some cells. The type of after-potentials was determined by inducing action potentials from the resting membrane potential. Extracellular application of dantrolene (10 mM) abolished after-depolarizations without affecting action potential properties. Furthermore, dantrolene significantly reduced repetitive action potentials after depolarizing current injection into these neurons, but had no significant effect on the steady-state current voltage relationship of calcium currents in these neurons. We conclude that dantrolene inhibits the induction of action potential after depolarizations by inhibiting CICR in cultured rat sensory neurons.

Mechanical stimuli induce intracellular calcium response in a subpopulation of cultured rat sensory neurons

Cultured dorsal root ganglion neurons from newborn rats were mechanically deformed with a fine-tipped glass capillary, and the change in the intracellular Ca 2+ concentration ([Ca 2] i) was recorded by Fura-2-based microfluorimetry. The deformation evoked elevation in [Ca 2+] i from 18.7±5.4 nM (mean±S.E.M., n=35) to 137.1±15.2 nM in some subpopulations of cells, especially those larger than 20 μm in diameter. The largest mechanosensitive cell group was that of cells 20–25 μm in diameter; 56% of the mechanosensitive cells were of this cell size. All of the cells larger than 25 μm in diameter displayed the Ca 2+ increase when prodded. The depletion of extracellular Ca 2+ diminished the Ca 2+ elevation. Verapamil and nickel, blockers of voltage-dependent Ca 2+ channels, did not influence the Ca 2+ response, whereas gadolinium, a relatively selective blocker of mechanosensitive channels, diminished the response. Na +-free conditions did not influence the response. We concluded that the mechanical stimulation induced a Ca 2+ influx in large dorsal root ganglion neurons through mechanosensitive Ca 2+-permeable channels.

Extracellular or intracellular application of argiotoxin-636 has inhibitory actions on membrane excitability and voltage-activated currents in cultured rat sensory neurones

The whole cell variant of the patch clamp technique was used to investigate the actions of the polyamine amide spider toxin, argiotoxin-636, on the excitability of cultured dorsal root ganglion neurones. Synthesized argiotoxin-636 (0.1–100 μM) reduced neuronal excitability when applied to the extracellular environment by low pressure ejection or to the intracellular environment via the patch pipette solution. The toxin prolonged the duration of evoked action potentials and reduced the peak amplitude of action potentials. Intracellular and extracellular application of argiotoxin-636 also decreased the number of action potentials evoked in response to 800-ms depolarizing current commands. This action of the toxin was mimicked by 100 μM tetraethylammonium. Extracellular application of argiotoxin-636 inhibited voltage-activated K + currents in a dose-dependent manner over the complete voltage range. This inhibition occurred without any significant changes in the voltage dependence of activation or inactivation. Intracellular application of argiotoxin-636, during 5–10 min of whole cell recording, also inhibited voltage-activated K + currents without changing the voltage dependence of activation or steady-state inactivation. Extracellular or intracellular spermidine (250 μM) reversibly attenuated the inhibitory actions of extracellular argiotoxin-636. Argiotoxin-636 also inhibited voltage-activated Na + currents; this effect was dependent on repeated activation of the currents and the period during which the neurones were in culture. We conclude that application of argiotoxin-636 to either the extracellular or intracellular environment reduced excitability of cultured sensory neurones from neonatal rats and that this involved inhibition of both voltage-activated K + and Na + currents. The data suggest that the toxin was more effective at attenuating action potentials when neurones were repeatedly excited, and that access to inhibitory sites of action on the voltage-activated ion channels can be achieved from the inside of the neurone.

Accelerated Formation of Nϵ-(carboxymethyl) Lysine, an Advanced Glycation End Product, by Glyoxal and 3-Deoxyglucosone in Cultured Rat Sensory Neurons

The formation of advanced glycation end products (AGEs) is associated with pathophysiological changes with aging and disease processes. In the neurodegeneration in Alzheimer's disease and other neurodegenerative diseases, AGEs are speculated to play a role in their pathogenesis. We provide the first evidence for the induction of AGEs in cultured neuronal cells. Glyoxal and 3-deoxyglucosone (3-DG), AGE precursors, induced Nϵ-(carboxymethyl) lysine (CML), a well characterized and major AGE structure, in cultured rat sensory neurons in a time- and dose-dependent manner. CML formation was prevented by addition of aminoguanidine, an inhibitor of AGE formation. This culture system provides a useful model to analyze the role of the glycoxidation reaction in neuronal aging and neurodegenerative disorder.

Cell death of neonatal rat sensory neurons is prevented by culture at clonal density.

We studied the trophic requirements of neonatal rat sensory neurons in single neuron and standard culture (9400 neurons/cm2). In agreement with previous studies, we found that survival of sensory neurons in standard culture is strictly dependent on nerve growth factor (NGF). Contrary to this, however, no difference between NGF-treated and untreated cultures with respect to survival and neurite formation was evident when neurons were plated as single cells. Approximately 70% of singly-seeded sensory neurons survived for 2 days independent of supply with NGF. Survival of large, mid-sized, and small sensory neurons in single neuron culture demonstrated that this phenomenon is not confined to a neuronal subpopulation. We speculate that survival in single neuron culture is due to the absence or inactivation of yet unidentified cell death-inducing factors that in standard culture are overridden or suppressed by NGF.

Cultured rat sensory neurones express functional tachykinin receptor subtypes 1, 2 and 3

The neuropeptide substance P (SP) is known to play a key role in peripheral nociceptive processes. We investigated the in vitro pharmacological characteristics of functional tachykinin receptors expressed in dorsal root ganglia (DRG) sensory neurones by analysing intracellular free calcium concentration changes induced after stimulation by SP or specific tachykinin agonists. We observed that about 37% of the tested neurones were responsive to either SP or an NK1-, NK2- or NK3-specific agonist. Tachykinin-responsive neurones had a small soma diameter (<20 μm) and were sensitive to capsaicin. These results suggest the presence of NK1, NK2 and NK3 receptors in noxious sensory neurones.

Regulation of opioid receptors in rat sensory neurons in culture.

To determine whether opioid receptors in sensory neurons are regulated by chronic exposure to opioids, we assessed the binding of various opioid ligands to membranes derived from isolated rat dorsal root ganglia neurons grown in culture. Equilibrium binding of [3H]diprenorphine onto membranes from cells grown for 13-15 days revealed a saturable binding site with a Kd value of 0.3 +/- 0.2 nM and an approximate Bmax value of 1300 +/- 200 fmol/mg of protein. [3H]Diprenorphine binding increased 3-fold from 1-15 days in culture. The mu receptors represent approximately 70 +/- 11% of the [3H]diprenorphine binding sites, as indicated by saturation binding of [3H]DAMGO. The kappa and delta receptors represent approximately 10 +/- 3% and approximately 5 +/- 2% of the [3H]diprenorphine binding sites, respectively. Preexposure of neurons to 10 microM naloxone for 48 hr up-regulated the receptors by 40%, whereas incubation with 100 nM to 10 microM DAMGO for 48 hr resulted in a significant decrease in the Bmax value of opioid receptors, with a maximum reduction of 70%. The identification of a high level of opioid receptors expressed in isolated sensory neurons and their modulation by opioids demonstrates that cultured sensory neurons are an excellent model with which to study opioid receptor regulation.

The Japanese herbal medicine 'Saiko-Keishi-To' activates GABAA receptors of rat sensory neurons in culture

The Japanese herbal medicine 'Saiko-Keishi-To' activates GABAA receptors of rat sensory neurons in culture

The Japanese herbal medicine ‘Saiko-Keishi-To’ activates GABA A receptors of rat sensory neurons in culture

The mechanism underlying the anticonvulsant action of ‘Saiko-Keishi-To’ (SK), the Japanese herbal medicine, was examined using whole-cell patch-clamp recording from cultured rat dorsal root ganglion cells. Neurons were held at −60 mV and perfused with an internal solution containing a high concentration of Cl −. Under these circumstances, SK produced an inward current which reversed at +8 mV, and was identical to the γ-aminobutyric acid (GABA)-induced chloride current (I Cl). The SK-induced current was completely blocked by bicuculline. SK depressed a high voltage-activated calcium current (HVA- I Ca), but this depression could not be antagonized by either phaclofen or saclofen. Of nine crude herbal drugs constituting SK, only Bupleuri radix, Ginseng radix, Zingiberis rhizoma and Paeoniae radix produced the inward currents blocked by bicuculline. These results suggest that SK, and four of its crude herbal drugs, activate the I Cl mediated by the GABA A receptor. In addition, SK depresses a HVA- I Ca through some mechanism other than an activation of GABA B receptors.

Activation of protein kinase C augments peptide release from rat sensory neurons.

To determine whether protein kinase C (PKC) mediates release of peptides from sensory neurons, we examined the effects of altering PKC activity on resting and evoked release of substance P (SP) and calcitonin gene-related peptide (CGRP). Exposing rat sensory neurons in culture to 10 or 50 nM phorbol 12,13-dibutyrate (PDBu) significantly increased SP and CGRP release at least 10-fold above resting levels, whereas the inactive 4alpha-PDBu analogue at 100 nM had no effect on release. Furthermore, 100 nM bradykinin increased peptide release approximately fivefold. Down-regulation of PKC significantly attenuated the release of peptides evoked by either PDBu or bradykinin. PDBu at 1 nM or 1-oleoyl-2-acetyl-sn-glycerol at 50 microM did not alter resting release of peptides, but augmented potassium- and capsaicin-stimulated release of both SP and CGRP approximately twofold. This sensitizing action of PKC activators on peptide release was significantly reduced by PKC down-regulation or by pretreating cultures with 10 nM staurosporine. These results establish that activation of PKC is important in the regulation of peptide release from sensory neurons. The PKC-induced enhancement of peptide release may be a mechanism underlying the neuronal sensitization that produces hyperalgesia.

A novel agonist, phorbol 12-phenylacetate 13-acetate 20-homovanillate, abolishes positive cooperativity of binding by the vanilloid receptor

Capsaicin binds to a specific recognition site, referred to as the vanilloid receptor, which it shares with the natural, ultrapotent agonist resiniferatoxin and with the competitive antagonist capsazepine. Upon binding to its receptor, capsaicin opens a cation channel leading to Ca 2+ influx. The binding of capsaicin or resiniferatoxin by the vanilloid receptor follows a sigmoidal saturation curve, indicative of positive cooperativity. The biological significance of this positive cooperative behaviour is unknown, as is the mechanism responsible for it. We have developed a novel ligand, phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV), which binds to cultured rat sensory neurons (with a K i of 3.1 ± 0.4 μM), and induces Ca 2+ uptake by them (with an ED 50 of 1.8 ± 0.3 μM) with similar affinities and in a non-cooperative manner (Hill coefficients are 0.99 and 1.06 for binding and Ca 2+ uptake, respectively). The behaviour of PPAHV thus contrasts with resiniferatoxin or capsaicin not only in the lack of cooperativity but also in the relative potencies for resiniferatoxin binding versus Ca 2+ uptake (resiniferatoxin is less potent and capsaicin is more potent for induction of Ca 2+ uptake than for binding). In further experiments in which the concentration of [ 3H]resiniferatoxin was varied, 1 μM PPAHV likewise reduced the cooperativity index that characterizes resiniferatoxin binding to rat spinal cord membranes from 2.3 ± 0.1 to 1.1 ± 0.2; in parallel experiments, neither capsaicin nor capsazepine (both at a concentration of 2 μM) affected binding cooperativity. Moreover, PPAHV (1 μM) turned the bi-phasic dissociation curve of resiniferatoxin into a monophasic curve, eliminating the second, slow-dissociation phase. The present results suggest that positive cooperativity is a ligand-induced feature rather than an inherent property of vanilloid receptors. A comparison of the spectrum of biological activity of ligands which bind to vanilloid receptors with different degrees of cooperativity may provide an approach to explore the functional significance of this binding behaviour.

Role of a Ca 2+-dependent slow afterhyperpolarization in prostaglandin E 2-induced sensitization of cultured rat sensory neurons

To determine if inhibition of a Ca 2+-dependent slow afterhyperpolarization (AHP slow) contributes to prostaglandin E 2 (PGE 2)induced sensitization of DRG neurons, we have used patch-clamp electrophysiological techniques on cultured dorsal root ganglion (DRG) neurons from the adult rat. In support of a role for AHPslow in sensitization of DRG neurons, we demonstrate that: (1) AHP slow expression is restricted to a subpopulation of putative nociceptors; (2) burst duration is controlled by AHP slow in these neurons; and (3) in some neurons, PGE 2 decreases AHP slow and produces a concomitant increase in the number of action potentials generated in response to depolarizing current injection. However, our results also demonstrate that AHP slow modulation is not sufficient to explain PGE 2-induced sensitization in the majority of DRG neurons because: (1) the size of the population of DRG neurons expressing AHP slow is less than half the size of the population of DRG neurons sensitized by PGE 2; (2) PGE 2 produces a decrease in action potential threshold as well as an increase in the number of action potentials in response to current injection, while inhibition of AHP slow has little effect on threshold; and (3) the sensitizing effects of PGE 2 are dissociated from its effects on AHP slow in more than half of neurons tested. We conclude that PGE 2-induced sensitization must involve the modulation of ionic currents in addition to that underlying AHP slow.

An analysis of an axonal gradient of phosphorylated MAP 1B in cultured rat sensory neurons.

The present study investigated the cellular distribution of a developmentally regulated phosphorylated form of MAP 1B recognized by monoclonal antibody (mAb) 150 in cultures of dorsal root ganglia. The cell soma and the whole axon, when it first appears, are labelled, but longer axons label with a proximodistal gradient, such that the cell soma and proximal axon become unlabelled, whilst the distal axon and growth cone label strongly. Double-labelling experiments with mAb 150 and a polyclonal antibody (N1-15) that recognizes all forms of MAP 1B demonstrated that MAP 1B is distributed along the entire length of axons with gradients, so the gradient of phosphorylated MAP 1B is not due to a loss or absence of MAP 1B from the proximal axon. The proportion of axons from 20 h cultures that were labelled with a mAb 150 gradient was at least 80% and this proportion was independent of the nerve growth factor concentration of the culture medium. Analysis of axons ranging in length from 100 to 700 microm and labelled with a gradient showed that the unlabelled proximal portions of axons increased in length more slowly than the labelled distal axon. Axons labelled along their entire length accounted for no more than 19% of th axonal population and analysis of these showed them to be frequently < 400 microm long. After simultaneously fixing and detergent-extracting cultures this proportion rose significantly to 93%, suggesting that in the proximal axon the mAb 150 epitope is masked by some factor(s) that is removed by detergent extraction. The possibility that mAb 150 could not access the epitope in the proximal axon was discounted because another IgM, mAb 125, which recognizes a different phosphorylation epitope on MAP 1B, labelled the proximal axon of conventionally fixed cultures. In growth cones of fixed and extracted neurons examined by immunofluorescence, the mAb 150 labelling strongly colocalized to bundled microtubules in the distal axon shaft and the C-domain. In the P-domain, mAb 150 staining was weaker and more widely distributed than the microtubules. Immunogold electron microscopy confirmed that antibody N1-15 and mAb 150 strongly labelled the bundled microtubules in the C-domain and also showed that individual microtubules in the P-domain, some of which lie alongside actin filament bundles of filopodia, were labelled lightly and discontinuously with both antibodies. This suggests that the phosphorylated isoform of MAP 1B recognized by mAb 150 may be microtubules and actin filaments in the P-domain.

The Local Anesthetic n-Butyl-p-Aminobenzoate Selectively Affects Inactivation of Fast Sodium Currents in Cultured Rat Sensory Neurons

Aqueous suspensions of the local anesthetic n-butyl-p-aminobenzoate (BAB), epidurally applied in terminal cancer patients, resulted in a sensory blockade, lasting up to several months. To investigate the mechanism of action on the cellular level, the effect of 100 microM BAB on Na+ action potentials and on Na+ currents in dorsal root ganglion neurons from neonatal rats was studied. Small neurons grown in cell culture were selected for patch-clamp measurements. Both Na+ action potentials, evoked by current pulses of increasing amplitude (current clamp) and Na+ currents, activated at different membrane potentials (voltage clamp), were investigated in the absence and presence of 100 microM BAB. The local anesthetic was applied by external perfusion for 2 or 10 min. In the presence of 100 microM BAB, either the firing threshold was raised or the action potential was abolished. The maximal peak conductances, underlying the fast sodium current INa,F and the slow sodium current INa,5, were not changed. However, the inactivation of INa,F was increased by BAB. The sigmoid inactivation curve shifted 12 mV toward hyperpolarizing membrane voltages, whereas no changes were found for the inactivation of the slow Na+ current. Only at short exposure times of 2 min, the effects of BAB could be reversed during a 10-min wash-out. BAB dramatically increased the firing threshold, and in part of the sensory neurons, it blocked the action potential. The inactivation of the fast Na+ channels, but not of the slow Na+ channels, was increased by BAB. Thus, the block of fast Na+ channels by BAB may contribute to epidural analgesia. At exposure times of 10 min, the effect of BAB was not reversible. This probably originates from its high lipid-solubility, which may be an important factor in determining the duration of the block in vivo.

Noradrenaline effects on cultured rat sensory neurones exposed to morphine in utero or in vitro

Dorsal root ganglion (DRG) neurones cultured from newborn rats were chronically exposed to 1 μM morphine. Untreated cells and in vitro morphine treated cells were cultured for 1 or 2 weeks from control offspring while in utero morphine treated cells were cultured for 1 week from offspring of morphine dependent rats. Noradrenaline (1 μM was applied by pressure ejection. Intracellular recordings measured the change in action potential duration (APD). Noradrenaline significantly decreased the APD of 1-week-old untreated cells while the 2-week-old untreated cells and the groups of morphine treated cells showed no significant change of APD following the drug application. Thus, newborn rat DRG neurones demonstrated a different sensitivity to noradrenaline with the culture duration or with morphine exposure.

Activation of Ca(2+)‐dependent Cl‐ currents in cultured rat sensory neurones by flash photolysis of DM‐nitrophen.

1. Voltage-gated Ca2+ currents (ICa) and Ca(2+)-activated Cl- currents (ICl(Ca)) were recorded from cultured rat dorsal root ganglion (DRG) neurones using the whole-cell configuration of the patch clamp technique. Intracellular photorelease of Ca2+ by flash photolysis of DM-nitrophen elicited transient inward currents only in those cells which possessed Ca(2+)-activated Cl- tail currents following ICa. The reversal potential of the flash responses was hyperpolarized when extracellular Cl- was replaced by SCN-. The flash responses and the Ca(2+)-activated Cl- tail currents were inhibited by the Cl- channel blockers niflumic acid (10-100 microM) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (10 microM). 2. After activation by ICa, the Ca(2+)-activated Cl- current could be reactivated during its decay by photorelease of caged Ca2+. Experiments carried out on neurones held at 0 mV demonstrated that ICl(Ca) could be chronically activated due to residual Ca2+ influx. These data directly demonstrated that the decay of ICl(Ca) is not due to inactivation but rather to deactivation as a result of removal of the Ca2+ load from the cell cytoplasm. 3. Photorelease of caged inositol 1,4,5-trisphosphate (IP3) failed to activate any Ca(2+)-dependent current responses in cultured DRG neurones, although application of caffeine elicited transient inward currents, and responses to photoreleased IP3 could be obtained from freshly dissociated smooth muscle cells. 4. Photorelease of Ca2+ provides a useful method for investigating the properties of ICl(Ca) independently from other physiological parameters. In addition, we have directly demonstrated that ICl(Ca) in DRG neurones does not inactivate, and so may continue to modulate membrane excitability as long as the intracellular Ca2+ concentration ([Ca2+]i) close to the cell membrane is elevated.(ABSTRACT TRUNCATED AT 250 WORDS)