Small interfering RNA-mediated selective knockdown of Na(V)1.8 tetrodotoxin-resistant sodium channel reverses mechanical allodynia in neuropathic rats.

The biophysical properties of a tetrodotoxin resistant (TTXr) sodium channel, Na(V)1.8, and its restricted expression to the peripheral sensory neurons suggest that blocking this channel might have therapeutic potential in various pain states and may offer improved tolerability compared with existing sodium channel blockers. However, the role of Na(V)1.8 in nociception cannot be tested using a traditional pharmacological approach with small molecules because currently available sodium channel blockers do not distinguish between sodium channel subtypes. We sought to determine whether small interfering RNAs (siRNAs) might be capable of achieving the desired selectivity. Using Northern blot analysis and membrane potential measurement, several siRNAs were identified that were capable of a highly-selective attenuation of Na(V)1.8 message as well as functional expression in clonal ND7/23 cells which were stably transfected with the rat Na(V)1.8 gene. Functional knockdown of the channel was confirmed using whole-cell voltage-clamp electrophysiology. One of the siRNA probes showing a robust knockdown of Na(V)1.8 current was evaluated for in vivo efficacy in reversing an established tactile allodynia in the rat chronic constriction nerve-injury (CCI) model. The siRNA, which was delivered to lumbar dorsal root ganglia (DRG) via an indwelling epidural cannula, caused a significant reduction of Na(V)1.8 mRNA expression in lumbar 4 and 5 (L4-L5) DRG neurons and consequently reversed mechanical allodynia in CCI rats. We conclude that silencing of Na(V)1.8 channel using a siRNA approach is capable of producing pain relief in the CCI model and further support a role for Na(V)1.8 in pathological sensory dysfunction.

Voltage-gated sodium channels and pain.

Voltage-gated sodium channels are highly specialized molecular transducers that play a significant role in the creation and transmission of electrical activity throughout the neuraxis. These ion channels are fundamentally involved in sensory neuron physiology and pathophysiology; a complete but localized suspension of their normal function can prevent all sensation--including that perceived as pain. Soft-tissue injuries that result in inflammation or direct damage to nerve fibers have each been shown to result in abnormal sodium channel function and, in many cases, to lead to pathological hyperexcitability in the sensory afferent nerves that innervate the injured dermatome or visceral organ. Abrogating abnormal activity whilst leaving normal sensation unaffected would represent a powerful approach to pain relief. This article reviews the evidence supporting abnormal sodium channel biology in various pathological contexts, the opportunities that this presents for novel therapeutics and progress towards realizing this goal.

The effect of baclofen and somatostatin on neuronal activity in the rat ventromedial hypothalamic nucleus in vitro.

The electrical properties of neurones within the ventromedial hypothalamic nucleus of the rat were studied in an in vitro slice preparation, using conventional intracellular recording techniques. A detailed analysis of 36 intracellular recordings appeared to suggest 3 cell types, based on membrane capacitance and resistance characteristics, confirming previous reports of a diversity of cell types within this nucleus. The responsiveness of each cell type to exogenously-applied baclofen and somatostatin was also investigated. The inhibitory responses to both of these drugs were concentration-related (over the range 100 nM to 1 microM), tetrodotoxin-resistant and consisted of a membrane hyperpolarization (mean +/- SEM = 6.7 +/- 1 and 10.7 +/- 1 mV for 1 microM somatostatin and baclofen, respectively) and an associated reduction in the firing frequency of spontaneously active cells. These agonist-evoked responses probably represented direct postsynaptic actions but they were not restricted to any single type of cell. Evidence for an additional presynaptic effect of baclofen was also obtained. Responses to baclofen were extremely robust and readily quantifiable, whereas those to somatostatin showed pronounced long-lasting desensitization, which was particularly marked a larger concentrations. These data support previous contentions, based on in vivo studies, that somatostatin and GABA are likely to participate in the control of complex functions by the ventromedial hypothalamic nucleus.

In vivo evidence for the selectivity of ICI 154129 for the delta-opioid receptor.

The in vivo selectivity of the novel delta opioid-receptor antagonist N,N-bisallyl-Tyr-Gly-Gly-psi-(CH2S)-Phe-Leu-OH (ICI 154129) was examined in several opioid-selective models. Antagonism at the delta receptor was demonstrated in the striatal head-turn model in the rat. Intrapallidal injection of the relatively selective delta-receptor agonist D-Ala2,D-Leu5-enkephalin (0.5 micrograms) slowed the head-turn time and this effect was completely prevented by prior subcutaneous administration of ICI 154129 (30 mg/kg). The role of delta receptors in two classical test situations was studied using the mixed opioid agonist etorphine and the antagonists naloxone and ICI 154129. The drug ICI 154129 (30 mg/kg, s.c.) failed to prevent the antinociceptive effects and stimulation of locomotor activity produced by etorphine, whereas the relatively selective mu-opioid receptor antagonist, naloxone was effective in both test situations. The possible involvement of delta receptors in morphine-induced dependence was studied by monitoring the abstinence behaviour precipitated in rats given pellets of morphine by either ICI 154129 or naloxone. Naloxone (0.5 mg/kg, i.p.) precipitated a characteristic withdrawal syndrome in conscious rats and, at a much smaller dose (0.02 mg/kg, i.p.), induced shaking behaviour in pentobarbitone-anaesthetised rats. No withdrawal signs were observed in either model after injection of ICI 154129 (30 mg/kg, s.c.), suggesting that the delta receptors are not involved in dependence on morphine.

Electrophysiological characterisation of the antagonist properties of two novel NMDA receptor glycine site antagonists, L-695,902 and L-701,324.

The pharmacological effects of two novel N-methyl-D-aspartate (NMDA) receptor glycine site antagonists, L-701,324 and L-695,902 were examined on whole-cell voltage-clamped cells and compared to a prototypic antagonist, 7-chlorokynurenic acid. Both L-701,324 and L-695,902 non-competitively antagonised NMDA responses elicited in rat cultured cortical neurones, this was shown to be due to a competitive interaction at the glycine co-agonist site on the receptor complex (Kb values: 19 nM and 2.6 microM, respectively). Inhibition curves for the antagonism of responses to combined applications of NMDA and glycine showed that both antagonists were devoid of any intrinsic activity i.e. "full" antagonists and were, therefore, capable of completely abolishing inward currents. Despite this fact, both of these novel antagonists apparently modulated glutamate affinity for its recognition site-a property hitherto associated only with glycine site partial agonists. Human recombinant NMDA receptors comprising NR1a/NR2A and NR1a/NR2B subunits expressed in mouse fibroblast cells were also used to determine whether L-701,324 and L-695,902 were capable of discriminating between subtypes of NMDA receptor. Inhibition curves to each antagonist showed no difference in affinity for either of these subunit assemblies (mK1 values L-701,324 = 0.005 microM on both assemblies; L-695,902 = 4.37 and 3.7 microM on NR1a/NR2A and NR1a/NR2B, respectively). Kinetic analysis of the off-rates of antagonism with L-701,324 revealed that the high affinity of this compound compared to 7-chlorokynurenic acid were attributable to an exceptionally slow dissociation of the antagonist from the receptor.

In vitro propagation and inducible differentiation of multipotential progenitor cells from human fetal brain.

Central nervous system neurons and glia arise from undifferentiated embryonic neuroepithelial cells. Such progenitor cells from the human fetal forebrain can be propagated in vitro for extended periods, when grown on non-adhesive substrates in medium containing epidermal growth factor and insulin-like growth factor-1. These actively-dividing cells can be induced to differentiate into a variety of histochemically-characterized neurons and glia consistent with their forebrain origin. Electrophysiological recording indicates that differentiated neurons derived from these progenitors mature slowly, and display a range of glutamate- and GABA-mediated conductances characteristic of normal mammalian forebrain neurons. Our observations support a role for these trophic factors in normal development of the human brain. The methods described here may provide abundant normal, untransformed human forebrain neurons and glia for research and therapeutic applications.

Pharmacological differences between two muscarinic responses of the rat superior cervical ganglion in vitro.

1 Pharmacological differences have been observed between the muscarinic agonist-induced depolarizing and hyperpolarizing responses of the rat isolated superior cervical ganglion. 2 Pirenzepine (0.3 microM) selectively reduced the depolarizing response and unmasked the hyperpolarizing response. No such selectivity was observed with a concentration of N-methylatropine which was equipotent with pirenzepine in antagonizing the depolarizing response. 3 The neuromuscular blocking agents gallamine (10 microM) and pancuronium (3 microM) exhibited the oppositive selectivity to pirenzepine, both dramatically reduced the hyperpolarization but only slightly antagonized the depolarization. 4 The potencies of a range of agonists in evoking the depolarizing and hyperpolarizing responses, the latter in the presence of 0.3 microM pirenzepine, have been determined. Methylfurmethide failed to hyperpolarize the ganglion at concentrations which evoked maximal depolarizations. 5 The muscarinic hyperpolarization did not appear to be mediated by the secondary release of catecholamines. 6 It was concluded that the two muscarinic responses on the rat superior cervical ganglion, the slow depolarization and faster hyperpolarization, are mediated by different muscarinic receptor subtypes.

The inhibitory effect of somatostatin peptides on the rat anococcygeus muscle in vitro.

1. Electrically evoked contractions of the rat anococcygeus muscle were inhibited in a concentration-dependent manner by somatostatin-14 (SS14), -28 (SS28) and two synthetic hexapeptide analogues: L-363,301 (Pro-Phe-D-Trp-Lys-Thr-Phe) and L-363,586 (N-Me-Ala-Tyr-D-Trp-Lys-Val-Phe), with pIC50 values of 7.41, 7.38, 7.07 and 8.34, respectively. 2. The inhibitory effects of SS14 were dependent on stimulation frequency and external calcium ion concentration. Calcium behaved as a non-competitive antagonist of SS14, it reduced the maximal inhibitory effect of the peptide and at a concentration of 5.08 mM it significantly affected the pIC50 value. 3. SS14 (3 x 10(-7) M) did not affect the tonic actions of bath-applied noradrenaline in the absence of field stimulation. 4. The effects of SS14 persisted in naloxone (10(-5) M) and were, therefore, not due to an action at opiate receptors. Furthermore, experiments involving the lyophilization of bath contents, showed no evidence to support an indirect mechanism involving the release of an endogenous inhibitory substance. 5. High concentrations (10(-5) M) of SS14 or L-363,301 inhibited the relaxation response evoked by electrical stimulation of guanethidine (3 x 10(-4) M)-treated preparations. 6. These results are consistent with similar actions of SS14 on other smooth muscle preparations and are presumed to reflect a presynaptic inhibition of transmitter release by a direct action on somatostatin receptors. The antagonistic effect of calcium on this response is discussed with reference to a possible role in receptor desensitization.

A 5-HT1-like receptor mediates a pertussis toxin-sensitive inhibition of rat ventromedial hypothalamic neurones in vitro.

5-Hydroxytryptamine (5-HT), 5-carboxamidotryptamine and 8-hydroxy-2-(di-n-propylamino)-tetralin inhibited rat ventromedial hypothalamic neurones in vitro in a concentration-dependent manner. The agonist-induced inhibition was reduced by spiperone (1 microM) and by pertussis toxin , but not by MDL 72222 (10 microM) or ketanserin (1 microM). The inhibition appeared to be mediated via 5-HT1A-receptors and a pertussis toxin-sensitive pathway.

Antagonism of responses to excitatory amino acids on rat cortical neurones by the spider toxin, argiotoxin636.

1. Several low molecular weight spider toxins have recently been shown to block potently glutamatergic neuromuscular transmission at the invertebrate neuromuscular junction. The aim of the present investigation was to evaluate the effects of one such toxin, argiotoxin636, on excitatory amino acid receptor-mediated responses in mammalian neurones. 2. Membrane currents were recorded from rat cortical neurones after 2-6 weeks in cell culture, by the whole-cell variant of the patch-clamp technique. N-methyl-D-aspartate (NMDA) and kainate were used as selective agonists for their respective receptor subtypes. alpha-Amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) was used as a selective agonist for the quisqualate receptor subtype. 3. Responses to these agonists were characterised with respect to their concentration and voltage-dependence. Argiotoxin636 (3-30 microM) was found to attenuate markedly responses to NMDA in an agonist- and voltage-dependent manner. Thus, argiotoxin636 progressively reduced successive responses to NMDA when membrane potentials were voltage clamped between -40mV to -100 mV. The more negative the membrane potential the more rapid the development of the block of inward current. 4. The antagonism of NMDA-induced currents by argiotoxin636 could be reversed by clamping the membrane at positive potentials (+20 to +60 mV) and reapplying NMDA. 5. Responses to AMPA and kainate were less affected by argiotoxin636, with an antagonist action only becoming evident at a concentration of 100 microM. 6. These results suggest that argiotoxin636 is an open-channel blocker of the NMDA activated ion-channel in mammalian neurones. Furthermore, our results indicate at least a 30 fold selectivity for NMDA over the quisqualate- and kainate-activated ion-channels.

L-687,414, a low efficacy NMDA receptor glycine site partial agonist in vitro, does not prevent hippocampal LTP in vivo at plasma levels known to be neuroprotective.

N-methyl-D-aspartic acid (NMDA) receptors are known to play a key role in the induction phase of long-term potentiation (LTP) at certain hippocampal synapses and to represent some component of spatial learning in animals. The ability of NMDA receptor antagonists (or gene knockout) to impair LTP has led to the suggestion that the therapeutic use of such antagonists may impair cognitive function in humans. The present study compares the effects on LTP of NMDA receptor ion channel block by MK-801 and glycine-site antagonism by 3R(+)cis-4-methyl-pyrrollid-2-one (L-687,414). In vitro experiments using rat cortical slices revealed L-687,414 to be approximately 3.6 fold more potent than its parent analogue, R(+)HA-966 at antagonizing NMDA-evoked population depolarizations (apparent Kbs: 15 microM and 55 microM, respectively). Whole-cell voltage-clamp experiments using rat cultured cortical neurones revealed L-687,414 to shift to the right the concentration-response relationship for NMDA-evoked inward current responses (pKb=6.2+/-0.12). L-687,414 affinity for the glycine site on the NMDA receptor complex was also determined from concentration-inhibition curves, pKi=6.1+/-0.09. In the latter experiments, L-687,414 and R(+)HA-966 were unable to completely abolish inward current responses suggesting each compound to be a low efficacy partial agonist (estimated intrinsic activity = approximately 10 and 20% of glycine, respectively). L-687,414 and MK-801 were compared for their effects on NMDA receptor-dependent LTP in the dentate gyrus of anaethestized rats following high frequency stimulation of the medial perforant path (mPP) afferents. Control rats, administered saline (0.4 ml kg(-1) followed by 0.0298 ml min(-1)), showed a robust augmentation of the population e.p.s.p. risetime (LTP) recorded in the dentate hilus following tetanic stimulation of the mPP. LTP was effectively abolished in a separate group of rats treated with an MK-801 dosing regimen (0.12 mg kg(-1) i.v. followed by 1.8 microg kg(-1) h(-1)) known to produce maximal neuroprotection in a rat stroke model but, by contrast, remained largely intact in a third group of animals given a similarly neuroprotective L-687,414 treatment (28 mg kg(-1) i.v. followed by 28 mg kg(-1) h(-1)). These experiments suggest that a low level of intrinsic activity at the glycine site may be sufficient to support NMDA receptor-dependent LTP but in circumstances where there is likely to be an excessive NMDA receptor activation the agonism associated with a low efficacy partial agonist, such as L-687,414, is dominated by the antagonist properties. Thus, an NMDA receptor partial agonist profile may offer a therapeutic advantage over neutral antagonists by permitting an acceptable level of 'normal' synaptic transmission whilst curtailing excessive receptor activation.

Time-dependent changes in NMDA receptor expression in neurones cultured from rat brain.

Several studies have shown marked changes in the regional expression pattern of N-methyl-D-aspartate (NMDA) receptor subunit composition in vivo in the developing brain. Similar developmental changes may also occur in vitro. Thus, displacement of [3H]MK-801 binding by the subunit-selective NMDA antagonist, ifenprodil, in membrane homogenates prepared from cultured neurones, has been shown to comprise two components, the relative proportion of which changed with time in culture [11]. In the present experiments we have used electrophysiological methods to determine the influence of time in culture on NMDA receptor subtype expression pattern in individual neurones. Shortly after plating (13 days in vitro (DIV)), approximately 70% of total NMDA-induced inward current in voltage-clamped rat cortical neurones is antagonised by ifenprodil with relatively high affinity (approximately 1 microM). By 65 DIV, however, the high-affinity component contributed to only approximately 20% of the overall antagonism. Cerebellar granule cells also appear to undergo a similar change in their NMDA receptor expression with the exception that, in general, they appear initially to show considerably less of the ifenprodil high-affinity component and this disappears completely by 15 DIV. These experiments suggest that individual cortical and granule cells express at least two different NMDA receptor subtypes and that their relative proportion changes with time in culture.

Subtypes of NMDA receptor in neurones cultured from rat brain.

The non-competitive N-methyl-D-aspartate (NMDA) antagonist, ifenprodil, discriminates two receptor populations, each of which shows a reciprocal abundance in cultured cortical and cerebellar granule cells. Thus approximately 70% of NMDA-gated membrane current was antagonized with high affinity (IC50 = 1.4 +/- 0.9 microM) in cortical neurones whereas only approximately 20% was antagonized with high affinity (IC50 = 1.3 +/- 0.3 microM) in granule cells. Inhibition curves for CGS 19755 appeared relatively monophasic: this competitive NMDA antagonist had a significantly higher affinity for the granule cell receptor (Ki = 0.8 +/- 0.2 microM) compared with that on cortical neurones (Ki = 2 +/- 0.6 microM). The data suggest that these two antagonists may be of value in identifying the expression of subpopulations of native NMDA receptors in other brain regions.

The effects of insulin-like growth factor analogues on survival of cultured cerebral cortex and cerebellar granule neurones.

Insulin and insulin-like growth factors (IGF-I, IGF-II) are closely related polypeptides which are found in the CNS and which promote neuronal survival and neurite outgrowth. They are each associated with specific cell surface receptors and several soluble binding proteins (IGFBPs) which are involved in regulating function and availability. Two analogues of IGF-I were produced by site directed mutagenesis: (Gln3, Ala4, Tyr15, (Leu16)IGF-1 (QAYL-IGF) and a B-chain mutant in which the first 16 amino acids of IGF-1 were replaced by the first 17 amino acids of insulin. These analogues have significantly reduced binding affinity for IGFBPs. Using glucose deprivation as a damaging stimulus and assaying lactate dehydrogenase released from cultures as a marker for cell death, we have investigated the effect of IGF analogues on cell death of cerebrocortical and cerebellar granule cell cultures. In the presence of IGF-I, QAYL-IGF or B-chain mutant, the amount of LDH released from cortical and cerebellar granule cell cultures was significantly reduced compared to control (no glucose), indicating that these molecules promote survival. Both QAYL and B-chain mutants, which have reduced affinity for IGFBPs, are as effective as IGF-I in promoting cell survival in conditions of glucose deprivation and their reduced affinity for IGFBPs has no apparent deleterious effect on their neuroprotective function. We also show that the neuroprotective effect of the IGF analogues is due to a direct effect on the neurones in these cultures and is independent of the presence of glia.

The effect of NMDA receptor glycine site antagonists on hypoxia-induced neurodegeneration of rat cortical cell cultures.

The neuroprotective potential of an antagonist (7-chlorokynurenic acid (7-CIKYNA)) and a low efficacy partial agonist (HA-966) for the glycine modulatory site on the N-methyl-D-aspartate (NMDA) receptor complex has been examined using a neuronal cell culture/hypoxia model of neurodegeneration. Their effects were compared to those of the potent uncompetitive NMDA antagonist, MK-801. Hypoxic cell injury was assessed visually and quantified by measuring the appearance of two cytosolic enzymes, lactate dehydrogenase (LDH) and neurone specific enolase (NSE), in the culture medium. MK-801 prevented the hypoxia-induced cell mortality in a concentration-related manner with an IC50 of 15 nM against increases in LDH levels. HA-966 and 7-CIKYNA also produced concentration-related protective effects with IC50s of 175 and 18 microM, respectively. Although both glycine antagonists were considerably weaker than MK-801 their maximum neuroprotective effects were comparable to that produced by MK-801, i.e. complete protection. This indicates that the level of NMDA receptor activation which can take place in the presence of the partial agonist HA-966 is insufficient to cause permanent neuronal damage. Concentration-effect curves were similar when NSE was used as the marker enzyme, supporting previous observations that the increases in LDH levels accurately and specifically reflect neuronal cell death. These results provide further evidence that hypoxia-induced injury to cortical neuronal cultures is mediated by an excessive stimulation of NMDA receptors and that glycine-site antagonists and partial agonists may have therapeutic potential in conditions where pathologically high levels of NMDA receptor activation are thought to occur.

Kinetic study of the interactions between the glutamate and glycine recognition sites on the N-methyl-D-aspartic acid receptor complex.

The N-methyl-D-aspartate (NMDA) receptor is unique among the ligand-gated ion channels, in that the gating process requires the binding of two independent coagonists, glutamate and glycine. Receptor binding experiments have suggested that the coagonist recognition sites interact with one another in an allosteric manner, and previous work in this laboratory has provided additional functional support in favor of an allosteric coupling; the affinity of glutamate for its recognition site was reduced when a partial agonist, (+)-HA-966, occupied the glycine site, compared with the affinity when glycine itself was bound to the receptor. The present experiments have taken these observations a step further and compare the effects of several glycine site ligands with different affinities and intrinsic activities (determined from equilibrium concentration-response curves) on glutamate off-rate. Thus, the dissociation rate for the decay of glutamate-activated membrane currents in voltage-clamped rat cortical neurons was fastest (160 +/- 28 msec) in the presence of saturating concentrations of (+)-HA-966 and progressively slower in the presence of D-cycloserine (258 +/- 27 msec), aminocyclopropanecarboxylic acid (330 +/- 21 msec), L-alanine (375 +/- 28 msec), and glycine (502 +/- 42 msec). We have also measured the affinities and intrinsic activities of several NMDA receptor ligands and report that a reciprocal interaction exists, such that the off-rate of glycine is influenced by the properties of the agonist occupying the glutamate coagonist site. Thus, the time constant for current decay after a brief exposure to glycine was fastest in the presence of a saturating concentration of cis-2,3-piperidinedicarboxylic acid (449 +/- 26 msec) and progressively slower in the presence of quinolinate (689 +/- 73 msec), NMDA (721 +/- 36 msec), and L-glutamate (1260 +/- 36 msec). The data suggested that the extent of the modulation of one site by the other is related to the intrinsic activity of the agonist, rather than its affinity. Specifically, we suggest that a partial agonist occupying one of the agonist recognition sites produces a conformational change that results in an accelerated off-rate for coagonist dissociation from the receptor; the lower the intrinsic activity, the greater is the effect on coagonist off-rate.

Effects of (+)-HA-966 and 7-chlorokynurenic acid on the kinetics of N-methyl-D-aspartate receptor agonist responses in rat cultured cortical neurons.

It has been suggested that one of the effects of glycine at the N-methyl-D-aspartate (NMDA) receptor complex is to reduce the amount of apparent receptor desensitization. Thus, blockade with a glycine site antagonist results in NMDA responses that show an increased amount of fade. In agreement with this, we found that antagonism of NMDA-evoked whole-cell currents by 7-chlorokynurenic acid (7-Cl-KYNA) indeed resulted in NMDA responses that displayed an increased amount of fade. However, those responses that were antagonized by (+)-HA-966 showed the opposite, i.e., less tendency to fade. On examination of these responses, it appeared that those produced in the presence of (+)-HA-966 were slower in onset and faster in offset than control responses recorded in the presence of glycine alone. Kinetic analysis of the on- and off-rates of NMDA- and glutamate-evoked NMDA receptor-mediated responses revealed that these were markedly affected by (+)-HA-966 but only slightly by 7-Cl-KYNA. The decrease of the glutamate response decay time constant and the increase of the response rise time constant produced by (+)-HA-966 indicated that it reduced the affinity of glutamate for its recognition site on the NMDA receptor by 5-fold. These results suggest that binding of (+)-HA-966 to the glycine site on the NMDA receptor complex produces an allosteric reduction in the affinity of agonists for the glutamate recognition site, whereas 7-Cl-KYNA has relatively little effect and, thus, acts more as a pure antagonist at the glycine site.