Cellular and molecular action of the putative GABA-mimetic, gabapentin.

Gabapentin was originally designed as an anti-convulsant gamma-aminobutyric acid (GABA) mimetic capable of crossing the blood-brain barrier. In the present review we show that although gabapentin is not a GABA mimetic, it has great utility as an add-on therapy for epilepsy and as a first-line treatment for neuropathic pain. We summarise the studies that have been performed which demonstrate that gabapentin appears to interact with a novel binding site expressed at high density within the central nervous system (CNS), namely the alpha2delta voltage-dependent calcium channel subunit. The review continues by examining the effects of gabapentin on calcium channel function and neurotransmitter release before, in the latter part of the review, summarising the more recently discovered actions of gabapentin in relation to intracellular signalling.

Identification of MEK1 as a novel target for the treatment of neuropathic pain.

(1) In the present study we have attempted to identify changes in gene expression which are associated with neuropathic pain using subtractive suppression hybridization analysis of the lumbar spinal cord of animals suffering streptozocin induced diabetic neuropathy. (2) Using this approach, we found a significant up-regulation of several key components of the extracellular signal-regulated kinase (ERK) cascade. These findings were confirmed by Western blot analysis, which demonstrated that the levels of active ERK1 and 2 correlated with the onset of streptozocin-induced hyperalgesia. (3) Intrathecal administration of the selective MAPK/ERK-kinase (MEK) inhibitor PD 198306 dose-dependently (1-30 micro g) blocked static allodynia in both the streptozocin and the chronic constriction injury (CCI) models of neuropathic pain. (4) The antihyperalgesic effects of PD 198306, in both the streptozocin and CCI models of neuropathic pain, correlated with a reduction in the elevated levels of active ERK1 and 2 in lumbar spinal cord. (5) Intraplantar administration of PD 198306 had no effect in either model of hyperalgesia, indicating that changes in the activation of ERKs and the effect of MEK inhibition are localized to the central nervous system. (6) In summary, we have demonstrated for the first time that the development of neuropathic pain is associated with an increase in the activity of the MAPK/ERK-kinase cascade within the spinal cord and that enzymes in this pathway represent potential targets for the treatment of this condition.

Hydrogen-peroxide-induced toxicity of rat striatal neurones involves activation of a non-selective cation channel.

Striatal neurones are particularly vulnerable to hypoxia/ischaemia-induced damage, and free radicals are thought to be prime mediators of this neuronal destruction. It has been shown that hydrogen peroxide (H2O2), through the production of free radicals, induces rat insulinoma cell death by activation of a non-selective cation channel, which leads to irreversible cell depolarization and unregulated Ca2+ entry into the cell. In the study presented here, we demonstrate that a subpopulation of striatal neurones (medium spiny neurones) is depolarized by H2O2 through the production of free radicals. Cell-attached recordings from rat cultured striatal neurones demonstrate that exposure to H2O2 opens a large-conductance channel that is characterized by extremely long open times (seconds). Inside-out recordings show that cytoplasmically applied beta-nicotinamide adenine dinucleotide activates a channel with little voltage dependence, a linear current-voltage relationship and a single-channel conductance of between 70 and 90 pS. This channel is permeable to Na+, K+ and Ca2+ ions. Fura-2 imaging from cultured striatal neurones reveals that H2O2 exposure induces a biphasic intracellular Ca2+ increase in a subpopulation of neurones, the second, later phase resulting in Ca2+ overload. This later component of the Ca2+ response is dependent on the presence of extracellular Ca2+ and is independent of synaptic activity or voltage-gated Ca2+ channel opening. Consequently, this channel may be an important contributor to free radical-induced selective striatal neurone destruction. These results are remarkably similar to those observed for insulinoma cells and suggest that this family of non-selective cation channels has a widespread distribution in mammalian tissues.

Gabapentin-mediated inhibition of voltage-activated Ca2+ channel currents in cultured sensory neurones is dependent on culture conditions and channel subunit expression.

We have used the whole cell patch clamp method and fura-2 fluorescence imaging to study the actions of gabapentin (1-(aminoethyl) cyclohexane acetic acid) on voltage-activated Ca(2+) entry into neonatal cultured dorsal root ganglion (DRG) neurones and differentiated F-11 (embryonic rat DRG x neuroblastoma hybrid) cells. Gabapentin (2.5 microM) in contrast to GABA (10 microM) did not influence resting membrane potential or input resistance. In current clamp mode gabapentin failed to influence the properties of evoked single action potentials but did reduce the duration of action potentials prolonged by Ba(2+). Gabapentin attenuated high voltage-activated Ca(2+) channel currents in a dose- and voltage- dependent manner in DRG neurones and reduced Ca(2+) influx evoked by K(+) depolarisation in differentiated F-11 cells loaded with fura-2. The sensitivity of DRG neurones to gabapentin was not changed by the GABA(B) receptor antagonist saclofen but pertussis toxin pre-treatment reduced the inhibitory effects of gabapentin. Experiments following pre-treatment of DRG neurones with a PKA-activator and a PKA-inhibitor implicated change in phosphorylation state as a mechanism, which influenced gabapentin action. Sp- and Rp-analogues of cAMP significantly increased or decreased gabapentin-mediated inhibition of voltage-activated Ca(2+) channel currents. Culture conditions used to maintain DRG neurones and passage number of differentiated F-11 cells also influenced the sensitivity of Ca(2+) channels to gabapentin. We analysed the Ca(2+) channel subunits expressed in populations of DRG neurones and F-11 cells that responded to gabapentin had low sensitivity to gabapentin or were insensitive to gabapentin, by Quantitative TaqMan PCR. The data obtained from this analysis suggested that the relative abundance of the Ca(2+) channel beta(2) and alpha(2)delta subunit expressed was a key determinant of gabapentin sensitivity of both cultured DRG neurones and differentiated F-11 cells. In conclusion, gabapentin inhibited part of the high voltage-activated Ca(2+) current in neonatal rat cultured DRG neurones via a mechanism that was independent of GABA receptor activation, but was sensitive to pertussis toxin. Gabapentin responses identified in this study implicated Ca(2+) channel beta(2) subunit type as critically important to drug sensitivity and interactions with alpha(1) and alpha(2)delta subunits may be implicated in antihyperalgesic therapeutic action for this compound.

Gabapentin inhibits high-threshold calcium channel currents in cultured rat dorsal root ganglion neurones.

1. This study examined the action of gabapentin (gabapentin,1-(aminomethyl) cyclohexane acetic acid (Neurontin) on voltage-gated calcium (Ca(2+)) channel influx recorded in cultured rat dorsal root ganglion (DRG) neurones. 2. Voltage-gated Ca(2+) influx was monitored using both fura-2 based fluorescence Ca(2+) imaging and the whole-cell patch clamp technique. 3. Imaging of intracellular Ca(2+) transients revealed that gabapentin inhibited KCl (30 mM)-evoked voltage-dependent Ca(2+) influx. Both the duration for 50% of the maximum response (W50) and total Ca(2+) influx were significantly reduced by approximately 25-30% in the presence of gabapentin (25 microM). 4. Gabapentin potently inhibited the peak whole-cell Ca(2+) channel current (I(Ba)) in a dose-dependent manner with an estimated IC(50) value of 167 nM. Block was incomplete and saturated at a maximal concentration of 25 microM. 5. Inhibition was significantly decreased in the presence of the neutral amino acid L-isoleucine (25 microM) but unaffected by application of the GABA(B) antagonist, saclofen (200 microM), suggesting a direct action on the alpha(2)delta subunit of the Ca(2+) channel. 6. Gabapentin inhibition was voltage-dependent, producing an approximately 7 mV hyperpolarizing shift in current voltage properties and reducing a non-inactivating component of whole-cell current activated at relatively depolarized potentials. 7. The use of specific Ca(2+) channel antagonists revealed a mixed pharmacology of the gabapentin-sensitive current (N-, L- and P/Q-type), which is dominated by N-type current. 8. The present study is the first to demonstrate that gabapentin directly mediates inhibition of voltage-gated Ca(2+) influx in DRG neurones, providing a potential means for gabapentin to effectively mediate spinal anti-nociception.

Streptozocin-induced neuropathy is associated with altered expression of voltage-gated calcium channel subunit mRNAs in rat dorsal root ganglion neurones.

Voltage-gated calcium channels (VGCCs) within sensory neurones are believed to perform an important role in neuropathic pain. In the present study we examine the changes in VGCC mRNA which occur following streptozocin- (STZ) induced diabetic neuropathy using in situ hybridization. STZ caused a significant increase in alpha(2)delta(1), alpha(2)delta(2), and alpha(2)delta(3) mRNA levels in all neuronal cell types. Similarly, mRNA levels of alpha(1F), alpha(1I), and alpha(1S) were increased in all cell types studied whilst alpha(1A) and alpha(1G) mRNAs were specifically upregulated in medium and large diameter neurones. In conclusion, we demonstrate that the induction of diabetic neuropathy is associated with dramatic changes in the expression of VGCCs.

Adenosine exerts multiple effects in dorsal horn neurones of the adult rat spinal cord.

In the present study, we have examined the effects of adenosine and its analogues on the electrophysiological properties of dorsal horn neurones in the rat adult spinal cord. Adenosine and the A1 receptor agonist R-phenylisopropyl adenosine (RPIA) reversibly hyperpolarised these neurones via the generation of an outward current at -60 mV that was inhibited by pre-application of barium or Rp-adenosine 3', 5'-cyclic monophosphothioate triethylamine. In contrast, the A2a receptor agonist 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680) had no effect on the resting membrane properties of these neurones. Stimulation of the dorsal root evoked non-NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) at -60 mV that were completely abolished by 2,3-dihydroxy-6-nitro-7-sulophamoyl-benzo(F)quinoxalone (NBQX). Bath application of adenosine or RPIA reversibly inhibited these EPSCs in a concentration-dependent manner via a presynaptic action. In contrast, CGS21680 increased the amplitude of the EPSC in 20% of neurones tested and decreased the EPSC amplitude in 30% of neurones tested. It is concluded that adenosine exerts multiple effects upon the electrophysiological properties of dorsal horn neurones in the adult spinal cord via interaction with multiple receptors. These findings have important implications in the understanding of adenosine action in preclinical models of pain.

Expression of voltage-gated calcium channel subunits in rat dorsal root ganglion neurons.

In the present study, we have used in situ hybridisation to examine the distribution of calcium channel subunits in rat dorsal root ganglion (DRG) neurons. Within DRG neurons, the calcium channel alpha subunit mRNAs alpha(1A), alpha(1B), alpha(1C), alpha(1D), alpha(1E), alpha(1I) and alpha(1S) were readily detected in small (<25 microm), medium (25-45 microm) and large (>45 microm) diameter neurons. alpha(1F) was present at very low levels in these neurons whilst alpha(1G) was virtually undetectable. The calcium channel auxiliary subunits alpha(2)delta(1) and alpha(2)delta(2) showed a complementary pattern of distribution to that of alpha(2)delta(3) in DRG neurons. alpha(2)delta(1) and alpha(2)delta(2) transcripts were expressed predominantly in small c-type sensory neurons and were present at lower levels in large Abeta-type sensory neurons. In contrast, alpha(2)delta(3) mRNA was present in high quantities in the large-diameter cells but was expressed at lower levels in small-diameter neurons of the DRG. The present study provides an insight into the molecular profile of calcium channel alpha(1) and alpha(2)delta subunits in the neurons responsible for transmitting sensory information.

Developmental expression of the novel voltage-gated sodium channel auxiliary subunit beta3, in rat CNS.

1. We have compared the mRNA distribution of sodium channel alpha subunits known to be expressed during development with the known auxiliary subunits Nabeta1.1 and Nabeta2.1 and the novel, recently cloned subunit, beta3. 2. In situ hybridisation studies demonstrated high levels of Nav1.2, Nav1.3, Nav1.6 and beta3 mRNA at embryonic stages whilst Nabeta1.1 and Nabeta2.1 mRNA was absent throughout this period. 3. Nabeta1.1 and Nabeta2.1 expression occurred after postnatal day 3 (P3), increasing steadily in most brain regions until adulthood. beta3 expression differentially decreased after P3 in certain areas but remained high in the hippocampus and striatum. 4. Emulsion-dipped slides showed co-localisation of beta3 with Nav1.3 mRNA in areas of the CNS suggesting that these subunits may be capable of functional interaction. 5. Co-expression in Xenopus oocytes revealed that beta3 could modify the properties of Nav1.3; beta3 changed the equilibrium of Nav1.3 between the fast and slow gating modes and caused a negative shift in the voltage dependence of activation and inactivation. 6. In conclusion, beta3 is shown to be the predominant beta subunit expressed during development and is capable of modulating the kinetic properties of the embryonic Nav1.3 subunit. These findings provide new information regarding the nature and properties of voltage-gated sodium channels during development.

Beta3, a novel auxiliary subunit for the voltage gated sodium channel is upregulated in sensory neurones following streptozocin induced diabetic neuropathy in rat.

In the present study we have used in situ hybridization to examine the changes in mRNA expression of the voltage gated sodium channel subunits beta1 and beta3, which occur in response to streptozocin induced diabetic neuropathy. Under control conditions beta1 mRNA was detected throughout the spinal cord and in large dorsal root ganglion (DRG) Abeta fibres whilst beta3 mRNA was expressed exclusively in the layers I/II and X of the spinal cord and in small DRG c-fibres. Following streptozocin treatment, the expression of beta1 mRNA remained unchanged in both the spinal cord and DRG whilst beta3 message was significantly increased in both the spinal cord and in medium diameter Adelta type DRG neurones. In conclusion, the present study illustrates that the development of the neuropathic pain state is associated with distinct changes in the pattern of beta3 subunit expression and that these changes appear to be specific to the neuropathic pain state induced.

Tissue distribution and functional expression of the human voltage-gated sodium channel beta3 subunit.

This study investigated the distribution of beta3 in human tissues and the functional effects of the human beta3 subunit on the gating properties of brain and skeletal muscle alpha subunits. Using RT-PCR of human cDNA panels, beta3 message was detected in brain, heart, kidney, lung, pancreas and skeletal muscle. Both alphaIIA and SkM1 expressed in Xenopus oocytes inactivated with a time course described by two exponential components representing fast and slow gating modes, while co-expression of human beta3 with alphaIIA or SkM1 significantly increased the proportion of channels operating by the fast gating mode. In the presence of beta3 a greater proportion of alphaIIA or SkM1 current was described by the fast time constant for both inactivation and recovery from inactivation. beta3 caused a hyperpolarizing shift in the voltage dependence of inactivation of alphaIIIA and reduced the slope factor. The voltage dependence of inactivation of SkM1 was described by a double Boltzmann equation. However, SkM1 co-expressed with beta3 was described by a single Boltzmann equation similar to one of the Boltzmann components for SkM1 expressed alone, with a small positive shift in V1/2 value and reduced slope factor. This is the first study demonstrating that beta3 is expressed in adult mammalian skeletal muscle and can functionally couple to the skeletal muscle alpha subunit, SkM1.

beta3, a novel auxiliary subunit for the voltage-gated sodium channel, is expressed preferentially in sensory neurons and is upregulated in the chronic constriction injury model of neuropathic pain.

Adult dorsal root ganglia (DRG) have been shown to express a wide range of voltage-gated sodium channel alpha-subunits. However, of the auxiliary subunits, beta1 is expressed preferentially in only large- and medium-diameter neurons of the DRG while beta2 is absent in all DRG cells. In view of this, we have compared the distribution of beta1 in rat DRG and spinal cord with a novel, recently cloned beta1-like subunit, beta3. In situ hybridization studies demonstrated high levels of beta3 mRNA in small-diameter c-fibres, while beta1 mRNA was virtually absent in these cell types but was expressed in 100% of large-diameter neurons. In the spinal cord, beta3 transcript was present specifically in layers I/II (substantia gelatinosa) and layer X, while beta1 mRNA was expressed in all laminae throughout the grey matter. Since the pattern of beta3 expression in DRG appears to correlate with the TTX-resistant voltage-gated sodium channel subunit PN3, we co-expressed the two subunits in Xenopus oocytes. In this system, beta3 caused a 5-mV hyperpolarizing shift in the threshold of activation of PN3, and a threefold increase in the peak current amplitude when compared with PN3 expressed alone. On the basis of these results, we examined the expression of beta-subunits in the chronic constriction injury model of neuropathic pain. Results revealed a significant increase in beta3 mRNA expression in small-diameter sensory neurons of the ipsilateral DRG. These results show that beta3 is the dominant auxiliary sodium channel subunit in small-diameter neurons of the rat DRG and that it is significantly upregulated in a model of neuropathic pain.

Gabapentin inhibits excitatory synaptic transmission in the hyperalgesic spinal cord.

In the present study we tested the effects of the antihyperalgesic compound gabapentin on dorsal horn neurones in adult spinal cord. Slices were taken from control and hyperalgesic animals suffering from streptozocin-induced diabetic neuropathy. At concentrations up to 100 microM, bath application failed to affect the resting membrane properties of dorsal horn neurones taken from both groups of animal. In contrast, bath application of gabapentin dramatically reduced the magnitude of the excitatory postsynaptic current (EPSC) in neurones taken from hyperalgesic animals without altering the magnitude of the EPSC in control animals. Using a paired pulse stimulation protocol, together with analysis of miniature EPSC's, it was possible to demonstrate that gabapentin mediated these effects via a pre-synaptic site of action.

NK-3 receptors are expressed on mouse striatal gamma-aminobutyric acid-ergic interneurones and evoke [(3)H] gamma-aminobutyric acid release.

In the present study the ability of tachykinin agonists and antagonists to modulate gamma-aminobutyric acid (GABA) release has been correlated with tachykinin receptor expression in the mouse striatum. Significant GABA release was observed when striatal slices were challenged with the NK-3 receptor agonist senktide, the selectivity of which was confirmed using the NK-3 receptor antagonist SR142801. In situ hybridisation revealed co-expression of NK-3 receptors with nitric oxide synthase (NOS)/preprosomatostatin containing GABAergic interneurones. These findings suggest that tachykinins modulate GABA release within the striatum via interaction with NK-3 receptors on somatostatin/NOS interneurones.

Gene-expression analysis at the single-cell level.

The manner in which a cell responds to and influences its environment is ultimately determined by the genes that it expresses. To fully understand and manipulate cellular function, identification of these expressed genes is essential. Techniques such as RT-PCR enable examination of gene expression at the tissue level. However, the study of complex heterogeneous tissue, such as the CNS or immune system, requires gene analysis to be performed at much higher resolution. In this article, the various methods that have been developed to enable RT-PCR to be performed at the level of the single cell are reviewed. In addition, how, when carried out in combination with techniques such as patch-clamp recording, single-cell gene-expression studies extend our understanding of biological systems is discussed.

beta 3: an additional auxiliary subunit of the voltage-sensitive sodium channel that modulates channel gating with distinct kinetics.

The voltage-sensitive sodium channel confers electrical excitability on neurons, a fundamental property required for higher processes including cognition. The ion-conducting alpha-subunit of the channel is regulated by two known auxiliary subunits, beta1 and beta2. We have identified rat and human forms of an additional subunit, beta3. It is most closely related to beta1 and is the product of a separate gene localized to human chromosome 11q23.3. When expressed in Xenopus oocytes, beta3 inactivates sodium channel opening more slowly than beta1 does. Structural modeling has identified an amino acid residue in the putative alpha-subunit binding site of beta3 that may play a role in this difference. The expression of beta3 within the central nervous system differs significantly from beta1. Our results strongly suggest that beta3 performs a distinct neurophysiological function.

Tachykinins increase [3H]acetylcholine release in mouse striatum through multiple receptor subtypes.

Tachykinins have been suggested to play a significant role in the mammalian striatum, at least in part by the control of acetylcholine release from cholinergic interneurons. In the present study, we have examined the ability of known tachykinin agonists and antagonists to modulate the activity of these interneurons in mouse striatal slices. Using whole-cell patch-clamp recordings, the selective neurokinin-1, neurokinin-2 and neurokinin-3 receptor agonists [sar9,Met(O2)11]substance P, [beta-ala8]neurokinin A(4-10) and senktide each produced a dose-dependent depolarization of visually identified cholinergic interneurons that was retained under conditions designed to interrupt synaptic transmission. The nature of these neurons and the expression of multiple tachykinin receptors was confirmed using single-cell reverse transcriptase-polymerase chain reaction analysis. Using in vitro superfusion techniques, the selective neurokinin-1, neurokinin-2 and neurokinin-3 receptor agonists [sar9,Met(O2)11]substance P, [beta-ala8]neurokinin A(4-10) and senktide, respectively, each produced a dose-dependent increase in acetylcholine release, the selectivity of which was confirmed using the neurokinin-1, neurokinin-2 and neurokinin-3 receptor antagonists SR140333, GR94800 and SR142801 (100 nM). U73122 (10 microM), a phospholipase C inhibitor, blocked [sar9,Met(O2)11]substance P- and senktide-induced acetylcholine release, but had no effect on [beta-ala8]neurokinin A(4-10)-induced release. The protein kinase C inhibitors chelerythrine and Ro-31-8220 (both 1 microM) significantly inhibited responses induced by all three agonists. These findings indicate that tachykinins modulate the activity of mouse striatal cholinergic interneurons. Furthermore, neurokinin-2 receptors are shown to perform a role in mouse that has not been identified previously in other species.

Correlating physiology with gene expression in striatal cholinergic neurones.

The expression of 34 transmitter-related genes has been examined in the cholinergic neurones of rat striatal brain slices, with the aim of correlating gene expression with functional activity. The mRNAs encoding types I, II/IIA, and III alpha subunits of the voltage-sensitive sodium channels were detected, suggesting the presence of these three types of sodium channel. Similarly, mRNAs encoding all four alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-type glutamate receptor subunits and the NR1 and NR2A, 2B, and 2D subunits of the NMDA-type glutamate receptors were detected, suggesting that various combinations of these subunits mediate the cellular response to synaptically released glutamate. Other mRNAs detected included the NK1 and NK3 tachykinin receptors, all four known adenosine receptors, and the GABA-synthesising enzyme glutamate decarboxylase. Subpopulations of these cholinergic neurones have been identified on the basis of the expression of the NK3 tachykinin receptor in 5% and the trkC neurotrophin receptor in 12% of the cells investigated.

Serotonin depolarizes hippocampal interneurones in the rat stratum oriens by interaction with 5HT2 receptors.

Patch-clamp recording techniques were used to examine the effect of serotonin (5HT) upon interneurones contained in the stratum oriens layer of hippocampal slices. Bath application of 1-20 microM 5HT depolarized neurones by the induction of an inward current at -60 mV. This inward current was Na+-dependent in nature, was mimicked by the 5HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and was inhibited by pre-incubation with the 5HT2 receptor antagonist ritanserin.

Bombesin-like peptides depolarize rat hippocampal interneurones through interaction with subtype 2 bombesin receptors.

1. Whole-cell patch-clamp recordings were made from visually identified hippocampal interneurones in slices of rat brain tissue in vitro. Bath application of the bombesin-like neuropeptides gastrin-releasing peptide (GRP) or neuromedin B (NMB) produced a large membrane depolarization that was blocked by pre-incubation with the subtype 2 bombesin (BB2) receptor antagonist [D-Phe6, Des-Met14]bombesin-(6-14)ethyl amide. 2. The inward current elicited by NMB or GRP was unaffected by K+ channel blockade with external Ba2+ or by replacement of potassium gluconate in the electrode solution with caesium acetate. 3. Replacement of external NaCl with Tris-HCl significantly reduced the magnitude of the GRP-induced current at -60 mV. In contrast, replacement of external NaCl with LiCl had no effect on the magnitude of this current. 4. Photorelease of caged GTPgammaS inside neurones irreversibly potentiated the GRP-induced current at -60 mV. Similarly, bath application of the phospholipase C (PLC) inhibitor U-73122 significantly reduced the size of the inward current induced by GRP. 5. Reverse transcription followed by the polymerase chain reaction using cytoplasm from single hippocampal interneurones demonstrated the expression of BB2 receptor mRNA together with glutamate decarboxylase (GAD67). 6. Although bath application of GRP or NMB had little or no effect on the resting membrane properties of CA1 pyramidal cells per se, these neuropeptides produced a dramatic increase in the number and amplitude of miniature inhibitory postsynaptic currents in these cells in a TTX-sensitive manner.