Martin D. Hall

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.

Changes in apomorphine-induced stereotypy as a result of subacute neuroleptic treatment correlates with increased D-2 receptors, but not with increases in D-1 receptors.

Administration of haloperidol (5 mg/kg i.p.), cis-flupenthixol (2.5 mg/kg i.p.) or sulpiride (2 X 100 mg/kg i.p.) daily for 21 days followed by a 3-day drug withdrawal period caused equivalent cerebral dopamine receptor supersensitivity as judged by enhanced apomorphine-induced stereotypy. These treatments also produced equivalent rises in the number of adenylate cyclase-independent dopamine receptors (D-2) in both striatal and mesolimbic tissue as assessed by specific [3H]spiperone and [3H]N,n-propylnorapomorphine (NPA) binding. No change in the dissociation constant (KD) was apparent in response to neuroleptic treatment. However, only repeated administration of cis-flupenthixol caused an increase in the number of adenylate cyclase-linked dopamine receptors (D-1) in striatum as assessed by enhanced [3H]piflutixol binding and increased dopamine-stimulated cyclic AMP formation. The dissociation constant for [3H]piflutixol binding was unchanged by cis-flupenthixol administration. No change in D-1 receptor numbers or dopamine stimulation of adenylate cyclase occurred in mesolimbic tissue. Repeated treatment with sulpiride or haloperidol was without effect on either [3H]piflutixol binding to D-1 receptors or cyclic AMP formation. In conclusion, increased apomorphine-induced stereotypy following subacute neuroleptic treatment correlates with changes in D-2 receptor numbers, but not with changes in D-1 receptors.

Differential alterations in striatal dopamine receptor sensitivity induced by repeated administration of clinically equivalent doses of haloperidol, sulpiride or clozapine in rats

Rats received therapeutically equivalent doses of either haloperidol (1.7–1.9 mg/kg/day), sulpiride (112–116 mg/kg/day) or clozapine (30–35 mg/kg/day) continuously for 4 weeks. Treatment with haloperidol, but not sulpiride or clozapine, caused inhibition of stereotyped behaviour induced by apomorphine (0.125–0.25 mg/kg SC). Following drug withdrawal for up to 7 days, haloperidol and sulpiride, but not clozapine treatment caused an exaggeration of stereotyped behaviour induced by apomorphine.Bmax values for striatal 3H-spiperione binding were erevated in animals treated for 2 and 4 weeks with haloperidol, but not with sulpiride or clozapine. Following drug withdrawal, haloperidol, but not sulpiride or clozapine, treatment caused an increase in Bmax for striatal 3H-piperone binding.Bmax values for striatal 3H-NPA binding revealed no change during haloperidol or clozapine treatment. Sulpiride treatment for 1 week caused an increase in Bmax for 3H-NPA binding, which returned to control levels at 2 and 4 weeks. Following drug withdrawal, there was an increase in Bmax for 3H-NPA binding in rats treated with haloperidol and sulpiride, but not clozapine.On continuous treatment and following withdrawal from haloperidol, sulpiride, or clozapine the ability of dopamine to stimulate striatal adenylate cyclase activity did not differ from that in control animals.Repeated administration of sulpiride or clozapine may not induce striatal dopamine receptor supersensitivity when given in clinically relevant doses, although haloperidol does.

Differential labelling of dopamine receptors in rat brain in vivo: Comparison of [3H]piribedil, [3H]S 3608 and [3H]N,n-propylnorapomorphine

Abstract The in vivo distribution of radioactivity in brain and labelling of cerebral dopamine receptors in rats derived from the administration of the atypical dopamine agonists [ 3 H]piribedil and [ 3 H]S 3608 has been compared to that of the classical dopamine agonist [ 3 H]N,n-propylnorapomorphine (NPA). Radioactivity derived from [ 3 H]piribedil accumulated in the substantia nigra, nucleus accumbens and cervical spinal cord, and this accumulation was prevented by administration of (+)-butaclamol and apomorphine only in substantia nigra and the nucleus accumbens. Radioactivity derived from [ 3 H]S 3608 accumulated in the same areas and, additionally, in the frontal cortex and tuberculum olfactorium; this accumulation was prevented by administration of (+)-butaclamol and apomorphine only in substantia nigra, nucleus accumbens and tuberculum olfactorium. Neither ligand caused accumulation of radioactivity in the striatum. In contrast, radioactivity derived from [ 3 H]NPA accumulated in the substantia nigra, nucleus accumbens, striatum, and tuberculum olfactorium. Radioactivity derived from [ 3 H]NPA was prevented from accumulating in all these areas by (+)-butaclamol and by apomorphine, but piribedil only prevented accumulation in the substantia nigra and nucleus accumbens and S 3608 only prevented accumulation in substantia nigra, tuberculum olfactorium and nucleus accumbens. Neither piribedil nor S 3608 prevented accumulation of radioactivity derived from [ 3 H]NPA in the striatum. Piribedil and S 3608 showed equal capacity in vitro to displace [ 3 H]spiperone and [ 3 H]NPA from striatal or nucleus accumbens tissue preparations. These results suggest that, in vivo piribedil and S 3608 selectively interact with dopamine receptors in the substantia nigra and nucleus accumbens, but not with those in striatum, perhaps due to differential distribution within brain.

Repeated administration of sulpiride for three weeks produces behavioural and biochemical evidence for cerebral dopamine receptor supersensitivity.

Administration of sulpiride (2 X 100 mg/kg i.p.) or haloperidol (5 mg/kg i.p.) to rats for 3 weeks with subsequent withdrawal for 3 or 4 days induced cerebral dopamine receptor supersensitivity. Apomorphine-induced stereotyped behaviour after drug withdrawal was enhanced by pretreatment with either haloperidol or sulpiride both of which increased the number of specific striatal binding sites (Bmax) for [3H]spiperone, [3H]N,n-propylnorapomorphine and [3H]sulpiride. Neither drug altered the dissociation constant (KD) for the ligand binding assays. Striatal dopamine sensitive adenylate cyclase activity was unaltered by such a pretreatment with either haloperidol or sulpiride. The data show that sulpiride, like haloperidol, is capable of inducing behavioural and biochemical supersensitivity of cerebral dopamine receptors.

Hypophysectomy does not prevent development of striatal dopamine receptor supersensitivity induced by repeated neuroleptic treatment

Hruska et al. (1980) reported that hypophysectomy prevented the onset of dopamine receptor supersensitivity. We have repeated this investigation administering haloperidol (0.75 mg/day) or sulpiride (2 X 15 mg/day) or saline for 17 days, followed by a 3 day drug washout period, to sham-operated or hypophysectomised rats. Haloperidol or sulpiride pretreatment caused an enhancement of apomorphine-induced stereotyped behaviour and increased the number of specific striatal [3H]spiperone binding sites (Bmax) in both hypophysectomised and sham-operated animals compared to their respective saline controls. We conclude that hypophysectomy does not prevent the onset of striatal dopamine receptor supersensitivity induced by repeated neuroleptic treatment in the rat.

Mesolimbic dopamine function is not altered during continuous chronic treatment of rats with typical or atypical neuroleptic drugs

Rats were treated continuously for up to 20 months with either haloperidol (1.4–1.6 mg/kg/day), sulpiride (102–109 mg/kg/day) or clozapine (24–27 mg/kg/day). Bmax for specific mesolimbic binding of3H-spiperone,3H-N,n-propylnorapomorphine or3H-piflutixol did not differ in tissue taken from animals treated for up to 12 months with haloperidol, sulpiride or clozapine by comparison to age-matched control rats. Mesolimbic dopamine (50 ΜM)-stimulated adenylate cyclase activity was not altered in any drug treatment group. Spontaneous locomotor activity was transiently decreased during treatment with haloperidol for 1 or 3 months, but not by chronic sulpiride or clozapine treatment. Locomotor activity was not consistently increased in any drug treatment group. After 20 months of continuous drug treatment, focal bilateral application of dopamine (12.5 or 25 Μg) into the nucleus accumbens caused equivalent increases in locomotor activity in control rats and in animals receiving haloperidol, sulpiride or clozapine. These findings suggest that dopamine receptor blockade is not maintained in the mesolimbic area following chronic treatment with haloperidol, sulpiride or clozapine, and indicate that, under these conditions, clozapine and sulpiride may not act selectively on mesolimbic dopamine receptors.

Behavioural and biochemical alterations in the function of dopamine receptors following repeated administration of l-DOPA to rats

Rats received L-DOPA (40 or 200 mg/kg, i.p.) for 14 days, followed by a 3 day withdrawal period. Spontaneous locomotor activity was not altered by repeated administration of L-DOPA. Rats treated with L-DOPA (200 mg/kg) showed identical locomotor hypoactivity in response to small doses of apomorphine when compared to saline-treated control animals. However, hyperactivity induced by large doses of apomorphine was reduced by prior treatment with L-DOPA (200 mg/kg). The smaller dose of L-DOPA (40 mg/kg) did not alter the locomotion induced by apomorphine. Stereotyped behaviour induced by apomorphine was enhanced by prior treatment with both 40 and 200 mg/kg of L-DOPA. The treatment regimes with L-DOPA had no effect on the concentrations of apomorphine in the striatum. Administration of L-DOPA (40 or 200 mg/kg) followed by withdrawal for 3 days, had no effect on the concentrations of dopamine, homovanillic acid (HVA) or 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum. The Bmax and KD for the binding of [3H]spiperone, [3H]N,n-propylnorapomorphine (NPA) and [3H] piflutixol in the striatum was not altered by drug treatment. Similarly, the formation of dopamine-stimulated cyclic AMP in homogenates of striatum was unaltered by repeated administration of L-DOPA. Repeated administration of L-DOPA for 14 days in the rat appears to result in altered behaviour mediated by dopamine in the absence of any apparent change in the function of dopamine receptors in the striatum.

Alterations in different populations of striatal dopamine receptors produced by 18 months continuous administration of cis- or trans-flupenthixol to rats.

Administration of cis-flupenthixol (0.8-1.2 mg/kg per day) for 18 months enhanced stereotyped behaviour induced by apomorphine, bromocriptine and lergotrile, but not that induced by amphetamine or lisuride. Catalepsy induced by acute administration of haloperidol, trifluoperazine or cis-flupenthixol was reduced by continuous chronic intake of cis-flupenthixol. The number (Bmax) and dissociation constant (KD) of specific [3H]spiperone binding sites on striatal membranes was increased by chronic administration of cis-flupenthixol, but not trans-flupenthixol. In contrast, the Bmax and KD for specific binding of [3H]N,n-propylnorapomorphine were decreased by administration of cis-flupenthixol compared to the effect of the trans-isomer. Specific binding of [3H]piflutixol was unaffected by chronic administration of cis- or trans-flupenthixol, but chronic administration of cis-flupenthixol enhanced stimulation by dopamine of the activity of striatal adenylate cyclase. As a result of chronic continuous administration of cis-flupenthixol dopamine receptors in the striatum appeared to be supersensitive to most dopamine agonists but sub-sensitive to dopamine antagonists. This was reflected by increased numbers of D-2 antagonist receptor sites of decreased affinity, but by a decreased number of agonist sites of higher affinity. The D-1 recognition sites appeared to be unaltered, but activity of adenylate cyclase stimulated by dopamine was enhanced, suggesting post-junctional changes. The D-2 receptors appear to be primarily concerned with altered function of dopamine receptors.

Turnover of specific [3H]spiperone and [3H]N, n-propylnorapomorphine binding sites in rat striatum following phenoxybenzamine administration

Inclusion of phenoxybenzamine into incubates containing rat striatal preparations equipotently displaced specific striatal [3H]spiperone and [3H]NPA binding. Pre-incubation of striatal membranes with phenoxybenzamine followed by extensive washing equipotently inhibited the subsequent specific [3H]spiperone or [3H]NPA binding. In both displacement and pre-incubation experiments phenoxybenzamine caused complete inhibition of specific [3H]spiperone binding to rat striatal membranes, but only partially inhibited specific [3H]NPA binding. Following parenteral administration to rats, phenoxybenzamine caused a marked inhibition of ex vivo specific [3H]spiperone binding in striatal tissue preparations from these animals which lasted approximately 24 hr following in vivo drug administration. In contrast, administration of phenoxybenzamine caused only a transient change in ex vivo specific [3H]NPA binding. Phenoxybenzamine causes irreversible inhibition of [3H]spiperone and [3H]NPA binding in vitro. In vivo administration of phenoxybenzamine discriminates between [3H]spiperone and [3H]NPA in ex vivo studies suggesting that these binding sites have different turnover rates.