Neonatal Rat Motoneurones Research Articles

Prochlorperazine Increases KCC2 Function and Reduces Spasticity after Spinal Cord Injury.

In mature neurons, low intracellular chloride level required for inhibition is maintained by the potassium-chloride cotransporter, KCC2. Impairment of Cl- extrusion after KCC2 dysfunction has been involved in many central nervous system disorders, such as seizures, neuropathic pain, or spasticity, after a spinal cord injury (SCI). This makes KCC2 an appealing drug target for restoring Cl- homeostasis and inhibition in pathological conditions. In the present study, we screen the Prestwick Chemical Library® and identify conventional antipsychotics phenothiazine derivatives as enhancers of KCC2 activity. Among them, prochlorperazine hyperpolarizes the Cl- equilibrium potential in motoneurons of neonatal rats and restores the reciprocal inhibition post-SCI. The compound alleviates spasticity in chronic adult SCI rats with an efficacy equivalent to the antispastic agent, baclofen, and rescues the SCI-induced downregulation of KCC2 in motoneurons below the lesion. These pre-clinical data support prochlorperazine for a new therapeutic indication in the treatment of spasticity post-SCI and neurological disorders involving a KCC2 dysfunction.

The effects of developmental nicotine exposure on hypoglossal motoneuron morphology and electrophysiology (871.2)

Developmental nicotine exposure (DNE) is known to have significant effects on motoneuron electrophysiology and morphology. Previous work in hypoglossal motoneurons (XIIMN) of neonatal rats exposed to nicotine in utero has shown decreased excitatory synaptic input, desensitized nicotinic acetylcholine receptors, increased input resistance, a left‐shifted frequency‐current curve as well as increased gain, and preliminary evidence for a decreased soma size. To further examine the effects that DNE has on XIIMNs, we have used whole‐cell patch clamp techniques to record responses to injected sine‐wave currents while neurobiotin passively fills the neurons. Filled cells were visualized using a DAB reaction followed by a detailed morphological reconstruction using the Neurolucida system. Response to sinusoidal current injection was used to record spike firing resonance, which is indicative of spike‐timing precision and variability. These measurements are useful for examining potential changes of the input‐output efficiency of XIIMNs by DNE. Preliminary data indicate significantly increased size of dendritic surface area in DNE cells (n=8 control; 2 DNE). Nicotine exposed cells also appear to be less responsive to sine wave inputs, and more variable in their response as well (n=4 control; 7 DNE).Grant Funding Source: Supported by NIH R01HD071302, AHA 12GRXIT120S0345

Sodium-Mediated Plateau Potentials in Lumbar Motoneurons of Neonatal Rats

The development and the ionic nature of bistable behavior in lumbar motoneurons were investigated in rats. One week after birth, almost all (∼80%) ankle extensor motoneurons recorded in whole-cell configuration displayed self-sustained spiking in response to a brief depolarization that emerged when the temperature was raised >30°C. The effect of L-type Ca(2+) channel blockers on self-sustained spiking was variable, whereas blockade of the persistent sodium current (I(NaP)) abolished them. When hyperpolarized, bistable motoneurons displayed a characteristic slow afterdepolarization (sADP). The sADPs generated by repeated depolarizing pulses summed to promote a plateau potential. The sADP was tightly associated with the emergence of Ca(2+) spikes. Substitution of extracellular Na(+) or chelation of intracellular Ca(2+) abolished both sADP and the plateau potential without affecting Ca(2+) spikes. These data suggest a key role of a Ca(2+)-activated nonselective cation conductance ((CaN)) in generating the plateau potential. In line with this, the blockade of (CaN) by flufenamate abolished both sADP and plateau potentials. Furthermore, 2-aminoethoxydiphenyl borate (2-APB), a common activator of thermo-sensitive vanilloid transient receptor potential (TRPV) cation channels, promoted the sADP. Among TRPV channels, only the selective activation of TRPV2 channels by probenecid promoted the sADP to generate a plateau potential. To conclude, bistable behaviors are, to a large extent, determined by the interplay between three currents: L-type I(Ca), I(NaP), and a Na(+)-mediated I(CaN) flowing through putative TRPV2 channels.

Modulation of voltage-gated sodium channels hyperpolarizes the voltage threshold for activation in spinal motoneurones

Previous work has shown that motoneurone excitability is enhanced by a hyperpolarization of the membrane potential at which an action potential is initiated (V(th)) at the onset, and throughout brainstem-evoked fictive locomotion in the adult decerebrate cat and neonatal rat. Modeling work has suggested the modulation of Na(+) conductance as a putative mechanism underlying this state-dependent change in excitability. This study sought to determine whether modulation of voltage-gated sodium channels could induce V(th) hyperpolarization. Whole-cell patch-clamp recordings were made from antidromically identified lumbar spinal motoneurones in an isolated neonatal rat spinal cord preparation. Recordings were made with and without the bath application of veratridine, a plant alkaloid neurotoxin that acts as a sodium channel modulator. As seen in HEK 293 cells expressing Nav1.2 channels, veratridine-modified channels demonstrated a hyperpolarizing shift in their voltage-dependence of activation and a slowing of inactivation that resulted in an enhanced inward current in response to voltage ramp stimulations. In the native rat motoneurones, veratridine-modified sodium channels induced a hyperpolarization of V(th) in all 29 neonatal rat motoneurones examined (mean hyperpolarization: -6.6 ± 4.3 mV). V(th) hyperpolarization was not due to the effects on Ca(2+) and/or K(+) channels as blockade of these currents did not alter V(th). Veratridine also significantly increased the amplitude of persistent inward currents (PICs; mean increase: 72.5 ± 98.5 pA) evoked in response to slow depolarizing current ramps. However, the enhancement of the PIC amplitude had a slower time course than the hyperpolarization of V(th), and the PIC onset voltage could be either depolarized or hyperpolarized, suggesting that PIC facilitation did not mediate the V(th) hyperpolarization. We therefore suggest that central neuronal circuitry in mammals could affect V(th) in a mechanism similar to that of veratridine, by inducing a negative shift in the activation voltage of sodium channels. Furthermore, this shift appears to be independent of the enhancement of PICs.

The possible additional role of the cystic fibrosis transmembrane regulator to motoneuron inhibition produced by glycine effects

In the present work we study the contribution of the chloride channel of the Cystic Fibrosis Transmembrane Regulator (CFTR) in the postsynaptic inhibition of somatic motoneurons during rapid-eye-movement (REM) sleep atonia. Postsynaptic inhibition of motoneurons is partially responsible for the atonia that occurs during REM sleep. Disfacilitation is an additional mechanism that lowers motoneuron excitability in this state. Postsynaptic inhibition is mediated by the release of glycine from synaptic terminals on motoneurons, and by GABA that plays a complementary role to that of glycine. In this work we look in brain stem motoneurons of neonatal rats at a mechanism unrelated to the actions of glycine, GABA or to disfacilitation which depends on the chloride channel of the CFTR. We studied the presence of CFTR by immunocytochemistry. In electrophysiological experiments utilizing whole cell recordings in in vitro slices we examined the consequences of blocking this chloride channel. The effects on motoneurons of the application of glycine, of the application of glibenclamide (a CFTR blocker) and again of glycine during the effects of glibenclamide were studied. Glycine produced an hyperpolarization, a decrease in motoneuron excitability and a decrease in input resistance, all characteristic changes of the postsynaptic inhibition produced by this neurotransmitter. Glibenclamide produced an increase in input resistance and in motoneurons' repetitive discharge as well as a shift in the equilibrium potential for chloride ions as indicated by the displacement of the reversal potential for glycinergic actions. In motoneurons treated with glibenclamide, glycine produced postsynaptic inhibition but this effect was smaller when compared to that elicited by glycine in control conditions. The fact that blocking of the CFTR-chloride channel in brain stem motoneurons influences glycinergic inhibition suggests that this channel may play a complementary role in the glycinergic inhibition that occurs during REM sleep.

Input‐specific plasticity of N‐methyl‐d‐aspartate receptor‐mediated synaptic responses in neonatal rat motoneurons

Lumbar motoneurons can be activated monosynaptically by two glutamatergic synaptic inputs: the segmental dorsal root (DR) and the descending ventrolateral funiculus (VLF). To determine whether their N-methyl-d-aspartate (NMDA) receptors are independent, we used (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine-hydrogen-maleate (MK-801), known to induce a use-dependent irreversible block of NMDA receptors (NMDARs). In the presence of MK-801 (in bath) and non-NMDA antagonists (in bath, to isolate NMDARs pharmacologically), we first stimulated the DR. After MK-801 blockade of DR synaptic input, the VLF was stimulated. Its response was found to be not significantly different from its control value, suggesting that the DR stimulus activated very few, if any, receptors also activated by VLF stimulation. Similar findings were obtained if the stimulation order was reversed. Both inputs also elicited a polysynaptic NMDAR-mediated response. Evoking the DR polysynaptic response in the presence of MK-801 eliminated the corresponding VLF response; the reverse did not occur. Surprisingly, when MK-801 was washed from the bath, both the DR and the VLF responses could recover, although the recovery of the DR monosynaptic and polysynaptic responses was reliably greater than those associated with the VLF. Recovery was prevented if extrasynaptic receptors were activated by bath-applied NMDA in the presence of MK-801, consistent with the possibility that recovery was due to movement of extrasynaptic receptors into parts of the membrane accessible to transmitter released by DR and VLF stimulation. These novel findings suggest that segmental glutamatergic inputs to motoneurons are more susceptible to plastic changes than those from central nervous system white matter inputs at this developmental stage.

The potential role of phrenic nucleus glutamate receptor subunits in mediating spontaneous crossed phrenic activity in neonatal rat

Cervical spinal cord hemisection rostral to the phrenic nucleus leads to paralysis of the ipsilateral hemidiaphragm in adult rats. Respiratory function can be restored to the paralyzed hemidiaphragm by activating a latent respiratory motor pathway. The latent pathway is called the crossed phrenic pathway. In adult rats, the pathway can be activated by drug-induced upregulation of NMDA receptor NR2A subunit and AMPA receptor GluR1 subunit in the phrenic nucleus following hemisection. In neonatal rats, this pathway is not latent as shown by the spontaneous expression of activity in the ipsilateral hemidiaphragm following hemisection. We hypothesized that the NR2A and GluR1 subunits may be highly expressed naturally on phrenic motoneurons of neonatal rats and may play a potential role in mediating the spontaneous expression of activity in the ipsilateral hemidiaphragm after hemisection. To test this hypothesis, the protein levels of NR2A and GluR1 in different age rats were assessed via Western blot analysis immediately following C2 hemisection and EMG recording of crossed phrenic activity. The protein levels of NR2A and GluR1 were transiently high in postnatal day 2 (P2) rats and then was significantly reduced in P7 and P35 animals. An immunofluorescence study qualitatively supported these findings. The present results indicate that the developmental downregulation of the phrenic nucleus glutamate receptor subunits correlates with the conversion of the crossed phrenic pathway in older postnatal animals from an active state to a latent state.

Modulation of voltage-dependent potassium currents by opiates in facial motoneurons of neonatal rats

We examined the modulation of rat facial motoneurons (FMNs) by opiates in a slice preparation (7–15 days old) using whole-cell patch clamp techniques. Although application of methionine enkephalin (ME) did not change the peak value of the transient outward current (A-current, IA), it reduced the persistent voltage-dependent K + currents (IKs) in a dose-dependent manner. The reduction was antagonized by naloxone (40 μM). IKs were reduced only by μ-selective agonist [ d-Ala 2, N-Me-Phe 4,Gly 5-ol]enkephalin (DAMGO, 2–121.6 μM). This reduction was antagonized by naloxone (40 μM) or the μ-selective antagonist, d-Phe-Cys-Tyr- d-Trp-Orn-Thr-Phe-Thr-NH 2 (CTOP, 1 μM). Agonists for other opiate receptors (δ- and κ-opiate receptor) showed no effect on IKs. In accord with the effects on IKs, DAMGO (100 μM) prolonged the duration of the action potential evoked in Ca 2+-free external solution containing 4-aminopiridine (1 mM). These results suggest that the activation of μ-opiate receptors contributes to signal transduction in FMNs primarily by modulating action potential duration.

Endogenous expression of inhibitor of apoptosis proteins in facial motoneurons of neonatal and adult rats following axotomy

The inhibitor of apoptosis protein family members inhibit cell death resulting from a variety of apoptotic stimuli. However, the endogenous expression of neuronal inhibitor of apoptosis proteins following axonal injury has not been thoroughly examined. Neonatal facial motoneurons are highly susceptible to axotomy-induced apoptosis, whereas adult facial motoneurons survive axotomy. We hypothesized that the endogenous expression of inhibitor of apoptosis proteins may be involved in the differential susceptibility of adult and neonatal facial motoneurons to axonal injury. In this study, we examined the expression of two endogenous inhibitor of apoptosis proteins, neuronal apoptosis inhibitory protein and x-linked inhibitory apoptosis protein, in adult and neonatal rat facial motoneurons following axotomy. Analyses using reverse-transcription polymerase chain reaction and in situ hybridization indicated that neuronal apoptosis inhibitory protein mRNA was increased in neonatal facial nuclei 24 h post axotomy. In the adult, neuronal apoptosis inhibitory protein mRNA expression increased at 1, 3, 7 and 14 days post axotomy, while little change in the expression of X-linked inhibitory apoptosis protein mRNA was detected at any age or time point time point analyzed. Interestingly, immunohistochemistry using antibodies for neuronal apoptosis inhibitory protein and X-linked inhibitory apoptosis protein, revealed the level of these proteins was higher in the neonatal motoneurons when compared with the adult. Furthermore, immunohistochemistry and western blot for neuronal apoptosis inhibitory protein revealed, in contrast to the observed increase in neuronal apoptosis inhibitory protein mRNA, a decline in the expression of neuronal apoptosis inhibitory protein following axotomy in the adult, whereas no change in neuronal apoptosis inhibitory protein was detected in neonatal facial motoneurons. X-linked inhibitory apoptosis protein, as analyzed by immunohistochemistry and western blot, remained unchanged by axotomy in neonatal motoneurons and adult motoneurons. These results indicate differential expression and/or turnover of inhibitor of apoptosis proteins in neonatal versus adult facial motoneurons, and suggest the level of inhibitor of apoptosis protein expression alone is not an indicator of cell fate following axotomy.

Increased incidence of gap junctional coupling between spinal motoneurones following transient blockade of NMDA receptors in neonatal rats.

Neonatal rat motoneurones are electrically coupled via gap junctions and the incidence of this coupling declines during postnatal development. The mechanisms involved in this developmental regulation of gap junctional communication are largely unknown. Here we have studied the role of NMDA receptor-mediated glutamatergic synaptic activity in the regulation of motoneurone coupling. Gap junctional coupling was demonstrated by the presence of graded, short latency depolarising potentials following ventral root stimulation, and by the transfer of the low molecular weight tracer Neurobiotin to neighbouring motoneurones. Sites of close apposition between the somata and/or dendrites of the dye-coupled motoneurones were identified as potential sites of gap junctional coupling. Early postnatal blockade of the NMDA subtype of glutamate receptors using the non-competitive antagonist dizocilpine maleate (MK801) arrested the developmental decrease in electrotonic and dye coupling during the first postnatal week. These results suggest that the postnatal increase in glutamatergic synaptic activity associated with the onset of locomotion promote the loss of gap junctional connections between developing motoneurones.

Neuroprotective action of melatonin on neonatal rat motoneurons after sciatic nerve transection

The neuronal isoform of nitric oxide synthase (nNOS), a NADPH-dependent diaphorase, is considered to play a role in motoneuron death induced by sciatic nerve transection in neonatal rats. Neuronal loss in these circumstances has been correlated with nitric oxide (NO) production and NADPH-diaphorase positivity in motoneurons after axotomy. In the present study we looked for a possible protective effect of melatonin, an antioxidant agent and inhibitor of nNOS, on spinal motoneurons after axonal injury. Neonatal Wistar rats (P2) were submitted to sciatic nerve transection and allowed to survive to P7. Melatonin at doses of 1, 5, 10, 50 and 100 mg/kg was given subcutaneously before and at intervals after the surgery. Controls operated in the same way received dilution vehicle or no treatment. The animals were killed by perfusion of fixative and the spinal cord was examined in serial paraffin sections. The motoneurons of the sciatic pool were counted in the axotomized and contralateral sides. Immunohistochemistry for nNOS and glial fibrillary acidic protein was used to evaluate nNOS expression in the axotomized cells and the astrocytic response. We found that melatonin at doses of 1–50 mg/kg decreased neuronal death. Astrocytic hypertrophy in melatonin treated animals was less intense. There were no differences in nNOS expression between treated and control rats, and surviving motoneurons of the sciatic pool did not express the enzyme, suggesting that nNOS may not be involved in neuronal death or survival in these experimental conditions. Possible mechanisms of melatonin neuroprotection, which was equally effective at doses of 1–50 mg/kg, are discussed. Doses of 50 and 100 mg/kg caused failure to thrive, seizures or death. The fact that neuroprotective doses were far smaller than toxic ones should encourage testing of melatonin in neurologic diseases.

Caspase‐3 is activated following axotomy of neonatal facial motoneurons and caspase‐3 gene deletion delays axotomy‐induced cell death in rodents

In this report, we examined the possible functions of the cell death protease, caspase-3, in the axotomy-induced apoptosis of facial motoneurons in newborn rodents. Using in situ hybridization and Western blot, we found higher levels of caspase-3 mRNA and pro-caspase-3 protein expression in motoneurons of neonatal and 2-week-old rats than adult rats. Following facial motoneuron axotomy, caspase-3 mRNA and protein expression increased in motoneurons of both neonatal and adult rats. However, using an antibody directed to the activated form of the caspase-3 protease, we found that catalytically active caspase-3 was present only in axotomized neonatal motoneurons. As motoneurons in neonatal but not adult rodents are susceptible to axotomy-induced apoptosis, we hypothesized that caspase-3 may play a role in their demise. To determine the necessity of caspase-3 activation in axotomy-induced apoptosis, we counted the number of surviving motoneurons at 4 and 7 days following axotomy in wild type mice and caspase-3 gene-deleted mice. There were nearly three times more surviving motoneurons in caspase-3 gene-deleted mice than in wild type mice at both 4 days (mean 1074 vs. 464, P<0.005) and 7 days (mean 469 vs. 190, P<0.005) following injury, indicating a slower rate of death. Examination of the dying motoneurons using TUNEL staining (for fragmented DNA) and bisbenzimide staining (for nuclear morphology) revealed incomplete nuclear condensation in caspase-3-deficient motoneurons. These results demonstrate that caspase-3 activation plays important roles in the rapid demise of axotomized neonatal motoneurons.

Induction of noradrenergic supersensitivity following cordotomy in neonatal rat spinal motoneurons

Patients with spinal cord lesions frequently show autonomic hyperreflexia. The mechanism of autonomic hyperreflexia has been thought to be an acute general autonomic overactivity in response to cutaneous or visceral stimuli, but it remains uncertain. Several kinds of experiments suggest that amplified spinal sympathetic reflexes in the decentralized cord are attributable to the denervation supersensitivity of denervated neurons, which is a well-known phenomenon in denervated muscle fibers. In the present study, changes in the supersensitivity of motoneurons after cordotomy were studied in the spinal cord of neonatal rats. Responses to bath-applied noradrenaline (NA) were recorded from a ventral root of the isolated spinal cord of 6-day-old rats. In normal spinal cords, NA induced depolarization in motoneurons dose-dependently. alpha 1-antagonist prazosin (3 microM) inhibited the deporalization induced by NA, and alpha 2-antagonist rauwolscine (1 microM) potentiated it. In one group of rats, cordotomy was performed 4 days after birth by complete transection of the spinal cord at vertebrate 8th-10th thoracic level, and NA response was examined two days later (when they were 6 days old). In cordotomized rats, NA-induced depolarization was increased with respect to both amplitude and duration. alpha 1- as well as alpha 2-antagonists inhibited the NA response in the spinalized rats. Especially, both antagonists shortened the duration of NA response as compared to normal level. It is concluded that the denervation supersensitivity to NA appears 2 days after cordotomy in the spinal motoneurons of neonatal rats and that the supersensitivity to NA is attributable to the upregulation of both alpha 1- and alpha 2-adrenoceptors on the motoneurons, indicating that a new type of alpha 2-adrenoceptor function appears.

Postinhibitory rebound during locomotor-like activity in neonatal rat motoneurons in vitro.

The aim of this study was to establish how a membrane property contributes to the neuronal discharge during ongoing behavior. We therefore studied the role of the postinhibitory rebound (PIR) in the bursting discharge of lumbar motoneurons intracellularly recorded in newborn rat in vitro brain stem/spinal cord preparation. The PIR is a transient depolarization that occurs after a hyperpolarization. We first investigated how it was expressed during experimentally induced hyperpolarizations. Its amplitude increased with the inhibition and was voltage dependent. The Ca2+ channel blockers Mn2+ and Co2+ partly suppressed the PIR in a few of the motoneurons tested. When hyperpolarized, the motoneurons exhibited a sag that was associated with the PIR. Adding caesium to the bath abolished both sag and rebound, which suggested that the PIR in the lumbar motoneurons was mainly due to the activation of the inward rectifying current IQ. In the second part, we studied the physiological involvement of PIR during fictive locomotion induced by bath application of N-methyl-D-L-aspartate and serotonin. We established that experimentally induced PIR could initiate or modulate the bursting discharge of motoneurons during fictive locomotion. We then studied whether the firing patterns of the motoneurons were correlated in one way with the synaptic inhibition. When the monosynaptic inhibitory input to the motoneurons was abolished with the glycinergic blocker strychnine, these neurons stopped discharging (although they still were depolarized rhythmically). The firing of action potentials was restored by applying negative current pulses. This study provides evidence as to how one membrane property in mammals is involved in a complex type of behavior, namely locomotion.

Dicarboxyphenylglycines antagonize AMPA- but not kainate-induced depolarizations in neonatal rat motoneurones

Ionotropic glutamate receptors have been categorized into three main groups according to the selective agonists that activate them, the N-methyl- d-aspartate (NMDA), ( S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propanoic acid (AMPA) and (2 S,3 S,4 S)-3-carboxymethyl-4-isopropenylpyrrolidine-2-carboxylic acid (kainate) receptors. Both AMPA and kainate induce depolarizations in neonatal rat spinal motoneurones. However, selective antagonists capable of discriminating between the effects of these two antagonists are not widely available. As part of a search for such compounds we report the actions of ( RS)-3,4-dicarboxyphenylglycine (DCPG) and ( RS)-3,5-dicarboxyphenylglycine on agonist-induced motoneuronal depolarizations in the neonatal rat spinal cord preparation. In addition, the actions of ( R)- and ( S)-3,4-DCPG are also described. ( RS)-3,4-DCPG and ( RS)-3,5-DCPG antagonized AMPA-induced depolarizations (apparent K d=137 μM ( n=3) and 167 μM ( n=5), respectively). However, ( RS)-3,5-DCPG (1 mM) potentiated responses due to kainate ( n=5) while ( RS)-3,4-DCPG (1 mM) displayed weak antagonism of these responses (apparent K d>12 mM, n=3). ( RS)-3,4- and ( RS)-3,5-DCPG at 500 μM both displayed antagonism at the NMDA receptor (apparent K d=472 μM and 346 μM, respectively) and a postsynaptic subgroup I metabotropic glutamate receptor activated by (1 S,3 R)-ACPD. The AMPA receptor antagonist activity of ( RS)-3,4-DCPG was shown to reside in the ( R)-enantiomer (apparent K d=77 μM, n=3). The same isomer was responsible for the NMDA receptor antagonism while showing little or no antagonism of kainate-induced depolarizations (apparent K d>3 mM, n=3), and a weak antagonistic effect at (1 S,3 R)-ACPD receptors. ( S)-3,4-DCPG (500 μM) was unable to antagonize kainate-induced depolarizations, showed weak or no antagonism of NMDA- and AMPA-induced depolarizations, but antagonized (1 S,3 R)-ACPD-induced depolarizations. Thus ( RS)-3,4-, ( RS)-3,5- and ( R)-3,4-DCPG demonstrate useful discrimination of responses due to AMPA and kainate, strongly suggesting that pharmacologically distinct AMPA and kainate receptors exist in motoneurones in the neonatal rat spinal cord.

Modulation of IH by 5-HT in Neonatal Rat Motoneurones In Vitro: Mediation through a Phosphorylation Independent Action of cAMP

The depolarization of adult and neonatal rat facial and spinal motoneurones by 5-hydroxytryptamine (5-HT) in part involves an enhancement of the hyperpolarization-activated, inward-rectifier, IH. Under experimental conditions which promote this action, 5-HT evokes an inward current which can be mimicked by intracellularly applied adenosine 3′:5′-cyclic monophosphate (cAMP) and potentiated by the cAMP-specific phosphodiesterase inhibitor Ro 20-1724. In this study, we show that this action of 5-HT can be blocked by the adenylyl cyclase inhibitors 2′3′-dideoxyadenosine (2′,3′-DDA), 5′-adenylimidodiphosphate (AMP-PNP) and SQ-22536 (9-(tetrahydro-2-furyl)adenine), but not by external or internal application of the protein kinase inhibitors H-7, staurosporine and chelerythrine. The most recently cloned 5-HT receptor subtypes, 5-HT4, 5-HT6 and 5-HT7, can all stimulate adenylyl cyclase when activated. In the presence of internal GTP-γ-S, 5-HT irreversibly enhanced IH. The 5-HT-induced inward current could be reversibly blocked by methysergide, but not by the 5-HT4 receptor antagonist GR-113808A, the 5-HT6 and 5-HT7 antagonist clozapine and the 5-HT1A antagonist WAY-100365. 5-Methoxytryptamine (5-MeOT) and 5-carboxamidotryptamine (5-CT) mimicked the action of 5-HT with a rank order of potency of 5-HT = 5MeOT > 5-CT. Surprisingly, 8-hydroxy-2-(di-N-propylamino)-tetralin (8-OH DPAT), a 5-HT1A and 5-HT7 agonist was inactive on facial motoneurones unlike its reported agonist action on spinal motoneurones. It is proposed that cAMP produced by 5-HT-mediated stimulation of adenylyl cyclase acts in a phosphorylation-independent manner, possibly directly, on the IH channel. The 5-HT receptor subtype mediating this response cannot be correlated with any of the classified 5-HT receptor subtypes that stimulate adenylyl cyclase. © 1997 Elsevier Science Ltd.

α-Methyl Derivatives of Serine-O-Phosphate as Novel, Selective Competitive Metabotropic Glutamate Receptor Antagonists

The antagonist selectivity and potency of two novel serine-O-phosphate derivatives (RS)-α-methylserine-O-phosphate (MSOP) and the monophenylester (RS)-α-methylserine-O-phosphate monophenyl-phosphoryl ester (MSOPPE) was investigated against l-2-amino-4-phosphonobutyrate (L-AP4)- and (1S,3S)-1-aminocyclopentane-1,3-dicarboxylate (ACPD)-induced depressions of the monosynaptic excitation of neonatal rat motoneurones, mediated via metabotropic glutamate receptors (mGluRs). MSOP was shown to be a selective antagonist for the L-AP4-sensitive presynaptic mGluR, displaying an apparent KD of 51 μM, compared to >700 μM for the (1S,3S)-ACPD-sensitive presynaptic mGluR. In contrast, MSOPPE displayed antagonist activity at both presynaptic mGluRs, with a three times greater selectivity for the (1S,3S)-ACPD-sensitive receptor over the L-AP4-sensitive mGluR (apparent KD values 73 μM and 221 μM, respectively). Therefore, on addition of an α-methyl group to the mGluR agonist serine-O-phosphate, we have developed an mGluR antagonist which is selective for the presynaptic L-AP4-sensitive receptor. In contrast, monoesterification of MSOP to give the monophenylphosphoryl ester (MSOPPE), confers a degree of selectivity for the (1S,3S)-ACPD-over the L-AP4-sensitive presynaptic mGluR. Neither MSOP nor MSOPPE had any activity on either postsynaptic mGluRs or ionotropic receptors.

α-methyl derivatives of serine-O-phosphate as novel, selective competitive metabotropic glutamate receptor antagonists

The antagonist selectivity and potency of two novel serine-O-phosphate derivatives ( RS)-α-methylserine-O-phosphate (MSOP) and the monophenylester ( RS)-α-methylserine-O-phosphate monophenyl-phosphoryl ester (MSOPPE) was investigated against l-2-amino-4-phosphonobutyrate (L-AP4)- and (1 S,3 S)-1-aminocyclopentane-1,3-dicarboxylate (ACPD)-induced depressions of the monosynaptic excitation of neonatal rat motoneurones, mediated via metabotropic glutamate receptors (mGluRs). MSOP was shown to be a selective antagonist for the L-AP4-sensitive presynaptic mGluR, displaying an apparent K D of 51 μM, compared to >700 μM for the (1 S,3 S)-ACPD-sensitive presynaptic mGluR. In contrast, MSOPPE displayed antagonist activity at both presynaptic mGluRs, with a three times greater selectivity for the (1 S,3 S)-ACPD-sensitive receptor over the L-AP4-sensitive mGluR (apparent K D values 73 μM and 221 μM, respectively). Therefore, on addition of an α-methyl group to the mGluR agonist serine-O-phosphate, we have developed an mGluR antagonist which is selective for the presynaptic L-AP4-sensitive receptor. In contrast, monoesterification of MSOP to give the monophenylphosphoryl ester (MSOPPE), confers a degree of selectivity for the (1 S,3 S)-ACPD-over the L-AP4-sensitive presynaptic mGluR. Neither MSOP nor MSOPPE had any activity on either postsynaptic mGluRs or ionotropic receptors.

Diastereoselective synthesis of all four isomers of 3-(4-chlorophenyl) glutamic acid: identification of the isomers responsible for the potentiation of L-homocysteic acid-evoked depolarizations in neonatal rat motoneurons.

All four isomers of 3-(4-chlorophenyl)glutamic acid (5-8) were prepared by diastereoselective synthesis. Addition of (6S)-(+)-bis-lactim ether 15 to cis-4-chlorocinnamate 12 gave a mixture comprising mainly the (2R,3S)- and (2R,3R)-isomers 5 and 6, respectively (in a ratio of 56:40), while addition of (6R)-(-)-bis-lactim ether 16 to 4-chlorocinnamate 12 gave a mixture comprising mainly the (2S,3R)- and (2S,3S)-isomers 8 and 7, respectively (in a ratio of 56:42). The four stereoisomers (5-8) were therefore conveniently prepared by addition of either 3-lithio-(6S)- or -(6R)-bis-lactim ether (15 or 16, respectively) to 4-chlorocinnamate 12 and separation of the resultant mixtures of diastereoisomers (23-26) by flash silica gel chromatography. The absolute configurations of 6 and 7 were confirmed by X-ray crystallography. Both the (2S,3S)- and (2S,3R)-isomers (7 and 8, respectively) at a concentration of 100 microM significantly potentiated depolarizations induced by 10 microM L-homocysteic acid (L-HCA) (% control +/- sem: 130.4 +/- 3.6, n = 20 and 114.5 +/- 2.4, n = 11, respectively) while the (2R,3S)-isomer 5 significantly reduced L-HCA response amplitude (94.2 +/- 1.4, n = 9) and the (2R,3R)-isomer 6 was inactive. Experiments designed to compare the agonist-potentiating actions of 7 and 8 in the neonatal rat spinal cord with L-trans-pyrrolidine-2,4-dicarboxylic acid, the well-known L-Glu uptake inhibitor, provided additional evidence for the selective enhancement of depolarizations due to L-HCA and not those due to L-Glu. This selective action supports the existence of multiple excitatory amino acid uptake sites.

Differential actions of 3-(4-chlorophenyl) glutamic acid stereoisomers and l- trans-pyrrolidine-2,4-dicarboxylic acid upon l-homocysteic acid- and l-glutamic acid-induced responses from rat spinal motoneurones

The four recently synthesized Stereoisomers of 3-(4-chlorophenyl) glutamic acid (chlorpheg) were individually examined for their abilities to potentiate depolarizations of neonatal rat motoneurones evoked by l-homocysteic acid ( l-HCA, 10 μM). This property had previously been observed using the racemate and is believed to be mediated by uptake inhibition. Both the (2 S,3 S)- and (2 S,3 R)- isomers were selective potentiators of l-HCA- (vs l-Glu) induced depolarizations although the (2 S,3 S)- isomer was more effective. The (2 R,3 S)- isomer had a slight but significant depressant action which could be attributed to N-methyl- d-aspartate (NMDA) receptor antagonism. Comparison of the potentiating properties of (2 S,3 S)- and (2 S,3 R)-chlorpheg with those of l- trans-pyrrolidine-2,4-dicarboxylic acid ( tPDC, a l-Glu uptake inhibitor) upon l-HCA- and l-Glu-evoked responses revealed that both chlorpheg isomers (500 μM each) selectively potentiated responses evoked by l-HCA (10 μM) but had no significant effect upon those evoked by l-Glu (50 μM). On the other hand, use of tPDC at the same concentration significantly enhanced the depolarizations evoked by both amino acids, although its action on l-Glu-evoked responses was greater. It is concluded that (i) the (2 S,3 S)- isomer and to a lesser extent, the (2 S,3 R)- isomer of chlorpheg are responsible for the potentiating actions seen with the chlorpheg racemate used in previous studies and (ii) (2 R,3 S)-chlorpheg is a weak NMDA antagonist. The apparently selective action of (2 S,3 S)- and (2 S,3 R)-chlorpheg upon l-HCA- relative to l-Glu-induced depolarizations supports the existence of multiple excitatory amino acid uptake sites, some of which may yet be unidentified.