Neuroprotective actions in vivo and electrophysiological actions in vitro of 202W92.

202W92 (R-(-)-2,4-diamino-6-(fluromethyl)-5-(2,3,5-trichlorophenyl)pyrimidine) is a novel compound in the same chemical series as the antiepileptic drug lamotrigine and the neuroprotective sipatrigine. Here 202W92 was quantitatively assessed as a neuroprotective agent in focal cerebral ischaemia, and as an inhibitor of sodium and calcium channels and of synaptic transmission. In the rat permanent middle cerebral artery occlusion (MCAO) model of acute focal ischaemia, 202W92 reduced infarct volume by 75% in cortex and by 80% in basal ganglia, with ED(50) approximately 2 mg/kg (single i.v. dose, 10 min post-occlusion). In whole-cell current recordings from single cells, 202W92 completely and reversibly inhibited voltage gated sodium channels (IC(50) 3 x 10(-6) M) in rat freshly-isolated cortical neurons and in the GH(3) pituitary cell line. 202W92 also inhibited a nifedipine-sensitive fraction (approximately 35%) of native high-voltage-activated (HVA) calcium current in rat cortical neurons (IC(50) 15 x 10(-6) M) and weakly inhibited low-voltage-activated (LVA) calcium currents of the recombinant alpha1I-mediated T-type (IC(50)>100 x 10(-6) M). The drug inhibited the amplitude and frequency of 4-aminopyridine-evoked glutamatergic excitatory post-synaptic currents (EPSCs). In conclusion, 202W92 is an effective neuroprotective agent when administered post-ischaemia and a potent sodium channel inhibitor in vitro.

Effects of extracellular pH on the interaction of sipatrigine and lamotrigine with high-voltage-activated (HVA) calcium channels in dissociated neurones of rat cortex.

Acidic extracellular pH reduced high-voltage-activated (HVA) currents in freshly isolated cortical pyramidal neurones of adult rats, shifting activation to more positive voltages (V(1/2)=-18 mV at pH 7.4, -11 mV at pH 6.4). Sipatrigine inhibited HVA currents, with decreasing potency at acidic pH (IC(50) 8 microM at pH 7.4, 19 microM at pH 6.4) but the degree of maximal inhibition was >80% in all cases (pH 6.4-8.0). Sipatrigine has two basic groups (pK(A) values 4.2, 7.7) and at pH 7.4 is 68% in monovalent cationic form and 32% uncharged. From simple binding theory, the pH dependence of sipatrigine inhibition indicates a protonated group with pK(A) 6.6. Sipatrigine (50 microM) shifted the voltage dependence of channel activation at pH 7.4 (-7.6 mV shift) but not at pH 6.4. Lamotrigine has one basic site (pK(A) 5.5) and inhibited 34% of the HVA current, with similar potency over the pH range 6.4--7.4 (IC(50) 7.5--9 microM). These data suggest that the sipatrigine binding site on HVA calcium channels binds both cationic and neutral forms of sipatrigine, interacts with a group with pK(A)=6.6 and with the channel activation process, and differs from that for lamotrigine.

The activation of mu opioid receptors promotes a small modulation of calcium currents in rat pallidal neurons.

Globus pallidus receives, from dorsal neostriatum, a dense enkephalinergic innervation whose role is still uncertain. We examined the possibility that the activation of mu, delta or k opioid receptors modulate high-voltage-activated calcium currents in isolated GP neurons. Neither dynorphin nor DPEPE inhibited calcium current, whilst DAMGO produced a small (-16%) but consistent response, selectively antagonized by CTOP. The mu-mediated modulation required the activation of G-proteins but was voltage-independent. The pre-incubation in omega-conotoxinVIA abolished the response, implying the involvement of N-type calcium channels. These findings suggest that enkephalin may exert a direct influence on GP excitability also through post-synaptic effects. In degenerative conditions as Parkinsonism, an excessive stimulation of mu binding sites might induce a pathological inhibition of calcium signals, thus contributing to modify the GP firing pattern and transmitter release.

The effects of gabapentin on different ligand- and voltage-gated currents in isolated cortical neurons.

A clear picture of the mechanisms of action of the anti-epileptic agent gabapentin is far from being accomplished. We have analyzed the effects of gabapentin on ligand- and voltage-gated currents in isolated adult rat cortical neurons. Gabapentin failed to modify glutamate currents and produced a slight reduction of GABA responses. Negligible inhibition of sodium, but consistent inhibition of high-voltage-activated calcium conductance was promoted by gabapentin. In addition, gabapentin reduced calcium current sensitivity to dihydropyridine agonist and antagonists. Interestingly, gabapentin also decreased a not-inactivating, cadmium-sensitive, potassium current. These unconventional effects might underlie its efficacy in a variety of diseases which involve periodic discharge patterns as neuropathic pain or essential tremor.

Selective vulnerability of pallidal neurons in the early phases of manganese intoxication.

Prolonged exposure to manganese in mammals may cause an extrapyramidal disorder characterized by dystonia and rigidity. Gliosis in the pallidal segments underlies the well-established phase of the intoxication. The early phase of the intoxication may be characterized by psychic, nonmotor signs, and its morphological and electrophysiological correlates are less defined. In a rat model of manganese intoxication (20 mg/ml in drinking water for 3 months), neither neuronal loss nor gliosis was detected in globus pallidus (GP). However, a striking vulnerability of manganese-treated GP neurons emerged. The majority of GP neurons isolated from manganese-treated rats died following brief incubation in standard dissociation media. In addition, patch-clamp recordings in the whole-cell configuration were not tolerated by surviving GP neurons. Neither coeval but untreated GP neurons nor striatal ones manifested analogous susceptibility. Using the perforated-patch mode of recording we attempted at identifying the functional hallmarks of GP vulnerability: in particular, voltage-gated calcium currents and glutamate-induced currents were examined. Manganese-treated GP neurons exhibited calcium currents similar to control cells aside from a slight reduction in the dihydropyridine-sensitive current facilitation. Strikingly, manganese-treated GP cells--but not striatal ones--manifested peculiar responses to glutamate, since repeated applications of the excitatory amino acid, at concentrations which commonly promote desensitizing responses, produced instead an irreversible cell damage. Possible mechanisms are discussed.

The modulation of calcium current by GABA metabotropic receptors in a sub-population of pallidal neurons.

Globus pallidus (GP) receives an abundant GABAergic (gamma-aminobutyric acid) pathway from the corpus striatum. Several evidences suggested that alterations of this pathway might underlie the development of movement disorders. Classical models on Parkinsonism are centred on the increased excitability of GABAergic striatofugal neurons impinging GP and, therefore, on the presumed hypoactivity of GP neurons, but very few electrophysiological studies have addressed the activation of GABA receptors in mammalian GP. We have isolated calcium currents in GP neurons dissociated from the adult rat brain and analysed GABA-mediated responses. In the presence of bicuculline, the fast, chloride-mediated, ionotropic responses were obscured and GABA produced a large (>/= 35%) inhibition of calcium currents. The GABA-induced inhibition of calcium currents strongly desensitized was mimicked by baclofen and prevented by hydroxy-saclofen, supporting the involvement of GABAB receptors. The baclofen-mediated modulation was: (i) associated with slowing of activation kinetics; (ii) relieved by prepulse facilitation; and (iii) G-protein-mediated. The response was slow in onset, requiring the mobilization of intracellular cAMP, and was abolished by the combination of N-type and P-type calcium channel blockers. The GABAB-mediated effect, however, was confined to a particular subtype of GP neurons, identified by relatively small to medium soma. Differently, in cells characterized by larger somata and capacitance, the baclofen response was negligible. Intriguingly, these baclofen-resistant, larger neurons manifested a consistent low-voltage-activated (LVA) calcium current, not detected in baclofen-sensitive cells, at least when recorded in whole-cell mode. This study demonstrates that GP neurons express functional GABAA and GABAB receptors. In a subset of GP neurons, the activation of GABAB receptors induces a large modulation of high-voltage-activated (HVA) calcium currents, which may strongly influence basal ganglia circuitry and partially explain some discrepancies of classical models of extrapyramidal disorders.

Brain excitability and electroencephalographic activation: non-invasive evaluation in healthy humans via transcranial magnetic stimulation.

Excitability changes of the central motor tracts as a function of the electroencephalographic (EEG) characteristics has been investigated in 10 healthy volunteers. Transcranial magnetic stimulation (TCS) was administered to the right motor cortex with an intensity 5-10% above threshold for the elicitation of motor evoked potentials (MEPs) in the left forearm muscles. Simultaneously, the right median nerve was stimulated to provoke an H-reflex in the forearm flexors and EEG activity was recorded from the left hemiscalp. Subjects were completely relaxed and were asked at random either to keep the eyes closed while maintaining mental inactivity (A) or to open their eyes and perform mental arithmetics (B). Latencies and amplitudes of MEPs and H-reflexes were statistically matched with the spectral content of the EEG. In condition A, MEPs of 119 +/- 61 microV, with up to 36% of missing responses and background EEG activity dominated by rhythms in the alpha range were found. In condition B, MEPs of 219 +/- 66 microV (P less than 0.001), with less than 16% of missing responses, 'blocking' of the background alpha rhythms, and a potentiation of the faster ones' relative power were observed. Changes of the H-reflex characteristics were neither statistically significant nor related to MEP amplitude and EEG spectral profile fluctuations.

Analysis of sympathetic skin responses in a group of healthy subjects and in patients affected by sphincter disorders and impotence.

Bilateral palmar and plantar sympathetic skin responses (SSRs) to pudendal and median nerves stimulation have been recorded in a population of 10 healthy adult subjects. A group of 11 patients affected by sphincter disorders and/or impotence of neurological origin (diabetic neuropathy, ischaemic myelopathy, myelitis, cauda aequina syndrome) was investigated and results were compared with normal values obtained from the control group. The technique is focused on the vegetative component of innervation and is shown to give complementary information on sympathetic dysfunction, in addition to traditional techniques relating to somatic innervation (electroneurography, electromyography, somatosensory evoked potentials.

Monitoring of subcortical and cortical somatosensory evoked potentials during carotid endarterectomy: comparison with stump pressure levels.

Monitoring of multichannel somatosensory evoked potentials (SEPs) has been performed in 40 cases of carotid endarterectomy (CEA). SEPs were obtained after median nerve stimulation at wrist, recording from 2nd cervical and from the scalp parietal (ipsi- and contralateral) and central (contralateral) positions. The reduction of CBF due to clamping of the carotid artery provoked SEP abnormalities in 10 of the 40 cases. None of the 30 patients with unmodified SEPs developed post-surgical neurological sequelae. SEP alterations were characterized exclusively by amplitude decrements and latency increases of the cortical components, the subcortical ones being unaffected. In 5 of these patients, SEPs returned to normal values before the end of the intervention and no neurological deficit was observed on awakening. In the remaining 5 cases SEPs retained their abnormalities and patients developed post-surgery neurological sequelae (4 immediately, 1 the day after). SEP alterations affected parietal and central components to a similar extent; however, in a few cases cerebral blood flow deficits provoked by carotid clamping modified differently the central P22 and the parietal N20-P25 waves. Comparisons with stump (back) pressure in the carotid artery revealed a higher sensitivity of the SEP technique in detecting vascularization problems due to carotid clamping. The time course of the appearance of SEP abnormalities seems to discriminate alterations secondary to collateral revascularization from those determined by embolization.

Non-clinical tests for the diagnosis of multiple sclerosis.

Liquoral, Neurophysiological and Nuclear Imaging data were investigated in 29 patients suffering from Definite (11 cases), Probable (11 cases) and Possible (7 cases) Multiple Sclerosis (MS). The most sensitive tests were: Visual Evoked Potentials (VEPs), altered in 75% of patients, Motor Evoked Potentials (MEPs), abnormal in 75%, Nuclear Magnetic Resonance Imaging (MRI) in 72.4%. Somatosensory Evoked Potentials (SEPs) were altered in 69.2% in PN-SEPs and in 53.6% in MN-SEPs, whilst Brainstem Evoked Potentials (BAEPs) were altered in 30.8% of cases. The results suggest that patients with suspected forms of MS should be submitted, as routine diagnostic protocol, to a battery of EPs, including as a first choice VEPs, MEPs and PN-SEPs. MRI should be performed to confirm a diagnosis not supported by EP data, or for a comprehensive localization of the demyelinating plaques.