Postsynaptic factors in the expression of long-term potentiation (LTP): increased glutamate receptor binding following LTP induction in vivo

Several lines of evidence indicate that LTP in the hippocampus is associated with a change in the properties of postsynaptic glutamate receptors. In the present study, we used quantitative autoradiography to examine the binding properties of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) and N-methyl-D-aspartate subclasses of glutamate receptors in frozen brain sections obtained from rats in which perforant-path LTP was induced in vivo. Induction of LTP resulted in a selective increase in [3H]AMPA binding in those hippocampal subfields receiving perforant-path axons. Increases in [3H]AMPA binding in dentate gyrus (stratum moleculare) were highly correlated with the magnitude of LTP recorded in this structure. Scatchard analyses of [3H]AMPA and 6-cyano-7-nitro-[3H]quinoxaline-2,3-dione (an AMPA receptor antagonist) binding in the dentate gyrus indicated that LTP induction resulted in an increase in the number of AMPA receptor binding sites. No changes in the binding of 3H-labeled N-[1-(thienyl)cyclohexyl]piperidine (an N-methyl-D-aspartate receptor antagonist) were observed in any hippocampal subfield. These results suggest that a modification in postsynaptic AMPA receptors plays a role in the expression of synaptic enhancement following LTP induction in the hippocampus.     

Metabotropic glutamate receptors are involved in long-term potentiation in isolated slices of rat medial frontal cortex

The prelimbic region of medial frontal cortex in the rat receives a direct input from the hippocampus and this functional connection is essential for aspects of spatial memory. Activity-dependent changes in the effectiveness of synaptic transmission in the medial frontal cortex, namely long-term potentiation (LTP) and long-term depression (LTD) can persist for tens of minutes or hours and may be the basis of learning and memory storage. Glutamatergic activation of ionotropic receptors is required to induce both LTP and LTD. We now present evidence of the involvement of metabotropic glutamate receptors in LTP in isolated slices of frontal cortex. Repetitive bursts of stimulation at theta frequencies (TBS) were applied to layer II, and monosynaptic EPSPs were monitored in layer V neurons of the prelimbic area. TBS was found to be more effective at inducing LTP than tetanic stimulation at 100 Hz and produced LTP that lasted >30 min in 8 out of 14 neurons. Tetanic stimulation at 100 Hz in the presence of the N-methyl--aspartate (NMDA)-antagonist 2-amino-5-phosphonopentanoate (AP5) was reported to be a reliable method of inducing LTD in prelimbic cortex (). However we found that this protocol did not facilitate the induction of LTD. The role of metabotropic glutamate receptors (mGluR) in LTP was assessed by using the selective, broad-spectrum antagonist (R, S)-alpha-methyl-4- carboxyphenylglycine (MCPG). This drug significantly reduced the incidence of LTP after TBS to only 1 of 14 neurons (P < 0.02, chi2 test). The pooled responses to TBS in MCPG showed significantly reduced potentiation [(P < 0.02, analysis of variance (ANOVA)]. The broad-spectrum mGluR agonist (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) and the selective group I agonist S-3 hydroxyphenylglycine(S-3HPG) both produced membrane depolarization, an increase in number of spikes evoked by depolarizing current pulses, and a reduction in the afterhyperpolarization. Similar effects were produced by these agonists even when synaptic transmission was blocked by use of the gamma-aminobutyric acid-B (GABAB) receptor agonist, 200 microM baclofen, which suggests that group I mGluRs are present on layer V neurons. We conclude that mGluRs participate in the production of LTP in prelimbic cortex, and that this excitatory effect could be mediated by the postsynaptic group I mGluRs.     

Membrane currents evoked by ionotropic glutamate receptor agonists in rod bipolar cells in the rat retinal slice preparation

1. With the use of the whole cell voltage-clamp technique, I have recorded the current responses to ionotropic glutamate receptor agonists of rod bipolar cells in vertical slices of rat retina. Rod bipolar cells constitute a single population of cells and were visualized by infrared differential interference contrast video microscopy. They were targeted by the position of their cell bodies in the inner nuclear layer and, after recording, were visualized in their entirety by labeling with the fluorescent dye Lucifer yellow, which was included in the recording pipette. To study current-voltage relationships of evoked currents, voltage-gated potassium currents were blocked by including Cs+ and tetraethylammonium+ in the recording pipette. 2. Pressure application of the non-N-methyl-D-aspartate (non-NMDA) receptor agonists kainate and (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) from puffer pipettes evoked a long-latency conductance increase selective for chloride ions. When the intracellular chloride concentration was increased, the reversal potential changed, corresponding to the change in equilibrium potential for chloride. The response was evoked in the presence of 5 mM Co2+ and nominally O mM Ca2+ in the extracellular solution, presumably blocking all external Ca2(+)-dependent release of neurotransmitter. 3. The long latency of kainate-evoked currents in bipolar cells contrasted with the short-latency currents evoked by gamma-aminobutyric acid (GABA) and glycine in rod bipolar cells and by kainate in amacrine cells. 4. Application of NMDA evoked no response in rod bipolar cells. 5. Coapplication of AMPA with cyclothiazide, a blocker of agonist-evoked desensitization of AMPA receptors, enhanced the conductance increase compared with application of AMPA alone. Coapplication of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocked the response to kainate and AMPA, indicating that the response was mediated by conventional ionotropic glutamate receptors. 6. The conductance increase evoked by non-NMDA receptor agonists could not be blocked by a combination of 100 microM picrotoxin and 10 microM strychnine. Application of the GABAC receptor antagonist 3-aminopropyl (methyl)phosphinic acid (3-APMPA) strongly reduced the response, and coapplication of 500 microM 3-APMPA and 100 microM picrotoxin completely blocked the response. These results suggested that the conductance increase evoked by non-NMDA receptor agonists was mediated by release of GABA and activation of GABAC receptors, and most likely also GABAA receptors, on rod bipolar cells. 7. Kainate responses like those described above could not be evoked in bipolar cells in which the axon had been cut somewhere along its passage to the inner plexiform layer during the slicing procedure. This suggests that the response was dependent on the integrity of the axon terminal in the inner plexiform layer, known to receive GABAergic synaptic input from amacrine cells. 8. The results indicate that ionotropic glutamate receptors are not involved in mediating synaptic input from photoreceptors to rod bipolar cells and that an unconventional mechanism of GABA release from amacrine cells might operate in the inner plexiform layer.     

Differential expression of glutamate decarboxylase messenger RNA in cerebellar Purkinje cells and deep cerebellar nuclei of the genetically dystonic rat

The genetically dystonic rat exhibits a motor syndrome that closely resembles the human disease, generalized idiopathic dystonia. Although in humans dystonia is often the result of pathology in the basal ganglia, previous studies have revealed electrophysiological abnormalities and alterations in glutamate decarboxylase, the synthetic enzyme for GABA, in the cerebellum of dystonic rats. In this study, we further characterized the alterations in cerebellar GABAergic transmission in these mutants by examining the expression of the messenger RNA encoding glutamate decarboxylase (67000 mol. wt) with in situ hybridization histochemistry at the single cell level in Purkinje cells and neurons of the deep cerebellar nuclei. Glutamate decarboxylase (67000 mol. wt) messenger RNA levels were increased in the Purkinje cells and decreased in the deep cerebellar nuclei of dystonic rats compared to control littermates, suggesting opposite changes in GABAergic transmission in Purkinje cells and in their target neurons in the deep cerebellar nuclei. In contrast, levels of glutamate decarboxylase (67000 mol. wt) messenger RNA in the pallidum, and of enkephalin messenger RNA in the striatum, were unaffected in dystonic rats. The data indicate that both the Purkinje cells and GABAergic neurons of the deep cerebellar nuclei are the site of significant functional abnormality in the dystonic rat.     

Subtype-specific involvement of metabotropic glutamate receptors in two forms of long-term potentiation in the dentate gyrus of freely moving rats

In this study, the role of metabotropic glutamate receptors in N-methyl-D-aspartate receptor-dependent and voltage-gated calcium channel-dependent long-term potentiation in the dentate gyrus of freely moving rats was investigated. Antagonists for group 1 metabotropic glutamate receptors ((S)-4-carboxyphenylglycine), group 1/2 metabotropic glutamate receptors ((RS)-alpha-methyl-4-carboxyphenylglycine) and group 2 metabotropic glutamate receptors ((RS)-alpha-methylserine O-phosphate monophenylester) were used. The N-methyl-D-aspartate receptor antagonist, D(-)-2-amino-5-phosphonopentanoic acid, and the L-type voltage-gated calcium channel antagonist, methoxyverapamil were used to investigate the N-methyl-D-aspartate receptor and voltage-gated calcium channel contribution to the long-term potentiation recorded. Field excitatory postsynaptic potential slope and population spike amplitude were measured. Drugs were applied, prior to tetanus, via a cannula implanted into the lateral cerebral ventricle. 200 Hz tetanization produces a long-term potentiation which is inhibited by application of D(-)-2-amino-5-phosphonopentanoic acid and (RS)-alpha-methyl-4-carboxyphenylglycine. In this study, a dose-dependent inhibition of 200 Hz long-term potentiation expression was obtained with (S)-4-carboxyphenylglycine. Long-term potentiation induced by 400 Hz tetanization was not inhibited by D(-)-2-amino-5-phosphonopentanoic acid, although the amplitude of short-term potentiation was reduced. (RS)-alpha-methyl-4-carboxyphenylglycine and (S)-4-carboxyphenylglycine, both in the presence and absence of D(-)-2-amino-5-phosphonopentanoic acid, inhibited the development of 400 Hz long-term potentiation. (RS)-alpha-methylserine O-phosphate monophenylester had no significant effect on long-term potentiation induced by either 200 or 400 Hz tetanization. Application of methoxyverapamil significantly inhibited 400 Hz long-term potentiation, but had no effect on 200 Hz long-term potentiation. These data suggest that 400 Hz long-term potentiation, induced in the presence of D(-)-2-amino-5-phosphonopentanoic acid, requires activation of L-type calcium channels. Furthermore, these results strongly support a critical role for group 1 metabotropic glutamate receptors in both N-methyl-D-aspartate receptor- and voltage-gated calcium channel-dependent long-term potentiation.     

Dihydrokainate-sensitive neuronal glutamate transport is required for protection of rat cortical neurons in culture against synaptically released glutamate

Glutamate transport in nearly pure rat cortical neurons in culture (less than 0.2% astrocytes) is potently inhibited by dihydrokainate, l-serine-O-sulphate, but not by l-alpha-amino-adipate. This system allows for a test of the hypothesis that glutamate transport is important for protecting neurons against the toxicity of endogenous synaptically released glutamate. In support of this hypothesis, a 20-24 h exposure to 1 mm dihydrokainate reduced cell survival to only 14.8 +/- 9.8% in neuronal cultures (P < 0.001; n = 3), although it had no effect on neuronal survival in astrocyte-rich cultures (P > 0.05; n = 3). Dihydrokainate also significantly caused accumulation of glutamate in the extracellular medium of cortical neuronal cultures (6.6 +/- 4.9 micrometer, compared to 1.2 +/- 0.3 micrometer in control, n = 14, P < 0.01). The neurotoxicity of dihydrokainate was blocked by 10 micrometer MK-801, 10 micrometer tetrodotoxin, and an enzyme system that degrades extracellular glutamate. The latter two also abolished the accumulation of glutamate in the extracellular medium. Dihydrokainate (1 mm) inhibited the 45calcium uptake stimulated by 30 micrometer N-methyl-d-aspartate (NMDA), but not by higher concentrations consistent with a weak antagonist action of dihydrokainate at the NMDA receptor. Whole cell recordings showed that 1 mm dihydrokainate produced approximately 25% inhibition of 30 micrometer NMDA-induced current in cortical neurons. Dihydrokainate (1 mm) alone generated a small current (17% of the current produced by 30 micrometer NMDA) that was blocked by 30 micrometer 5,7-dichlorokynurenate and only weakly by 10 micrometer cyano-7-nitroquinoxaline-2,3-dione (CNQX). These results suggest that the toxicity of dihydrokainate in neuronal cultures is due to its ability to block glutamate transport in these cultures, and that dihydrokainate-sensitive neuronal glutamate transport may be important in protecting neurons against the toxicity of synaptically released glutamate.     

Development of cerebellar somatosensory evoked potentials in the rat

The effect of radio and microwave radiation on dietary atherosclerosis of rabbits was tested. 16 New Zealand white rabbits were exposed to microwave (2.45 GHz) radiation at a power density of 20--30 mW/cm2 for 4 h a day, 5 days a week for 8 to 10 weeks. Irradiated animals had serum cholesterol concentrations, aortic wall cholesterol concentrations and percentage of intimal surface involved in atherosclerotic lesions which were not different from age and weight matched controls. Continuous radio frequency irradiation (1 MHz) for 8 to 11 weeks with a field strength of 30 V/cm also failed to change these indices of atherogenesis. We conclude that under the conditions of these experiments radio and microwave irradiation do not affect the course of diet induced atherogenesis.     

Different central and peripheral responses to leptin in rhesus monkeys: brain transport may be limited

The purpose of this experiment was to determine the effect of leptin administration on food intake and energy expenditure in rhesus monkeys. Four adult male rhesus monkeys, cannulated in the left lateral cerebral ventricle, were used for all phases of this experiment. Food intake was measured following intracerebroventricular injections of vehicle or three doses (500 ng, 2 micrograms, and 22 micrograms) leptin. Leptin administration resulted in a dose-dependent decrease in food intake (P < 0.05), with food intake decreased by an average of 54% at 22 micrograms leptin. Energy expenditure was also measured at two intracerebroventricular doses of leptin. Energy expenditure was not different (P > 0.10) between placebo and leptin injections at either dose. Food intake was also measured following i.v. injection of 3 mg leptin. In this case, leptin did not alter (P > 0.10) food intake, despite increasing serum leptin levels by as much as 100-fold. These results suggest that leptin is a potent inhibitor of food intake in rhesus monkeys, but this effect requires elevation of leptin concentrations in the cerebrospinal fluid or critical brain sites. The transport system for movement of leptin across the blood-brain barrier may limit the influence of circulating leptin on food intake in monkeys.     

Effects of nocodazole and taxol on glycine evoked currents on rat spinal cord neurones in culture

We examined the effect of altering the cytoskeleton polymerization state by treatment with nocodazole and taxol on glycine-evoked currents in patch-clamp recordings from cultured spinal cord neurones. Adding ATP and GTP to the pipette solution did not prevent the rundown of the peak current. In the absence or in the presence of ATP, the proportion of the non-desensitizing part of the glycine evoked-current declined with time. Adding intracellular GTP and ATP stabilized glycine-evoked responses although the proportion of non-inactivating current was reduced. Nocodazole reduced by itself the proportion of the non-inactivating current whereas taxol (with ATP and GTP) had an opposite effect. These results suggest that the polymerization state of microtubules has functional consequences on glycine receptors.     

Two different forms of long-term potentiation in the hippocampus

Several forms of long-term potentiation (LTP), a putative cellular mechanism for memory storage, have been described in the hippocampus. In this review, I discuss the mechanisms of induction and expression of LTP at the Schaffer collateral synapses and at the mossy fiber pathway. The early biochemical steps responsible for LTP at these two pathways are well understood. However, future studies should transcend the study of signal transduction systems and focus on the identification of the synaptic proteins that experience activity-dependent modifications, ultimate effectors of the plastic changes.     

Calretinin expression as a critical component in the control of dentate gyrus long-term potentiation induction in mice

We have recently reported that mice homozygous (Cr-/-) for a null mutation in the calretinin gene have impaired long-term potentiation (LTP) induction in the dentate gyrus (S. Schurmans et al. (1997) Proc. Natl. Acad. Sci. USA, 94, 10415 ). Here, we investigated dentate LTP induction in mice heterozygous (Cr+/-) for the same mutation. Despite the presence of calretinin in neurons of these mice, although at reduced levels as compared with normal mice, LTP induction in dentate gyrus was totally impaired. Spatial memory and learning were found unaffected in Cr+/- mice, such as in Cr-/- mice. Altogether, our results suggest that calretinin is a critical component in the control of dentate synaptic plasticity in mice, and that levels of calretinin higher than those observed in Cr+/- mice are required to induce LTP in this area. The possible mechanisms leading to the absence of correlation between gene dosage and biological effects are discussed.     

Hippocampal long-term potentiation preserves the fidelity of postsynaptic responses to presynaptic bursts

Hippocampal cells often fire prolonged bursts of action potentials, resulting in dynamic modulation of postsynaptic responses; yet long-term potentiation (LTP) has routinely been studied using only single presynaptic stimuli given at low frequency. Recent work on neocortical synapses has suggested that LTP may cause a "redistribution of synaptic strength" in which synaptic responses to the first stimulus of a presynaptic burst of action potentials are potentiated with later responses depressed. We have examined whether this redistribution occurs at hippocampal synapses during LTP. Using prolonged bursts that result in maximal short-term depression of later responses within the burst, we found that LTP resulted in a uniform potentiation of individual responses throughout the burst rather than a redistribution of synaptic strength. This occurred both at Schaffer collateral-CA1 synapses and at CA3-CA3 synapses, the latter being activated and monitored using paired recordings. Thus in the hippocampus, LTP preserves the fidelity of postsynaptic responses to presynaptic bursts by a uniform increase rather than a redistribution of synaptic strength, a finding that suggests there are important differences between neocortex and hippocampus in how long-term changes in synaptic strength are used to encode new information.     

Group 1 metabotropic glutamate receptors contribute to slow-onset potentiation in the rat CA1 region in vivo

It has been demonstrated in the CA1 region of the hippocampus in vitro, and in the dentate gyrus and CA1 region in vivo, that application of the metabotropic glutamate receptor (mGluR) agonist, 1S, 3R-amino cyclopentane 2,3-dicarboxylic acid triggers a slow-onset potentiation of synaptic transmission in the hippocampus. This study examined the involvement of group 1 and 2 mGluRs in this phenomenon in the CA1 region of freely moving rats. Drugs were applied via the lateral cerebral ventricle, and measurements were obtained from the CA1 region via permanently implanted electrodes. The group 1 mGluR agonists, 3,5-dihydroxyphenylglycine (DHPG, 20-100 nmol/5 microl) and trans-azetidine-2,4-dicarboxylic acid (ADA, 100 nmol-1 micromol/5 microl) induced a dose-dependent potentiation of basal synaptic transmission. The mGluR antagonist R,S-alpha-methyl-carboxyphenylglycine (MCPG, 1 micromol), and the group 1 mGluR antagonist, S-4-carboxyphenylglycine (4CPG, 100 nmol) completely inhibited the effects of both DHPG and ADA. The group 2 mGluR agonist, (S)-4-carboxy-3-hydroxy phenylglycine (4C3H-PG, 50-200 nmol/5 microl) induced a dose-dependent decrease of basal synaptic transmission. These results suggest that in the CA1 region in vivo, slow-onset potentiation may be mediated by group 1 mGluRs.     

Experimental brain injury induces expression of amyloid precursor protein, which may be related to neuronal loss in the hippocampus

Previous reports have demonstrated that some focal brain injuries increase amyloid precursor protein (APP) immunoreactivity in the region surrounding the injury where it was localized, in damaged axons and in pre-alpha 2 cells of the entorhinal cortex. However, to date, APP expression in the hippocampus remote from the impact site has not been comprehensively studied. Therefore, we have evaluated APP expression not only in the locally injured cerebral cortex but also in the hippocampus remote from the impact site. In the present paper, diffuse axonal injury was induced in rats in midline fluid percussion injury. APP expression was examined post injury using Western blot analysis and immunohistochemistry. Western blot analysis demonstrated that the expression of 100-kd APP was increased in both the cerebral cortex and hippocampus 24 h after injury. It then decreased in the hippocampus, but did not change in the cerebral cortex, 7 days after injury. Immunohistochemical studies showed increased immunoreactivity of APP in the neuronal perikarya and reactive astrocytes near the region of injury in the cerebral cortex 24 h to 7 days after injury. In the hippocampus, APP accumulated in the CA3 neurons 24 h and 3 days after injury, although no hemorrhagic lesions were seen at that site. The APP positive neurons in CA3 showed shrunken cell bodies and pyknotic nuclei 3 days after injury, and some of the neurons in CA3 had disappeared by 7 days postinjury. The results of present study suggest that traumatic brain injury induces overexpression and accumulation of APP in neuronal perikarya and that these events are followed by degeneration of CA3 neurons. Further, the decline in APP expression in the hippocampus is thought to be due to neuronal loss in CA3 subsector.     

A comparison of the effects of selective metabotropic glutamate receptor agonists on synaptically evoked whole cell currents of rat spinal ventral horn neurones in vitro

1. Whole cell synaptic currents were recorded under voltage clamp from a total of 54 ventral horn neurones held near to their resting potential by the patch clamp technique in immature rat spinal cord preparations in vitro. Twenty eight neurones were identified, by antidromic invasion from ventral roots, as motoneurones. Excitatory postsynaptic currents (e.p.s.cs) of peak amplitude -480 pA +/- 66 s.e. mean and -829 +/- 124 pA were evoked respectively from the unidentified ventral horn neurones and the motoneurones in response to maximal activation of the segmental dorsal root. 2. The e.p.s.cs were depressed reversibly by the metabotropic glutamate agonists 1S3S-1-aminocyclopentane-1,3-dicarboxylate (1S3S-ACPD) (EC50 17.1 microM +/- 0.3 s.e. mean, n = 14) and L-2-amino-4-phosphonobutanoate (L-AP4) (EC50 = 2.19 +/- 0.19 microM, n = 15). Since both agonists independently produced more than 90% depression it is likely that the receptors that mediate their effects are present on the same presynaptic terminals. 3. When the Mg2+ concentration was raised from 0.75 mM to 2.75 mM together with the addition of 50 microM D-2-amino-5-phosphonopentanoate (AP5), a treatment which would increase the proportion of monosynaptic component in the e.p.s.c. the concentration-effect plots for both 1S3S-ACPD (EC50 1.95 +/- 0.4 microM, n = 8) and L-AP4 (EC50 0.55 +/- 0.20 microM, n = 7) were shifted to the left, suggesting that monosynaptic e.p.cs of primary afferents to ventral horn neurones are more susceptible to L-AP4 and 1S3S-ACPD than are other synapses in polysynaptic pathways. 4. lS3S-ACPD (20 and 50 microM) also caused mean sustained inward currents of 95 +/- 31 pA (n = 6) and248 +/- 49 pA (n = 10) respectively. In the combined presence of AP5 (50 microM) and Mg2+ (2.75 mM) themean response to 50 microM lS3S-ACPD was reduced to 106+/- 18 pA (n = 4). In the presence of tetrodotoxin(1 microM) the corresponding value was 48 +/- 6 pA (n = 4). Similar sustained inward currents produced by N-methyl-D-aspartate (NMDA) were almost abolished to < 10 pA in the presence of AP5 and 2.75 mMMg2+. In the presence of tetrodotoxin the maximum inward current produced by NMDA was undiminished. Thus a large component of the excitatory action of lS3S-ACPD was mediated at non-NMDA receptors both directly at the patch-clamped neurones and indirectly by synaptic relay.     

Nitric oxide acts directly in the presynaptic neuron to produce long-term potentiation in cultured hippocampal neurons

Nitric oxide (NO) has been proposed to act as a retrograde messenger during long-term potentiation (LTP) in the CA1 region of hippocampus, but the inaccessibility of the presynaptic terminal has prevented a definitive test of this hypothesis. Because both sides of the synapse are accessible in cultured hippocampal neurons, we have used this preparation to investigate the role of NO. We examined LTP following intra- or extracellular application of an NO scavenger, an inhibitor of NO synthase, and a membrane-impermeant NO donor that releases NO only upon photolysis with UV light. Our results indicate that NO is produced in the postsynaptic neuron, travels through the extracellular space, and acts directly in the presynaptic neuron to produce long-term potentiation, supporting the hypothesis that NO acts as a retrograde messenger during LTP.     

Usefulness of trigeminal somatosensory evoked potentials to detect subclinical trigeminal impairment in multiple sclerosis patients

Trigeminal somatosensory evoked potentials (TSEPs) by surface electric pulse stimulation were recorded in 30 normal subjects and in 70 multiple sclerosis (MS) patients, 13 of whom presenting clinical trigeminal impairment. We observed significant prolongation of all TSEPs parameters in MS group. TSEPs were abnormal in 45 patients (64.3%). Clinical and neurophysiological data agreed in 36 patients (51%) on 84 sides (60%). TSEPs were able to detect clinically silent lesions 54 times. TSEPs recording proves to be an additional useful test in MS multimodal evoked potential protocols.