Moderate hypoglycemia aggravates effects of hypoxia in hippocampal slices from diabetic rats.

We recorded the effects of hypoxia combined with relative hypoglycemia on pre- and post-synaptic potentials in the CA1 area of slices from 4-month-old control and diabetic (streptozotocin-treated) Wistar rats. In experiments on slices kept in 10 or 4 mM glucose (at 33 degrees C), hypoxia was applied until the pre-synaptic afferent volley disappeared--after 12-13 min in most slices, but much earlier (5+/-0.8 min) in diabetic slices kept in 4 mM glucose. When oxygenation was resumed, the afferent volley returned in all slices, for an overall mean recovery of 86.5% (+/-8.8%). Field post-synaptic potentials were fully blocked within 2-3 min of the onset of hypoxia. After the end of hypoxia, they failed to reappear in some slices: overall, their recovery varied between 62 and 68% in control slices, as well as in diabetic slices kept in 10 mM glucose; but recovery was very poor in diabetic slices kept in 4 mM glucose (only 15+/-0.94%). In the latter, hypoxic injury discharges occurred earlier (4.2+/-0.68 min vs. 6.5-8 min for other groups). We conclude that diabetes appears to make hippocampal slices more prone to irreversible loss of synaptic function and early block of axonal conduction when temporary hypoxia is combined with moderate hypoglycemia.

Hypoxia on hippocampal slices from mice deficient in dystrophin (mdx) and isoforms (mdx3cv).

Slices from control C57, mdx, and mdx3cv mice were made hypoxic until both field excitatory postsynaptic potential (fEPSP) and presynaptic afferent volley (AV) disappeared (H1). After reoxygenation and recovery of fEPSP, a second and longer hypoxic test (H2) lasted 3 minutes beyond the time required to block AV. When slices were kept in 10 mmol/L glucose, HI abolished AV 37 and 19% earlier in slices from mdr and mdx3cv mutants than in control slices (where HI = 12 +/- 4.6 minutes, mean +/- SD). During H2 or when slices were kept in 4 mmol/L glucose, AV vanished even more quickly, but the times to block did not differ significantly between slices from controls and mutants. After reoxygenation, AV fully recovered in most slices. Rates of blockade of fEPSPs were comparable in all slices, and most fEPSPs recovered fully after HI. But even in the presence of 10 mmol/L glucose, the second hypoxia suppressed fEPSPs irreversibly in some slices: 2 of 10 from control, 3 of 7 from mdx, and 1 of 6 from mdx3cv mice. Most slices in 4 mmol/L glucose showed no recovery at all: six of seven from control, three of five from mdx, and four of five from mdx3cv mice. Thus, slices from mdx mice were more susceptible than other slices to irreversible hypoxic failure when slices were kept in 10 mmol/L glucose, but they were less susceptible than other slices when kept in 4 mmol/L glucose. In conclusion, the lack of full-length dystrophin (427 kDa) predisposes to quicker loss of nerve conduction in slices from mdx and mdx3cv mutants and improved posthypoxic recovery of fEPSPs in 4 mmol/L glucose in slices from mdx but not mdx3cv mutants, perhaps because the 70-kDa and other C-terminal isoforms are still present in mdx mice.

Long-term potentiation in mice lacking the neural cell adhesion molecule L1.

Genetic evidence indicates that cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) are critical for activity-dependent synapse formation at the neuromuscular junction in Drosophila and have also been implicated in synaptic remodelling during learning in Aplysia (see [1] for review). In mammals, a widely adopted model for the process of learning at the cellular level is long-term potentiation (LTP) in the hippocampal formation. Studies in vitro have shown that antibodies to the IgCAMs L1 and NCAM reduce LTP in CA1 neurons of rat hippocampus, suggesting a role for these molecules in the modulation of synaptic efficacy, perhaps by regulating synaptic remodelling [2]. A role for NCAM in LTP has been confirmed in mice lacking NCAM [3] (but see [4]), but similar studies have not been reported for L1. Here we examine LTP in the hippocampus of mice lacking L1 [5,6], using different experimental protocols in three different laboratories. In tests of LTP in vitro and in vivo we found no significant differences between mutant animals and controls. Thus, contrary to expectation, our data suggest that L1 function is not necessary for the establishment or maintenance of LTP in the hippocampus. Impaired performance in spatial learning exhibited by L1 mutants may therefore not be due to hippocampal dysfunction [6].

Facilitation of a nociceptive flexion reflex in man by nonnoxious radiant heat produced by a laser.

Electromyographic recordings were made in healthy volunteers from the knee-flexor biceps femoris muscle of the nociceptive RIII reflex elicited by electrical stimulation of the cutaneous sural nerve. The stimulus intensity was adjusted to produce a moderate pricking-pain sensation. The test responses were conditioned by a nonnoxious thermal (

Mice lacking the gene encoding tissue-type plasminogen activator show a selective interference with late-phase long-term potentiation in both Schaffer collateral and mossy fiber pathways.

The gene encoding tissue-type plasminogen activator (t-PA) is an immediate response gene, downstream from CREB-1 and other constitutively expressed transcription factors, which is induced in the hippocampus during the late phase of long-term potentiation (L-LTP). Mice in which the t-PA gene has been ablated (t-PA-/-) showed no gross anatomical, electrophysiological, sensory, or motor abnormalities but manifest a selective reduction in L-LTP in hippocampal slices in both the Schaffer collateral-CA1 and mossy fiber-CA3 pathways. t-PA-/- mice also exhibit reduced potentiation by cAMP analogs and D1/D5 agonists. By contrast, hippocampal-dependent learning and memory were not affected in these mice, whereas performance was impaired on two-way active avoidance, a striatum-dependent task. These results provide genetic evidence that t-PA is a downstream effector gene important for L-LTP and show that modest impairment of L-LTP in CA1 and CA3 does not result in hippocampus-dependent behavioral phenotypes.

Long-term potentiation in the hippocampus of fragile X knockout mice.

To gain more insight in the physiological function of the fragile X gene (FMR1) and the mechanisms leading to fragile X syndrome, the Fmr1 gene has been inactivated in mice by gene targeting techniques. In the Morris water maze test, the Fmr1 knockout mice learn to find the hidden platform nearly as well as the control animals, but show impaired performance after the position of the platform has been modified. As malperformance in the Morris water maze test has been associated with impaired long-term potentiation (LTP), electrophysiological studies were performed in hippocampal slices of Fmr1 knockout mice to check for the presence of LTP. Judged by field extracellular excitatory postsynaptic potential recordings in the CA1 hippocampal area, Fmr1 knockout mice express LTP to a similar extent as their wild type littermates during the first 1-2 hr after high frequency stimulation. Also, short-term potentiation (STP) was similar in both types of mice. To investigate whether Fmr1 is involved in the latter stages of LTP as an immediate early gene, we compared Fmr1 mRNA quantities on northern blots after chemical induction of seizures. A transient increase in the transcription of immediate early genes is thought to be essential for the maintenance of LTP. As no increase in Fmr1 mRNA could be detected, neither in cortex nor in total brain, during the first 2 1/2 hr after pentylenetetrazol-induced seizures, it is unlikely that Fmr1 is an immediate early gene in mice. In conclusion, we found no evidence for a function of FMR1 in STP or LTP.

Internal Ca2+ stores involved in anoxic responses of rat hippocampal neurons.

1. During whole-cell recordings from CA1 neurons of rat brain slices with electrodes containing only KMeSO4 and Hepes, brief anoxia (2-3 min) consistently evoked a hyperpolarization (delta V approximately 14 mV) and reduction in input resistance (delta R approximately -20%). 2. As in previous intracellular recordings, Dantrolene sodium (10 microM) suppressed the anoxic delta V and delta R, confirming the release of internal Ca2+ is a major component of the anoxic response. 3. To identify the relevant intracellular Ca2+ store, other blockers of Ca2+ release were applied either externally (in the bath) or internally, by addition to the contents of the recording electrode. 4. The anoxic hyperpolarization was abolished or much reduced by heparin (10-20 micrograms ml-1, internal), thapsigargin (10 microM, external), Ruthenium Red (50 microM, internal) and external procaine (0.5-2 mM), but not by internal procaine (0.5-1 mM) or ryanodine (10 microM, external). 5. The anoxic fall in resistance was also abolished or reduced by heparin, thapsigargin and external procaine, but not by ryanodine, internal procaine or Ruthenium Red. 6. In addition, external procaine (0.5-2 mM) eliminated the early (transient) depolarization and reduced the post-anoxic hyperpolarization by 60 +/- 22%. 7. None of these agents consistently changed the resting potential, but the input resistance was significantly increased by Dantrolene and external procaine. 8. In view of the marked effects of heparin and thapsigargin, but not ryanodine and internal procaine, we conclude that the anoxic response seen in such whole-cell recordings is initiated predominantly by Ca2+ release from an internal store that is InsP3 sensitive rather than Ca2+ sensitive. 9. Comparable but less pronounced effects of external procaine were seen during intracellular recordings with 3 M KCl-containing electrodes. The dose-dependent suppression of various features of the anoxic response by external procaine (EC50 approximately 0.2 mM) is presumed to be mediated by a superficial membrane trigger or modulating site.

Specificity of protein kinase inhibitor peptides and induction of long-term potentiation.

Previous studies have used synthetic peptide analogs, corresponding to sequences within the pseudosubstrate domain of protein kinase C (PKC) or the autoregulatory domain of Ca2+/calmodulin-dependent protein kinase II (CaMKII), in attempts to define the contribution of each of these protein kinases to induction of long-term potentiation (LTP). However, the specificity of these inhibitor peptides is not absolute. Using intracellular delivery to rat CA1 hippocampal neurons, we have determined the relative potency of two protein kinase inhibitor peptides, PKC-(19-36) and [Ala286]CaMKII-(281-302), as inhibitors of the induction of LTP. Both peptides blocked the induction of LTP; however, PKC-(19-36) was 30-fold more potent than [Ala286]CaMKII-(281-302). The relative specificity of PKC-(19-36), [Ala286]CaMKII-(281-302), and several other CaMKII peptide analogs for protein kinase inhibition in vitro was also determined. A comparison of the potencies of PKC-(19-36) and [Ala286]CaMKII-(281-302) in the physiological assay with their Ki values for protein kinase inhibition in vitro indicates that the blockade of induction of LTP observed for each peptide is attributable to inhibition of PKC.

Heterotopic nociceptive conditioning stimuli and mental task modulate differently the perception and physiological correlates of short CO2 laser stimuli.

The present study was aimed at examining the specificity of the action of heterotopic nociceptive conditioning stimulation (HNCS) by comparing its effects of those induced by a mental task (MT). Five test stimuli made from short CO2 laser pulses (duration: 40 msec; diameter: 10 mm; intensity: 0.25-0.8 Joules) were delivered every 30 to 45 sec at random to 4 different spots on the skin of the upper lip in 3 groups of 10 healthy subjects. The two most intense stimuli were perceived as painful, the two least intense stimuli as warm, and the intermediate stimulus as hot or near painful. Perception (VAS), reaction time (T) and cerebral evoked potentials (CEPs) were monitored before, during and after conditioning stimulation consisting either of HNCS (hand submerged in cold water) or of MT (arithmetic subtraction). Pain perception (first pain) threshold was increased in both conditioning stimulations; however, the stimulus-response curve and the neurophysiological correlates were differently affected. During HNCS, the stimulus-response curve was depressed and T was increased mainly for the intermediate stimulus, whilst CEP power density was reduced for all stimulus intensities; discrimination performance near pain threshold was dramatically depressed. During MT, the stimulus-response curve was shifted down toward higher stimulus intensities, T was equally increased for all stimulus intensities, whereas CEP power density was not changed; discrimination performance remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Tolbutamide suppresses anoxic outward current of hippocampal neurons.

In hippocampal slices, 2-3 min of hypoxia often evokes a hyperpolarisation or outward current. In the presence of tetrodotoxin and kynurenic acid (to minimize indirect effects of the drugs), we applied two sulphonylureas to detect a possible involvement of ATP-sensitive K (KATP) channels. In all 9 cells tested, tolbutamide (TOLB, 0.1-1 mM) greatly reduced both the hypoxic current (by 81.3 +/- 9.4%) and the conductance increase (by 77.2 +/- 10.2%). By contrast, glibenclamide (GLIB, 10-30 microM) tested on 5 cells, had no comparable effects. We therefore conclude that if KATP channels play a role in the hypoxic response, they are likely to be of the low affinity type found in neocortical and hypothalamic neurons.

Microionophoretic study with milacemide, a glycine precursor, on mammalian central nervous system cells.

1. The effect of milacemide, a glycine percursor known to increase gamma-aminobutyric acid (GABA) and glycine content in the brain, and to have anticonvulsant properties, was tested by ionophoresis on 247 neurones situated in the cerebral cortex and in deeper structures of cats and rats anaesthetized with urethane. 2. Virtually all the neurones, either firing spontaneously or exogenously driven by the excitatory amino acids, glutamate, N-methyl-D-aspartate (NMDA), kainate and quisqualate or by acetylcholine, were reversibly depressed in a dose-dependent fashion. The same depressant effect was observed in animals pretreated with the monoamine oxidase B inhibitor (IMAO-B) deprenyl which is known to reduce milacemide metabolism into glycinamide and glycine. Intravenous administration of milacemide (10 to 100 mg kg-1) also depressed the firing induced by glutamate, NMDA and acetylcholine. 3. When compared to GABA, milacemide was a weaker depressant. However, its effect could still be observed in the presence of the reversible GABAA antagonist, SR 95531, and thus milacemide is unlikely to act through GABA receptors. In addition, on cells unaffected by glycine, milacemide also had a depressant effect, and on cells inhibited by glycine, it was still capable of depressing cell firing during reversible blockade by strychnine of the glycine inhibitory action; thus milacemide is unlikely to act through glycine receptors. Simultaneous release of milacemide and GABA or of milacemide and glycine, did not show potentiation of the inhibitory amino acid action. However, the depressant effect of milacemide was additive with that of GABA and glycine. 4. No consistent depression of glutamate-induced firing was obtained by ionophoresis of glycinamide, the first metabolite of milacemide. 5. It is concluded that milacemide by itself is a depressant agent and that its depressant effect does not necessarily require its metabolism into glycine, or its stimulator effect on the production of GABA.

Intrasomatic and intradendritic recordings of plateau potentials in slices of the dentate gyrus maintained in vitro.

Intrasomatic and intradendritic recordings were performed in slices of the dentate gyrus maintained in vitro. When barium ions (2.4 mM) were substituted for calcium ions in the perfusing medium, plateau potentials appeared with an amplitude of 20-40 mV which lasted from 40 ms to more than one min; during these plateau potentials, the input membrane resistance was decreased. In the soma, plateau potentials were also observed in a medium containing barium ions + tetrodotoxin (0.3 or 0.6 microM); whereas, in the dendrites, the barium-induced plateau potentials were abolished after addition of tetrodotoxin to the barium containing perfusion. The somatic plateau potentials had a duration which appeared to be dependent on the stimulus frequency. After being in contact with the barium-tetrodotoxin solution for a long period, the soma membrane potential was observed to jump between two relatively stable levels: a resting state and a depolarized state. In conclusion, calcium conductances appear to be present both at the soma and the dendrites of dentate granule cells; however, at the dendritic level, it appears that, when sodium channel permeability is blocked by tetrodotoxin, there is insufficient inward current to support the generation of action potentials.

A simple and inexpensive device for applying a calibration pulse when performing intracellular recording.

A simple and low-cost device is described which facilitates the calibration of intracellular DC recordings in amplitude and time. A current source, optically coupled to a conventional stimulator, injects a pulse of current, across a 1-omega resistor connecting the preparation to ground. Amplitude and duration of the current pulse are determined by adjusting the voltage output of the conventional stimulator. By linking the preparation to ground through a resistor of 1 omega the background noise and the DC shifts in the voltage trace of the intracellular recording are kept to a minimum.

Possible role of acetylcholine in the mediation of non-retinal input to the lateral geniculate nucleus region.

In these series of experiments, the effects of acetylcholine and related substances administered by iontophoresis or by intravenous injection, have been examined on neurones of the lateral geniculate nucleus region to see whether non-retinal inputs on these cells use acetylcholine as a mediator. Quantitative analysis has shown that the somatic induced facilitation of the visual responses evoked in the lateral geniculate nucleus is suppressed after intravenous administration of atropine. This observation favors the intervention of muscarinic cholinergic receptors in the mediation of non-retinal inputs to the lateral geniculate nucleus region.

Acetylcholine and somatically evoked inhibition on perigeniculate neurones in the cat.

1 Perigeniculate neurones in cats were found to be inhibited by iontophoretically applied acetylcholine (ACh) and some of them by somatic sensory stimulation under certain experimental conditions. 2 Under chloralose anaesthesia, perigeniculate neurones could be divided into two groups with regard to their spontaneous activity, sensitivity to glutamate and reaction to sensory inputs. Somatic sensory stimulation clearly inhibited the glutamate discharges of those perigeniculate neurones which were characterized by a high sensitivity to glutamate and the absence of spontaneous activity. ACh had no clear inhibitory effect. 3 Under fluothane and urethane anaesthesia, no somatic sensory influence was noticed but ACh depressed almost all perigeniculate neurones. 4 In an unanaesthetized midpontine pretrigeminal preparation, the inhibitory effect of ACh was confirmed. 5 No conditions were found which the inhibitory influences of ACh and those of somatic sensory stimulation could be observed simultaneously on the same neurone. Therefore, it could not be established whether ACh mediates the somatic sensory influences on perigeniculate cells.