Priming of long-term potentiation mediated by ryanodine receptor activation in rat hippocampal slices.

Administration of the Group 1 metabotropic glutamate receptor (mGluR) agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) facilitates ("primes") subsequent long-term potentiation (LTP) through a phospholipase C signaling cascade that may involve release of Ca2+ from the endoplasmic reticulum (ER). We investigated the intracellular calcium pathways involved in this priming effect, recording field potentials from area CA1 of rat hippocampal slices before and after high-frequency stimulation. The priming of LTP by DHPG was prevented by co-administration of cyclopiazonic acid, which depletes ER Ca2+ stores. The priming effect was also blocked by the ryanodine receptor (RYR) antagonist ryanodine (RYA, 100 microM). In contrast, a low dose of RYA (10 microM) which opens the RYR channel, by itself primed LTP. In addition to RYR activation, entry of extracellular calcium through store-operated channels appears necessary for priming, since diverse treatments known to impede store-operated channel activity completely blocked both RYA and DHPG priming effects. Thus, RYR activation plays a critical role in the priming of LTP by Group 1 mGluRs, and this effect is coupled to the entry of extracellular calcium, probably through store-operated calcium channels.

Coupling of organotypic brain slice cultures to silicon-based arrays of electrodes.

Fetal or early postnatal brain tissue can be cultured in viable and healthy condition for several weeks with development and preservation of the basic cellular and connective organization as so-called organotypic brain slice cultures. Here we demonstrate and describe how it is possible to establish such hippocampal rat brain slice cultures on biocompatible silicon-based chips with arrays of electrodes with a histological organization comparable to that of conventional brain slice cultures grown by the roller drum technique and on semiporous membranes. Intracellular and extracellular recordings from neurons in the slice cultures show that the electroresponsive properties of the neurons and synaptic circuitry are in accordance with those described for cells in acutely prepared slices of the adult rat hippocampus. Based on the recordings and the possibilities of stimulating the cultured cells through the electrode arrays it is anticipated that the setup eventually will allow long-term studies of defined neuronal networks and provide valuable information on both normal and neurotoxicological and neuropathological conditions.

An array of Pt-tip microelectrodes for extracellular monitoring of activity of brain slices.

A microelectrode array (MEA) consisting of 34 silicon nitride passivated Pt-tip microelectrodes embedded on a perforated silicon substrate (porosity 35%) has been realized. The electrodes are 47 microns high, of which only the top 15 microns are exposed Pt-tips having a curvature of 0.5 micron. The MEA is intended for extracellular recordings of brain slices in vitro. Here we report the fabrication, characterization and initial electrophysiological evaluation of the first generation of Pt-tip MEAs.

NCAM-antibodies modulate induction of long-term potentiation in rat hippocampal CA1.

The neural cell adhesion molecule (NCAM) probably plays a role in neural plasticity in the adult vertebrate brain. We here present evidence that NCAM may be involved in long-term potentiation (LTP) in the CA1-region of rat hippocampal slices. It is shown that local application of antibodies against NCAM inhibits subsequent LTP-induction. Thus NCAM may be directly involved in the initial phase of LTP-induction. These results have important implications for the possible involvement of NCAM in learning and memory.

A spectral analysis of the integration of artificial synaptic potentials in mammalian central neurons.

In order to simulate the interaction between synaptic input and intrinsic membrane properties in mammalian central neurons a well-defined current was injected into the neurons through a recording electrode. The stimulus was white noise bandpass filtered at 0.5 and 75 Hz and the power spectra of the responses were calculated. Recordings were obtained from neurons of the neocortex, the hippocampus, the thalamus and the cerebellar cortex. The neurons were either located in newly cut slices from adult guinea pig brains or in 3-10-weeks-old slice cultures from brains of newborn rats. In hippocampal and cortical cells the passive membrane properties dominated the shape of the power spectra. In general, when the average membrane potential was made more positive the power of the response increased. When neurons had active subthreshold responses like delayed rectification, sag-and-hump responses or delayed depolarization there was a depression of the response power at frequencies below 10-20 Hz. The depression was voltage dependent in the same way as the current that produced the active subthreshold response. In thalamic cells with a low-threshold Ca2+ spike (lts) the power of the responses grew in the 3-20-Hz range with hyperpolarization. The spectra of the responses of thalamic neurons had multiple peaks indicating multiple frequencies of resonance. Purkinje cells of the cerebellar cortex have prominent plateau potentials. When these cells were stimulated with the white noise at levels where the plateau potentials could be activated the spectra were dominated by a large peak at the lowest frequencies, i.e., below 5 Hz. Few cells in our data base generated spontaneous membrane potential oscillations. When the current stimulus was injected into such neurons the intrinsic rhythm was unaffected by the input and the power spectrum showed a marked peak at the frequency of the intrinsic oscillations. We conclude that bandpass filtered white noise as simulation of synaptic input is valuable for quantification of how passive and active membrane properties affect synaptic integration. The technique can also provide information on the role of transmitters and modulators in the CNS.

NADPH-diaphorase (NOS) is induced in pyramidal neurones of hippocampal slices.

We found NADPH-diaphorase (presumably identical with nitric oxide synthase) in pyramidal neurones of the hippocampus in slices that stayed in a chamber for 30 min or longer. In some instances parallel slices showed normal membrane properties when studied electrophysiologically. In freshly made slices the pyramidal neurones were not stained. Thus, after induction of the enzyme, the hippocampal pyramidal neurones can synthesize nitric oxide which may serve as a retrograde messenger in long-term potentiation. The enzyme may also play a role in cell loss seen in slices which stayed in a chamber for 9-22 h before fixation.

Actions of acetylcholinesterase in the guinea-pig cerebellar cortex in vitro.

Acetylcholinesterase is released in a calcium-dependent manner when afferents of the cerebellar cortex are stimulated. Since cholinergic transmission is probably insignificant in the cerebellar cortex, the esterase itself might serve as a transmitter or modulator. Therefore, the effect of acetylcholinesterase in the cerebellum was investigated in slices of guinea-pig cerebella during intracellular recording from Purkinje cell somata or dendrites. Addition of acetylcholinesterase (20 U/ml) to the superfusion medium did not change the membrane potential or the input resistance of the Purkinje cells. Thus, esterase does not act like a classical transmitter. The threshold for Na+ spikes generated by intracellular current injection was unaffected, but the threshold for Ca2+ spikes was increased. This increase was abolished by tetrodotoxin (1 microM). Furthermore, when Ca2+ currents were blocked by substituting Mn2+ for Ca2+ (2 mM) a decrease in a Na+ plateau potential was seen in the presence of esterase. The effect of acetylcholinesterase of Ca2+ spikes is therefore most likely due to a reduction of the non-inactivating Na+ current of the Purkinje cell membrane. When present this current contributes to activation of Ca2+ spikes in dendrites. Acetylcholinesterase also enhanced the response of Purkinje cells to the excitatory amino acids glutamate and aspartate thought to be transmitters in the cerebellar cortex. The responses became larger and faster in the presence of esterase. Responses to climbing fibre stimulation were also enhanced by acetylcholinesterase. The late part of this synaptic response was increased. The potentiation by esterase of responses of Purkinje cells to excitatory amino acids and to climbing fibre stimulation may be mediated through interference with transmitter uptake, because it was prevented by treatment with DL-2-amino-4-phosphonobutyric acid (0.5 mM) and di-hydrokainate (0.1 mM). None of the effects of esterase was due to hydrolysis of acetylcholine because irreversible inhibition of the catalytic site of the enzyme with soman did not prevent the actions. The observations were specific for acetylcholinesterase. Butyrylcholinesterase (20-40 U/ml) showed none of the effects. It is concluded that acetylcholinesterase in the cerebellar cortex seems to mediate a novel type of modulation by two separate mechanisms. Esterase reduces the tendency towards Ca2+ spike generation in Purkinje cells. Ca2+ spikes are followed by afterhyperpolarizations and in their absence firing of Na+ spikes at higher frequencies is possible. Secondly, there is an enhancement of the action of excitatory transmitters so that the extended operating range can be utilized.

The effect of two lipophilic gamma-aminobutyric acid uptake blockers in CA1 of the rat hippocampal slice.

1. Drugs that increase inhibitory synaptic transmission in the central nervous system may be valuable tools in the treatment of seizures. Theoretically, substances that block the uptake of inhibitory transmitters such as gamma-aminobutyric acid (GABA) into intracellular compartments should also increase inhibition and therefore have potential value as antiepileptic drugs. However, most of these substances penetrate the blood-brain barrier poorly and have therefore until now had limited value. NO-05-0328 and NO-05-0329 are two new lipophilic GABA uptake inhibitors that readily enter the CNS from the blood. 2. We have investigated the effect of these two uptake inhibitors on the responses to exogenous GABA and on GABA-mediated inhibitory synaptic potentials in pyramidal neurones of the CA1 region in the rat hippocampal slice. 3. We found that both drugs increased the amplitude and duration of responses to exogenous GABA. Furthermore, the inhibitory synaptic potentials increased in amplitude. This increase was seen in both early and late phases of the synaptic potentials. We conclude that NO-05-0328 and NO-05-0329, at least in vitro, are more effective than older GABA uptake inhibitors such as nipecotic acid and they therefore deserve consideration for clinical use.

Electrophysiological characteristics of neurones in the guinea-pig deep cerebellar nuclei in vitro.

The properties of neurones of the guinea-pig deep cerebellar nuclei in a slice preparation were investigated by intracellular recording. The recorded population of cells did not differ morphologically from nuclear cells in vivo as judged from neurones stained with Lucifer Yellow. Fifty-two out of sixty cells were spontaneously active with a regular firing pattern and a mean frequency of 26 +/- 14 (mean +/- S.D.) impulses/s. The action potentials lasted 0.41 +/- 0.07 ms (n = 60) with an amplitude of 58 +/- 8 mV. Input resistance was 44 +/- 10 M omega and the time constant of the membrane 13 +/- 3 ms. When stimulated with intracellularly injected depolarizing current pulses the cells responded with trains of action potentials. Near the threshold for the spike the stimulation produced firing of constant frequency and from more hyperpolarized levels an initial acceleration sometimes followed by a deceleration was seen. At levels less than 15 mV from the spike threshold there was a rebound train of spikes as a response to a hyperpolarizing current injection. At more hyperpolarized levels there was only a small depolarizing potential after the hyperpolarizing stimulation. Three types of subthreshold potentials were recorded. Spikelets rose from base line as 3-10 mV depolarizing wavelets with a duration between 5 and 10 ms. They served as trigger potentials for the action potential. Plateau potentials were slow depolarizing potentials often reaching the spike threshold and thus generating long trains of action potentials. After-hyperpolarizations followed each spike with a time course dependent on the previous activity of the cell. Plots of the firing frequency versus injected current were linear at the first and second interspike interval, after 50 ms of activity and at steady state. Plots of the voltage versus injected current were upward concave demonstrating anomalous rectification of the cell membrane. It is concluded that neurones in the deep cerebellar nuclei in vitro are spontaneously active because of the electroresponsive properties of their membranes. The physiological importance may be that the cerebellar output from these cells can be rapidly and efficiently modulated by synaptic potentials generated by Purkinje cells and mossy and climbing fibres.

Extracellular activation and membrane conductances of neurones in the guinea-pig deep cerebellar nuclei in vitro.

The responses of cerebellar nuclear cells to extracellular stimulation in a slice preparation were studied and the ionic basis of their electroresponsiveness was investigated with blockers of membrane conductances and with ion substitutions in the extracellular medium. The cells could be activated antidromically from the cerebellar cortex and the white matter surrounding the nuclei. The dominating response to orthodromic stimulation was an inhibitory synaptic potential presumably produced by activation of Purkinje cell fibres. The action potentials and the subthreshold spikelets were shown to be Na+ dependent and are presumably generated by a voltage-dependent inactivating Na+ conductance. Plateau potentials with a low threshold were also Na+ dependent, but these long-lasting potentials are probably produced by activation of a voltage-dependent non-inactivating Na+ conductance. Plateau potentials with a high threshold and high-threshold spikelets were Ca2+ dependent and seem to be generated by non-inactivating and possibly inactivating Ca2+ conductances. The spike after-hyperpolarizations had an early voltage-dependent K+ component and a late Ca2+-dependent K+ component. They are therefore produced by voltage-sensitive and Ca2+-dependent K+ conductances. By analogy with the distribution of conductances in Purkinje cells it is proposed that the Na+ conductances are mainly located in the somatic and axonal membrane and that the Ca2+ conductances are located in the dendrites. The functional implications of the complex electroresponsive properties of cerebellar nuclear cells are discussed.

Uncoupling of CA3 pyramidal neurons by propionate.

The influence of cytoplasmic acidification on dye-coupling between CA3 pyramidal neurons was examined by Lucifer yellow injections in guinea pig hippocampal brain slices. Neurons were believed to be acidified by exposure to 100 mM propionate. Dye-coupling was reduced significantly to 6% (n = 35) in propionate versus 28% (n = 39) in control solution (P less than 2%). Propionate may be a useful tool for experimental manipulation of coupling in the mammalian CNS.

Electrophysiological properties of guinea-pig thalamic neurones: an in vitro study.

The electroresponsive properties of guinea-pig thalamic neurones were studied using an in vitro slice preparation. A total of 650 cells were recorded intracellularly comprising all regions of the thalamus; of these 229 fulfilled our criterion for recording stability and were used as the data base for this report. The resting membrane potential for thirty-four representative neurones which were analysed in detail was -64 +/- 5 mV (mean +/- S.D.), input resistance 42 +/- 18 M omega, and action potential amplitude 80 +/- 7 mV. Intracellular staining with horseradish peroxidase and Lucifer Yellow revealed that the recorded cells had different morphology. In some their axonal trajectory characterized them as thalamo-cortical relay cells. Two main types of neuronal firing were observed. From a membrane potential negative to -60 mV, anti- or orthodromic and direct activation generated a single burst of spikes, consisting of a low-threshold spike (l.t.s.) of low amplitude and a set of fast superimposed spikes. Tonic repetitive firing was observed if the neurones were activated from a more positive membrane potential; this was a constant finding in all but two of the cells which fulfilled the stability criteria. The l.t.s. response was totally inactivated at membrane potentials positive to -55 mV. As the membrane was hyperpolarized from this level the amplitude of the l.t.s. increased and became fully developed at potentials negative to -70 mV. This increase is due to a de-inactivation of the ionic conductance generating this response. After activation the l.t.s. showed refractoriness for approximately 170 ms. Deinactivation of l.t.s. is a voltage- and time-dependent process; full de-inactivation after a step hyperpolarization to maximal l.t.s. amplitude (-75 to -80 mV) requires 150-180 ms. Membrane depolarization positive to -55 mV generated sudden sustained depolarizing 'plateau potentials', capable of supporting repetitive firing (each action potential being followed by a marked after-hyperpolarization, a.h.p.). The a.h.p. and the plateau potential controlled the voltage trajectory during the interspike interval and, with the fast spike, constitute a functional state where the thalamic neurone displayed oscillatory properties. Frequency-current (f-I) plots from different initial levels of membrane potential were obtained by the application of square current pulses of long duration (2s). From resting membrane potential and from hyperpolarized levels a rather stereotyped onset firing rate was observed due to the presence of the l.t.s.(ABSTRACT TRUNCATED AT 400 WORDS)

Ionic basis for the electro-responsiveness and oscillatory properties of guinea-pig thalamic neurones in vitro.

The ionic requirements for electro-responsiveness in thalamic neurones were studied using in vitro slice preparations of the guinea-pig diencephalon. Analysis of the current-voltage relationship in these neurones revealed delayed and anomalous rectification. Substitution of Na+ with choline in the bath or addition of tetrodotoxin (TTX) abolished the fast spikes and the plateau potentials, described in the accompanying paper. Ca2+ conductance blockage with Co2+, Cd2+ or Mn2+, or replacement of Ca2+ by Mg2+ abolished the low-threshold spikes (l.t.s.). Substitution with Ba2+ did not significantly increase the duration of the l.t.s., suggesting that under normal conditions the falling phase of this response is brought about by inactivation of the Ca2+ conductance. The after-hyperpolarization (a.h.p.) following fast spikes was markedly reduced in amplitude and duration by bath application of Cd2+, Co2+ or Mn2+, indicating that a large component of this response is generated by a Ca2+-dependent K+ conductance (gK[Ca]). Following hyperpolarizing current pulses, the membrane potential showed a delayed return to base line. This delay is produced by a transient K+ conductance as it can be modified by changing the drive force for K+. Presumptive intra-dendritic recording demonstrated high-threshold Ca2+ spikes (h.t.s.s.) which activate a gK[Ca]. Such h.t.s.s. were also seen at the somatic level when K+ conductance was blocked with 4-aminopyridine. It is proposed that the intrinsic biophysical properties of thalamic neurones allow them to serve as relay systems and as single cell oscillators at two distinct frequencies, 9-10 and 5-6 Hz. These frequencies coincide with the alpha and theta rhythms of the e.e.g. and, in the latter case, with the frequency of Parkinson's tremor.

Voltage-dependent burst-to-tonic switching of thalamic cell activity: an in vitro study.

The electroresponsiveness of mammalian thalamic neurons was studied in a slice preparation of the guinea pig diencephalon. Although the morphology of the cells varied, their electroresponsive properties were the same. Stimulation of thalamic cells at a membrane potential more negative than--60 mV produced burst responses and stimulation of more depolarized levels produced tonic firing of fast spikes. The burst response is generated by an inactivating Ca++-conductance. It is seen as a slow Ca++-spike which in turn triggers fast Na+-spikes. The Ca++-conductance is deinactivated by hyperpolarization beyond--60 mV. The membranes of thalamic neurons contain a number of other conductances including a Ca++-dependent K+-conductance producing spike afterhyperpolarization and a non-inactivating Na+-conductance which plays an important role during tonic activity of the cells. The early part of a response to a long-lasting stimulus given at rest or at a hyperpolarized level is dominated by the burst and thus is is independent of the stimulus amplitude. During the late part of such a response the firing rate is highly dependent of the stimulus intensity. Current-frequency plots for the first inter-spike intervals after the burst during long stimuli are upward convex, but after "steady-state" is reached the plots are almost linear.

Electrophysiology of pars compacta cells in the in vitro substantia nigra--a possible mechanism for dendritic release.

The electrophysiological properties of neurons in the pars compacta of the substantia nigra have been examined in vitro in guinea pig mesencephalic slices. These cells display a set of voltage- and Ca2+-dependent ionic conductances which confer upon them rather unique electrophysiological characteristics. In particular, the results indicate the presence of two separate dendritic Ca2+ conductances. One is inactivated at rest membrane potential and is de-inactivated by membrane hyperpolarization. The second resembles that responsible for dendritic spikes in other neurons. These conductances fulfil most of the physiological and pharmacological requirements for the ionic mechanisms underlying Ca2+-dependent dendritic release.

Anoxia increases potassium conductance in hippocampal nerve cells.

The effect of anoxia on nerve cell function was studied by intra- and extracellular microelectrode recordings from the CA1 and CA3 region in guinea pig hippocampal slices. Hyperpolarization and concomitant reduction of the nerve cell input resistance was observed early during anoxia. During this period the spontaneous activity first disappeared, then the evoked activity gradually disappeared. The hyperpolarization was followed by depolarization and an absence of a measurable input resistance. All the induced changes were reversed when the slice was reoxygenated. Reversal of the electro-chemical gradient for Cl- across the nerve cell membrane did not affect the course of events during anoxia. Aminopyridines blocked the anoxic hyperpolarization and attenuated the decrease of membrane resistance, but had no effect on the later depolarization. Blockers of synaptic transmission. Mn++, Mg++ and of Na+-channels (TTX) were without effect on the nerve cell changes during anoxia. It is suggested that the reduction of nerve cell excitability in anoxia is primarily due to increased K+-conductance. Thus, the nerve cells are hyperpolarized and the input resistance reduced, causing higher threshold and reduction of synaptic potentials. The mechanism of the K+-conductance activation is unknown at present.

The dendritic response to GABA in CA1 of the hippocampal slice.

Application of GABA in the dendritic region of pyramidal cells elicits a depolarization which, in fact, is the sum of a hyperpolarizing and a depolarizing process. At the reversal potential of the depolarizing response (-42 mV) the GABA-induced current fluctuations do not have a minimum. Consequently, a conductance change to more than one ion is involved. Cl- is in part responsible, Ca2+ is not because Mn2+ and Mg2+ do not change the response. Whether Na+ is involved is uncertain. Substitution with choline had no effect but choline may permeate through the membrane during the depolarizing response. Nipecotic acid inhibits a Na+-GABA uptake mechanism but does not change the dendritic response.