Guinea Pig Hippocampal Slices Research Articles

Genesis of MEG signals in a mammalian CNS structure

Neuromagnetic signals of guinea pig hippocampal slices were characterized and compared with the extracellular field potential to elucidate the genesis of magnetoencephalographic signals in a mammalian CNS structure. The spatial distribution of magnetic evoked field (MEF) directed normal to bath surface was similar for transverse CA1, longitudinal CA1 and longitudinal CA3 slices in the presence of 0.1 mM picrotoxin (PTX) which blocks inhibitory synaptic transmission. Their MEFs were produced by currents along the longitudinal axis of the pyramidal cells. Comparisons of the MEF with the laminar potential profile revealed that the MEF was generated by intracellular longitudinal currents. The dipolar component of the intracellular currents was the dominant factor generating the MEF even at a distance of 2 mm from the slice. The MEF from a slice in Ringer's solution without PTX became similar in temporal waveform with time to the MEF in the presence of PTX, indicating the predominance of excitatory connections in generating the MEF and the existence of highly synchronous population activities across the slice even in PTX-free Ringer's solution. The presence of such highly synchronous population activities underlying the MEFs was verified directly with field potentials recorded across the slice. A systematic variation of the stimulation site revealed a characteristic waveform for each site. The variation of the waveform with stimulation site suggested the contribution of many factors, both synaptic and voltage-sensitive conductances, to the overall waveform of the MEF.

Role of group I metabotropic glutamate receptors in the patterning of epileptiform activities in vitro.

In guinea pig hippocampal slices, picrotoxin elicited spontaneous epileptiform bursts 300-550 ms in duration. Additional application of (R,S)-3,5-dihydroxyphenylglycine or (S)-3-hydroxyphenylglycine, agonists specific for group I metabotropic glutamate receptors (mGluRs), or (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid, a broad-spectrum mGluR agonist, converted picrotoxin-induced interictal bursts into prolonged discharges measured on the order of seconds. The prolonged discharges induced by selective group I mGluR agonist continued to be produced for hours after agonist removal. The antagonists (S)-4-carboxyphenylglycine and (+)-alpha-methyl-4-carboxyphenylglycine had no effect on the duration of picrotoxin-induced interictal bursts. However, after agonist exposure, the persistent prolonged discharges occurring in the absence of agonist were reversibly suppressed by the antagonists, suggesting that the activity is maintained via endogenous activation of group I mGluRs by synaptically released glutamate. Our results suggest that, under some conditions, activation of group I mGluRs produces long-lasting enhancement of synaptic responses, mediated at least in part by autopotentiation of the group I mGluR response itself, which may result in the production of seizure discharges and contribute to epileptogenesis.

8-Cyclopentyltheophylline, an adenosine A 1 receptor antagonist, inhibits the reversal of long-term potentiation in hippocampal CA1 neurons

The effects of an adenosine A 1 receptor antagonist, 8-cyclopentyltheophylline (8-CPT, 1 μM), on the reduction of long-term potentiation were studied in CA1 neurons of guinea pig hippocampal slices. Reduction of long-term potentiation (depotentiation) was achieved by delivering a train of low-frequency afferent stimuli (low-frequency stimulation, 1000 pulses, 1 Hz) 20 min after the tetanus (100 Hz, 100 pulses). In control experiments, low-frequency stimulation reduced the potentiated component of the slope of the field EPSP and the amplitude of the population spike by 68.5±14.4% and 80.1±8.8%, respectively ( n=6); these values were significantly reduced to 13.4±9.7% and 9.0±10.9% ( n=7) when the low-frequency stimulation was applied during the perfusion with 8-CPT (1 μM). These results indicate that activation of adenosine A 1 receptors enhances the depotentiation of long-term potentiation.

Effects of temperature on neuronal bursting in guinea-pig hippocampal slices

In guinea-pig hippocampal slices, activity of CA1 and CA3 neurons (pyramidal layer) during temperature changes was extracellularly recorded. A sharp increase in the rate of spontaneous bursts of action potentials as well as in the number of spikes in a burst during low-amplitude warming in a functional temperature range (36–40°C) was found. Cessation of warming was followed by a recovery of these parameters to the initial values irrespective of the temperature (between 32 and 40°C) at which stabilization occurred. In a functional temperature range (36–40°C), continuous slow cooling inhibited neuronal bursting activity. It is suggested that temporal extension of neuronal bursting in response to continuous temperature rise may play a role in the generation of febrile of febrile seizures.

Regulation of [ 3H]norepinephrine release from guinea pig hippocampus by σ 2 receptors

The binding profile of the σ 2 receptor ligand endo- N-(8-methyl-8-azabicyclo[3.2.1.]oct-3-yl)-2,3-dihydro-(1-methyl)ethyl-2-oxo-1 H-benzimidazole-1-carboxamidehydrochloride (BIMU-8) had previously been determined, but its agonist/antagonist status at σ 2 receptors had not been identified. We therefore investigated the effects of BIMU-8 for its ability to regulate the stimulated release of [ 3H]norepinephrine from slices of guinea pig hippocampus. BIMU-8 alone, at a concentration chosen to occupy 50% of σ 2 receptors, had no significant effect on N-methyl- d-aspartate (NMDA)-stimulated release of [ 3H]norepinephrine. We have shown previously that the σ receptor agonist (+)-pentazocine inhibits NMDA-stimulated release in a concentration-dependent manner, producing a biphasic inhibition curve. Similarly, the σ receptor agonist 1 S,2 R-(−)- cis- N-[2-(3,4-dichlorophenyl)ethyl]- N-methyl-2-(1-pyrrolidinyl)cyclohexylamine (BD737) produced a broad inhibition curve. The inhibition by low concentrations of (+)-pentazocine or BD737 that selectively activated σ 1 receptors was reversed by the σ 1-selective receptor antagonist (1-(cyclopropylmethyl)-4-2′-oxoethyl)piperidine HBr (DuP 734). In the current study, when the σ 1 component of inhibition by (+)-pentazocine was blocked by DuP 734, the remaining component of inhibition mediated by σ 2 receptors was reversed by BIMU-8. Our results suggest that (1) BIMU-8 is an antagonist at σ 2 receptors and that (2) σ 2 receptors contribute to regulation of norepinephrine release in guinea pig hippocampus.

Effects of hypothermia on the neuronal activity, [Ca 2+] i accumulation and ATP levels during oxygen and/or glucose deprivation in hippocampal slices of guinea pigs

The protective effects of mild hypothermia (33 and 29°C) on the neuronal activity, intracellular Ca 2+ accumulation and ATP levels during deprivation of oxygen and/or glucose were investigated using guinea pig hippocampal slices. During oxygen and glucose deprivation and oxygen deprivation only, mild hypothermia had only minor effects on the maintenance of neuronal activity and ATP levels, but it suppressed Ca 2+ accumulation and drastically improved the reversibility of neuronal activity. During glucose deprivation only, mild hypothermia could maintain the neuronal activity as well as ATP levels.

θ-activity-dependent and -independent muscarinic facilitation of long-term potentiation in guinea pig hippocampal slices.

Cholinergic input to the hippocampus originates in the septum and diagonal band. Guinea pig hippocampal slices in a bath of carbachol, a cholinergic agonist, displayed different patterns of rhythmical activities depending on the carbachol concentration. Exposure to 50 or 100 μM led to intermittent induction of θ-like activities (TLAs). Long-term potentiation (LTP), induced by tetanus, was facilitated at concentrations within the optimum range for generating TLAs. This facilitation was blocked by the muscarinic receptor antagonist atropine. Augmentation of LTP during TLAs was greater than that during the rest period of TLAs which, in turn, was greater than that induced without activation of the muscarinic receptors. These results suggest that there are two muscarinic facilitation processes of LTP, one dependent on and the other independent of TLAs, with the former being more easily facilitated than the latter.

Effects of serotonin through serotonin 1A and serotonin 4 receptors on inhibition in the guinea-pig dentate gyrus in vitro

The role of serotonin 1A and serotonin 4 receptors in the modulation of synaptic inhibition in the dentate gyrus of guinea-pig hippocampal slices was studied. The effects of serotonin (5-hydroxytryptamine) on hilar neurons and on inhibitory postsynaptic potentials in granule cells were compared using intracellular recording in the presence of glutamatergic receptor antagonists. On the basis of electrophysiological properties hilar neurons were classified as type I neurons (presumably inhibitory) and type II neurons (presumably excitatory). Serotonin hyperpolarized a proportion of type I hilar neurons (60%) and decreased their input resistance through activation of a K +-conductance. This effect was mediated by serotonin 1A receptors since it was mimicked by the selective serotonin 1A receptor agonist (±)-8-hydroxy-dipropylaminotetralin hydrobromide and blocked by the selective serotonin 1A receptor antagonist (+) WAY 100135. In some type I hilar neurons (40%) neither serotonin nor (±)-8-hydroxy-dipropylaminotetralin hydrobromide induced a membrane hyperpolarization. Instead, serotonin induced an excitatory response, depolarizing the cells and blocking the slow afterhyperpolarization. Similar effects were seen in all hilar neurons after blockade of serotonin 1A receptors. They were mimicked by the serotonin 4 receptor agonist zacopride. Serotonin induced either decreases or increases in the frequency of spontaneous GABA A receptor-mediated inhibitory postsynaptic potentials in granule cells via activation of serotonin 1A and of serotonin 4 receptors, respectively. 4-aminopyridine-evoked GABA B receptor-mediated inhibitory postsynaptic potentials were inhibited by serotonin via activation of serotonin 1A receptors. However, after blockade of serotonin 1A receptors, serotonin increased the frequency of GABA B-inhibitory postsynaptic potentials through the activation of serotonin 4 receptors. We conclude that a proportion of inhibitory neurons in the dentate area does not express serotonin 1A receptors and is excited by serotonin. Other inhibitory neurons express serotonin 1A receptors and are inhibited by serotonin.

Presynaptic Calcium Dynamics and Transmitter Release Evoked by Single Action Potentials at Mammalian Central Synapses

The relationship between presynaptic calcium transients ([Ca2+]t) and transmitter release evoked by a single stimulus was both investigated experimentally and modeled at a mammalian central synapse, the CA3 to CA1 pyramidal cell synapse in guinea pig hippocampal slices. In the present study, we compared the low-affinity calcium indicator furaptra with the higher-affinity indicator fura-2. The 10-90% rise time of the furaptra transient was 2.4 ms compared to 7.8 ms with fura-2; the half-decay time (tau 1/2) was 30 ms for furaptra, compared to 238 ms for fura-2. The half-width of the calcium influx was 1.8 ms with furaptra, which provides an upper limit to the duration of the calcium current (ICa) evoked by an action potential. Modeling the decay time course of the furaptra transients led to the conclusion that the predominant endogenous calcium buffer in these terminals must have relatively slow kinetics (kon < 10(5)/M.s), although the presence of small amounts of fast buffers cannot be excluded. The relationship between the [Ca2+]t measured with furaptra and the postsynaptic response was the same as previously observed with fura-2: the postsynaptic response was proportional to about the fourth power (m approximately 4) of the amplitude of either [Ca2+]t or calcium influx. Thus, although fura-2 may be locally saturated by the high local [Ca2+] responsible for transmitter release, the volume-averaged fura-2 signal accurately reflects changes in this local concentration. The result that both indicators gave similar values for the power m constrains the amplitude of calcium influx in our model: Ica < 1 pA for 1 ms.

Involvement of M 2 receptor in an enhancement of long-term potentiation by carbachol in Schaffer collateral-CA1 synapses of hippocampal slices

We examined effects of carbachol (CCh), muscarinic receptor agonist, on long-term potentiation (LTP) of field excitatory postsynaptic potential (fEPSP) at Schaffer collateral-CA1 synapse of guinea pig hippocampal slices using extracellular recording technique. Application of 0.1 μM CCh to the slices significantly augmented the magnitude of LTP without significant change in the amplitude of pretetanus fEPSP. The enhancement of LTP by 0.1 μM CCh was significantly attenuated by 0.1 μM AF-DX 116, M 2 receptor antagonist, but not by 0.1 μM pirenzepine, M 1 receptor antagonist. Ten micromolar of carbachol reduced the amplitude of pretetanus fEPSP, while the magnitude of LTP was significantly larger than that in control slices to which tetanus was applied in a stimulus intensity producing pretetanus fEPSPs with an amplitude comparable to those during administration of 10 μM CCh. Neither 0.1 μM pirenzepine nor 0.1 μM AF-DX 116 had significant effect on the enhancement of LTP by 10 μM CCh. These results suggest that the induction of LTP at Schaffer collateral-CA1 synapse was enhanced through the activation of M 2 receptors by CCh at a lower concentration.

Spatiotemporal Distribution of Intracellular Calcium Transients During Epileptiform Activity in Guinea Pig Hippocampal Slices

Calcium ions are known to play an important role in epileptogenesis. Although there is clear evidence for increased neuronal calcium influx during epileptiform potentials, direct measurements of the corresponding intracellular calcium transients are rare and the origin of calcium influx is not known. Therefore the spatial and temporal distribution of intracellular calcium transients during epileptiform activity in guinea pig hippocampal slices was monitored with the use of the indicator Calcium-Green and a fast optical recording method. Two models of epilepsy (bicuculline and low Mg2+) were compared. In both models, single epileptiform events were evoked by electrical stimulation of the Schaffer collaterals in CA1 or of stratum pyramidale in area CA3. Intracellular calcium transients during epileptiform activity were approximately 5 times larger than during control stimulation. Calcium transients during epileptiform activity were present across at least the entire CA1 area, whereas presynaptic calcium transients from stimulated fibers were only seen at a distance up to 1 mm from the stimulation site. DL-2-amino-5-phosphonovaleric acid (APV), a specific antagonist of the N-methyl-D-aspartate (NMDA) receptor, abolished low-Mg2+ epileptiform activity and reduced bicuculline-induced epileptiform activity; it reduced calcium transients following stimulation of CA1 by only 29% (bicuculline) and 38% (low Mg2+). For comparison, calcium transients during control stimulation were 78% (bicuculline) and 69% (low Mg2+) smaller than epileptiform calcium transients. At a distance from the stimulation site, calcium transients and their NMDA-receptor-dependent components were largest in stratum pyramidale in the bicuculline model and in stratum oriens in the low-Mg2+ model. In both models, minimal onset latencies of calcium influx shifted with increasing distance to the stimulation electrode from stratum radiatum to stratum oriens. APV reduced the extent of spread of calcium transients in the low-Mg2+ model. In the bicuculline model, the spatial extent of spread of epileptiform calcium transients was not affected by application of APV; however, the mean velocity of spread was reduced from 0.20 to 0.12 m/s. In conclusion, the large size of calcium transients and of their NMDA-receptor-dependent components in stratum pyramidale or stratum oriens as well as shortest onset latencies of calcium transients at these sites suggest an important role of cell somata, basal dendrites, and possibly local circuit excitatory interactions for the generation and spread of epileptiform activity.

Characterizing the Site and Mode of Action of Dynorphin at Hippocampal Mossy Fiber Synapses in the Guinea Pig

Extracellular field potential recordings from the CA3 region in guinea pig hippocampal slices were used to study the release and action of dynorphin at the mossy fiber synapse. Dynorphin A(1-17) or U69593 inhibited mossy fiber synaptic responses in preparations in which the CA3 region was surgically isolated from the rest of the hippocampus. This inhibition was completely reversed by the kappa 1 selective antagonist nor-BNI, thus establishing the presence of functional kappa 1 receptors in CA3. Inhibitory effects of dynorphin on mossy fiber responses were unaltered in the presence of the N- or P-type Ca2+ channel blockers, omega-CgTx or omega-Aga IVA, respectively. This indicates that the action of dynorphin is independent of the particular type of Ca2+ channel that mediates transmitter release at the mossy fiber terminal. Heterosynaptic inhibition of mossy fiber responses was observed in the presence of nifedipine, omega-CgTx, or omega-Aga IVA, indicating that dynorphin release does not depend specifically on L-, N-, or P-type Ca2+ channels. The blockade of heterosynaptic inhibition by the membrane-permeant Ca2+ chelator EGTA-AM suggests the involvement of a slow Ca(2+)-dependent process in dynorphin release. On the basis of a variety of experimental evidence, we propose that the time course of heterosynaptic inhibition is determined primarily by the time course of clearance of dynorphin in the extracellular space.

Effects of glutamate application on the rhythm of low magnesium-induced epileptiform activity in hippocampal slices of guinea-pigs.

The extracellular concentration of glutamate has previously been reported to increase to more than 10-fold the basal level during seizure activity. In the present study, we tested whether localized increases in extracellular glutamate concentration influence the rhythm of epileptiform discharges in the low-magnesium epilepsy model. In hippocampal slices of guinea-pigs, epileptiform activity was induced by omission of magnesium from the bath fluid. Glutamate and its subreceptor agonists N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were ejected into different strata of the CA3 and CA1 regions using microiontophoretic and micropressure application. Glutamate, NMDA and AMPA applied to the CA3 region, but not to the CA1 region, induced a short-lasting increase in epileptiform discharge frequency, often followed by a transient reduction. The effect was most pronounced with application into the stratum lacunosum-moleculare of the CA3 region and could only be evoked in slices exceeding 400 microns in thickness. The effects on the rhythm of epileptiform discharges induced by NMDA and AMPA were blocked by their specific receptor antagonists. They were not influenced by application of GABAA and GABAB receptor antagonists. Changes in somatic membrane potential of CA3 pyramidal neurons did not correlate with changes in the rhythm of epileptiform discharges elicited in this region. The transient suppression of epileptiform discharges that followed the increase in discharge frequency was abolished by an adenosine A1 receptor antagonist. We propose that localized increases in extracellular glutamate concentration modify the rhythm of epileptiform discharges due to changes in neuronal network activity.

The long-term suppressive effect of prior activation of synaptic inputs by low-frequency stimulation on induction of long-term potentiation in CA1 neurons of guinea pig hippocampal slices

We have investigated the effects of prior activation of afferent inputs by a train of low-frequency stimulation (LFS) on the induction of long-term potentiation (LTP) induced by high-frequency stimulation (tetanus, 100 Hz, 100 pulses) in CA1 neurons of guinea pig hippocampal slices. The parameters of the LFS were altered systematically: the frequency (1 or 5 Hz); the number of pulses (80, 200 or 1000); and the time lag from the LFS to the tetanus (20, 60 or 100 min). Conditioning effects of the LFS on the induction of LTP were evaluated in terms of the slope of the field excitatory postsynaptic potential (S-EPSP) and the amplitude of the population spike (A-PS). LTP could reliably be induced by 100 Hz tetanic stimulation delivered to a naive slice. In contrast, the attempt to induce LTP 60 min after LFS of 200 or 1000 pulses at 1 Hz resulted only in short-term potentiation while the LFS itself produced no significant change in the responses. The suppressive effect on LTP was significantly reduced for 1 Hz LFS with a smaller number of pulses (80 pulses), or a shorter (20 min) or longer (100 min) time lag from the LFS to the tetanus, or with LFS at a higher frequency (5 Hz). When the LFS of 1000 pulses at 1 Hz was delivered in the presence of the N-methyl-D-aspartate (NMDA) receptor antagonist AP5 (D,L-4-amino-5-phosphonovalerate, 50 microM), which was washed out after the end of the LFS, the tetanus given 60 min after application of the LFS produced stable LTP, indicating the involvement of NMDA receptor/channels in the mechanisms of this particular form of synaptic plasticity-long-term suppression of LTP.

Galanin inhibits long-term potentiation at Schaffer collateral-CA1 synapses in guinea-pig hippocampal slices

The role of galanin on long-term potentiation (LTP) of the field excitatory postsynaptic potentials (fEPSPs) at Schaffer collateral-CA1 synapses was investigated in guinea-pig hippocampal slices. Application of galanin (10 −7 M) inhibited the magnitude of LTP without any changes in the amplitude of fEPSP prior to tetanic stimulation. A putative central nervous system (CNS) type galanin receptor antagonist, galanin(1–13)-Pro-Pro-(Ala-Leu) 2-Ala-Amide (M40), did not modify the magnitude of LTP when applied alone. However, M40 significantly reversed the inhibition by galanin when applied with galanin simultaneously. These findings suggest that galanin plays an inhibitory role through the CNS type galanin receptors.

Reduced probability of orthodromically evoked action potential firing in CA1 pyramidal cells of guinea pig hippocampal slices after acute thallium exposure.

We investigated the effect of thallium ions on extracellular field potentials in the CA1 region of guinea pig hippocampal slices in a matched-pair experimental setup. Somatic and dendritic responses evoked by paired-pulse stimulation of the Schaffer collateral-commissural pathway were recorded before, during and after acute thallium exposure and compared to field potentials from nontreated control slices recorded simultaneously. Thallium reduced the orthodromically evoked population spike reversibly in a clear concentration-effect relationship. In contrast, the field excitatory postsynaptic potential fEPSP, as well as the presynaptic fiber volley of the afferent pathway, were not affected by thallium. Furthermore, the paired-pulse facilitation was reversibly reduced during thallium exposure. Input-output relations clearly demonstrated that thallium did not interfere with the presynaptic transmitter release mechanisms or the postsynaptic transmitter receptor sensitivity, but had a predominant postsynaptic target site. Additionally, any influence of thallium ions on the somatic and/or axonal membrane excitability could be excluded, as the antidromically evoked responses after alvear stimulation were not diminished by thallium. Therefore, the main effect of thallium was a decoupling of the somatic from the dendritic activity at the CA1 pyramidal cells. We conclude that the toxic influence of thallium ions in the guinea pig hippocampus must be confined to intracellular somatic mechanisms. Interactions with intracellular organelles and an impairment of their calcium storage capacity are supposed.

A metabotropic glutamate receptor agonist DCG-IV suppresses synaptic transmission at mossy fiber pathway of the guinea pig hippocampus

The effects of specific metabotropic glutamate receptor (mGluR) agonists on field excitatory postsynaptic potentials (fEPSPs) at mossy fiber-CA3 synapses were examined in guinea pig hippocampal slice preparations. Application of a novel and potent group II-selective mGluR agonist (2 S,1′ R,2′ R,3′ R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV; 0.1 μM) reversibly reduced the fEPSPs. Both the group III-selective agonist DL-2-amino-4-phosphonobutyric acid (AP4; 50 μM) and the broad-spectrum agonist 1 S,3 R-1-aminocyclopentane-1,3-dicarboxylic acid (1 S,3 R-ACPD; 5 μM) also reversibly suppressed the fEPSPs. These results suggest that multiple mGluR subtypes (belonging to groups II and III)_are expressed at mossy fiber synapses of the guinea pig hippocampus and activation of the receptors reduces the synaptic excitation, although we cannot exclude the possibility that guinea pig mossy fiber-CA3 synapses express a single class of mGluRs with unique pharmacological profiles.

The Effects of Rhythmical Waves Induced by Carbachol on LTP in Guinea Pig Hippocampal Slices

The cholinergic input to the hippocampus originates in the septum and diagonal band. A bath application of carbachol, a cholinergic agonist, induced different patterns of rhythmical waves depending on its concentration in guinea pig hippocampal slices. Tetanus-induced long-term potentiation (LTP) was differentially facilitated under several concentrations of carbachol. Under the concentrations, which were within the optimum range for generating theta-like waves, LTP was most facilitated. In addition, the higher augmentation of LTP occurred when LTP was induced during the generation of theta-like wave. These results suggested that depending on the activity of the cholinergic septal input to hippocampus, different patterns of rhythmical waves are induced and the plasticity in hippocampal neural networks can be modulated by them.

2337 Effects of long-term potentiation(LTP) on carbachol-induced theta-like activities in guinea pig hippocampal slices

2337 Effects of long-term potentiation(LTP) on carbachol-induced theta-like activities in guinea pig hippocampal slices

238 The effects of low-frequency stimulation (1 hz) on the synaptic transmission in ca1 neurons of guinea pig hippocampal slices

238 The effects of low-frequency stimulation (1 hz) on the synaptic transmission in ca1 neurons of guinea pig hippocampal slices