Perfusion Of Hippocampal Slices Research Articles

Mecamylamine inhibits seizure-like activity in CA1-CA3 hippocampus through antagonism to nicotinic receptors.

Cholinergic modulation of hippocampal network function is implicated in multiple behavioral and cognitive states. Activation of nicotinic and muscarinic acetylcholine receptors affects neuronal excitability, synaptic transmission and rhythmic oscillations in the hippocampus. In this work, we studied the ability of the cholinergic system to sustain hippocampal epileptiform activity independently from glutamate and GABA transmission. Simultaneous CA3 and CA1 field potential recordings were obtained during the perfusion of hippocampal slices with the aCSF containing AMPA, NMDA and GABA receptor antagonists. Under these conditions, spontaneous epileptiform discharges synchronous between CA3 and CA1 were recorded. Epileptiform discharges were blocked by addition of the calcium-channel blocker Cd2+ and disappeared in CA1 after a surgical cut between CA3 and CA1. Cholinergic antagonist mecamylamine abolished CA3-CA1 synchronous epileptiform discharges, while antagonists of α7 and α4β2 nAChRs, MLA and DhβE, had no effect. Our results suggest that activation of nicotinic acetylcholine receptors can sustain CA3-CA1 synchronous epileptiform activity independently from AMPA, NMDA and GABA transmission. In addition, mecamylamine, but not α7 and α4β2 nAChRs antagonists, reduced bicuculline-induced seizure-like activity. The ability of mecamylamine to decrease hippocampal network synchronization might be associated with its therapeutic effects in a wide variety of CNS disorders including addiction, depression and anxiety.

Mecamylamine modulates epileptiform discharges in low-Mg2+ model of epilepsy

Mecamylamine is a nonselective antagonist of nicotinic acetylcholine receptors that was developed as an antihypertensive medication and is now being studied for its beneficial effects in several pathological conditions, such as substance abuse, depression, anxiety and epilepsy. In this work, we investigate the effect of mecamylamine on the manifestations of seizure-like activity evoked by perfusion of hippocampal slices with low-Mg2+ solution of artificial cerebrospinal fluid. Reducing Mg2+ concentration in extracellular solution induced two distinct types of epileptiform activity: recurring seizure-like activity and continuous discharges. Application of mecamylamine significantly increased internal frequency of recurring seizurelike activity and significantly decreased inter-event intervals between continuous discharges. We also show that mecamylamine significantly decreased internal frequency of continuous epileptiform discharges. The results of our work show that mecamylamine exerts modulatory effect on the low-Mg2+ epileptiform activity induced in hippocampal acute rat brain slices. Additionally, obtained results indicate the role of nicotinic acetylcholine receptors in the modulation of hippocampal network activity, which might explain some of the therapeutic effects of mecamylamine in CNS.

Calcium-Sensitive Translocation of Calmodulin and Neurogranin between Soma and Dendrites of Mouse Hippocampal CA1 Neurons

Calmodulin (CaM) and neurogranin (Ng) are two abundant neuronal proteins whose interactions are implicated in the regulation of synaptic responses and plasticity. We employed the “low-calcium” model of epilepsy in hippocampal slices to investigate the mobilization of these two proteins in CA1 pyramidal neurons. Perfusion of mouse hippocampal slices with Ca2+-free artificial CSF (ACSF) caused a suppression of synaptic transmission and generation of epileptic activity; these responses could be reversed by normal Ca2+-containing ACSF. Fluorescence immunochemical staining of control hippocampal slices bathed in normal ACSF revealed that CaM and Ng were more concentrated in soma than in dendrites; especially for CaM, it was concentrated in the nucleus. Perfusion of hippocampal slices with Ca2+-free ACSF caused translocation of these two proteins from soma to dendrites, and this trafficking was also reversed by Ca2+-containing buffer. A reduction of ∼15 and 40 nM intracellular Ca2+, [Ca2+]i, caused half-maximum translocation of Ng and CaM, respectively. Hippocampal CA1 pyramidal neurons were the most responsive to this Ca2+-sensitive translocation as compared to those from other areas of the hippocampus. These results illustrated the unique feature of hippocampal CA1 pyramidal neurons in sequestering high concentrations of CaM and Ng in soma and releasing them to distal dendrites at reducing level of [Ca2+]i.

A role for monomeric G-proteins in synaptic plasticity in the rat dentate gyrus in vitro

Recent studies have implicated Ras signalling in synaptic plasticity. In this study we have investigated a role for the low molecular weight G proteins Ras, Rap, Ra1 and Rac in long-term potentiation and depression using Clostridium Sordelli Lethal Toxin-82 (LT-82), which inactivates Ras, Rap, Ra1 and Rac, and manumycin A, a Ras inhibitor. Perfusion of hippocampal slices with LT-82 (200 ng/ml) attenuated LTP (83±10%, n=5, P<0.01, compared with controls of 160±11% at 60 min post HFS, n=5). LT-82 had no effect on LTD (63±1% at 100 ng/ml, n=5 and 66±1% at 200 ng/ml, n=4, compared to controls of 56±6%, n=6). Manumycin A (2μM) had no effect on LTP (162±2%, n=5, compared to controls of 167±13%, n=5), but significantly attenuated LTD (88±6%, n=5, P<0.01, compared to controls of 63±9%, n=7). LT-82 (200 ng/ml) significantly increased the amplitude of the isolated NMDA-EPSP at 60 min post-drug application (240±40%, n=5, P<0.01, compared with controls of 100±4%, n=5). However, manumycin A, had no significant effect on NMDAR-EPSP amplitude (92±2%, n=5, compared with controls). These results demonstrate an important role for Ras in LTD and a role for Rap, Ra1 and Rac in LTP.

Selective suppression of hippocampal region hyperexcitability related to seizure susceptibility in epileptic El mice by the GABA-transporter inhibitor tiagabine

High seizure susceptibility in El mice is associated with disinhibition in the dentate gyrus (DG) and paired-pulse facilitation in the CA3 area in hippocampal slices [Brain Res. 745 (1997) 165; Brain Res. 779 (1998) 324]. A decrease in γ-aminobutyric acid (GABA)-mediated inhibition and an increase in excitatory inputs to the major neurons seem to be the responsible mechanisms, respectively, for these phenomena. In this study, we examined the effects of tiagabine, an inhibitor of GABA transporter, on hyperexcitation in vivo and in slice preparations. Tiagabine (0.3–0.5 mg/kg) suppressed the occurrence of seizures to about 20% of controls with an ED 50 value of about 0.17 mg/kg. In addition, perfusion of hippocampal slices with tiagabine (20 μM) counteracted the paired-pulse facilitation in the CA3 region over the entire range of interpulse intervals ( P<0.05, two-way ANOVA) and reduced the disinhibition in the DG measured at 10 and 20 ms during short interpulse intervals ( P<0.005, paired t-test). The CA1 region in the El mice, as well as in a non-epileptic parental strain of ddY mice did not respond to the drug. However, frequency potentiation of CA3 was enhanced in both strains ( P<0.05, paired t-test). Our results suggest that within the hippocampus the antiepileptic action of tiagabine is selectively suppressive for hyperexcitability of DG and CA3, which are responsible for seizure-susceptibility in El mice.

Glial protein S100B modulates long-term neuronal synaptic plasticity.

Glial cells are traditionally regarded as elements for structural support and ionic homeostasis, but have recently attracted attention as putative integral elements of the machinery involved in synaptic transmission and plasticity. Here, we demonstrate that calcium-binding protein S100B, which is synthesized in considerable amounts in astrocytes (a major glial cell subtype), modulates long-term synaptic plasticity. Mutant mice devoid of S100B developed normally and had no detectable abnormalities in the cytoarchitecture of the brain. These mutant mice, however, had strengthened synaptic plasticity as identified by enhanced long-term potentiation (LTP) in the hippocampal CA1 region. Perfusion of hippocampal slices with recombinant S100B proteins reversed the levels of LTP in the mutant slices to those of the wild-type slices, indicating that S100B might act extracellularly. In addition to enhanced LTP, mutant mice had enhanced spatial memory in the Morris water maze test and enhanced fear memory in the contextual fear conditioning. The results indicate that S100B is a glial modulator of neuronal synaptic plasticity and strengthen the notion that glial-neuronal interaction is important for information processing in the brain.

Effects of postsynaptic GABA B receptor activation on epileptiform activity in hippocampal slices

GABA B receptor agonists have been reported to have both pro- and antiepileptic properties. Here, the effects of a GABA B receptor agonist, baclofen, were studied on epileptiform activity induced in the absence of synaptic transmission — to focus on the postsynaptic effects. Perfusion of hippocampal slices with 0-added calcium and high potassium induced field bursts in CA1 and the dentate gyrus. Addition of baclofen caused a transient suppression of the field bursts in CA1 and the dentate gyrus. The duration of the suppression was dependent on the concentration of baclofen and when the bursts reappeared they had a larger amplitude than before baclofen. Baclofen also suppressed the multiple population spikes evoked by antidromic stimulation in the dentate gyrus. This effect also decreased with continued baclofen perfusion. The effects of baclofen on the amplitude of the spontaneous field bursts and on the stimulation-induced multiple population spikes were blocked by the GABA B receptor antagonist SCH 50911, suggesting that these effects of baclofen are mediated by GABA B receptor activation. Baclofen significantly increased the peak extracellular K + concentration during each field burst in the dentate gyrus but did not change the baseline level of K + between field bursts. The results suggest that postsynaptic GABA B receptor activation by baclofen has transient antiepileptic effects followed by a rebound increase in excitability.

Role of Tissue Plasminogen Activator Receptor LRP in Hippocampal Long-Term Potentiation

The low-density lipoprotein (LDL) receptor-related protein (LRP) is a multifunctional endocytic receptor that is expressed abundantly in neurons of the CNS. Both LRP and several of its ligands, including tissue plasminogen activator (tPA), apolipoprotein E/lipoproteins, alpha(2)-macroglobulin, and the beta-amyloid precursor protein, have been implicated in various neuronal functions and in the pathogenesis of Alzheimer's disease. It has been reported that induction of tPA expression may contribute to activity-dependent synaptic plasticity in the hippocampus and cerebellum. In addition, long-term potentiation (LTP) is significantly decreased in mice lacking tPA. Here we demonstrate that tPA receptor LRP is abundantly expressed in hippocampal neurons and participates in hippocampal LTP. Perfusion of hippocampal slices with receptor-associated protein (RAP), an antagonist for ligand interactions with LRP, significantly reduced late-phase LTP (L-LTP). In addition, RAP also blocked the enhancing effect of synaptic potentiation by exogenous tPA in hippocampal slices prepared from tPA knock-out mice. Metabolic labeling and ligand binding analyses showed that both tPA and LRP are synthesized by hippocampal neurons and that LRP is the major cell surface receptor that binds tPA. Finally, we found that tPA binding to LRP in hippocampal neurons enhances the activity of cyclic AMP-dependent protein kinase, a key molecule that is known to be involved in L-LTP. Taken together, our results demonstrate that interactions between tPA and cell surface LRP are important for hippocampal L-LTP.

Oxygen/Glucose Deprivation in Hippocampal Slices: Altered Intraneuronal Elemental Composition Predicts Structural and Functional Damage

Effects of oxygen/glucose deprivation (OGD) on subcellular elemental composition and water content were determined in nerve cell bodies from CA1 areas of rat hippocampal slices. Electron probe x-ray microanalysis was used to measure percentage water and concentrations of Na, P, K, Cl, Mg, and Ca in cytoplasm, nucleus, and mitochondria of cells exposed to normal and oxygen/glucose deficient medium. As an early (2 min) consequence of OGD, evoked synaptic potentials were lost, and K, Cl, P, and Mg concentrations decreased significantly in all morphological compartments. As exposure to in vitro OGD continued, a negative DC shift in interstitial voltage occurred ( approximately 5 min), whereas general elemental disruption worsened in cytoplasm and nucleus (5-42 min). Similar elemental changes were noted in mitochondria, except that Ca levels increased during the first 5 min of OGD and then decreased over the remaining experimental period (12-42 min). Compartmental water content decreased early (2 min), returned to control after 12 min of OGD, and then exceeded control levels at 42 min. After OGD (12 min), perfusion of hippocampal slices with control oxygenated solutions (reoxygenation) for 30 min did not restore synaptic function or improve disrupted elemental composition. Notably, reoxygenated CA1 cell compartments exhibited significantly elevated Ca levels relative to those associated with 42 min of OGD. When slices were incubated at 31 degreesC (hypothermia) during OGD/reoxygenation, neuronal dysfunction and elemental deregulation were minimal. Results show that in vitro OGD causes loss of transmembrane Na, K, and Ca gradients in CA1 neurons of hippocampal slices and that hypothermia can obtund this damaging process and preserve neuronal function.

On the Respective Roles of Nitric Oxide and Carbon Monoxide in Long-Term Potentiation in the Hippocampus

Perfusion of hippocampal slices with an inhibitor nitric oxide (NO) synthase blocked induction of long-term potentiation (LTP) produced by a one-train tetanus and significantly reduced LTP by a two-train tetanus, but only slightly reduced LTP by a four-train tetanus. Inhibitors of heme oxygenase, the synthetic enzyme for carbon monoxide (CO), significantly reduced LTP by either a two-train or four-train tetanus. These results suggest that NO and CO are both involved in LTP but may play somewhat different roles. One possibility is that NO serves a phasic, signaling role, whereas CO provides tonic, background stimulation. Another possibility is that NO and CO are phasically activated under somewhat different circumstances, perhaps involving different receptors and second messengers. Because NO is known to be activated by stimulation of NMDA receptors during tetanus, we investigated the possibility that CO might be activated by stimulation of metabotropic glutamate receptors (mGluRs). Consistent with this idea, long-lasting potentiation by the mGluR agonist tACPD was blocked by inhibitors of heme oxygenase but not NO synthase. Potentiation by tACPD was also blocked by inhibitors of soluble guanylyl cyclase (a target of both NO and CO) or cGMP-dependent protein kinase, and guanylyl cyclase was activated by tACPD in hippocampal slices. However, biochemical assays indicate that whereas heme oxygenase is constitutively active in hippocampus, it does not appear to be stimulated by either tetanus or tACPD. These results are most consistent with the possibility that constitutive (tonic) rather than stimulated (phasic) heme oxygenase activity is necessary for potentiation by tetanus or tACPD, and suggest that mGluR activation stimulates guanylyl cyclase phasically through some other pathway.

On the Respective Roles of Nitric Oxide and Carbon Monoxide in Long-Term Potentiation in the Hippocampus

Perfusion of hippocampal slices with an inhibitor nitric oxide (NO) synthase blocked induction of long-term potentiation (LTP) produced by a one-train tetanus and significantly reduced LTP by a two-train tetanus, but only slightly reduced LTP by a four-train tetanus. Inhibitors of heme oxygenase, the synthetic enzyme for carbon monoxide (CO), significantly reduced LTP by either a two-train or four-train tetanus. These results suggest that NO and CO are both involved in LTP but may play somewhat different roles. One possibility is that NO serves a phasic, signaling role, whereas CO provides tonic, background stimulation. Another possibility is that NO and CO are phasically activated under somewhat different circumstances, perhaps involving different receptors and second messengers. Because NO is known to be activated by stimulation of NMDA receptors during tetanus, we investigated the possibility that CO might be activated by stimulation of metabotropic glutamate receptors (mGluRs). Consistent with this idea, long-lasting potentiation by the mGluR agonist tACPD was blocked by inhibitors of heme oxygenase but not NO synthase. Potentiation by tACPD was also blocked by inhibitors of soluble guanylyl cyclase (a target of both NO and CO) or cGMP-dependent protein kinase, and guanylyl cyclase was activated by tACPD in hippocampal slices. However, biochemical assays indicate that whereas heme oxygenase is constitutively active in hippocampus, it does not appear to be stimulated by either tetanus or tACPD. These results are most consistent with the possibility that constitutive (tonic) rather than stimulated (phasic) heme oxygenase activity is necessary for potentiation by tetanus or tACPD, and suggest that mGluR activation stimulates guanylyl cyclase phasically through some other pathway.

Balysum-2 inhibitis evoked activity of the pyramidal neurons in hippocampus

The effects of Balysum-2 on the responsiveness of field CA1 hippocampal pyramidal neurons was studied in experiments with cultured cerebral slices. Addition to the medium of 10−4M Balysum-2 or its active ingredient (comenic acid) led to a reversible decrease in the peak amplitude of focal response (pop-spike) recorded in the pyramidal layer CA1 during stimulation of the radial layer. Perfusion of hippocampal slices with a solution containing the noncompetitive GABAA-antagonist picrotoxin prevented the effects of Balysum-2 and comenic acid. Inhibition of hippocampal pyramids by Balysum-2 and comenic acid is probably caused by an incease in the inhibitory effect mediated by GABAA-receptors.

Pathway specificity of noradrenergic plasticity in the dentate gyrus.

Previous experiments have described highly specific effects of noradrenergic agonists on synaptic transmission in the dentate gyrus (DG). For example, perfusion of hippocampal slices with the beta-noradrenergic agonist isoproterenol induces a long-lasting potentiation (LLP) of extracellularly recorded responses following stimulation of the medial perforant path (PP), and long-lasting depression (LLD) of responses evoked by stimulation of the lateral PP (Dahl D, Sarvey JM, 1989, Proc Natl Acad Sci USA 86:4776-4780). To examine the possible interactions of LLP, LLD, and long-term potentiation induced by tetanic stimulation (LTP), the authors recorded extracellular field potentials evoked in the DG by stimulation of the lateral or medial perforant path following LTP and LLP or LLD, invoked in different orders. After establishment of LLP or LLD by bath application of isoproterenol, subsequent tetanization of the respective afferents resulted in additional potentiation of the medial PP-evoked response and return of the lateral PP-evoked response to baseline levels. In other slices, application of isoproterenol after establishment of LTP resulted in further potentiation of medial PP-evoked responses but no change in the potentiated response evoked by lateral PP stimulation. Thus the pathway specificity was maintained irrespective of the history of previous potentiation or depression. Experiments using the specific beta 1 antagonist metoprolol further confirmed pathway specificity. Perfusion with 20 microM of metoprolol appeared to reduce LTP evoked by stimulation of the medial but not lateral PP. In a subsequent experiment, metoprolol in the absence of tetanization produced LLD of the medial PP-evoked response and LLP of the lateral PP-evoked response, opposite to the effects of ISO.(ABSTRACT TRUNCATED AT 250 WORDS)

Low sodium injury in the hippocampal slice is mediated through NMDA receptors

Perfusion of hippocampal slices with normoxic medium containing no added sodium resulted in a rapid loss of the CA1 population spike, with only 35 ± 10% (S.E.M.)( P <0.001) recovery after a 15 min exposure. This injury was prevented by the non-competitive N- methyl- d-aspartate (NMDA) antagonist MK-801 (93 ± 4% recovery, P <0.001f), suggesting that low sodium injury may be mediated by opening of the NMDA receptor-associated ionic channels, possibly secondary to the well known sodium dependency of transmitter uptake systems. By contrast, slice perfusion with a medium moderately low in sodium (26 mM) produced only slight injury to the CA1 population spike under normoxic conditions (76 ± 8% recovery) and provided no protection against hypoxic injury. Low chloride medium also provided no protection against hypoxaxic injury.

Fibroblast Growth Factor Enhances Long-term Potentiation in the Hippocampal Slice.

Recently we reported that perfusion of hippocampal slices with epidermal growth factor (EGF) lead to enhancement of potentiated responses after tetanic stimulation. In the present study we report that basic fibroblast growth factor (FGF) can also lead to an enhancement of potentiated responses. FGF is a mitogen for several cell types and exhibits neurotrophic effects on neurons of the central nervous system (CNS). Rat hippocampal slices were perfused with FGF at a concentration of 10-9 M. During extra- and intracellular recordings in the CA1-region, the addition of FGF to the perfusing medium produced no change in evoked responses if single pulse or paired pulse stimulation was used. Furthermore FGF had no influence on the resting membrane potential and input resistance. However, after tetanic stimulation, FGF-treated slices showed an increase in the magnitude of potentiation compared to control slices. Taken together with the EGF data these results support the hypothesis that growth factors like FGF with neurotrophic potential on CNS-neurons can influence synaptic efficacy. Furthermore these results show that factors which are able to modulate developmental plasticity and regenerative plasticity can also modulate synaptic plasticity.

NMDA-receptor-independent effects of low magnesium: involvement of adenosine

Perfusion of hippocampal slices with magnesium-free media elicits epileptiform activity attributable partly to the activity of N-methyl- D-aspartate (NMDA) receptors, but recent reports have documented an NMDA-independent enhancement of orthodromic potentials by moderate reductions in magnesium concentration. The present experiments indicate that this enhancement is comparable with that produced by perfusion with an adenosine antagonist, 8-phenyltheophylline, and that superfusion with this compound or adenosine deaminase precludes any enhancement of potential size in low magnesium solutions. The low magnesium enhancement is probably attributable to the recently described magnesium dependency of presynaptic inhibition by adenosine.