Neuroprotective effects of extracellular glutamate are absent in hippocampal organotypic cultures treated with the amyloid peptide Abeta(25-35).

Hippocampal cells are particularly vulnerable in Alzheimer's disease but the cause of cell death is unknown. Amyloid toxicity has been implicated in hippocampal cell death, but its specific mechanisms are poorly understood. We used confocal microscopy to examine the effects of the amyloid peptide fragment 25-35 (Abeta(25-35)) on cell death in organotypic hippocampal slice cultures. Addition of glutamate to the culture medium significantly improved nerve cell survival in cultures subjected to consecutive medium exchanges. This effect was lost if cultures were treated with the amyloid peptide fragment Abeta(25-35) but not the inactive peptide 35-25. These data suggest that one of the mechanisms responsible for amyloid toxicity may be inhibition of the survival promoting effects of extracellular glutamate.

Glutamate-mediated neuroprotection against N-methyl-D-aspartate toxicity: a role for metabotropic glutamate receptors.

We studied N-methyl-D-aspartate-induced cell death in organotypic hippocampal slices from seven-day-old Wistar rat pups cultured for 12-14 days in a medium containing no added glutamate. Propidium iodide fluorescence intensity was used as an indicator of cell death measured with the help of confocal microscopy. Exposure of slices for 2h to L-glutamate (1-500 microM) prior to the N-methyl-D-aspartate challenge significantly reduced N-methyl-D-aspartate-induced cell death. Glutamate at 10 and 500 microM concentrations was highly protective against N-methyl-D-aspartate-induced cell death, but was less protective at the 1 microM concentration. The protection was not blocked by the Na(+) channel blocker tetrodotoxin (1 microM), the N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonopentanoic acid (20 microM) or the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (20 microM). 1S, 3R-1-Aminocyclopentane-trans-1,3-dicarboxylic acid, an agonist at metabotropic glutamate receptor types 1, 2/3 and 5, was protective at 100 microM but not at 50 microM. In contrast, the ionotropic glutamate receptor agonist aspartate (250 microM) facilitated N-methyl-D-aspartate toxicity. Treatment of slices with the protein kinase C inhibitor staurosporine (0.2 microM) or antisense oligonucleotide (10nM, 72 h) that selectively inhibits metabotropic glutamate receptor type 5 synthesis significantly reduced glutamate protection. These results suggest that ambient glutamate may reduce nerve cell susceptibility to injury caused by excessive N-methyl-D-aspartate receptor activation by acting at metabotropic glutamate receptors linked to protein kinase C.

Down-regulation of mGluR5 by antisense deoxynucleotides alters pharmacological responses to applications of ACPD in the rat hippocampus.

Metabotropic glutamate receptors are thought to be important regulators of synaptic transmission and plasticity in the hippocampus. The metabotropic glutamate receptor subtype mGluR5 is expressed in hippocampal pyramidal neurons but its function remains unknown due to the lack of selective pharmacological blockers. We inhibited the synthesis of mGluR5 with antisense oligonucleotides injected into the hippocampus in vivo. The functional effects of altered mGluR5 expression were measured electrophysiologically in the CA1 region of the hippocampus during applications of the metabotropic agonist 1S,3R-ACPD (50 microM) to hippocampal slices from injected animals. The results show a concomitant reduction of the mGluR5 receptor protein and physiological effects in the hippocampus. The major effect found in the antisensetreated animals was the lack of an excitatory action normally produced by 1S,3R-ACPD. Another effect attributed to metabotropic glutamate receptors, depression of synaptic transmission, had a more rapid onset, but unchanged magnitude, while long-term potentiation remained unchanged. The specificity and effectiveness of the antisense treatment were confirmed using mismatched oligonucleotides and immunoblotting. We conclude that the metabotropic glutamate receptor subtype mGluR5 plays a major role in the regulation of cell excitability in the hippocampus without directly affecting synaptic transmission or long-term potentiation. Moreover, in vivo applications of antisense deoxynucleotides are a useful approach in studies of neurotransmitter receptor subtypes.

Brain-derived peptides inhibit synaptic transmission via presynaptic GABAB receptors in CA1 area of rat hippocampal slices.

GABAB receptors form the basis of a powerful and versatile inhibitory system in the mammalian brain. Presynaptic and postsynaptic actions of GABAB receptors have been described in various brain regions, including the hippocampus. We report here on a novel pharmacological agent, presumably a peptide, which inhibits synaptic transmission in the CA1 area of the rat hippocampus via GABAB receptors. The agent is a component of a nootropic drug, Cerebrolysin, obtained from pig's brain extract. In contrast to other, presently known agonists, such as baclofen or GABA, Cerebrolysin acts preferentially on presynaptic GABAB receptors and has no detectable postsynaptic inhibitory effects. Additional, postsynaptic depolarizing action of the drug resulting in increased excitability is pharmacologically distinct from the GABAB response and partially masked by the inhibition. The presynaptic GABAB agonist may add to clinical effects of Cerebrolysin in treatment of brain injuries. Moreover, it promises to be a useful experimental agent in further studies of many possible functional roles of GABAB receptors.

Brain tissue hydrolysate acts on presynaptic adenosine receptors in the rat hippocampus.

Adenosine is a potent inhibitory modulator in the brain. It suppresses glutamatergic synaptic transmission and possibly acts as a brain endogenous neuroprotective agent. In this study we have examined the effects of a clinically used porcine brain tissue hydrolysate, Cerebrolysin, on synaptic transmission in the CA1 area of rat hippocampal slices. A major effect of the drug at doses approximating those administered clinically to demented patients was a depression of synaptic transmission at the Schaffer collateral-commissural pathway in CA1. Detailed analysis showed that the inhibition is presynaptic and can be reduced by low doses of a specific blocker of adenosine A1 receptors, 8-cyclopentyltheophylline. Because Cerebrolysin does not contain a detectable amount of adenosine, the effect on adenosine A1 receptors must be indirect, perhaps by release of the endogenous agonist. This action of Cerebrolysin is consistent with a putative neuroprotective action underlying its clinical usage.

Effects of brain tissue hydrolysate on synaptic transmission in the hippocampus.

A mix of peptides and amino acids obtained from porcine brain tissue (Cerebrolysin) has been shown to affect passive avoidance behavior in neonatal rats. To identify the active components and mechanisms of action, Cerebrolysin effects were studied in in vitro hippocampal slices. Cerebrolysin induced dose-dependent suppression followed by a small rebound increase of synaptic responses in the CA1 but not dentate gyrus neurons. These actions may be due to peptides present in Cerebrolysin and may contribute to its reported behavioural effects.

Haloperidol and loxapine but not clozapine increase synaptic responses in the hippocampus.

Effects of two typical neuroleptics haloperidol and loxapine, and an atypical antipsychotic clozapine on excitatory synaptic transmission were examined in the CA1 area of rat hippocampal slices. Haloperidol (10 mumol) and loxapine (10 mumol) increased extracelllar evoked field potentials by an average of 57 and 125% of the control level respectively. Clozapine (50 mumol) induced a transient depression (21% of control) of the response followed by a small augmentation (10%). Examination of haloperidol actions using the whole cell clamp technique showed an increase in the amplitude of the excitatory postsynaptic current (e.p.s.c.) in response to the drug without any apparent changes in the resting membrane current. These data suggest that haloperidol and loxapine may act by enhancing excitatory synaptic transmission in some areas of the brain, and that novel antipsychotic clozapine differs in its mechanism of action.

Rearrangement of centromeric satellite DNA in hippocampal neurons exhibiting long-term potentiation.

In situ hybridization in conjunction with three-dimensional reconstruction was used to examine the topology of satellite DNA (sDNA) sequences in hippocampal CA1 neurons. In slices fixed immediately after preparation, 4-5 signals/nucleus were detected in CA1, CA3 and dentate neurons. 70-80% of 154 neurons examined in these 3 areas displayed all signals at the nuclear periphery. In the remaining fraction of neurons, sDNA signals were divided between the nucleolus and the nuclear periphery. sDNA signals were consistently localized to the nuclear midplane. Slices left to equilibrate in artificial cerebral spinal fluid for 1 h, in the absence of potentiation, exhibited a significant increase in the total number of signals/nucleus in CA1 and dentate neurons. This increase in the number of signals occurred in both nucleolar and peripheral compartments, with the number of the nucleolar compartment nearly doubling. The total number of signals/nucleus was found to be consistently reduced in tetanized CA1 neurons (4.89 +/- 0.09 signals/nucleus, n = 195, P less than 0.05) as compared to neurons from unpotentiated slices (5.27 +/- 0.10 signals/nucleus, n = 81). A similar decrease in the total number of signals/nucleus was also observed in CA1 neurons exposed to N-methyl-D-aspartate (NMDA), from 5.27 +/- 0.10 signals/nucleus (n = 81) to 5.00 +/- 0.08 signals/nucleus (n = 215, P less than 0.05). In contrast, dentate neurons, employed as internal controls, did not exhibit any change in number and compartmentalization of sDNA signals.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabotropic receptors and 'slow' excitatory actions of glutamate agonists in the hippocampus.

The actions of glutamate in the CNS can be divided into ionotropic and metabotropic effects. The ionotropic receptors participate in synaptic transmission by directly opening nonselective cation channels. Recently, a so-called 'metabotropic effect' of glutamate has been described and is attributed to a novel metabotropic glutamate receptor. This effect consists of increased hydrolysis of membrane phosphoinositides, production of the second messengers diacylglycerol and inositol 1,4,5-trisphosphate, and mobilization of intracellular Ca2+. Activation of metabotropic glutamate receptors blocks the slow Ca(2+)-dependent K+ conductance and increases the membrane excitability of neurones. In addition, metabotropic agonists block the excitatory synaptic transmission supported by the ionotropic glutamate receptor, and may therefore play a critical role in synaptic plasticity. However, intracellular mechanisms linking metabotropic glutamate receptors with ionic channels remain unclear. This article discusses recent findings concerning metabotropic agonist effects on membrane currents and synaptic transmission, the pharmacology of the agonists and the roles played by G proteins and second messengers in mediating their effects.

Agonists at metabotropic glutamate receptors presynaptically inhibit EPSCs in neonatal rat hippocampus.

1. The effects of metabotropic glutamate receptor agonists on excitatory synaptic transmission in the CA1 region of rat hippocampal slices (11-30 days) were studied using extracellular and whole-cell patch-clamp recording techniques. 2. Trans-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD; 25-100 microM) reversibly depressed excitatory postsynaptic currents (EPSCs) without affecting presynaptic fibre excitability or EPSC reversal potential. 3. Ibotenate (25 microM) or L-glutamate (250 microM), in the presence of the N-methyl-D-aspartate (NMDA) receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV, 50-75 microM), depressed the EPSC amplitude while inducing no detectable inward current. L-2-Amino-4-phosphonobutyrate (L-AP4, 25-100 microM), the phosphonic derivative of glutamate, also depressed EPSC amplitude and caused no detectable inward current. 4. The NMDA receptor-mediated component of the EPSC recorded in the presence of the non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20-30 microM) was depressed by trans-ACPD, L-AP4, or quisqualate (1-2 microM). 5. The response to ionophoretic application of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) was unaffected by trans-ACPD or L-AP4 although the simultaneously recorded EPSC was strongly depressed. In addition, paired-pulse facilitation (50-75 ms interstimulus interval) was reversibly enhanced by trans-ACPD or L-AP4. These results indicate that the depression of synaptic transmission likely was mediated by a presynaptic 'autoreceptor'. 6. The effects of trans-ACPD or L-AP4 on synaptic transmission decreased significantly over ages 12-30 days and were minimal in adult (greater than 80 days) slices. 7. The depression of synaptic transmission caused by trans-ACPD or L-AP4 was not altered following the induction of long-term potentiation (LTP). 8. The results indicate that metabotropic glutamate receptor agonists suppress excitatory synaptic transmission in CA1 pyramidal cells by an action at a presynaptic site. This effect is developmentally regulated and is maximally expressed during the first postnatal month.

Long-term potentiation of synaptic responses in the rat dentate gyrus is due to increased quantal content.

Long-term potentiation (LTP) of synaptic responses in the dentate gyrus neurons of the rat hippocampus was studied in in vitro slices with the use of intracellular recordings. The goal of the study was to determine if the expression of LTP is pre- or postsynaptic. LTP was induced by tetanic stimulation of the perforant pathway in the presence of bicuculline. The expression of potentiation was measured during low-intensity stimulation at 1-5 Hz. It was found that a 104% (S.E.M. +/- 35, n = 5) increase in the amplitude of evoked synaptic potentials was associated with a reduction in the number of transmission failures to 38% (S.E.M. +/- 15, n = 5) of the control values. The size of quantal responses was determined on the basis of asynchronous release from stimulated synapses. The average size of the quanta remained unchanged during LTP. The evident increase of quantal content suggests a presynaptic locus for expression of LTP.

NMDA depolarizations and long-term potentiation are reduced in the aged rat neocortex.

N-Methyl-D-aspartate (NMDA) responses were recorded intracellularly in layer V neocortical neurons in in vitro slices taken from young (4-6 months) and aged (27-29 months) Fischer 344 rats. Increasing amounts of NMDA produced membrane depolarizations in both groups of cells. The regression analysis showed significantly reduced sensitivity to NMDA in old neurons compared to young. A significant long-term potentiation of the field potential evoked by subcortical white matter stimulation was present in young but not in old slices. These results suggest that aging results in a decreased sensitivity to NMDA and impaired synaptic plasticity in the neocortex.

NMDA and quisqualate reduce a Ca-dependent K+ current by a protein kinase-mediated mechanism.

Stimulation of N-methyl-D-aspartate (NMDA) or quisqualate (Quis) receptors by submicromolar concentrations of NMDA or Quis but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) reduced post-spike train after hyperpolarizations (AHPs) and blocked the underlying Iahp in dentate granule (DG) neurones in vitro. The NMDA but not Quis action was blocked by the NMDA receptor blocker 2-D,L-aminophosphonovaleric acid (APV). Actions of both NMDA and Quis were abolished by isoquinolinesulphonyl-2-methyl-piperazine dihydrochloride (H-7), an inhibitor of several protein kinases. These data suggest that there is a link between excitatory amino acid receptor activation, the protein kinase system, and neuronal excitability.

Modulatory actions of serotonin on ionic conductances of hippocampal dentate granule cells.

Pressure ejection of serotonin (2 x 10(-4) M) onto dentate granule neurons in vitro produced a short-lasting membrane hyperpolarization associated with a 10-30% decrease in the input resistance. The hyperpolarization magnitude depended on the extracellular K+ concentration but not on the extra or intracellular Ca2+ concentration. It was followed by a depolarization, especially when serotonin was applied onto the perisomatic area of the neuron. The post-spike-train afterhyperpolarization, which represents a Ca2+-dependent K+ conductance, was decreased by serotonin by 10-100% and remained reduced for 2-10 min following the serotonin-induced hyperpolarization. Decreased adaptation of cell firing was also observed following serotonin application. Ca2+ action potentials evoked by intracellular depolarizing current pulses in the presence of the Na+ channel blocker tetrodotoxin and the K+ channel blocker tetraethylammonium were followed by a large afterhyperpolarization, which was markedly reduced for several minutes following serotonin application. The preceding Ca2+ action potential was either unaffected or prolonged. The hyperpolarization occurring in response to localized application of serotonin, and the reduction of the afterhyperpolarization, may represent two different mechanisms of serotonin action, probably mediated by different mechanisms. The slow time course of the late depolarization and the afterhyperpolarization depression represent modulatory effects of serotonin on dentate granule neurons.

The effects of serotonin on N-methyl-D-aspartate and synaptically evoked depolarizations in rat neocortical neurons.

The effects of serotonin (5-HT) on neuronal responses to the excitatory amino acid agonist N-methyl-D-aspartate (NMDA) were examined in neocortical slices of the Fischer rat using current-clamp and single-electrode voltage-clamp techniques. Layer V neocortical neurons responded to application of NMDA by depolarization with no change or an apparent increase in input resistance. Following perfusion with 10(-5) M 5-HT, the response of these neurons to NMDA was significantly increased in both amplitude and duration, whereas neuronal responses to quisqualic acid and acetylcholine were not altered by 5-HT. Furthermore, the enhanced response to NMDA in 5-HT was long-lasting, and could not be reversed during the course of the experiment. Resting membrane potential and the postspike train afterhyperpolarization were not significantly altered by 5-HT, although the input resistance was decreased by 5-HT. Excitatory postsynaptic potentials (EPSPs) were usually not affected or reversibly decreased by 5-HT. However, in a few cells exhibiting a complex voltage-dependent EPSP, 5-HT produced a long-lasting enhancement in the amplitude of the EPSP. Under voltage-clamp conditions, with Na+- and K+-channels blocked, 5-HT enhanced the inward current stimulated by application of NMDA. It is suggested that 5-HT selectively enhances the voltage- and Ca2+-dependent NMDA response.

Reversed ethanol effects on potassium conductances in aged hippocampal dentate granule neurons.

The effect of low dose (20 mM) ethanol superfusion on the membrane and synaptic properties of dentate granule neurons was studied in hippocampal slices from young-mature (6-8 months) and old (25-29 months) Fischer-344 rats. In young neurons, ethanol hyperpolarized the resting membrane potential (RMP) and prolonged the post-spike train afterhyperpolarization (AHP). By contrast, ethanol depolarized old neurons and decreased their AHPs, in addition to reducing IPSP amplitudes and spike frequency adaptation. These effects can be explained by ethanol-enhancing potassium conductance (gK) in young neurons and diminishing gK in old neurons.

Altered modulatory actions of serotonin on dentate granule cells of aged rats.

The effects of serotonin (5-HT) on dentate granule (DG) neurons in hippocampal slices taken from young mature (6-8 months) and old (25-29 months) rats were compared. Intracellular measurements of membrane potential, cell input resistance and slow postspike afterhyperpolarization did not differ significantly between young and old neurons. Neurons recorded in slices taken from old animals responded with less hyperpolarization to increasing doses of the drug, and their responses were significantly reduced after repeated applications of 5-HT. Serotonin-mediated reduction of the slow afterhyperpolarization in young DG neurons was less prominent or totally absent in the old cells. It is concluded that serotonergic postsynaptic actions are impaired in old age.