Persistent epileptiform activity induced by low Mg2+ in intact immature brain structures.

We have determined the properties of seizures induced in vitro during the first postnatal days using intact rat cortico-hippocampal formations (CHFs) and extracellular recordings. Two main patterns of activity were generated by nominally Mg2+-free ACSF in hippocampal and cortical regions: ictal-like events (ILEs) and late recurrent interictal discharges (LRDs). They were elicited at distinct developmental periods and displayed different pharmacological properties. ILEs were first observed in P1 CHFs 52 +/- 7 min after application of low-Mg2+ ACSF (frequency 1.5 +/- 0.3 h-1, duration 86 +/- 3 s). There is a progressive age-dependent maturation of ILEs characterized by a decrease in their onset and an increase in their frequency and duration. ILEs were abolished by d-APV and Mg2+ ions. From P7, ILEs were followed by LRDs that appeared 89 +/- 8 min after application of low-Mg2+ ACSF (frequency approximately 1 Hz, duration 0.66 s, amplitude 0.31 +/- 0.03 mV). LRDs were no longer sensitive to d-APV or Mg2+ ions and persisted for at least 24 h in low-Mg2+ or in normal ACSF. ILEs and LRDs were synchronized in limbic and cortical regions with 10-40 ms latency between the onsets of seizures. Using a double chamber that enables independent superfusion of two interconnected CHFs, we report that ILEs and LRDs generated in one CHF propagated readily to the other one that was being kept in ACSF. Therefore, at a critical period of brain development, recurrent seizures induce a permanent form of hyperactivity in intact brain structures and this preparation provides a unique opportunity to study the consequences of seizures at early developmental stages.

Late embryonic expression of AMPA receptor function in the CA1 region of the intact hippocampus in vitro.

Studies in slices suggest that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated synaptic currents are not present in CA1 (Cornu ammonis) pyramidal neurons at birth (P0). We have re-examined this issue in the rat intact hippocampal formation (IHF) in vitro. Injections of biocytin or carbocyanine show that the temporo-ammonic, commissural and Schaffer collateral pathways are present at birth in the marginal zone of CA1. Electrical stimulation of these pathways evoked field excitatory postsynaptic potentials (fEPSPs) in the marginal zone of CA1 from embryonic day 19 (E19) to postnatal day 9 (P9). These fEPSPs are mediated by synaptic AMPA receptors as they are reduced or completely blocked by: (i) tetrodotoxin; (ii) high divalent cation concentrations; (iii) the adenosine A1 receptor agonist CPA; (iv) anoxic episodes; (v) the selective AMPA receptor antagonist 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7, 8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine (GYKI-53655) or the mixed AMPA-kainate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX). The amplitude of the fEPSPs is also reduced by D(-)-2-amino-5-phosphonopentanoic acid (D-APV) and its duration is increased by bicuculline suggesting the participation of N-methyl-D-aspartate (NMDA) and GABAA (gamma-aminobutyric acid) receptors. Finally, AMPA receptor-mediated fEPSPs are also recorded in P0 slices, but they are smaller and more labile than in the IHF. Our results suggest that in embryonic CA1 neurons, glutamate acting on AMPA receptors already provides a substantial part of the excitatory drive and may play an important role in the activity-dependent development of the hippocampus. Furthermore, the IHF may be a convenient preparation to investigate the properties of the developing hippocampus.

Gene expression in developing rat hippocampal pyramidal neurons appears independent of mossy fiber innervation.

In the rat, neonatal gamma-irradiation of the hippocampus induces a selective destruction of dentate granule cells and prevents the development of the mossy fiber-CA3 pyramidal cell connection. In the absence of mossy fiber input, the CA3 pyramidal neurons exhibit morphological alterations and rats deprived of dentate granule cells fail to develop kainate-induced epileptic activity in the CA3 pyramidal neurons. Neonatal elimination of the granule cells also impairs learning and memory tasks in adult rats. In the present work, we assessed by in situ hybridization and semi-quantitative RT-PCR, whether in the pyramidal layers, the absence of mossy fiber input alters the expression of a number of genes involved in activity-dependent signal transduction, in GABAergic neurotransmitter signaling and in neurite development via microtubule organization. Surprisingly, we show that the expression and the developmentally regulated alternative splicing of the genes we examined in the developing hippocampus are not altered in the pyramidal neurons, whether the dentate granule afferents are present or absent. Our results suggest that in the CA3 pyramidal layer, the developmental expression patterns of the mRNAs we studied are independent of extrinsic cues provided by mossy fiber input.

Neonatal gamma-ray irradiation impairs learning and memory of an olfactory associative task in adult rats.

Adult neonatally gamma-irradiated rats were compared with control animals in a non-spatial olfactory associative task using two different procedures. Irradiation induced a clear reduction in the total mean area of the olfactory bulbs and hippocampus but not of the orbital prefrontal cortex, diagonal band and cell layers of the entorhinal and piriform cortex. The gamma-irradiation affected the granule cells of the olfactory bulbs and differentially altered the cell layers of the subfields of the ammonic fields and the dorsal and ventral blades of the dentate gyrus. In the CA1 ammonic field, dorsal and ventral blades of the dentate gyrus, the cellular loss was significant in comparison with control adult rats. The behavioural data indicated that irradiated rats were deeply disturbed in learning the odour-reward association, and substantially impaired in a reversal experiment, but not in the discrimination of the odours per se. The cellular loss in the olfactory bulbs, in the CA1 and in the ventral blade of the gyrus dentatus was positively correlated with the deficit in behavioural performance. The data support the findings that the hippocampal system participates in the odour-reward associations and facilitates the long-term storage of associations after learning is achieved in this olfactory associative task.

Contributions of AMPA and GABA(A) receptors to the induction of NMDAR-dependent LTP in CA1.

The contributions of (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and gamma-aminobutyric acid (GABA[A]) receptors in the induction of long-term potentiation (LTP) have been studied in the CA1 region of the rat hippocampus. The results suggest that: (1) in physiological conditions, AMPARs are necessary for the induction of N-methyl-D-aspartate receptor (NMDAR)-dependent LTP since LTP cannot be elicited in the presence of the AMPAR antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Although a NMDAR-dependent LTP occurs in the presence of a GABA(A) antagonist and high concentrations of divalents cations, blockade of AMPARs leads to a voltage-dependent calcium channels (VDCC)-dependent LTP since its induction is blocked by nifedipine and not by APV. (2) The bicarbonate-induced GABA(A) receptor-mediated depolarizing response is not necessary in the induction of NMDAR-dependent or VDCC-dependent LTP since induction of these two types of LTP were not blocked by acetazolamide or in a nominally bicarbonate-free solution.

In CA1 hippocampal neurons, the redox state of NMDA receptors determines LTP expressed by NMDA but not by AMPA receptors.

1. Using extracellular recording techniques in the CA1 region of the rat hippocampus, we have evaluated the effects of the redox reagents 5,5O-dithiobis-2-nitrobenzoic acid (DTNB) and tris (carboxyethyl) phosphine (TCEP) on long-term potentiation (LTP) expressed by alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors. In physiological conditions a high-frequency stimulation (HFS) of Schaffer collateral-commissural fibers induced a LTP expressed by a persistent increase (73 +/- 13%, mean +/- SE, n = 8/10) of AMPA field potentials (LTPA). In the presence of 10 microM of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and reduced concentration of Mg2+ (0.1 mM) to boost NMDA receptors, the HFS induced LTP of NMDA field potentials (LTPN; 62 +/- 11%, n = 8/10). 2. The thiol-oxidizing reagent DTNB (200 microM) reduced, by 46 +/- 5% (n = 24), NMDA-receptor field potentials (NMDA-FP), and this effect could not be reversed by extensive washing. The disulfide-reducing agent TCEP (200 microM) slightly increased AMPA-FP and reversed the DTNB-induced inhibition of NMDA-FP. 3. DTNB (200 microM, 10 min), and TCEP (200 microM, 20 min), had no effect on AMPA-FP (98 +/- 3% and 101 +/- 5%, respectively, n = 12). 4. DTNB (200 microM, 15 min) did not prevent the induction or expression of LTPA (-12 and -5%, respectively, n = 8/8). Similar results were observed with TCEP (200 microM, 20 min).(ABSTRACT TRUNCATED AT 250 WORDS)

Major differences between long-term potentiation and ACPD-induced slow onset potentiation in hippocampus.

We examined the effects of a long-lasting application of the selective metabotropic glutamate receptor (mGluR) agonist 1S-3R, 1-amino cyclopentane-1,3-dicarboxylic acid (ACPD) on synaptic potentials recorded from the CA1 and CA3 subfields in hippocampal slices maintained in a superfusion slice chamber. In 25% of the slices, ACPD generated an slow onset potentiation (SOP) of population EPSPs (pEPSPs) in CA1. In contrast to long-term potentiation (LTP) induced by a tetanic train, SOP was accompanied by an increase in the magnitude of the presynaptic fiber volley. Potentiation of the isolated afferent volley suggests that the expression of SOP is due to a recruitment of additional presynaptic fibers by the test stimulus caused by the persistent block of K+ channels by ACPD.

Anoxic LTP is mediated by the redox modulatory site of the NMDA receptor.

1. The effects of redox reagents, 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and tris(carboxyethyl)phosphine (TCEP), on anoxia-induced long-term potentiation (LTP) were investigated in CA1 hippocampal neurons using extracellular recording techniques. Experiments were performed in the presence of 0.1 mM MgCl2 and 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to pharmacologically isolate N-methyl-D-aspartate (NMDA) receptor-mediated responses. 2. DTNB (200 microM), a thiol oxidizing reagent, reduces by 52 +/- 9% (mean +/- SE) (n = 9/9) NMDA-receptor field potentials evoked by electrical stimulation of Schaffer collaterals and this effect could not be reversed by extensive washing. Nearly the same reduction of the initial response was obtained with different concentrations of DTNB (100 and 500 microM), but the time required to reach the maximal inhibition was concentration-dependent. 3. In keeping with an earlier study oxygen and glucose deprivation for 2-3 min induced a long-term potentiation (LTP) of the NMDA receptor response (+65 +/- 16%, n = 4/6). This potentiation was reversed by DTNB (100-500 microM) (-47 +/- 18%; n = 4/4) and the initial LTP could not be restored upon extensive washing of the drug. 4. TCEP (200 microM), a reagent which reduces S-S bond, amplified the electrically evoked NMDA-receptor EPSP (+27 +/- 12%; n = 3). In addition, TCEP (200 microM), nearly completely reversed the effect of DTNB (200 microM) on anoxia-induced LTP (+56 +/- 19%; n = 3/3). Preliminary results also indicate that TCEP occlude anoxic-LTP (n = 3/4). 5. Following DTNB (200 microM) treatment, oxygen and glucose deprivation did not generate anoxic LTP and extensive washing did not restore a potentiated NMDA field potential. 6. These observations strongly suggest that the redox site of the NMDA receptor is involved in the induction and the maintenance of the anoxic LTP of the NMDA receptor-mediated response in CA1.

[NMDA receptor and long-term potentiation]. / Récepteur NMDA et potentialisation à long terme.

In order to evaluate the role of the NMDA receptor redox site in long-term potentiation (LTP), we have investigated the effects of two redox reagents, 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and tris(carboxyethyl)phosphine (TCEP) on the induction and expression of various forms of LTP. DTNB a thiol-oxidizing agent, irreversibly reduces by 50% NMDA receptor EPSP. In the presence of DTNB, the induction of tetanic and anoxic LTP are prevented. When tetanic or anoxic LTP were generated first, DTNB completely reverses the potentiation and TCEP a disulfide-reducing agent restores LTP to its initial level. These redox agents have no effect on AMPA synaptic transmission and did not significantly modify the induction and the expression of tetanic AMPA-LTP. These results suggest that thiol-oxidizing compounds might be useful for the treatment of cerebral ischemia.

NMDA redox site modulates long-term potentiation of NMDA but not of AMPA receptors.

We have compared the effects of redox drugs on long-term potentiation mediated by AMPA or NMDA receptors. A reducing and an oxidizing agent had no effect on long-term potentiation mediated by AMPA receptors. In contrast, the induction of long-term potentiation mediated by NMDA receptors was prevented by a thiol oxidizing drug and restored by a disulfide reducing agent.

(RS)-alpha-methyl-4-carboxyphenylglycine neither prevents induction of LTP nor antagonizes metabotropic glutamate receptors in CA1 hippocampal neurons.

1. The effects of the putative antagonist of metabotropic glutamate receptors (mGluR), (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG), were investigated in CA1 hippocampal neurons using intracellular and extracellular recordings. 2. MCPG (0.5 mM) did not antagonize the characteristic block of the slow afterhyperpolarization and spike accomodation produced by the selective mGluR agonist, 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) (30 microM). 3. MCPG (0.5 mM) did not prevent the inward current produced by 1S,3R-ACPD (30 microM) [240 +/- 14 and 255 +/- 21 pA (mean +/- SD) in the absence and in presence of MCPG, respectively]. 4. MCPG (0.5 mM, 10 min) did not prevent the presynaptically mediated reduction by 1S,3R-ACPD (50 microM, 10 min) of the field excitatory postsynaptic potential (EPSP) (51 +/- 7 and 64 +/- 10% in the absence and in presence of MCPG, respectively). 5. MCPG (0.5 mM) did not prevent the induction of long-term potentiation by a high-frequency tetanic stimulation of Schaffer collaterals (100 Hz, 1 s) (+61 +/- 5 and +67 +/- 16% increase in the absence and presence of MCPG, respectively). 6. These observations suggest that MCPG is not an antagonist of the subtypes of mGlu receptors that are present in CA1 pyramidal neuron. Possible selectivity of this compound for specific mGluRs is discussed in view of the regional distribution of metabotropic receptors in the hippocampus.

A selective LTP of NMDA receptor-mediated currents induced by anoxia in CA1 hippocampal neurons.

1. The possibility of long-lasting modifications of glutamatergic responses after anoxic-aglycemic (AA) episodes was investigated in CA1 hippocampal neurons of adult slices. Bicuculline (10 microM) was continuously bath applied to block GABAA receptor-mediated currents. AA episodes were induced by brief (1.30-3 min) perfusions with a glucose free artificial-cerebro-spinal-fluid (ACSF) saturated with 95% N2-5% CO2. 2. In presence of (0.6 mM) Mg2+ and a low concentration of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 1 microM), the Schaffer collateral field EPSPs consisted of an early AMPA receptor-mediated component and a late N-methyl-D-aspartate (NMDA) receptor-mediated component. The former was blocked by (10 microM) CNQX and the latter by (50) microM D-2-amino-5-phosphonovalerate (D-APV). The AA episode induced a selective long-term potentiation (LTP) of the NMDA receptor-mediated component [+70 +/- 13% (mean +/- SE), P < or = 0.008, n = 9] without affecting significantly the AMPA receptor-mediated component (+2 +/- 4, P < or = 0.86 n = 9). This selective LTP is due to an enhanced efficacy of synaptic transmission and will be referred to as anoxic LTP. 3. In slices perfused with an ACSF containing a physiological concentration of (1.3 mM) Mg2+ and no CNQX, the intracellularly recorded excitatory postsynaptic potential (EPSP) was mixed (AMPA/NMDA) at -65 mV and exclusively mediated by AMPA receptors at -100 mV. At -65 mV, the AA episode induced a persistent potentiation of the EPSP (peak amplitude potentiated by 43 +/- 6%, P < or = 0.008, n = 9, 1 h after return to control ACSF). This potentiated component of the EPSP was fully sensitive to (50 microM) D-APV. The CNQX-sensitive AMPA receptor-mediated component was not affected by the AA episode (-5.7 +/- 6%, P < or = 0.123, n = 9). Furthermore, at -100 mV a large APV-sensitive component appeared after the AA episode (+58 +/- 18% of the peak amplitude, P < or = 0.018, n = 9). Therefore, the AA episode induced a selective LTP of the NMDA receptor-mediated component of the EPSP. 4. A robust LTP (+50.0 +/- 7.5%, P < or = 0.008, n = 12) of the NMDA receptor-mediated intracellular EPSP was also observed when AMPA receptors were fully and continuously blocked by (15 microM) CNQX.(ABSTRACT TRUNCATED AT 400 WORDS)