Developmentally regulated alternative splicing of mRNAs encoding N-terminal tau variants in the rat hippocampus: structural and functional implications.

Tau protein variants are axonal microtubule-associated phosphoproteins whose expression correlates with developmentally regulated neurite outgrowth. A single gene encodes multiple tau transcripts via complex alternative splicing. We studied the expression of the mRNAs encoding N-terminal variants of tau, and we showed distinct alternative splicing of exons 2 and 3 in nervous tissues of the adult rat, including the inner ear, hippocampus, cortex, striatum, brainstem, cerebellum, olfactory bulb and retina.

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.

Differential expression of fibronectin, tenascin-C and NCAMs in cultured hippocampal astrocytes activated by kainate, bacterial lipopolysaccharide or basic fibroblast growth factor.

Different reports demonstrated that reactive glial cells express increased amounts of adhesion and matrix molecules. Despite a wealth of information on the expression of these molecules during development and after lesion, very little is known of how this expression is regulated. In the present report we used Western blots and immunocytochemistry to investigate the expression of neural cell adhesion molecule (NCAM), fibronectin and tenascin-C in cultured astrocytes from rat hippocampus.

GABAA, NMDA and AMPA receptors: a developmentally regulated 'ménage à trois'.

The main ionotropic receptors (GABAA, NMDA and AMPA) display a sequential participation in neuronal excitation in the neonatal hippocampus. GABA, the principal inhibitory transmitter in the adult CNS, acts as an excitatory transmitter in early postnatal stage. Glutamatergic synaptic transmission is first purely NMDA-receptor based and lacks functional AMPA receptors.

A novel in vitro preparation: the intact hippocampal formation.

The intact hippocampal formation (IHF) of neonatal or young rats can be kept alive for an extended period in a fully submerged chamber with excellent morphological preservation. Field or patch-clamp recordings, intracellular Ca2+ measurements, and 3-D reconstruction of biocytin-filled neurons can be performed routinely. The generation and propagation of network-driven activities can be studied within the IHF or between connected intact structures such as the septum and the hippocampus or two hippocampi, and the use of a dual chamber enables the application of drugs separately to each structure.

Operative GABAergic inhibition in hippocampal CA1 pyramidal neurons in experimental epilepsy.

Patch-clamp recordings of CA1 interneurons and pyramidal cells were performed in hippocampal slices from kainate- or pilocarpine-treated rat models of temporal lobe epilepsy. We report that gamma-aminobutyric acid (GABA)ergic inhibition in pyramidal neurons is still functional in temporal lobe epilepsy because: (i) the frequency of spontaneous GABAergic currents is similar to that of control and (ii) focal electrical stimulation of interneurons evokes a hyperpolarization that prevents the generation of action potentials. In paired recordings of interneurons and pyramidal cells, synchronous interictal activities were recorded.

A long-lasting calcium-activated nonselective cationic current is generated by synaptic stimulation or exogenous activation of group I metabotropic glutamate receptors in CA1 pyramidal neurons.

We have shown previously that a selective metabotropic glutamate receptor (mGluR) agonist, 1S,3R-1-aminocyclo-pentane-1, 3-dicarboxylate (1S,3R-ACPD), evokes an inward current in CA1 pyramidal neurons of rat hippocampal slices in the presence of K+ channel blockers (). This current has been characterized as a Ca2+-activated nonselective cationic (CAN) current. Using whole-cell patch-clamp recordings and intracellular dialysis, we now have identified the mGluR subtype and the mechanisms underlying the CAN current (ICAN) and report for the first time the presence of a synaptic ICAN in the mammalian CNS.

Effects of tenascin-C in cultured hippocampal astrocytes: NCAM and fibronectin immunoreactivity changes.

Tenascin-C is an extracellular matrix glycoprotein with trophic and repulsive properties on neuronal cells, involved in migratory processes of immature neurons. Previous reports demonstrated that this molecule is produced and secreted by astrocytes, in vitro after activation by bFGF or in vivo after CNS lesion. In injured brain the expression of tenascin-C has been correlated with the glial reaction since it was observed in regions suffering a dramatic glial proliferation and hypertrophy.

FGF-2 induces nerve growth factor expression in cultured rat hippocampal neurons.

Basic fibroblast growth factor (FGF-2) is expressed in the hippocampus and has been demonstrated to promote neurotrophic effects on hippocampal neurons in vitro. We show that these neurons, even at the embryonic stage, express the mRNAs encoding the FGF receptors, bek and flg. We have characterized the effects of FGF-2 on the expression of nerve growth factor (NGF) using the reverse transcription-coupled polymerase chain reaction, in situ hybridization and immunocytochemistry.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is differentially induced in neurons and astrocytes after seizures: evidence for developmental, immediate early gene, and lesion response.

We investigated in vivo the expression of the tissue inhibitor of metalloproteinases-1 (TIMP-1) in the rat CNS after kainate (KA)-induced excitotoxic seizures. In situ hybridization revealed that TIMP-1 mRNA is induced rapidly and massively in most regions of the adult forebrain after KA treatment. Neuronal activity seems to be necessary but not sufficient to trigger TIMP-1 induction, because it is not observed in seizing 10-d-old pups, unlike what is observed in 21- and 35-d-old animals after seizures.

[Consequences of recurrent seizures during development].

While many children with recurrent seizures have a good prognosis, a small percentage of children with intractable epilepsy have a more ominous course with a gradual decline in cognitive abilities over time. While the reasons for this cognitive decline may be multifactorial, there is evidence both from human and animal studies that recurrent seizures may lead to gradual cognitive impairment in some children. Laboratory studies have also demonstrated that recurrent seizures can lead to deficits in learning and memory as well as structural changes in the brain.

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.

Blocking GABA(A) inhibition reveals AMPA- and NMDA-receptor-mediated polysynaptic responses in the CA1 region of the rat hippocampus.

We have investigated the conditions required to evoke polysynaptic responses in the isolated CA1 region of hippocampal slices from Wistar adult rats. Experiments were performed with extracellular and whole cell recording techniques. In the presence of bicuculline (10 microM), 6-cyano-7-nitroquinoxaline-2-3-dione (10 microM), glycine (10 microM), and a low external concentration of Mg2+ (0.

Large amplitude miniature excitatory postsynaptic currents in hippocampal CA3 pyramidal neurons are of mossy fiber origin.

Neonatal (P0) gamma-irradiation was used to lesion selectively the mossy fiber (MF) synaptic input to CA3 pyramidal cells. This lesion caused a > 85% reduction in the MF input as determined by quantitative assessment of the number of dynorphin immunoreactive MF boutons. The gamma-irradiation lesion caused a reduction in the mean number of miniature excitatory postsynaptic currents (mEPSCs) recorded from CA3 pyramidal cells (2,292 vs.

The calcium-dependent transient inactivation of recombinant NMDA receptor-channel does not involve the high affinity calmodulin binding site of the NR1 subunit.

N-methyl-D-aspartate (NMDA) receptor function can be regulated by direct binding of calmodulin to a low and high affinity (C1 exon cassette) site in the C-terminal region of the NR1 subunit. To evaluate the involvement of the high affinity binding site in the transient inactivation of the NMDA receptor-channels by intracellular calcium, several splice variants of the NR1 subunit have been individually co-transfected with the NR2A subunit in HEK 293 cells. The transient Ca2+ induced inactivation (40-50%) of the heteromeric receptors was similar whether the NR1 variants contained (NR1-1a, 1b) or lacked (NR1-2a, 2b, 4a, 4b) the C1 exon cassette bearing the high affinity binding site for calmodulin.

Redox modulation of synaptic responses and plasticity in rat CA1 hippocampal neurons.

Effects of redox reagents on excitatory and inhibitory synaptic responses as well as on the bidrectional plasticity of alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor-mediated synaptic responses were studied in CA1 pyramidal neurons in rat hippocampal slices. The oxidizing agent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, 200 microM) did not affect AMPA, GABAA or GABAB receptor-mediated synaptic responses or the activation of presynaptic metabotropic receptors. However, DTNB irreversibly decreased (by approximately 50%) currents evoked by focal application of NMDA.

Ca2+ oscillations mediated by the synergistic excitatory actions of GABA(A) and NMDA receptors in the neonatal hippocampus.

We asked whether GABA(A) and NMDA receptors may act in synergy in neonatal hippocampal slices, at a time when GABA exerts a depolarizing action. The GABA(A) receptor agonist isoguvacine reduced the voltage-dependent Mg2+ block of single NMDA channels recorded in cell-attached configuration from P(2-5) CA3 pyramidal neurons and potentiated the Ca2+ influx through NMDA channels. The synaptic response evoked by electrical stimulation of stratum radiatum was mediated by a synergistic interaction between GABA(A) and NMDA receptors.

Synchronization of GABAergic interneuronal network in CA3 subfield of neonatal rat hippocampal slices.

1. Cell-attached and whole-cell recordings from interneurons localized in the stratum radiatum of the CA3 subfield (SR-CA3) of neonatal (postnatal days 2-5) rat hippocampal slices were performed to study their activity during the generation of GABAergic giant depolarizing potentials (GDPs) in CA3 pyramidal cells. 2.

Bicuculline induces ictal seizures in the intact hippocampus recorded in vitro.

The effects of the gamma-aminobutyric acid (GABA) receptor antagonist bicuculline on rat hippocampal neurons recorded in slices and in the intact hippocampi kept in vitro were studied using whole-cell patch-clamp recordings. Bicuculline (10 microM) evoked ictal discharges in the intact hippocampus but only interictal discharges in conventional slices. Recording from the intact hippocampus in vitro is an alternative preparation to study the organization of the hippocampal neuronal network.

Down-regulation of striatal enkephalinergic (PPA) messenger RNA without prior apoptotic features following reversible focal ischemia in rat.

In order to determine whether striatal enkephalinergic neurons were affected by reversible focal ischemia, we have investigated the expression of the preproenkephalin (PPA) messenger by in situ hybridization (ISH) combined with TUNEL staining to display apoptosis in the same rat brain sections. Our data demonstrated a massive reduction of the number of PPA-mRNA containing neurons concomitant with the emergence of apoptotic cells. However, double-labeled neurons (ISH- and TUNEL-positive cells) were not detected, suggesting that either disruption of mRNA precedes DNA fragmentation or ischemia leads to a long lasting reduction of mRNA(s) without damage.

Regulation of apoptosis-associated proteins in cell death following transient focal ischemia in rat pups.

Apoptosis is a process whereby developmental or environmental stimuli activate a genetic programme to execute a specific series of events that culminate in the death and efficient disposal of a cell. Although a series of recent data suggested that neuronal death following cerebral ischemia occurs through an apoptotic pathway, additional work is needed to establish the existence of a causal relationship between gene expression and DNA breaks in neuronal death. We investigate the role of p53 and Bax proteins in the induction of apoptosis induced by a new transient focal ischemia model in the rat pup.

[Glutamatergic excitatory receptors and temporal lobe epilepsy].

Despite a considerable wealth of data, the mechanisms responsible for the generation of epileptic bursts in temporal lobe epilepsy (TLE) remain a mystery. Recently, research and therapy have focused on impairment of GABAergic inhibition in epilepsy. Several lines of evidence support this approach: 1-GABA is the main inhibitory neurotransmitter in the neuronal structures involved in TLE.

Model of spatio-temporal propagation of action potentials in the Schaffer collateral pathway of the CA1 area of the rat hippocampus.

There is a sharp contrast between the profuse in vivo axonal arborization of CA3 pyramidal cells in the CA1 area and the low probability of finding pairs of connected CA3-CA1 pyramidal cells in vitro. These anatomical differences contribute to a connectivity argument for discrepancies between electrophysiological data recorded in vitro and in vivo. In order to investigate this issue, we have developed a realistic computer model of the Schaffer collateral pathway of the hippocampus and analyzed the spatio-temporal distribution of action potentials along this pathway following three different types of electrical test stimulus.

Epileptiform activity but not synaptic plasticity is blocked by oxidation of NMDA receptors in a chronic model of temporal lobe epilepsy.

Simultaneous extracellular recordings were performed in stratum radiatum and stratum pyramidale of hippocampal slices 7 days following unilateral intracerebroventricular injections of kainic acid. In this ex vivo experimental model of human temporal lobe epilepsy, stimulation of the surviving commissural fibres in stratum radiatum produced graded epileptiform activity in the CA1 area. The oxidizing reagent 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) acting at NMDA receptors redox sites decreases NMDA receptor-mediated responses by half and suppresses evoked epileptiform discharges.