Anne Jouvenceau

Partial inhibition of PP1 alters bidirectional synaptic plasticity in the hippocampus

Synaptic plasticity is an important cellular mechanism that underlies memory formation. In brain areas involved in memory such as the hippocampus, long‐term synaptic plasticity is bidirectional. Major forms of bidirectional plasticity encompass long‐term potentiation (LTP), LTP reversal (depotentiation) and long‐term depression (LTD). Protein kinases and protein phosphatases are important players in the induction of both LTP and LTD, and the serine/threonine protein phosphatase‐1 (PP1), in particular, has emerged as a key phosphatase in these processes. The goal of the present study was to assess the contribution of PP1 to bidirectional plasticity and examine the impact of a partial inhibition of PP1 on LTP, LTD and depotentiation in the mouse hippocampus. For this, we used transgenic mice expressing an active PP1 inhibitor (I‐1*) inducibly in forebrain neurons. We show that partial inhibition of PP1 by I‐1* expression alters the properties of bidirectional plasticity by inducing a shift of synaptic responses towards potentiation. At low‐frequency stimulation, PP1 inhibition decreases LTD in a frequency‐dependent fashion. It favours potentiation over depression at intermediate frequencies and increases LTP at high frequency. Consistently, it also impairs depotentiation. These results indicate that the requirement of bidirectional plasticity for PP1 is frequency‐dependent and that a broad range of plasticity is negatively constrained by PP1, a feature that may correlate with the property of PP1 to constrain learning efficacy and promote forgetting.

Different phosphatase-dependent mechanisms mediate long-term depression and depotentiation of long-term potentiation in mouse hippocampal CA1 area

Two types of synaptic depression have been described in the hippocampus, long‐term depression and depotentiation of long‐term potentiation known to recruit the serine/threonine protein phosphatases PP1, PP2A and PP2B (calcineurin). The contribution of each of these protein phosphatases is controversial. To examine the role of the Ca2+/calmodulin‐dependent protein phosphatase calcineurin in long‐term depression and depotentiation, we analysed the effect of genetically inhibiting calcineurin reversibly in the hippocampus, using the doxycycline‐dependent rtTA system in transgenic mice. We show that reducing calcineurin activity has no effect on long‐term depression but reversibly affects depotentiation. Consistently, the calcineurin inhibitor FK‐506 reproduces the depotentiation impairment observed in the mutant mice but does not affect long‐term depression in control animals. In contrast, the PP1/PP2A inhibitor okadaic acid fully blocks both long‐term depression and depotentiation. These data demonstrate that the nature of signalling cascades induced by synaptic activity depends on the initial synaptic state. While depression of potentiated synaptic responses requires activation of PP1/PP2A and/or calcineurin, depression of basal synaptic responses depends only on PP1/PP2A activation.

Glutamatergic synaptic responses and long‐term potentiation are impaired in the CA1 hippocampal area of calbindin D28k‐deficient mice

The contribution of the cytosolic calcium binding protein calbindin D28K (CaBP) to glutamatergic neurotransmission and synaptic plasticity was investigated in hippocampal CA1 area of wild‐type and antisense transgenic CaBP‐deficient mice, with the use of extracellular recordings in the ex vivo slice preparation. The amplitude of non‐N‐methyl‐D‐aspartate receptor (non‐NMDAr)‐mediated extracellular field excitatory postsynaptic potentials (fEPSPs) recorded in control medium was significantly greater in CaBP‐deficient mice, whereas the afferent fiber volley was not affected. In contrast, the amplitude of NMDAr‐mediated fEPSPs isolated in a magnesium‐free medium after blockade of non‐NMDAr and GABAergic receptors was significantly depressed in these animals. No alteration in the magnitude of paired‐pulse facilitation was found, indicating that the presynaptic calcium mechanisms controlling glutamate release were not altered in CaBP‐deficient mice. The magnitude and time course of the short‐term potentiation (STP) of fEPSPs induced by a 30 Hz conditioning stimulation, which was blocked by the NMDAr antagonist 2‐amino‐5‐phosphonovalerate acid (2‐APV), was not impaired in the transgenic mice, whereas long‐term potentiation (LTP) induced by a 100 Hz tetanus was not maintained. The long‐term depression (LTD) induced by low‐frequency stimulation (1 Hz, 15 min) in the presence of the GABA antagonist bicuculline was not altered. These results argue for a contribution of CaBP to the mechanisms responsible for the maintenance of long‐term synaptic potentiation, at least in part by modulating the activation of NMDA receptors. Synapse 33:172–180, 1999.

Age-related alterations of GABAergic input to CA1 pyramidal neurons and its control by nicotinic acetylcholine receptors in rat hippocampus.

The aim of this study was to determine whether age-associated alterations in the GABAergic input to pyramidal neurons in the hippocampus are due to a dysfunction of GABAergic interneurons, and/or a decrease in their cholinergic control via nicotinic receptors (nAChRs). Electrophysiological recordings were obtained from pyramidal cells in the CA1 area of hippocampal slices from young (3-4 months old) and aged (25-30 months old) Sprague-Dawley rats. Synaptic GABA(A) receptor-mediated inhibitory postsynaptic currents and inhibitory postsynaptic potentials induced by stimulation of the stratum oriens were significantly smaller in aged rats. The frequency (but not amplitude) of spontaneous and miniature GABA inhibitory postsynaptic currents (IPSCs) was reduced in aged rats, suggesting a presynaptic alteration. Tetanic stimulation of cholinergic afferents to release endogenous acetylcholine, or an exogenous application of the nAChR agonist cytisine, increased the frequency of spontaneous IPSCs in young rats; however these effects were not evident in aged rats, indicating that the nicotinic control of GABA release is lowered during aging. None of these age-related alterations were reversed by a chronic treatment with donepezil, a cholinesterase inhibitor. Immunofluorescent labeling of GABA interneurons with somatostatin (SOM), parvalbumin (PV) or calbindin (CB), together with the vesicular acetylcholine transporter VAChT, revealed a selective loss of subpopulations of SOM and CB positive interneurons. This loss was associated with a general decrease in density of the cholinergic network in aged rats. Thus, the lower GABAergic inhibition observed in the aged rat hippocampus is due to a selective loss/dysfunction of subpopulations of GABAergic interneurons, associated with a widespread cholinergic deficit.

Alteration of NMDA receptor‐mediated synaptic responses in CA1 area of the aged rat hippocampus: Contribution of GABAergic and cholinergic deficits

Synaptic responses mediated by the N‐methyl‐D‐aspartate receptor (NMDAr) and non‐NMDAr activation were compared in CA1 hippocampal region of young (3–4 months old) and aged (25–33 months old) Sprague‐Dawley rats with the use of ex vivo extracellular recordings techniques. In aged rats, the amplitude of the NMDAr‐mediated field excitatory postsynaptic potentials (fEPSPs) was not altered, whereas their duration was significantly increased. In contrast, the magnitude of non‐NMDAr‐mediated fEPSPs was significantly smaller. The presynaptic fiber volley was not affected by age. Considering that the depression of non‐NMDAr‐mediated responses was previously attributed to fewer synaptic contacts between glutamatergic afferent fibers and pyramidal cells in aged animals (see Barnes et al., Hippocampus 1992;2:457–468), the absence of age‐related changes in the amplitude of NMDAr‐mediated fEPSPs suggests that compensatory mechanisms may occur. The contribution of γ‐aminobutyric acid (GABA) and acetylcholine to these mechanisms was addressed. The NMDAr‐mediated fEPSPs were then recorded (1) in young and aged rats before and after blockade of the GABAB receptor‐mediated inhibition by the specific antagonist CGP 55845 and (2) in young rats after a selective cholinergic denervation of the hippocampus by the immunotoxin 192 IgG‐saporin. The results did not indicate statistically relevant age‐related effects of CGP 55845. In contrast, the loss of the cholinergic innervation by the immunotoxin induced a significant increase in both the amplitude and duration of the NMDAr‐mediated fEPSPs. Our results indicate that the functional properties of the ionotropic glutamate receptor subtypes located on CA1 pyramidal cells are differentially affected by aging and suggest that the cholinergic deficit that occurs during aging may be involved in the maintenance of robust NMDAr‐mediated synaptic responses. Hippocampus 1998;8:627–637.

Protein phosphatase 1-dependent transcriptional programs for long-term memory and plasticity

Gene transcription is essential for the establishment and the maintenance of long-term memory (LTM) and for long-lasting forms of synaptic plasticity. The molecular mechanisms that control gene transcription in neuronal cells are complex and recruit multiple signaling pathways in the cytoplasm and the nucleus. Protein kinases (PKs) and phosphatases (PPs) are important players in these mechanisms. Protein serine/threonine phosphatase 1 (PP1), in particular, was recently shown to be important for transcription-dependent memory by regulating chromatin remodeling. However, the impact of PP1 on gene transcription in adult neurons remains not fully delineated. Here, we demonstrate that the nuclear pool of PP1 is associated with transcriptional events involving molecular components of signaling cascades acting as positive and negative regulators of memory and brain plasticity. The data show that inhibiting this pool selectively in forebrain neurons improves memory performance, enhances long-term potentiation (LTP), and modulates gene transcription. These findings highlight an important role for PP1 in the regulation of gene transcription in LTM and synaptic plasticity in the adult brain.

Decrease in calbindin content significantly alters LTP but not NMDA receptor and calcium channel properties.

The contribution of the cytosolic calcium binding protein calbindin D(28K) (CaBP) to the synaptic plasticity was investigated in hippocampal CA1 area of wild-type and antisense transgenic CaBP-deficient mice. We showed that long-term potentiation (LTP) induced by tetanic stimulation in CaBP-deficient mice was impaired. The fundamental biophysical properties of NMDA receptors and their number were not modified in CaBP-deficient mice. We also demonstrated that the physiological properties of calcium channels were identical between genotypes. An insufficient Ca(2+) entry through NMDA receptors or calcium channels, or a decrease in NMDA receptor density are unlikely to explain this impairment of LTP. Interestingly, we showed that the loss of LTP was not prevented by glycine but was restored in the presence of a low concentration of the NMDA receptor antagonist D-APV (5 microM) and of the calcium chelator BAPTA-AM (5 microM). Moreover, we observed a loss of LTP in the wild-type mice when the postsynaptic tetanic-induced [Ca(2+)](i) rise is excessively increased. Conversely, a weaker tetanus stimulation allowed LTP induction and maintenance in CaBP-deficient mice. These results suggest that a higher cytosol [Ca(2+)](i), due to the decrease of CaBP expression may impair LTP induction and maintenance mechanisms without affecting the mechanisms of calcium entry. Thus, CaBP plays a critical role in long term synaptic plasticity by limiting the elevation of calcium rise in the cytosol to some appropriate spatio-temporal pattern.

A role for the protein phosphatase 2B in altered hippocampal synaptic plasticity in the aged rat.

Synaptic plasticity following NMDA application on hippocampal slices from young (3-5 months) and aged (24-27 months) rats was compared. In young rats, NMDA (20 microM) induced opposite effects depending on the duration of the application. A short (1 min) or long (5 min) application induced a long-term depression of synaptic activity while a 3 min application induced a potentiation. In aged rats, however, NMDA application always induced depression, regardless of the duration. To identify mechanisms which could explain the difference observed between young and aged rats, we explored changes in NMDA receptor activation and changes in kinase/phosphatase balance. We first demonstrate that the potentiation present in slices from young rats was not restored in aged rats by exogenous application of the co-agonist of NMDA receptor d-serine (which compensates for the changes in NMDAR activation seen in aged rats). This suggested that alterations in synaptic plasticity activation mainly involve intracellular mechanisms. We next showed that the participation of the kinases PKA and CaMKII in the NMDA-induced potentiation in young rats is negligible. Finally, we determined the consequences of phosphatase inhibition in aged rats. Incubation of slices in okadaic acid (a PP1/PP2B antagonist) did not affect the depression induced by a 3min NMDA application in aged rats. The PP2B antagonist FK506 restored potentiation in aged rats (3 min NMDA application). In hippocampal neurons from aged rats, a depression is always observed, suggesting a preferential activation of PP2B by NMDA in these neurons.

NMDA Receptor Activation in the Aged Rat: Electrophysiological Investigations in the CA1 Area of the Hippocampal Slice Ex Vivo

The effects of aging on activation of N-methyl-D-aspartate (NMDA) receptors were studied in the CA1 field of hippocampal slices from young (2-4 months old) and aged (25-32 months old) Sprague-Dawley rats with the use of ex vivo extra- and intracellular electrophysiological recording techniques. No significant age-related changes of the unitary NMDA-receptor mediated excitatory postsynaptic potentials (EPSPs), recorded from the pyramidal cells after stimulation of the stratum radiatum in a magnesium-free medium and isolated in the presence of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, were found. Simultaneously, the magnitude of synaptic plasticity which involved NMDA receptor activation was not altered. No significant age-related modifications in the mechanisms controlling glutamate release and of postsynaptic NMDA receptor responsiveness were revealed. Considering the 30-40% decrease in NMDA binding sites in the aged hippocampus, our results suggest the occurrence of compensatory mechanisms which are discussed.

POTENTIATION OF GLUTAMATERGIC EPSPS IN RAT CA1 HIPPOCAMPAL NEURONS AFTER SELECTIVE CHOLINERGIC DENERVATION BY 192 IGG-SAPORIN

A complete and selective destruction of the basal forebrain cholinergic neurons projecting to the cerebral cortex and the hippocampus was induced in the rat by the toxin 192 IgG‐saporin. Using electrophysiologic techniques, we have investigated the consequences of this cholinergic denervation on inhibitory and excitatory synaptic responses of CA1 pyramidal cells in rat hippocampal slices ex vivo. Histochemical experiments were performed in slices from control and 192 IgG‐saporin–treated rats to check the efficacy of the intracerebroventricular injection of the immunotoxin.