Brain-derived neurotrophic factor upregulates and maintains AMPA receptor currents in neocortical GABAergic neurons

The regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors is implicated in synaptic plasticity. Although we have found that brain-derived neurotrophic factor (BDNF) triggers surface translocation of AMPA receptor proteins, the physiological significance of the BDNF effect remained to be determined. The present immunohistochemical studies revealed that cortical GABAergic neurons exhibited the most striking response to BDNF. Accordingly, we monitored AMPA-triggered currents through GABAergic neurons: Chronic BDNF treatment increased the AMPA-triggered currents but not NMDA-triggered currents in culture. In parallel, the amplitude, but not frequency, of spontaneous miniature excitatory postsynaptic currents (mEPSCs) was elevated in GABAergic neurons. In agreement, BDNF enhanced GABA release triggered by AMPA compared to the amount triggered by high potassium. Conversely, there was a significant decrease in the mEPSC amplitude of GABAergic neurons in heterozygous BDNF-knockout mice. These findings indicate that the neurotrophin enhances the input sensitivity of GABAergic neurons to facilitate their inhibitory function in the neocortex.     

Modulation of AMPA receptors in spinal motor neurons by the neuroprotective agent riluzole

We investigated the interaction of riluzole, a therapeutic agent used in amyotrophic lateral sclerosis (ALS), with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor channels in mouse spinal motor neurons in culture using whole-cell patch-clamp recording techniques. Kainate elicited concentration-dependent (EC(50) = 35 microM) inward currents in all the patched cells. These responses were mediated primarily through the activation of AMPA receptors with a negligible contribution from kainate receptors, because bath application of 100 microM GYKI53655, a potent noncompetitive AMPA receptor antagonist, completely blocked the kainate-induced currents. Riluzole (0.5-100 microM) reduced in a dose-dependent manner the kainate-induced currents with an IC(50) of 1.54 microM in all tested neurons (n = 25) and this effect was found to be reversible. The response to kainate decreased in the presence of 1 microM riluzole in all spinal motor neurons tested, without changing its EC(50), indicating a noncompetitive mechanism of inhibition. The amplitude of the responses induced by kainate under control condition and during riluzole was a linear function of the membrane potential. The reversal potential of the current was not significantly different in the two experimental conditions, whereas the total conductance of the motor neurons for the currents induced by 100 microM kainate was reduced significantly in the presence of 1 microM riluzole (P < 0.05). These results reveal an interaction of riluzole with glutamatergic neurotransmission in spinal cord motor neurons and can contribute to explain its beneficial effect in the ALS treatment.     

Differences in the properties of ionotropic glutamate synaptic currents in oxytocin and vasopressin neuroendocrine neurons.

Oxytocin (OT) and vasopressin (VP) hormone release from neurohypophysial terminals is controlled by the firing pattern of neurosecretory cells located in the hypothalamic supraoptic (SON) and paraventricular nuclei. Although glutamate is a key modulator of the electrical activity of both OT and VP neurons, a differential contribution of AMPA receptors (AMPARs) and NMDA receptors (NMDARs) has been proposed to mediate glutamatergic influences on these neurons. In the present study we examined the distribution and functional properties of synaptic currents mediated by AMPARs and NMDARs in immunoidentified SON neurons. Our results suggest that the properties of AMPA-mediated currents in SON neurons are controlled in a cell type-specific manner. OT neurons displayed AMPA-mediated miniature EPSCs (mEPSCs) with larger amplitude and faster decay kinetics than VP neurons. Furthermore, a peak-scaled nonstationary noise analysis of mEPSCs revealed a larger estimated single-channel conductance of AMPARs expressed in OT neurons. High-frequency summation of AMPA-mediated excitatory postsynaptic potentials was smaller in OT neurons. In both cell types, AMPA-mediated synaptic currents showed inward rectification, which was more pronounced in OT neurons, and displayed Ca2+ permeability. On the other hand, NMDA-mediated mEPSCs of both cell types had similar amplitude and kinetic properties. The cell type-specific expression of functionally different AMPARs can contribute to the adoption of different firing patterns by these neuroendocrine neurons in response to physiological stimuli.     

In vivo administration of epidermal growth factor and its homologue attenuates developmental maturation of functional excitatory synapses in cortical GABAergic neurons

The ErbB1 ligand family includes epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha), heparin-binding EGF-like growth factor, amphiregulin and betacellulin. Previously, we demonstrated that TGFalpha decreases alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors in cultured neocortical gamma-aminobutyric acid (GABA) neurons. In the present study, we examined in vivo effects of EGF and TGFalpha in the mouse neocortex using electrophysiological and biochemical techniques. In mouse neonates, subcutaneously administered EGF penetrated the blood-brain barrier and activated ErbB1 in the neocortex. Daily administration of EGF or TGFalpha attenuates developmental increases in expression of the AMPA receptor subunits (GluR1 and GluR2/3) in the neocortex of postnatal mice. Immunohistochemistry revealed that the reduction in AMPA receptor expression was significant in the GABAergic neurons, especially those positive for parvalbumin. Using cortical slices prepared from EGF-treated mice, we recorded miniature excitatory postsynaptic currents (mEPSCs) in both GABAergic and pyramidal neurons. Subchronic treatment with EGF decreased the amplitude and frequency of mEPSCs in GABAergic neurons, but its effects were negligible on pyramidal neurons. We conclude that EGF or other ErbB1 ligand(s) attenuates a developmental increase in AMPA receptor expression and function in cortical GABAergic neurons.     

Pregnenolone sulphate attenuates AMPA cytotoxicity on rat cortical neurons

Neuroactive steroids can modulate brain excitability by interaction with several neurotransmitter receptor-associated channels. These compounds may thus exert profound influences on excitotoxic injury, i.e. neuronal cell death triggered by over-activation of glutamate receptors. It has been reported that pregnenolone sulphate (PS) and pregnenolone hemisuccinate (PHS) augment N-methyl-D-aspartate (NMDA) neurotoxicity in rat cultured neurons. Here we show that the effects of neuroactive steroids on AMPA cytotoxicity display features distinct from those on NMDA cytotoxicity. Concomitant application of PS (30-300 microm) attenuated, rather than augmented, AMPA neurotoxicity in cortical slice cultures in a concentration-dependent manner, whereas various other steroids including pregnenolone and PHS had no effect. Inhibition of steroid sulphatase by estrone-3-O-sulphamate led to a shift of the minimum effective concentration of PS against AMPA cytotoxicity from 30 to 10 microm. The protective action of PS was not affected by inhibition of protein synthesis or by blockade of glucocorticoid receptors, GABAA receptors or sigma-receptors. In dissociated cortical neurons, PS attenuated AMPA-induced inward currents whereas pregnenolone and PHS exhibited no significant effect. Thus, with strict structural specificity, PS but not pregnenolone or PHS attenuates AMPA cytotoxicity, probably by inhibiting activities of AMPA receptor-associated channels.     

Substance P receptor activation induces downregulation of the AMPA receptor functionality in cortical neurons from a genetic model of Amyotrophic Lateral Sclerosis

Substance P (SP), a neuropeptide member of the tachykinin (TK) family, has a functional role both in physiological and pathological conditions, including Amyotrophic Lateral Sclerosis disease. One hypothesis of the selective motor neuron death in ALS involves the excitatory neurotransmitter glutamate, because these neurons are extremely susceptible to excessive stimulation of AMPA receptors. It has been reported that SP exerts its action against a variety of insults including excitotoxicity, and that altered levels of SP have been observed in the cerebrospinal fluid (CSF) of patients with ALS. Here we have analyzed the interaction between SP and AMPA receptor functionality, both in Control cortical neurons in culture and in those obtained from a genetic mouse model of ALS (G93A). Our studies demonstrate that SP reduces the kainate-activated currents in Control and G93A neurons and that this reduction is significantly higher in the mutated neurons. SP effect is mediated by its receptor NK1 because GR 82334 (5 μM), a NK1 competitive antagonist, is able to suppress the current reduction. Analysis of miniature excitatory postsynaptic currents (mEPSCs) in Control and G93A neurons indicates that SP (200 nM) is able to significantly decrease the mEPSC amplitudes in G93A neurons, whereas it is ineffective on Control mEPSCs. Western blotting experiments in cultures and cortical tissues show a higher NK1 expression level in G93A mice compared to that of Control. This is also confirmed by immunocytochemistry experiments in cultured neurons. In addition, the amount of GluR1 subunit AMPA receptors is not modified following SP exposure, indicating a non internalization of the AMPA receptors. Finally, toxicity experiments have revealed that SP is able to rescue G93A cortical cells whereas it is ineffective on those of Control. These findings provide the first evidence of SP having a physiological and protective role in the G93A mouse model of ALS, and may suggest the possible use of SP as a clinical therapeutic treatment.     

The antiepileptic drug levetiracetam stabilizes the human epileptic GABAA receptors upon repetitive activation

PURPOSE: GABAA receptors from the brain of patients afflicted with mesial temporal lobe epilepsy (MTLE) become less efficient (run-down) when repetitively activated by GABA. Experiments were designed to investigate whether the antiepileptic drug, levetiracetam (LEV), which is used as an adjunctive treatment for medically intractable MTLE, counteracts the GABAA receptor run-down. METHODS: GABAA receptors were microtransplanted from the brains of patients afflicted with MTLE into Xenopus oocytes. The GABA-current run-down, caused by repetitive applications of GABA, was investigated using the standard two-microelectrode voltage-clamp technique. Additionally, the GABA-current run-down was investigated directly on pyramidal neurons in human MTLE cortical slices. RESULTS: It was found that, in oocytes injected with membranes isolated from the MTLE neocortex, the GABA-current run-down was inhibited by a 3-h pretreatment with 0.5-100 microM LEV. Moreover, the GABAA receptors of pyramidal neurons in human neocortical slices exhibited a current run-down that was significantly reduced by 1 microM LEV. Interestingly, the run-down in oocytes injected with membranes isolated from the MTLE hippocampal subiculum was not affected by LEV. CONCLUSIONS: We report that the antiepileptic LEV strengthens GABA inhibition of neuronal circuits by blocking the receptor run-down in the cortex whilst leaving the run-down of GABAA receptors in the hippocampal subiculum unaltered. These findings point to the GABAA receptor run-down as an important event in epileptogenesis and as a possible target for testing and screening antiepileptic drugs.     

Member of the Ampakine class of memory enhancers prolongs the single channel open time of reconstituted AMPA receptors

Ampakines are small benzamide compounds that allosterically produce the positive modulation of AMPA receptors and improve performance on a variety of behavioral tasks. To test if the native synaptic membrane is necessary for the effects of such positive modulators, the mechanism of action of the Ampakine 1-(1,3-benzodioxol-5-ylcarbonyl)-1,2,3,6-tetrahydropyridine (CX509) was investigated in isolated rat brain AMPA receptors reconstituted in lipid bilayers. The drug increased the open time of AMPA-induced single channel current fluctuations with an EC(50) of 4 microM. The action of CX509 was highly selective since it had no effect on the amplitude or close time of channel events. The open time effect had a maximum enhancement of 70-fold and the modulated currents were blocked by CNQX. It is concluded that the synaptic membrane environment is not necessary for Ampakine effects. In fact, CX509 was about 100 times more potent on the reconstituted AMPA receptors than on receptors in their native membrane. These findings indicate that centrally active Ampakines modulate specific kinetic properties of AMPA currents. They also raise the possibility that AMPA receptors are regulated by factors present in situ, thus explaining the more efficient modulatory effects of CX509 when acting on receptors removed from their synaptic location.     

Caspase-mediated suppression of glutamate (AMPA) receptor channel activity in hippocampal neurons in response to DNA damage promotes apoptosis and prevents necrosis: implications for neurological side effects of cancer therapy and neurodegenerative disorders

DNA damage in neurons is implicated in the pathogenesis of several neurodegenerative disorders and may also contribute to the often severe neurological complications in cancer patients treated with chemotherapeutic agents. DNA damage can trigger apoptosis, a form of controlled cell death that involves activation of cysteine proteases called caspases. The excitatory neurotransmitter glutamate plays central roles in the activation of neurons and in processes such as learning and memory, but overactivation of ionotropic glutamate receptors can induce either apoptosis or necrosis. Glutamate receptors of the AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) type mediate such physiological and pathological processes in most neurons. We now report that DNA damage can alter glutamate receptor channel activity by a mechanism involving activation of caspases. Whole-cell patch clamp analyses revealed a marked decrease in AMPA-induced currents after exposure of neurons to camptothecin, a topoisomerase inhibitor that induces DNA damage; N-methyl-d-aspartate (NMDA)-induced currents were unaffected by camptothecin. The decrease in AMPA-induced current was accompanied by a decreased calcium response to AMPA. Pharmacological inhibition of caspases abolished the effects of camptothecin on AMPA-induced current and calcium responses, and promoted excitotoxic necrosis. Combined treatment with glutamate receptor antagonists and a caspase inhibitor prevented camptothecin-induced neuronal death. Caspase-mediated suppression of AMPA currents may allow neurons with damaged DNA to withdraw their participation in excitatory circuits and undergo apoptosis, thereby avoiding widespread necrosis. These findings have important implications for treatment of patients with cancer and neurodegenerative disorders.     

Presynaptic and postsynaptic modulation of glutamatergic synaptic transmission by activation of α1‐ and β‐adrenoceptors in layer V pyramidal neurons of rat cerebral cortex

Adrenergic agonists have different modulatory effects on excitatory synaptic transmission depending on the receptor subtypes involved. The present study examined the loci of α₁‐ and β‐adrenoceptor agonists, which have opposite effects on excitatory neural transmission, involved in modulation of glutamatergic transmission in layer V pyramidal cells of rat cerebral cortex. Phenylephrine, an α₁‐adrenoceptor agonist, suppressed the amplitude of AMPA receptor‐mediated excitatory postsynaptic currents evoked by repetitive electrical stimulation (eEPSCs, 10 pulses at 33 Hz). The coefficient of variation (CV) of the 1st eEPSC amplitude and paired‐pulse ratio (PPR), which were sensitive to extracellular Ca²⁺ concentration, were not affected by phenylephrine. Phenylephrine suppressed miniature EPSC (mEPSC) amplitude without changing its frequency. In contrast, isoproterenol, a β‐adrenoceptor agonist, strongly increased the amplitude of the 1st eEPSC compared with that of the 2nd to 10th eEPSCs, which resulted in a decrease in PPR. Isoproterenol‐induced enhancement of eEPSC amplitude was accompanied by a decrease in CV. Isoproterenol increased the frequency of mEPSCs without significant effect on amplitude. Phenylephrine suppressed inward currents evoked by puff application of glutamate, AMPA, or NMDA, whereas isoproterenol application was not accompanied by significant changes in these inward currents. These findings suggest that phenylephrine decreases eEPSCs through postsynaptic AMPA or NMDA receptors, while the effects of isoproterenol are mediated by facilitation of glutamate release from presynaptic terminals without effect on postsynaptic glutamate receptors. These two different mechanisms of modulation of excitatory synaptic transmission may improve the “signal‐to‐noise ratio” in cerebral cortex. Synapse 63:269–281, 2009. © 2008 Wiley‐Liss, Inc.