Presynaptic kainate and NMDA receptors are implicated in the modulation of GABA release from cortical and hippocampal nerve terminals

One of the pathways implicated in a fine-tuning control of synaptic transmission is activation of the receptors located at the presynaptic terminal. Here we investigated the intracellular events in rat brain cortical and hippocampal nerve terminals occurring under the activation of presynaptic glutamate receptors by exogenous glutamate and specific agonists of ionotropic receptors, NMDA and kainate. Involvement of synaptic vesicles in exocytotic process was assessed using [³H]GABA and pH-sensitive fluorescent dye acridine orange (AO). Glutamate as well as NMDA and kainate were revealed to induce [³H]GABA release that was not blocked by NO-711, a selective blocker of GABA transporters. AO-loaded nerve terminals responded to glutamate application by the development of a two-phase process. The first phase, a fluorescence transient completed in ∼1 min, was similar to the response to high K⁺. It was highly sensitive to extracellular Ca²⁺ and was decreased in the presence of the NMDA receptor antagonist, MK-801. The second phase, a long-lasting process, was absolutely dependent on extracellular Na⁺ and attenuated in the presence of CNQX, the kainate receptor antagonist. NMDA as well as kainate per se caused a rapid and abrupt neurosecretory process confirming that both glutamate receptors, NMDA and kainate, are involved in the control of neurotransmitter release. It could be suggested that at least two types ionotropic receptor are attributed to glutamate-induced two-phase process, which appears to reflect a rapid synchronous and a more prolonged asynchronous vesicle fusion.     

Subunit composition of kainate receptors in hippocampal interneurons

Kainate receptor activation affects GABAergic inhibition in the hippocampus by mechanisms that are thought to involve the GluR5 subunit. We report that disruption of the GluR5 subunit gene does not cause the loss of functional KARs in CA1 interneurons, nor does it prevent kainate-induced inhibition of evoked GABAergic synaptic transmission onto CA1 pyramidal cells. However, KAR function is abolished in mice lacking both GluR5 and GluR6 subunits, indicating that KARs in CA1 stratum radiatum interneurons are heteromeric receptors composed of both subunits. In addition, we show the presence of presynaptic KARs comprising the GluR6 but not the GluR5 subunit that modulate synaptic transmission between inhibitory interneurons. The existence of two separate populations of KARs in hippocampal interneurons adds to the complexity of KAR localization and function.     

Presynaptic kainate receptors are localized close to release sites in rat hippocampal synapses

The subsynaptic distribution of kainate receptors is still a matter of much debate given its importance to understand the way they influence neuronal communication. Here, we show that, in synapses of the rat hippocampus, presynaptic kainate receptors are localized within the presynaptic active zone close to neurotransmitter release sites. The activation of these receptors with low concentrations of agonists induces the release of [(3)H]glutamate in the absence of a depolarizing stimulus. Furthermore, this modulation of [(3)H]glutamate release by kainate is more efficient when compared with a KCl-evoked depolarization that causes a more than two-fold increase in the intra-terminal calcium concentration but no apparent release of [(3)H]glutamate, suggesting a direct receptor-mediated process. Using a selective synaptic fractionation technique that allows for a highly efficient separation of presynaptic, postsynaptic and non-synaptic proteins we confirmed that, presynaptically, kainate receptors are mainly localized within the active zone of hippocampal synapses where they are expected to be in a privileged position to modulate synaptic phenomena.     

Ripple-associated high-firing interneurons in the hippocampal CA1 region

By simultaneously recording the activity of individual neurons and field potentials in freely behaving mice, we found two types of interneurons firing at high frequency in the hippocampal CA1 region, which had high correlations with characteristic sharp wave-associated ripple oscillations (100–250 Hz) during slow-wave sleep. The firing of these two types of interneurons highly synchronized with ripple oscillations during slow-wave sleep, with strongly increased firing rates corresponding to individual ripple episodes. Interneuron type I had at most one spike in each sub-ripple cycle of ripple episodes and the peak firing rate was 310±33.17 Hz. Interneuron type II had one or two spikes in each sub-ripple cycle and the peak firing rate was 410±47.61 Hz. During active exploration, their firing was phase locked to theta oscillations with the highest probability at the trough of theta wave. Both two types of interneurons increased transiently their firing rates responding to the startling shake stimuli. The results showed that these two types of high-frequency interneurons in the hippocampal CA1 region were involved in the modulation of the hippocampal neural network during different states.     

Ripple-associated high-firing interneurons in the hippocampal CA1 region

By simultaneously recording the activity of individual neurons and field potentials in freely behaving mice, we found two types of interneurons firing at high frequency in the hippocampal CA1 region, which had high correlations with characteristic sharp wave-associated ripple oscillations (100―250 Hz) during slow-wave sleep. The firing of these two types of interneurons highly synchronized with ripple oscillations during slow-wave sleep, with strongly increased firing rates corresponding to individual ripple episodes. Interneuron type I had at most one spike in each sub-ripple cycle of ripple episodes and the peak firing rate was 310±33.17 Hz. Interneuron type II had one or two spikes in each sub-ripple cycle and the peak firing rate was 410±47.61 Hz. During active exploration, their firing was phase locked to theta oscillations with the highest probability at the trough of theta wave. Both two types of interneurons increased transiently their firing rates responding to the startling shake stimuli. The results showed that these two types of high-frequency interneurons in the hippocampal CA1 region were involved in the modulation of the hippocampal neural network during different states.     

Pertussis toxin prevents presynaptic inhibition by kainate receptors of rat hippocampal [(3)H]GABA release

Kainate receptors are ionotropic receptors, also reported to couple to G(i)/G(o) proteins, increasing neuronal excitability through disinhibition of neuronal circuits. We directly tested in hippocampal synaptosomes if kainate receptor-mediated inhibition of GABA release involved a metabotropic action. The kainate analogue, domoate (3 microM), inhibited by 24% [(3)H]GABA-evoked release, an effect reduced by 76% in synaptosomes pre-treated with pertussis toxin. Protein kinase C inhibition attenuated by 82% domoate-induced inhibition of GABA release whereas protein kinase C activation did not change kainate receptor binding. Thus, domoate inhibition of GABA release recruits G(i)/G(o) proteins and a protein kinase C pathway.     

Opioid inhibition of GABA release from presynaptic terminals of rat hippocampal interneurons.

Opiates and the opioid peptide enkephalin can cause indirect excitation of principal cortical neurons by reducing inhibitory synaptic transmission mediated by GABAergic interneurons. The mechanism by which opioids mediate these effects on interneurons is unknown, but enkephalin hyperpolarizes the somatic membrane potential of a variety of neurons in the brain, including hippocampal interneurons. We now report a new, more direct mechanism for the opioid-mediated reduction in synaptic inhibition. The enkephalin analog D-Ala2-Met5-enkephalinamide (DALA) decreases the frequency of miniature, action potential-independent, spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) without causing a change in their amplitude. Thus, we conclude that DALA inhibits the action potential-independent release of GABA through a direct action on interneuronal synaptic terminals. In contrast, DALA reduces the amplitude of action potential-evoked, GABA-mediated IPSCs, as well as decreases their frequency. This suggests that the opioid-mediated inhibition of non-action potential-dependent GABA release reveals a mechanism that contributes to reducing action potential-evoked GABA release, thereby decreasing synaptic inhibition.     

Presynaptic glycine receptors on hippocampal mossy fibers

Presynaptic glycine receptors (GlyRs) have been implicated in the regulation of glutamatergic synaptic transmission. Here, we characterized presynaptic GlyR-mediated currents by patch-clamp recording from mossy fiber boutons (MFBs) in rat hippocampal slices. In MFBs, focal puff-application of glycine-evoked chloride currents that were blocked by the GlyR antagonist strychnine. Their amplitudes declined substantially during postnatal development, from a mean conductance per MFB of ∼600 pS in young to ∼130 pS in adult animals. Single-channel analysis revealed multiple conductance states between ∼20 and ∼120 pS, consistent with expression of both homo- and hetero-oligomeric GlyRs. Accordingly, estimated GlyRs densities varied between 8-17 per young, and 1-3 per adult, MFB. Our results demonstrate that functional presynaptic GlyRs are present on hippocampal mossy fiber terminals and suggest a role of these receptors in the regulation of glutamate release during the development of the mossy fiber - CA3 synapse.     

Metabotropic glutamate receptors modulate GABA release from mouse hippocampal slices

The effects of metabotropic glutamate receptor agonists on the basal and potassium (50 mM K⁺)-stimulated release of [³H]GABA from mouse hippocampal slices were investigated using a superfusion system. The group I agonist (1±)-1-aminocyclopentane-trans-1,3-dicarboxylate enhanced the basal GABA release and reduced the K⁺-evoked release by a mechanism antagonized by (RS)-1-aminoindan-1,5-dicarboxylate in both cases. The group II agonist (2S,2R,3R)-2-(2,3-dicarboxycyclopropyl)glycine failed to have any effect on the basal release, but inhibited the stimulated release. This inhibition was not affected by the antagonist (2S)-2-ethylglutamate. The group III agonists L(+)-amino-4-phosphonobutyrate and O-phospho-L-serine inhibited the basal GABA release, which effects were blocked by the antagonist (RS)-2-cyclopropyl-4-phosphonophenylglycine. Moreover, the suppression of the K⁺-evoked release by L(+)2-amino-4-phosphonobutyrate was apparently receptor-mediated, being blocked by (RS)-2-cyclopropyl-4-phosphonophenylglycine. The results show that activation of metabotropic glutamate receptors of group I is able to potentiate the basal release of GABA, whereas activation of groups I and III receptors reduce K⁺-stimulated release in mouse hippocampal slices.     

The influence of myo-inositol on the ultrastructure of hippocampal CA1 area in kainate treated rats

The ultrastructure of neurons, synapses and astrocytes of hippocampal CA1 area in rats was investigated 14 days after: systemic injection of kainic acid and kainic acid and myo-Inositol. After injection of kainic acid numerous neurons with superficial and deep ultrastructural changes of cytoplasmic organelles were described. Among synapses numerous forms with osmiophilic active zone and single synaptic vesicles, also presynaptic terminals with core vesicles were often seen. After kainic acid + myo-Inositol injection the cells with superficial changes of organelles dominated and the synapsoarchitectonics of the area was close to normal. Thus, electrono-microscopic data indicate possible neuroprotective (antiepileptic?) features of myo-Inositol.     

Calcium extrusion mechanisms in dendrites of mouse hippocampal CA1 inhibitory interneurons

Local circuit GABAergic inhibitory interneurons control the integration and transfer of information in many brain regions. Several different forms of plasticity reported at interneuron excitatory synapses are triggered by cell- and synapse-specific postsynaptic calcium (Ca²⁺) mechanisms. To support this function, the spatiotemporal dynamics of dendritic Ca²⁺ elevations must be tightly regulated. While the dynamics of postsynaptic Ca²⁺ signaling through activation of different Ca²⁺ sources has been explored, the Ca²⁺ extrusion mechanisms that operate in interneuron dendrites during different patterns of activity remain largely unknown. Using a combination of whole-cell patch-clamp recordings and two-photon Ca²⁺ imaging in acute mouse hippocampal slices, we characterized the Ca²⁺ extrusion mechanisms activated by Ca²⁺ transients (CaTs) associated with backpropagating action potentials (bAPs) in dendrites of hippocampal CA1 stratum radiatum interneurons. Our data showed that Ca²⁺ clearance increased as a function of activity, pointing to an activity-dependent recruitment of specific Ca²⁺ extrusion mechanisms. bAP-CaTs were significantly prolonged in the presence of the plasma membrane Ca²⁺ ATPase (PMCA) and Na⁺/Ca²⁺ exchanger (NCX) inhibitors as well as the sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) and the mitochondria Ca²⁺ uniporter (MCU) blockers. While PMCA, NCX and SERCA pumps cooperated in the cytosolic Ca²⁺ removal at a wide range of concentrations, the MCU was only activated at higher Ca²⁺ loads produced by repetitive interneuron firing. These results identify a division of labor between distinct Ca²⁺ extrusion mechanisms shaping dendritic Ca²⁺ dynamics and possibly contributing to activity-dependent regulation of synaptic inputs in interneurons. In addition, the MCU activated by larger Ca²⁺ levels may be involved in the activity-dependent ATP production or interneuron-selective vulnerability associated with cytosolic Ca²⁺ overloads under pathological conditions.     

Characteristics of GABA release modified by glutamate receptors in mouse hippocampal slices

The major part of hippocampal innervation is glutamatergic, regulated by inhibitory GABA-releasing interneurons. The modulation of [(3)H]GABA release by ionotropic and metabotropic glutamate receptors and by nitric oxide was here characterized in superfused mouse hippocampal slices. The ionotropic glutamate receptor agonists kainate, N-methyl-D-aspartate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate potentiated the basal GABA release. These effects were blocked by their respective antagonists 6-nitro-7-cyanoquinoxaline-2,3-dione (CNQX), dizocilpine and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo(f)quinoxaline-7-sulfonamide (NBQX), indicating receptor-mediated mechanisms. The NO-generating compounds S-nitroso-N-acetylpenicillamine (SNAP), sodiumnitroprusside and hydroxylamine enhanced the basal GABA release. Particularly the sodiumnitroprusside-evoked release was attenuated by the NO synthase inhibitor N(G)-nitro-L-arginine (L-NNA) and the inhibitor of soluble guanylyl cyclase 1H-(1,2,4)oxadiazolo(4,3a)quinoxalin-1-one (ODQ), indicating the involvement of the NO/cGMP pathway. This inference is corroborated by the enhancing effect of zaprinast, a phosphodiesterase inhibitor, which is known to increase cGMP levels. The K(+)-stimulated hippocampal GABA release was reduced by the groups I and III agonists of metabotropic glutamate receptors (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylate (t-ACPD) and L-(+)-2-amino-4-phosphonobutyrate (L-AP4), which effects were abolished by their respective antagonists (RS)-1-aminoindan-1,5-dicarboxylate (AIDA) and (RS)-2-cyclopropyl-4-phosphonophenylglycine (CPPG), again indicating modification by receptor-mediated mechanisms.     

Selective block of AMPA/kainate receptors of hippocampal interneurons as a new approach to the investigation of inhibitory system

Effects of open channel blockers of AMPA/kainate receptors have been examined using whole cell recordings and kainate application in the neurons freshly isolated by vibrodissociation from the rat hippocampal slice preparation. Although the hippocampal neurons differed little in the voltage-current relations and sensitivity to kainate, a prominent difference was found in their susceptibility to the blocking action of adamantane derivatives studied. The pyramidal neurons had low sensitivity to the open channel blockers but the neurons which might be assigned most probably to the group of inhibitory interneurons proved to be highly sensitive. A group of neurons of intermediate sensitivity have also been found. The ability of the same blocking drugs to depress the excitatory inputs in the inhibitory interneurons has been demonstrated in the experiments on the hippocampal slice preparation. Enhancement of the field spike and excitatory postsynaptic potential amplitude was observed in the presence of adamantane derivatives. An additional treatment of the preparation with a GABA receptor antagonist, bicuculline, did not potentiate this effect. In conclusion, the observed difference in the pharmacological properties of inhibitory interneurons may be effectively used for detailed analysis of the brain synaptic transmission.     

Presynaptic kainate receptors in the hippocampus: slowly emerging from obscurity.

Kainate receptor agonists depress transmitter release at several synapses in the hippocampus. Distinct mechanisms appear to underlie this phenomenon at different synapses. Recently, it has emerged that presynaptic kainate receptors can also potentiate the release of both GABA and glutamate and that axonal kainate receptors can trigger ectopic action potentials in interneurons. Because synaptically released glutamate mimics many of the actions of exogenous agonists, presynaptic kainate receptors potentially play an extensive role in hippocampal signaling.     

Neurosteroids enhance spontaneous glutamate release in hippocampal neurons. Possible role of metabotropic sigma1-like receptors

Pregnenolone sulfate (PREGS), one of the most abundantly produced neurosteroids in the mammalian brain, improves cognitive performance in rodents. The mechanism of this effect has been attributed to its allosteric modulatory actions on glutamate- and gamma-aminobutyric acid-gated ion channels. Here we report a novel effect of PREGS that could also mediate some of its actions in the nervous system. We found that PREGS induces a robust potentiation of the frequency but not the amplitude of miniature excitatory postsynaptic currents (mEPSCs) mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors in cultured hippocampal neurons. PREGS also decreased paired pulse facilitation of autaptic EPSCs evoked by depolarization, indicating that it modulates glutamate release probability presynaptically. PREGS potentiation of mEPSCs was mimicked by dehydroepiandrosterone sulfate and (+)-pentazocine but not by (-)-pentazocine, the synthetic (-)-enantiomer of PREGS or the inactive steroid isopregnanolone. The sigma receptor antagonists, haloperidol and BD-1063, blocked the effect of PREGS on mEPSCs, as did pertussis toxin and the membrane-permeable Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl) ester. These results suggest that PREGS increases spontaneous glutamate release via activation of a presynaptic G(i/o)-coupled sigma receptor and an elevation in intracellular Ca2+ levels. We postulate that presynaptic actions of neurosteroids have a role in the maturation and/or maintenance of synaptic networks and the processing of information in the central nervous system.     

Synaptic activation of presynaptic kainate receptors on hippocampal mossy fiber synapses

Kainate receptors (KARs) are a poorly understood family of ionotropic glutamate receptors. A role for these receptors in the presynaptic control of transmitter release has been proposed but remains controversial. Here, KAR agonists are shown to enhance fiber excitability, and a number of experiments show that this is a direct effect of KARs on the presynaptic fibers. In addition, KAR activation inhibits evoked transmitter release from mossy fiber synapses. Synaptic release of glutamate from either neighboring mossy fiber synapses or associational/commisural (A/C) synapses results in the activation of these presynaptic ionotropic KARs. These results, along with previous studies, indicate that KARs, through the endogenous release of glutamate, mediate excitatory postsynaptic potentials (EPSPs), alter presynaptic excitability, and modulate transmitter release.     

Implication of ionotropic glutamate receptors in the release of noradrenaline in hippocampal CA1 and CA3 subregions under oxygen and glucose deprivation

A strong linkage between adrenergic and glutamatergic systems exists in the CNS but it is still unclear whether the excessive release of noradrenaline under ischemic conditions is modulated by excitatory amino acids. We studied the effect of selective glutamate receptor antagonists on the release of [3H]noradrenaline evoked by glucose and oxygen deprivation in hippocampal CA1, CA3 and dentate gyrus subregions. The release of glutamate, aspartate and GABA was measured by HPLC. Omission of oxygen and glucose increased the release of [3H]noradrenaline as well as the release of amino acids. Maximum effect on noradrenaline release was observed in CA1 region. The relative increase of the release after 30 min energy deprivation (R(2)) versus the basal release under normal conditions (R(1)), i.e. the R(2)/R(1) ratio was 7.1+/-1.0, 3.87+/-0.4 and 3.26+/-0.27 for CA1, CA3 and dentate gyrus, respectively. The [3H]noradrenaline outflow in response to glucose and oxygen deprivation was abolished at low temperature, but not by Ca(2+) removal, suggesting a cytoplasmic release process. In CA1 and CA3 [3H]noradrenaline release was significantly attenuated by MK-801, an NMDA receptor antagonist. The AMPA receptor antagonist GYKI-53784 had no effect in CA3, but partly reduced noradrenaline release in CA1.Our results suggest that ionotropic glutamate receptors seem to be implicated in the massive cytoplasmic release of noradrenaline in CA1 what may contribute to its selective vulnerability.     

Direct presynaptic regulation of GABA/glycine release by kainate receptors in the dorsal horn: an ionotropic mechanism.

In the spinal cord dorsal horn, excitatory sensory fibers terminate adjacent to interneuron terminals. Here, we show that kainate (KA) receptor activation triggered action potential-independent release of GABA and glycine from dorsal horn interneurons. This release was transient, because KA receptors desensitized, and it required Na+ entry and Ca2+ channel activation. KA modulated evoked inhibitory transmission in a dose-dependent, biphasic manner, with suppression being more prominent. In recordings from isolated neuron pairs, this suppression required GABA(B) receptor activation, suggesting that KA-triggered GABA release activated presynaptic GABA(B) autoreceptors. Finally, glutamate released from sensory fibers caused a KA and GABA(B) receptor-dependent suppression of inhibitory transmission in spinal slices. Thus, we show how presynaptic KA receptors are linked to changes in GABA/glycine release and highlight a novel role for these receptors in regulating sensory transmission.     

Kainate receptor activation enhances both tonic and phasic GABA-ergic inhibition in CA1 hippocampal interneurons

Kainate receptor agonists are powerful convulsants and excitotoxins. It has been a lot of controversy around functions of these receptors in the brain. It is shown in this article that kainate enhances evoked GABAergic IPSC (phasic currents) in CA1 interneurons in concentration-dependent manner. The phenomenon is likely to be due to kainate-mediated lowering of the threshold for action potential generation in interneuron axons and increased number of terminals responding to the same stimulus strength. Kainate application also induced an enhancement in tonic GABAergic conductance. This phenomenon can be attributed to massive extracellular GABA accumulation caused by interneuron firing in the presence of kainate. Extracellular GABA also shunts synaptic currents by activating tonic conductance as well as desensitizing synaptic GABAA receptors. Thus, the enhancement of the evoked IPSCs by 1 microM kainate was complicated by early and transient decrease. The kainate receptor-mediated enhancement of GABAergic tonic and phasic signalling to interneurons can contribute to the depression of GABAergic transmission to pyramidal neurons. The consequence of this phenomenon may play a major role in the epileptogenic action of this agent.