Kainate modulates presynaptic GABA release from two vesicle pools.

Inhibitory control of local neuronal circuits is critical for prefrontal cortical functioning. Modulation of inhibitory circuits by several neuromodulators has been demonstrated, but the underlying mechanisms are unclear. Neuromodulator effects on synaptic vesicle recycling have received little attention. Controversy also exists whether different pools of synaptic vesicles underlie spontaneous and activity-dependent vesicle recycling. We therefore investigated the effects of kainate receptor activation on GABA release in rat prefrontal neocortex using electrophysiological and styryl dye imaging techniques in acute neocortical slices. Electrophysiological studies demonstrated that activation of kainate receptors increased the frequency, but not the amplitude of miniature IPSCs, suggesting a presynaptic action. Using styryl dye staining and multiphoton excitation microscopy, we visualized vesicular release from inhibitory GABAergic terminals in prefrontal cortical slices and demonstrate that kainate facilitates GABA release from presynaptic terminals. Our findings also indicate the presence of two pools of GABA-containing vesicles within inhibitory terminals. Kainate modulates both pools but only when vesicles are endocytosed and exocytosed by matching protocols of dye loading, i.e., spontaneous or evoked afferent activity.

GABA vesicles at synapses: are there 2 distinct pools?

Fast synaptic inhibition in the neocortex is mediated by the neurotransmitter GABA, acting on GABA( A) receptors. Neurotransmitters, including GABA, are stored in synaptic vesicles at presynaptic nerve terminals. A long-held assumption has been that evoked and spontaneous neurotransmissions draw on the same pools of vesicles. We review the evidence from FM1-43 studies supporting the contention that at least 2 distinct pools of GABA vesicles are present at inhibitory synapses in the rat neocortex. FM1-43 uptake during spontaneous vesicle endocytosis labels a vesicle pool within neocortical inhibitory nerve terminals that is released much more slowly ("reluctant" pool) than those vesicles loaded by electrical stimulation of afferent fibers or hyperkalemic solutions. These multiple pools may play diverse roles in such processes as long-term depression and/or potentiating of inhibitory synaptic transmission, homeostatic plasticity of inhibitory activity, or developmental changes in inhibitory synaptic transmission.

Calcium release via activation of presynaptic IP3 receptors contributes to kainate-induced IPSC facilitation in rat neocortex.

We examined the mechanisms of kainate (KA) induced modulation of GABA release in rat prefrontal cortex. Pharmacologically isolated IPSCs were recorded from visually identified layer II/III pyramidal cells using whole-cell patch clamp techniques. KA produced an increase in evoked IPSC amplitude at low nanomolar concentrations (100-500 nM). The frequency but not the amplitude of miniature (m) IPSCs was also increased. The GluR5 subunit selective agonist (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA) caused an increase in mIPSC frequency whereas (3S,4aR,6S,8aR)-6-(4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid (LY382884), a selective GluR5 subunit antagonist, inhibited this facilitation. Philanthotoxin-433 (PhTx) blocked the effect of KA, indicating involvement of Ca(2+)-permeable GluR5 receptors. No IPSC facilitation was seen when Ca(2+) was omitted from the bathing solution. Facilitation was observed when slices were preincubated in ruthenium red or high concentrations of ryanodine, but was inhibited with application of thapsigargin. The IP3 receptor (IP3R) antagonists diphenylboric acid 2-amino-ethyl ester (2-APB) (15 microM) and Xestospongin C (XeC) blocked IPSC facilitation. These results show that activation of KA receptors (KARs) on GABAergic nerve terminals results is linked to intracellular Ca(2+) release via activation of IP3, but not ryanodine, receptors. This represents a new mechanism of presynaptic modulation whereby Ca(2+) entry through Ca(2+)-permeable GluR5 subunit containing KARs activates IP3Rs receptors leading to an increase in GABA release.

Pre- and postsynaptic effects of kainate on layer II/III pyramidal cells in rat neocortex.

Kainate receptors mediate both direct excitatory and indirect modulatory actions in the CNS. We report here that kainate has both pre- and postsynaptic actions in layer II/III pyramidal neurons of rat prefrontal cortex. Application of low concentration of kainate (50-500 nM) increased the amplitude of evoked excitatory postsynaptic currents (EPSCs) whereas higher concentrations (3 microM) caused a decrease. The frequency of spontaneous and miniature (action potential-independent) EPSCs was increased by low concentrations of kainate without affecting their amplitudes, suggesting a presynaptic mechanism of action. The facilitatory and inhibitory effects of kainate were mimicked by the GluR5 subunit selective agonist ATPA. In addition to decreasing EPSC amplitudes, high concentrations of kainate and ATPA induced an inward current which was not blocked by AMPA- or NMDA-receptor antagonists GYKI52466 and D-APV, respectively. The inward currents were blocked by the AMPA/KA receptor antagonist CNQX, indicating the presence of postsynaptic kainate receptors. Single shock stimulation in the presence of GYKI52466 and D-APV evoked an EPSC which was blocked by CNQX. The GluR5 antagonist LY382884 changed paired-pulse facilitation to paired pulse depression, indicating that synaptically released glutamate can activate presynaptic kainate receptors. These results suggest that kainate receptors containing GluR5 subunits play a major role in glutamatergic transmission in rat neocortex, having both presynaptic modulatory and direct postsynaptic excitatory actions.

Presynaptic NMDA receptors mediate IPSC potentiation at GABAergic synapses in developing rat neocortex.

BACKGROUND: NMDA receptors are traditionally viewed as being located postsynaptically, at both synaptic and extrasynaptic locations. However, both anatomical and physiological studies have indicated the presence of NMDA receptors located presynaptically. Physiological studies of presynaptic NMDA receptors on neocortical GABAergic terminals and their possible role in synaptic plasticity are lacking. METHODOLOGY/PRINCIPAL FINDINGS: We report here that presynaptic NMDA receptors are present on GABAergic terminals in developing (postnatal day (PND) 12-15) but not older (PND21-25) rat frontal cortex. Using MK-801 in the recording pipette to block postsynaptic NMDA receptors, evoked and miniature IPSCs were recorded in layer II/III pyramidal cells in the presence of AMPA/KA receptor antagonists. Bath application of NMDA or NMDA receptor antagonists produced increases and decreases in mIPSC frequency, respectively. Physiologically patterned stimulation (10 bursts of 10 stimuli at 25 Hz delivered at 1.25 Hz) induced potentiation at inhibitory synapses in PND12-15 animals. This consisted of an initial rapid, large increase in IPSC amplitude followed by a significant but smaller persistent increase. Similar changes were not observed in PND21-25 animals. When 20 mM BAPTA was included in the recording pipette, potentiation was still observed in the PND12-15 group indicating that postsynaptic increases in calcium were not required. Potentiation was not observed when patterned stimulation was given in the presence of D-APV or the NR2B subunit antagonist Ro25-6981. CONCLUSIONS/SIGNIFICANCE: The present results indicate that presynaptic NMDA receptors modulate GABA release onto neocortical pyramidal cells. Presynaptic NR2B subunit containing NMDA receptors are also involved in potentiation at developing GABAergic synapses in rat frontal cortex. Modulation of inhibitory GABAergic synapses by presynaptic NMDA receptors may be important for proper functioning of local cortical networks during development.