No abstract

2-Arachidonoyl-glycerol (2-AG) is an endocannabinoid that is released from postsynaptic neurons, acts retrogradely on presynaptic cannabinoid receptor CB1, and induces short-and long-term suppression of transmitter release. To understand the mechanisms of the 2-AG-mediated retrograde modulation, we investigated subcellular localization of a major 2-AG biosynthetic enzyme, diacylglycerol lipase-␣ (DAGL␣), by using immunofluorescence and immunoelectron microscopy in the mouse brain. In the cerebellum, DAGL␣ was predominantly expressed in Purkinje cells. DAGL␣ was detected on the dendritic surface and occasionally on the somatic surface, with a distal-to-proximal gradient from spiny branchlets toward somata. DAGL␣ was highly concentrated at the base of spine neck and also accumulated with much lower density on somatodendritic membrane around the spine neck. However, DAGL␣ was excluded from the main body of spine neck and head. In hippocampal pyramidal cells, DAGL␣ was also accumulated in spines. In contrast to the distribution in Purkinje cells, DAGL␣ was distributed in the spine head, neck, or both, whereas somatodendritic membrane was labeled very weakly. These results indicate that DAGL␣ is essentially targeted to postsynaptic spines in cerebellar and hippocampal neurons, but its fine distribution within and around spines is differently regulated between the two neurons. The preferential spine targeting should enable efficient 2-AG production on excitatory synaptic activity and its swift retrograde modulation onto nearby presynaptic terminals expressing CB1. Furthermore, different fine localization within and around spines suggests that the distance between postsynaptic 2-AG production site and presynaptic CB1 is differentially controlled depending on neuron types.

1. The effects of metabotropic glutamate receptor (mGluR) agonists on excitatory transmission at mossy fibre-CA3 synapses were studied in rat hippocampal slice preparations using both extracellular and whole-cell clamp recording techniques.2. Application of a novel and potent mGluR2/mGluR3-specific agonist (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV, 0-1 SM) reversibly suppressed field excitatory postsynaptic potentials evoked by mossy fibre stimulation. DCG-IV at the same concentration did not affect other glutamatergic excitatory transmissions at the commissural/associational input to CA3 or at the Schaffer collateral/commissural input to CAI regions. 3. This suppressing effect of DCG-IV on mossy fibre transmission was dose dependent and partly antagonized by a competitive mGluR antagonist (+)-methyl-4-carboxylphenylglycine (1 mM). 4. The field potential changes induced by pressure application of glutamate (01 mM) to the stratum lucidum of the CA3 region was unaffected by 0f IUM DCG-IV.5. In whole-cell clamp experiments, 0-1 /SM DCG-IV suppressed excitatory postsynaptic currents evoked by mossy fibre stimulation without inducing detectable inward current in CA3 neurons, and paired-pulse facilitation was enhanced by DCG-IV application. 6. These results suggest that mGluR2/mGluR3 are specifically expressed at mossy fibre synapses in the hippocampal CA3 region, and activation of the receptor suppresses synaptic transmission by an action on a presynaptic site.A family of metabotropic glutamate receptors was revealed by recent molecular cloning studies (Nakanishi, 1994), and at least eight subtypes, termed mGluR1-mGluR8, have been identified so far (Masu, Tanabe, Tsuchida, Shigemoto

At many synapses, the amount of transmitter released by action potentials increases progressively during a train of spikes. This enhancement of evoked transmitter release grows during tetanic stimulation with several time constants, each bearing a different name (facilitation: tens to hundreds of milliseconds; augmentation: several seconds; potentiation: several minutes), and the enhancement of release to test spikes after a tetanus decays with similar time constants. All these processes depend on presynaptic Ca2+ influx during the conditioning tetanus. It has often been proposed that these forms of synaptic plasticity are due to residual Ca2+ present in nerve terminals following conditioning activity. We tested this idea directly by using photolabile Ca2+ chelators to reduce residual Ca2+ following conditioning stimulation or to generate an artificial elevation in Ca2+ concentration, and observed the effects on synaptic transmission at crayfish neuromuscular junctions. We found that facilitation, augmentation and potentiation are caused by the continuing action of residual Ca2+. Augmentation and potentiation seem to arise from Ca2+ acting at a separate site from facilitation, and these sites are different from the molecular target triggering neurosecretion.

Hippocampal mossy fibers project preferentially to the stratum lucidum, the proximal-most lamina of the suprapyramidal region of CA3. The molecular mechanisms that govern this lamina-restricted projection are still unknown. We examined the projection pattern of mossy fibers in mutant mice for semaphorin receptors plexin-A2 and plexin-A4, and their ligand, the transmembrane semaphorin Sema6A. We found that plexin-A2 deficiency causes a shift of mossy fibers from the suprapyramidal region to the infra- and intrapyramidal regions, while plexin-A4 deficiency induces inappropriate spreading of mossy fibers within CA3. We also report that the plexin-A2 loss-of-function phenotype is genetically suppressed by Sema6A loss of function. Based on these results, we propose a model for the lamina-restricted projection of mossy fibers: the expression of plexin-A4 on mossy fibers prevents them from entering the Sema6A-expressing suprapyramidal region of CA3 and restricts them to the proximal-most part, where Sema6A repulsive activity is attenuated by plexin-A2.

The presynaptic action of kainate (KA) receptor activation at the mossy fibre‐CA3 synapse was examined using fluorescence measurement of presynaptic Ca2+ influx as well as electrophysiological recordings in mouse hippocampal slices. Bath application of a low concentration (0·2 μM) of KA reversibly increased the amplitude of presynaptic volley evoked by stimulation of mossy fibres to 146 ± 6 % of control (n= 6), whereas it reduced the field excitatory postsynaptic potential (EPSPs) to 30 ± 4 %. The potentiating effect of KA on the presynaptic volleys was also observed in Ca2+‐free solution, and was partly antagonized by (2S,4R)‐4‐methylglutamic acid (SYM 2081, 1 μM), which selectively desensitizes KA receptors. The antidromic population spike of dentate granule cells evoked by stimulation of mossy fibres was increased by application of 0·2 μM KA to 160 ± 10 % of control (n= 6). Whole‐cell current‐clamp recordings revealed that the stimulus threshold for generating antidromic spikes recorded from a single granule cell was lowered by KA application. Application of KA (0·2 μM) suppressed presynaptic Ca2+ influx to 78 ± 4 % of control (n= 6), whereas the amplitude of the presynaptic volley was increased. KA at 0·2 μM reversibly suppressed excitatory postsynaptic currents (EPSCs) evoked by mossy fibre simulation to 38 ± 9 % of control (n= 5). These results suggest that KA receptor activation enhances the excitability of mossy fibres, probably via axonal depolarization, and reduces action potential‐induced Ca2+ influx, thereby inhibiting mossy fibre EPSCs presynaptically. This novel presynaptic inhibitory action of KA at the mossy fibre‐CA3 synapse may regulate the excitability of highly interconnected CA3 networks.

gene. In the mutant CA1 region, synaptic and extrasynaptic AMPA receptors on dendrites and spines were severely reduced to 35-37% of control levels, whereas reduction was mild for extrasynaptic receptors on somata (74%) and no significant decrease was seen for intracellular receptors within spines. In the mutant CA3 region, synaptic AMPA receptors were reduced mildly at asymmetrical synapses in the stratum radiatum (67% of control level), and showed no significant decrease at mossy fiber-CA3 synapses. Therefore, γ-8 is abundantly distributed on hippocampal excitatory synapses and extrasynaptic membranes, and plays an important role in increasing the number of synaptic and extrasynaptic AMPA receptors on dendrites and spines, particularly, in the CA1 region.

The effect of a low concentration (1 μM) of kainate (kainic acid; KA) on presynaptic calcium (Ca2+) influx at the Schaffer collateral‐commissural (SCC) synapse was examined in rat hippocampal slices. Following selective loading of the presynaptic terminals with the fluorescent Ca2+ indicator rhod‐2 AM, transient increases in the presynaptic Ca2+ concentration (pre[Ca2+]t) and field excitatory postsynaptic potentials (EPSPs) evoked by electrical stimulation of the SCC pathway were recorded simultaneously. Bath application of 1 μM KA reversibly suppressed field EPSPs and pre[Ca2+]t to 37.7 ± 4.0 % and 72.9 ± 2.4 % of control, respectively. Excitatory postsynaptic currents (EPSCs) recorded with the use of the whole‐cell patch‐clamp technique were also suppressed by 1 μM KA to 42.6 ± 6.3 % of control. A quantitative analysis of the decreases in pre[Ca2+]t and the amplitude of field EPSP during KA application suggests that KA inhibits transmission primarily by reducing the pre[Ca2+]t. Consistent with a presynaptic site for these effects, paired‐pulse facilitation (PPF) was enhanced by 1 μM KA. A substantial KA‐induced suppression of NMDA receptor‐mediated EPSPs was detected when AMPA receptors were blocked by the AMPA receptor‐selective antagonist GYKI 52466 (100 μM). The suppressive effect of KA on field EPSPs and pre[Ca2+]t was antagonized by the KA antagonist NS‐102 (10 μM). These results suggest that the presynaptic inhibitory action of KA at the hippocampal CA1 synapse is primarily due to the inhibition of Ca2+ influx into the presynaptic terminals.