The mechanisms of Ca(2+) release from intracellular stores in CNS white matter remain undefined. In rat dorsal columns, electrophysiological recordings showed that in vitro ischemia caused severe injury, which persisted after removal of extracellular Ca(2+); Ca(2+) imaging confirmed that an axoplasmic Ca(2+) rise persisted in Ca(2+)-free perfusate. However, depletion of Ca(2+) stores or reduction of ischemic depolarization (low Na(+), TTX) were protective, but only in Ca(2+)-free bath. Ryanodine or blockers of L-type Ca(2+) channel voltage sensors (nimodipine, diltiazem, but not Cd(2+)) were also protective in zero Ca(2+), but their effects were not additive with ryanodine. Immunoprecipitation revealed an association between L-type Ca(2+) channels and RyRs, and immunohistochemistry confirmed colocalization of Ca(2+) channels and RyR clusters on axons. Similar to "excitation-contraction coupling" in skeletal muscle, these results indicate a functional coupling whereby depolarization sensed by L-type Ca(2+) channels activates RyRs, thus releasing damaging amounts of Ca(2+) under pathological conditions in white matter.

Objective: The deleterious effects of glutamate excitoxicity are well described for CNS gray matter. While overactivation of glutamate receptors also contributes to axonal injury, the mechanisms are poorly understood. Our goal was to elucidate the mechanisms of kainate receptordependent axonal Ca 2+ deregulation.Methods: dorsal column axons were loaded with a Ca 2+ indicator and imaged in vitro using confocal laser-scanning microscopy.Results: Activation of GluR6 kainate receptors promoted a substantial rise in axonal [Ca 2+ ]. This Ca 2+ accumulation was due not only to influx from the extracellular space, but a significant component originated from ryanodine-dependent intracellular stores, which in turn depended on activation of L-type Ca 2+ channels: ryanodine, nimodipine or nifedipine blocked the agonistinduced Ca 2+ rise. Also, GluR6 stimulation induced intra-axonal production of NO, which greatly enhanced the Ca 2+ response: quenching of NO with intra-axonal (but not extracellular) scavengers, or inhibition of nNOS with intra-axonal L-NAME, blocked the Ca 2+ rise. Loading axons with a peptide that mimics the C-terminal PDZ binding sequence of GluR6 -thus interfering with the coupling of GluR6 to downstream effectors -greatly reduced the agonistinduced axonal Ca 2+ increase. Immunohistochemistry showed GluR6/7 clusters on the axolemma co-localized with nNOS and Ca v 1.2.Interpretation: Myelinated spinal axons express functional GluR6-containing kainate receptors, forming part of novel signaling complexes reminiscent of post-synaptic membranes of glutamatergic synapses. The ability of such axonal "nanocomplexes" to release toxic amounts of Ca 2+ may represent a key mechanism of axonal degeneration in disorders such as multiple sclerosis where abnormal accumulation of glutamate and NO are known to occur. . In this report we tested the hypothesis that myelinated axons from rat spinal cord express functional kainate receptors capable of mediating a potentially deleterious axonal Ca 2+ rise. We found that GluR6-containing kainate receptors reside along the internodal axolemma in "nanocomplexes" together with nNOS, exerting control over L-type Ca 2+ channels and causing Ca 2+ release from intra-axonal Ca 2+ stores. These signaling molecules are organized in a surprisingly intricate arrangement (see Fig. 6) reminiscent of what is found at the post-synaptic membrane of conventional glutamatergic synapses. Materials and MethodsAll experiments were performed in accordance with institutional guidelines for the care and use of experimental animals. Additional details can be found in Supplementary Material. Ca 2+ imagingDorsal columns from deeply anesthetized adult Long Evans male rats were removed from the thoracic region and placed in cold oxygenated zero-Ca 2+ solution containing, loaded for 2 hours with Ca 2+ -insensitive reference dye (red dextran-conjugated Alexa 594, 250 μM) to allow identification of axon profiles (Fig. 1A) together with the dextran-conjugated Ca 2+ indicator Oregon Green BAPTA-1 (250 μ...

Whole-cell recordings were used to investigate long-term potentiation of inhibitory synaptic currents (IPSCs) in neurons of deep cerebellar nuclei (DCN) in slices. IPSCs were evoked by electrical stimulation of the white matter surrounding the DCN in the presence of non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (20 microM). High-frequency stimulation induced a long-term potentation (LTP) of the IPSC amplitude without changing its reversal potential, rise time, and decay-time constant. This LTP did not require the activation of postsynaptic gamma-aminobutyric acid-A (GABA(A)) receptors but depended on the activation of NMDA receptors. LTP of IPSCs in DCN neurons could also be induced by voltage-depolarizing pulses in postsynaptic neurons and appeared to depend on an increase in intracellular calcium as the LTP was blocked when the cells were loaded with a calcium chelator, 1,2-bis-(2-amino-phenoxy)-N,N,N', N'-tetraacetic acid (BAPTA, 10 mM). LTP of IPSCs was accompanied by an increase in the frequency of spontaneous IPSCs and miniature IPSCs (recorded in the presence of tetrodotoxin 1 microM), but there was no significant change in their amplitude. In addition, during the LTP, the amplitude of response to exogenously applied GABA(A) receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride was increased. Intracellular application of tetanus toxin, a powerful blocker of exocytosis, in DCN neuron prevented the induction of LTP of IPSCs. Our results suggest that the induction of LTP of IPSCs in the DCN neurons likely involves a postsynaptic locus. Plasticity of inhibitory synaptic transmission in DCN neurons may play a crucial role in cerebellar control of motor coordination and learning.

Objective-Glutamate receptors, which play a major role in the physiology and pathology of CNS gray matter, are also involved in the pathophysiology of white matter. However the cellular and molecular mechanisms responsible for excitotoxic damage to white matter elements are not fully understood. We explored the roles of AMPA and GluR5 kainate receptors in axonal Ca 2+ deregulation.Methods-Dorsal column axons were loaded with a Ca 2+ indicator and imaged in vitro using confocal microscopy.Results-Both AMPA and a GluR5 kainate receptor agonists increased intra-axonal Ca 2+ in myelinated rat dorsal column fibers. These responses were inhibited by selective antagonists of these glutamate receptors. The GluR5-mediated Ca 2+ rise was mediated by both canonical (i.e. ionotropic) and non-canonical (metabotropic) signalling, dependent on a pertussis toxin-sensitive G protein and a phospholipase C-dependent pathway, promoting Ca 2+ release from IP3-dependent stores. Additionally, the GluR5 response was significantly reduced by intra-axonal NO scavengers. In contrast, GluR4 AMPA receptors operated via Ca 2+ induced Ca 2+ release, dependent on ryanodine receptors, and unaffected by NO scavengers. Neither pathway depended on L-type Ca 2+ channels, in contrast to GlurR6 kainate receptor action 1. Immunohistochemistry confirmed the presence of GluR4 and GluR5 clustered at the surface of myelinated axons; GluR5 co-immunoprecipitated with nNOS and often co-localized with nNOS clusters on the internodal axon.Interpretation-Central myelinated axons express functional AMPA and GluR5 kainate receptors, and can directly respond to glutamate receptor agonists. These glutamate receptordependent signalling pathways promote an increase in intra-axonal Ca 2+ levels potentially contributing to axonal degeneration.The precise mechanisms of glutamate-mediated toxicity in white matter are not completely established. This transmitter likely causes damage to glia given that both astrocytes and oligodendrocytes express a variety of glutamate receptors 2 -8, with oligodendrocytes being indicator Oregon Green BAPTA-1 (250 μM) (both from Molecular Probes) using a suction electrode applied to the cut end. The final dye concentration in the axons was estimated at ≈ 2 μM. Tissue was transferred to a custom-built chamber on a Nikon C1 confocal microscope and imaged every 60 sec at 37°C with a 60× 1.0 NA water immersion lens warmed to 37°C. Green signal was ratioed against the Ca 2+ -insensitive red channel, and then percent change during exposure to various agents compared to control was calculated. PTX was first activated by adding ATP (1mM) and glutathione (2mM) and incubated at 37°C overnight. Final PTX concentration in the loading pipette was 5 μM. ImmunohistochemistryFor light microscopy, deeply anesthetized rats were perfused with saline then 4% paraformaldehyde in 0.1 M phosphate buffer. Dorsal columns were excised, post-fixed, and immersed in 20% sucrose overnight. 40 μm sections were cut with a freezing microtome and washed with Tris b...

1. We investigated long-term potentiation (LTP) of synaptic transmission in different populations of interneurons in the CA1 region of rat hippocampal slices using whole cell recordings. We elicited excitatory postsynaptic currents (EPSCs) in interneurons located in stratum oriens near the alveus (O/A) or in stratum lacunosum-moleculare near the stratum radiatum border (L-M) by electrical stimulation of nearby axons in stratum oriens and radiatum, respectively. 2. High-frequency stimulation (100 Hz, 1 s) of axons in conjunction with postsynaptic depolarization (to -20 mV) increased the peak amplitude of test EPSCs elicited at -80 mV in O/A interneurons. The mean peak amplitude of EPSCs was significantly potentiated relative to the control period at 10 min (39 +/- 7% increase, mean +/- SE; n = 11 cells) and 30 min (30 +/- 1% increase; n = 5 cells) after tetanization. Similar stimulation did not produce potentiation of EPSCs in L-M interneurons (n = 7 cells). 3. This selective LTP in O/A interneurons was reversibly blocked by the N-methyl-D-aspartate receptor antagonist (+/-)2-amino-5-phosphonopentanoic acid (AP-5). Tetanization in the presence of 25 microM AP-5 did not increase the amplitude of EPSCs (8 cells). After washout of AP-5 (4 cells), a second tetanization resulted in long-term potentiation of EPSCs. 4. LTP was dependent on the activation of metabotropic glutamate receptors. The peak amplitude of EPSCs was not increased 5-10 or 15-20 min after tetanization during bath application of the metabotropic glutamate receptor antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (500 microM) (n = 5 cells). 5. Inclusion of the Ca2+ chelator 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA; 25 mM) in the patch pipette blocked LTP in O/A interneurons. In five cells recorded with BAPTA-containing electrodes, the mean peak amplitude was not significantly increased after tetanization. Thus a rise in postsynaptic intracellular Ca2+ appeared necessary for the induction of LTP in these interneurons. 6. Incubation of slices with the inhibitor of nitric oxide synthase N omega-nitro-L-arginine methyl ester (100 microM) before and throughout the recording session also blocked the increase in EPSC amplitude at 5-10 min (5 cells) and 15-20 min (3 cells) after tetanization. NO synthesis may therefore be necessary for LTP in O/A interneurons. 7. These results suggest that LTP of excitatory synapses is selectively produced in O/A but not L-M interneurons, and that this LTP shares similar characteristics with LTP in hippocampal CA1 pyramidal cells.(ABSTRACT TRUNCATED AT 400 WORDS)