Akiko Momiyama

Functional correlation of fetal and adult forms of glycine receptors with developmental changes in inhibitory synaptic receptor channels

Functional maturation of the nicotinic acetylcholine receptor is executed by its gamma-to-epsilon subunit switching. The glycine receptor also has fetal (alpha 2) and adult (alpha 1) isoforms. However, whether subunit switching is responsible for developmental changes in glycine receptor function is not known. We recorded single-channel currents from homomeric glycine receptors expressed in Xenopus oocytes with cRNAs encoding the alpha 2 or alpha 1 subunits and compared them with those recorded from native glycine receptors in rat spinal neurons at various ontogenic periods. The mean channel life times of the alpha 1 and mature glycine receptors were equally short, whereas both the alpha 2 and fetal receptors showed a significantly longer open time. Consistent with these results, the decay time of the glycinergic inhibitory postsynaptic currents (IPSCs) in spinal neurons became shorter during postnatal development. We conclude that developmental switching of alpha subunits may accelerate the kinetics of IPSCs.

Number and density of AMPA receptors in single synapses in immature cerebellum.

The number of ionotropic receptors in synapses is an essential factor for determining the efficacy of fast transmission. We estimated the number of functional AMPA receptors at single postsynaptic sites by a combination of two-photon uncaging of glutamate and the nonstationary fluctuation analysis in immature rat Purkinje cells (PCs), which receive a single type of excitatory input from climbing fibers. Areas of postsynaptic membrane specialization at the recorded synapses were measured by reconstruction of serial ultrathin sections. The number of functional AMPA receptors was proportional to the synaptic area with a density of ∼1280 receptors/μm2. Moreover, highly sensitive freeze-fracture replica labeling revealed a homogeneous density of immunogold particles for AMPA receptors in synaptic sites (910 ± 36 particles/μm2) and much lower density in extrasynaptic sites (19 ± 2 particles/μm2) in the immature PCs. Our results indicate that in this developing synapse, the efficacy of transmission is determined by the synaptic area.

Re‐evaluation of calcium currents in pre‐ and postsynaptic neurones of the chick ciliary ganglion.

1. Presynaptic nerve terminals of ciliary ganglia of the chick embryo were identified by the accumulation of dextran‐tetramethylrhodamine applied to the cut end of the oculomotor nerve. Ca2+ currents were then recorded from the identified nerve terminals. 2. Whole‐cell recordings were carried out simultaneously from a presynaptic terminal and its postsynaptic cell. The generation of presynaptic Ca2+ currents induced a postsynaptic response with a short delay. Electrical coupling was present in eight of fifteen pairs. The coupling ratio did not exceed 5%. 3. High‐threshold Ba2+ currents were observed in presynaptic terminals without any evidence for the presence of low‐threshold Ca2+ channels. The Ba2+ current was completely blocked by 50 microM Cd2+. 4. The presynaptic Ca2+ current induced by a long depolarizing pulse showed inactivation, but this inactivation was diminished when Ca2+ was replaced with Ba2+. 5. The presynaptic Ba2+ current was insensitive to dihydropyridines (DHPs). omega‐Conotoxin GVIA (omega CgTX) suppressed a large fraction of the Ba2+ current irreversibly. About 10% of the Ba2+ current was resistant to both DHPs and omega CgTX. 6. The omega CgTX‐sensitive component was not sensitive to changes in the holding potential between ‐120 and ‐50 mV. The omega CgTX‐resistant component tended to be inactivated at depolarized holding potentials. 7. In some perisynaptic Schwann cells, small Ca2+ currents were observed. These Ca2+ currents increased monotonically with depolarization. 8. Only high‐threshold Ca2+ channel currents were observed in postsynaptic ciliary cells. Exposure to 50 microM Cd2+ completely abolished the Ca2+ current. 9. About 25% of the Ba2+ currents were blocked by nifedipine (10 microM) in ciliary cells. The nifedipine‐resistant component was partly blocked by omega CdTX (10 microM) leaving a small component (about 20%) which was resistant to both nifedipine and omega CgTX. 10. In ciliary cells, the fraction of Ba2+ currents blocked by omega CgTX was not affected by the presence or absence of nifedipine. Similarly, nifedipine blocked the Ba2+ currents to the same extent whether omega CgTX was present or not. The Ba2+ currents potentiated by Bay K 8644 were eliminated by nifedipine. 11. It is concluded that the presynaptic terminal of chick ciliary ganglion did not possess DHP‐sensitive Ca2+ channels in contrast with the postsynaptic cell. Two subpopulations of presynaptic Ca2+ channels were distinguishable by their sensitivity to omega CgTX and membrane potential.

Single-channel currents underlying glycinergic inhibitory postsynaptic responses in spinal neurons

Single-channel properties of glycine receptors have been characterized so far only in cultured neurons. To characterize the glycine receptor channels in situ, we applied the patch-clamp technique to spinal neurons in slice preparations. Glycine-gated, single-channel currents were recorded in outside-out patches excised from spinal neurons. In the falling phase of glycinergic inhibitory synaptic currents, single-channel currents were resolved as discrete steps. In both cases, the glycine-gated channels showed similar multiple conductance levels. These results suggest that the receptor channel properties are indistinguishable in the synaptic and extrasynaptic sites. We conclude that multiple conductance states of a receptor channel are the native feature of the glycine receptor in situ.

Presynaptic inhibitory action of a metabotropic glutamate receptor agonist on excitatory transmission in visual cortical neurons

A family of metabotropic glutamate receptors (mGluRs) has been elucidated by molecular cloning. To study the possible modulatory role of mGluRs in synaptic transmission, we tested the effect of a mGluR agonist, (±)-l-aminocyclopentane-trans-l,3-dicarboxylic acid (trans-ACPD), on the excitatory post-synaptic currents (EPSCS) recorded from neurons in thin slices of rat visual cortex, by using the whole-cell patch-clamp method. We found that trans-ACPD) markedly suppressed the evoked EPSCS without affecting the mean amplitude of spontaneous miniature EPSCS. This effect on the evoked EPSCS was blocked by a potassium channel blocker, 4-aminopyridine (4-AP) in a dose-dependent manner We suggest that trans-ACPD presynaptically inhibits EPSCS by a mechanism involving the 4-AP-sensitive channels.

Calcium channels responsible for potassium-induced transmitter release at rat cerebellar synapses.

The effects of calcium channel blockers on potassium‐induced transmitter release were studied in thin slices of cerebellum from neonatal rats using whole‐cell patch clamp methods. Miniature inhibitory postsynaptic currents (mIPSCs) mediated by gamma‐aminobutyric acid (GABA) were recorded from deep cerebellar nuclear neurones in the presence of tetrodotoxin. The frequency of mIPSCs was reproducibly increased by a brief application of high‐potassium solution. In the presence of the L‐type Ca2+ channel blocker nicardipine (10 microM), the potassium‐induced increase in mIPSC frequency was suppressed by 49%. Neither the mean amplitude nor the time course of mIPSCs was affected by the blocker. The N‐type Ca2+ channel blocker omega‐conotoxin GVIA (omega‐CgTX, 3 microM) had no effect on the frequency of potassium‐induced mIPSCs. The P‐type Ca2+ channel blocker omega‐Aga‐IVA (200 nM) suppressed the potassium‐induced increase in mIPSC frequency by 83% without affecting the mean amplitude or time course of mIPSCs. Comparing these data with previous studies of neurally evoked transmission, it is concluded that the Ca2+ channel subtypes responsible for potassium‐induced transmitter release may be different from those mediating fast synaptic transmission.