Hirokazu Hirai

New role of δ2-glutamate receptors in AMPA receptor trafficking and cerebellar function

Previous gene knockout studies have shown that the orphan glutamate receptor δ2 (GluRδ2) is critically involved in synaptogenesis between parallel fibers and Purkinje cells during development. However, the precise function of GluRδ2 and whether it is functional in the mature cerebellum remain unclear. To address these issues, we developed an antibody specific for the putative ligand-binding region of GluRδ2, and application of this antibody to cultured Purkinje cells induced AMPA receptor endocytosis, attenuated synaptic transmission and abrogated long-term depression. Moreover, injection of this antibody into the subarachnoidal supracerebellar space of adult mice caused transient cerebellar dysfunction, such as ataxic gait and poor performance in the rotorod test. These results indicate that GluRδ2 is involved in AMPA receptor trafficking and cerebellar function in adult mice.

Ipsilateral corticotectal pathway inhibits the formation of long-term potentiation (LTP) in the rat superior colliculus through GABAergic mechanism

The purpose of the present experiments was to clarify the possible mechanism for the depression of long term potentiation (LTP) induction in the superficial gray layer of the rat superior colliculus after optic nerve stimulation. A postsynaptic field potential was recorded in vitro in the superficial gray layer of superior colliculus slices after stimulation of the optic layer. Tetanic optic layer stimulation (50 Hz, 20 s) induced LTP of the postsynaptic field potential elicited in the superficial gray layer. The postsynaptic field potential, with unitary discharges, produced in the superficial gray layer by optic nerve stimulation in vivo was depressed by a conditioning stimulus to the visual cortex. Identical inhibition of the cortical response of the superficial gray layer was produced by optic nerve stimulation. The application of picrotoxin (2.5 mg/kg, i.p.), a GABAA antagonist or methoxypyridoxine (100 mg/kg, i.v.), an anti-glutamate decarboxylase agent which reduces GABA levels, blocked the inhibitory interaction between the optic nerve-superficial gray layer and visual cortex-superficial gray layer. Tetanic optic nerve stimulation (50 Hz, 20 s) failed to induce LTP in the superficial gray layer of the intact rat. LTP was only elicited by tetanic optic nerve stimulation when picrotoxin or methoxypyridoxine was administered prior to the tetanic stimulation and when the ipsilateral visual cortex was removed. These results indicate that GABAergic interneurons in the superficial gray layer activated by corticotectal input, may stop the formation of LTP in the superficial gray layer.

The release of glutamate and accumulation of intracellular calcium in the guinea pig hippocampal slices during glucose deprivation

Simultaneous recordings of the time course of change in the extracellular glutamate concentration ([Glu]O) and intracellular Ca2+ level [Ca2+]i during glucose deprivation were performed in the CA3 area of hippocampal slices from guinea pig. Direct measurement of [Glu]O with a glutamate sensitive sensor revealed that [Glu]0 showed a biphasic increase during glucose deprivation and decreased rapidly after re-introduction of glucose in the perfusing medium. The initial increase of [Glu]O preceded [Ca2+]i increase and thereafter a sustained increase of [Glu]O was observed concomitant with massive [Ca2+]i accumulation. Replacement of Na with Li markedly attenuated the rapid reduction of [Glu]O, suggesting that the reduction of [Glu]O was due to Na-dependent glutamate uptake system, which worked immediately after energy recovery.

Long-term potentiation of neurotransmission in the inferior colliculus of the rat.

Postsynaptic field potential was elicited in the central nucleus of the rat inferior colliculus after electrical stimulation to the lateral lemniscus. The inhibitory action of locally applied GABA on the potential was blocked by the systemic administration of picrotoxin, a GABAA antagonist (2.5 mg/kg, i.p.). After the administration of picrotoxin, the tetanic stimulation of 50 Hz for 20 s increased the amplitude of the field potential to over 120% of the original level in 10 min; this was maintained for 40 min, and formed the long-term potentiation (LTP). The tetanic stimulation alone, however, failed to enhance the amplitude. Thus, LTP was induced in the inferior colliculus only when GABAergic action was depressed.

Adenosine facilitates glutamate release in a protein kinase-dependent manner in superior colliculus slices

Experiments were performed to clarify the excitatory mechanism of adenosine in superior colliculus slices. Postsynaptic field potential was recorded in the superficial gray layer after stimulation to the optic layer. Application of adenosine to the perfusion medium at a concentration of 0.2 microM or 100 microM enhanced the amplitude of the postsynaptic field potential and the excitatory effect of adenosine was counteracted by application of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) (100 microM), a non-selective protein kinase inhibitor or N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004) (100 microM), a selective protein kinase A inhibitor. The presence of adenosine at a concentration of 0.2 microM or 100 microM during electrical stimulation to slices of the superficial gray layer increased the release of glutamate 2.5 and 2.3 times, respectively, and this increase was suppressed in Ca(2+)-free medium or by applying tetrodotoxin (1 microM) to the medium. Application of H-7 or HA1004 to the incubation medium inhibited the enhancement of the glutamate release. These results suggest that the excitatory effect of adenosine in the superior colliculus is due to an increase in glutamate release and involves the protein kinase system.

Excitatory and inhibitory effects of toluene on neural activity in guinea pig hippocampal slices

To investigate the effect of toluene and its derivatives on neural activity, postsynaptic field potential (population spike, PS) of granule cells as well as antidromic potential (AP) and presynaptic fiber potential (FP) (perforant path) were recorded in the guinea pig hippocampal slices. Toluene at the concentration of 0.2 ng/ml to 20 micrograms/ml in the perfusion medium increased the amplitude of PS to 109-150%. Toluene also increased the amplitude of FP and AP, although the most remarkable enhancement was observed in the PS. However, toluene at the concentrations over 1000 micrograms/ml completely depressed the PS, whereas it increased the amplitude of AP to 130% of the original level. These results indicate that toluene has excitatory and inhibitory biphasic effects on neurotransmission in the hippocampal slices according to concentration applied.

Inhibitory effect of GABA (γ-aminobutyric acid) on the induction of long-term potentiation in guinea pig superior colliculus slices

Superior colliculus (SC) slices were prepared from guinea pigs and postsynaptic potentials (PSPs) were recorded in the superficial grey layer of the SC after electrical stimulation to the optic layer (OL). Tetanic stimulation of 50 Hz frequency and 20 s duration to the OL induced long-term potentiation (LTP) in the PSP increasing the amplitude to 140% of the original level in 30 min. Tetanic stimulation failed to induce LTP after application of gamma-aminobutyric acid (GABA) (300 microM, 1 or 3 mM) to the perfusion medium. On the other hand, tetanic stimulation to the OL after application of bicuculline methiodide (1 microM), a GABAA receptor antagonist, facilitated the formation of LTP. These results indicate that GABA modulates the formation of LTP in the SC.

Exogenously applied gangliosides (GM1, GD1a and Gmix) fail to facilitate the induction of long-term potentiation (LTP) in the slices of hippocampus and superior colliculus of the guinea pig

To confirm the effect of gangliosides on the facilitation of the induction of long-term potentiation (LTP), slices of hippocampus and superior colliculus from guinea pig were prepared. One group of slices was incubated in the standard medium containing gangliosides (GM1, GD1a, or a mixture of gangliosides from bovine brain, each at a concentration of 70 microM) for 2 h. The other group of slices was incubated in the standard medium only. In both groups of slices, tetanic stimulation induced LTP in a similar manner. In low Ca2+ (1 mM) medium, LTP was not induced in either group of slices. Thus, the present experiments could not confirm previous reports indicating that exogenously applied gangliosides facilitate the induction of LTP.

Toluene inhibits synaptic transmission without causing gross morphological disturbances

The effects of toluene on synaptic transmission and neuronal morphology were investigated using guinea pig hippocampal slices. Population spikes (PS) were elicited in granule cell layer by stimulation of the perforant path and antidromic potentials (AP) were evoked in the same locii by stimulation of mossy fibers. Toluene at a concentration of 1000 micrograms/ml completely depressed post-synaptic responses within 15 min but increased the AP to 140% of its original value. The PS recovered completely when washout was begun 10 and 20 min after onset of depression, but exhibited only partial recovery following a 40 min depression. There were no evident changes in staining of axons or cell bodies after toluene treatment. These results indicate that toluene at high concentrations (1000 micrograms/ml) inhibits synaptic transmission selectively and with longer exposures causes lasting physiological effects unaccompanied by gross morphological changes.

Adenosine enhances neuronal damage during deprivation of oxygen and glucose in guinea pig superior collicular slices

The purpose of the present study was to clarify whether adenosine has a neuroprotective effect against neuronal damage during deprivation of oxygen and glucose in superior collicular slices. After deprivation of oxygen and glucose for 7 min, the concentration of ATP in slices incubated with adenosine (100 microM) for 60 min was significantly higher (6.43 +/- 0.16 nmol/mg protein, mean +/- S.E.M.) than that in slices incubated without adenosine (4.77 +/- 0.61). The postsynaptic field potential (PSP) recorded in the superficial gray layer of the SC slice completely disappeared within 7 min after deprivation of oxygen and glucose and it recovered to approximately 80% of the original amplitude in the medium without adenosine. But, in the presence of adenosine (100 microM) during and after oxygen and glucose removal, the PSP showed only approximately 40% recovery. These results indicate that in the superior colliculus adenosine has no protective effect on functional derangement caused by anoxia although it may facilitate the resynthesis of tissue ATP during recovery.