Age-related behavioral, morphological and physiological changes in the hippocampus of zitter rats.

Convulsive seizure is known to be associated with hippocampal abnormalities, such as hilar cell degeneration, abnormal mossy fiber sprouting in the dentate gyrus (DG) and abnormal expression of immediate early genes. However, whether these morphological changes are a cause or consequence of convulsive seizures has remained contentious. Zitter (zi/zi) rats carry a mutation of the attractin gene and display spongiform degeneration of the brain. Spontaneous convulsive seizures in zi/zi rats over 8 months (M) old were demonstrated using 24-h video monitoring. Spontaneous convulsive seizures did not occur before this age. The present study examined structural changes in the hippocampus of zi/zi rats at different ages. Fluoro-Jade B-positive cells first appeared in the hilus of 1-M zi/zi rats, indicating hilar cell degeneration. After 2 M, mossy fiber sprouting was observed in granular cell layers and in the inner molecular layer. After 10 M, granule cells showed Fos expression. In the hippocampal slices from 12-M zi/zi rats, abnormal population spikes in the DG were observed in the presence of bicuculline and strychnine. Conversely, Sprague-Dawley rats showed no aberrant zinc distribution, few Fos-positive cells, no Fluoro-Jade B-positive cells in the hippocampus and no abnormal population spikes in the DG. These data indicate that morphological changes in the hippocampus might contribute to epileptogenesis in this mutant rat.

Effects of pregabalin on spinal d-serine content and NMDA receptor-mediated synaptic transmission in mice with neuropathic pain.

Pregabalin (PGB) is a chemical derivative of the inhibitory neurotransmitter γ-aminobutyric acid, and is successfully used for the treatment of neuropathic pain. Substantial evidence suggests that d-serine, an endogenous co-agonist at the strychnine-insensitive glycine site of the NMDA receptor, counteracts the antinociceptive actions of PGB at the level of the spinal cord. In the present study, we examined the impact of PGB treatment on spinal d-serine content and NMDA receptor-mediated synaptic transmission in the superficial dorsal horn of peripheral nerve-ligated neuropathic mice. Mechanical allodynia was assessed using von Frey filaments. On post-surgical day 9 (after 5days of treatment with PGB [50mg/kg] or saline vehicle), the lumbar spinal cord was removed, homogenized, and ultrafiltrated. Supernatant samples were treated with Marfey's reagent and analyzed with liquid chromatography-mass spectrometry to measure d-serine content. In the electrophysiological experiments, tight-seal whole-cell recording was performed on neurons in the superficial dorsal horn of spinal cord slices. Partial sciatic nerve ligation increased spinal d-serine content, increased the NMDA/non-NMDA ratio of EPSC amplitudes, and slowed the decay phase of the NMDA component of EPSCs (NMDA-EPSCs). PGB treatment attenuated mechanical allodynia and reduced spinal d-serine content, decreased the NMDA/non-NMDA ratio, and shortened the decay time of NMDA-EPSCs. Furthermore, bath-applied d-serine attenuated the effects of PGB treatment. Although the precise mechanism for the effect of PGB on d-serine metabolism and abundance is unknown, the antinociceptive action of PGB likely involves the reduction of spinal d-serine content and subsequent attenuation of NMDA receptor-mediated synaptic transmission in the superficial dorsal horn.

Differential expression of NMDA receptors in serotonergic and/or GABAergic neurons in the midbrain periaqueductal gray of the mouse.

N-methyl-d-aspartate (NMDA) receptors expressed in the midbrain periaqueductal gray (PAG) exert various physiological functions. The PAG contains various neurotransmitter phenotypes, which include GABAergic neurons and serotonergic neurons. In the present experiments, we made tight-seal whole-cell recordings from GABAergic and/or serotonergic neurons in mouse PAG slices and analyzed NMDA and non-NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation. The NMDA/non-NMDA ratio of EPSC amplitude was high and the decay time course of NMDA-EPSC was slow in non-serotonergic/GABAergic neurons. In contrast, serotonergic neurons exhibited a low NMDA/non-NMDA ratio and a fast decay time course of NMDA-EPSC. Peripheral nerve ligation-induced chronic pain was associated with an increased NMDA/non-NMDA ratio in serotonergic neurons. Additionally, single-cell real-time RT-PCR analysis showed that peripheral nerve ligation up-regulated NR2B subunit expression in non-serotonergic/non-GABAergic neurons. Such changes in NMDA receptor expression in the PAG result in an alteration of the descending modulation of nociception, which might be an underlying mechanism for peripheral nerve injury-evoked persistent pain. Finally, the expression of NMDA receptors seems differentially regulated among neurons of different neurotransmitter phenotypes in the PAG.

Functional roles of endogenous D-serine in pain-induced ultrasonic vocalization.

The N-methyl-D-aspartate receptor (NMDAR) is crucial for pain-related behaviors. D-Serine is synthesized from L-serine by serine racemase (SR) and modulates NMDAR functions by acting as an agonist at the glycine-binding site. We analyzed noxious stimulus-induced ultrasonic vocalization and locomotor activity in the open-field test using SR knockout (SR-KO) mice to examine the role of endogenous D-serine in mammalian behaviors. SR-KO mice emitted less ultrasonic vocalization after noxious stimulation (VAS) than wild-type (WT) mice. The locomotor activity of WT mice decreased with repeated daily exposures to the open field, whereas that of SR-KO mice remained unchanged. VAS was significantly enhanced during arthritis in WT mice, whereas it was not enhanced during arthritis in SR-KO mice. These results indicate that mice lacking the ability to produce D-serine endogenously in the brain differ from normal mice with respect to the chronic pain-induced behavioral changes.

Functional roles of endogenous D-serine in the chronic pain-induced plasticity of NMDAR-mediated synaptic transmission in the central amygdala of mice.

The amygdala is implicated in chronic pain-induced emotional changes. Chronic pain induces plastic changes of the N-methyl-d-aspartate receptor (NMDAR) functions in the brain including the amygdala. d-Serine is synthesized endogenously by serine racemase and modulates NMDAR-mediated synaptic transmission as a coagonist of glycine binding site. To clarify the functional roles of endogenous d-serine in chronic pain-induced plasticity of NMDAR mediated synaptic transmission, we investigated the NMDAR-mediated excitatory synaptic current (EPSC) of neurons in the latero-capsular division of the central amygdala (CeLC) using brain slices from serine racemase knockout (SR-KO) mice with chronic pain induced by monoarthritis. The decay time of NMDAR-mediated EPSC was significantly elongated by monoarthritis in wild type (WT) mice, but not in SR-KO mice. The d-serine application-induced increase of NMDAR-mediated EPSC was significantly facilitated by monoarthritis in WT mice, but not in SR-KO mice. These results suggest that endogenous d-serine facilitates chronic pain-induced plastic changes of NMDAR mediated synaptic transmission in CeLC.

Facilitatory effects of subanesthetic sevoflurane on excitatory synaptic transmission and synaptic plasticity in the mouse hippocampal CA1 area.

Behavioral investigations have shown that general anesthetics at low concentration have enhancing effects on learning and memory in some animal models. In the present experiments, in order to elucidate the cellular mechanisms underlying such memory enhancement, the effects of anesthetics at low doses on synaptic plasticity in the hippocampus were investigated. Tight-seal whole-cell recordings were made from CA1 pyramidal cells in hippocampal slices prepared from adult male mice, and the effects of subanesthetic concentrations of the volatile anesthetic sevoflurane on the glutamatergic excitatory postsynaptic currents (EPSCs) were investigated. In addition, extracellular recordings of field excitatory postsynaptic potential (fEPSP) and population spike (PS) were made, and the effects of subanesthetic sevoflurane on long-term potentiation (LTP) of the fEPSP slope and on LTP of PS amplitude were analyzed. Sevoflurane at anesthetic concentration inhibited the amplitude of EPSCs with an increase in the paired-pulse facilitation (PPF) ratio. In contrast, subanesthetic sevoflurane increased the amplitude of EPSCs without any appreciable changes in the PPF ratio. Subanesthetic sevoflurane also showed facilitatory influences on LTP of PS amplitude but not on LTP of the fEPSP slope. These observations suggest that sevoflurane at anesthetic concentration presynaptically inhibits excitatory synaptic transmission and at subanesthetic concentration postsynaptically enhances excitatory synaptic transmission in the hippocampal CA1 region. Further, subanesthetic sevoflurane seems to exert facilitatory effects on the EPSP-to-spike coupling process in the postsynaptic neurons. These results might provide clues as to the cellular mechanism of light level of sevoflurane anesthesia.

D-amino-acid oxidase is involved in D-serine-induced nephrotoxicity.

D-serine is nephrotoxic in rats. Based on circumstantial evidence, it has been suspected that D-amino-acid oxidase is involved in this nephrotoxicity. Since we found that LEA/SENDAI rats lacked D-amino-acid oxidase, we examined whether this enzyme was associated with D-serine-induced nephrotoxicity using the LEA/SENDAI rats and control F344 rats. When d-propargylglycine, which is known to have a nephrotoxic effect through its metabolism by D-amino-acid oxidase, was injected intraperitoneally into the F344 rats, it caused glucosuria and polyuria. However, injection of d-propargylglycine into LEA/SENDAI rats did not cause any glucosuria or polyuria, indicating that D-amino-acid oxidase is definitely not functional in these rats. D-serine was then injected into the F344 and LEA/SENDAI rats. It caused glucosuria and polyuria in the F344 rats but not in the LEA/SENDAI rats. These results indicate clearly that D-amino-acid oxidase is responsible for the D-serine-induced nephrotoxicity.

Differential expression of NMDA receptor subunits between neurons containing and not containing enkephalin in the mouse embryo spinal cord.

We transfected cultures of mouse spinal cord slices with the enhanced green fluorescent protein (GFP) gene driven by the promoter for preproenkephalin, using the particle-mediated gene transfer system adapted for small neurons in the superficial dorsal horn, and observations were made after 4-6 days in vitro. A considerable number of cells in the superficial dorsal horn were observed to express GFP fluorescence, reminiscent of the previously reported distribution of enkephalinergic neurons in the spinal cord. The number of GFP-expressing neurons increased in response to forskolin application. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of single neurons revealed that the N-methyl-d-aspartate (NMDA) receptor NR2B subunit is expressed more frequently in enkephalinergic neurons, and the NR2A subunit more frequently in non-enkephalinergic neurons. These observations suggest that expression of NMDA receptor subunits is controlled differentially in distinct populations of neurochemically identified neurons in the spinal cord. Biolistic particle-mediated gene transfection seems useful for identifying neuronal phenotypes in organotypic cultures of the spinal cord.

Spatial learning and long-term potentiation of mutant mice lacking D-amino-acid oxidase.

We evaluated the role of D-amino-acid oxidase on spatial learning and long-term potentiation (LTP) in the hippocampus, since this enzyme metabolizes D-amino-acids, some of which enhance the N-methyl-D-aspartate receptor functions. The Morris water maze learning and the LTP in the CA1 area of the hippocampal slice were observed in wild-type mice and mutant mice lacking D-amino-acid oxidase. The mutant mice showed significantly shorter platform search times in the water maze and significantly larger hippocampal LTPs than the wild-type mice. These results suggest that the abundant D-amino-acids in the mutant mouse brain facilitate hippocampal LTP and spatial learning.