Neuropeptide Y and the Calcitonin Gene-related Peptide Attenuate Learning Impairments Induced by MK-801 via a Sigma Receptor-related mechanism

It has been shown recently that low doses of sigma (σ) receptor ligands like 1,3-di-(2-tolyl)guanidine (DTG), (+) N -allylnormetazocine [(+)SKF 10 047] and (+)pentazocine can antagonize learning impairments induced by dizocilpine (MK-801), a non-competitive antagonist at the NMDA receptor channel. This antagonism has been proposed to involve σ receptor sites since it is blocked by the administration of purported o antagonists such as NE-100 and BMY-14802. It has also been demonstrated that peptides of the neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP) families modulate, in vivo , é labelling and electrophysiological effects in the hippocampal formation. Accordingly, we investigated if NPY- and CGRP-related peptides modulate cognitive processes by interacting with σ sites in mice. In order to test this hypothesis, a step-down passive avoidance task was used. Interestingly, similarly to various σ agonists, NPY, peptide YY (PYY) and the Y₁ agonist [Leu³¹Pro³⁴]NPY (but not NPY13–36, a purported Y₂ agonist), as well as hCGRPα and the purported CGRP₂ agonist [CyS(ACM)^2–7]hCGRPα (but not CGRP_8–37, a CGRP₁ receptor antagonist), significantly attenuated learning impairments induced by MK-801. Furthermore, the effects of NPY, [Leu³¹ Pro³⁴]NPY, hCGRPα and [CYS(ACM)^2–7]hCGRPα were blocked by the administration of the σ antagonist, BMY-14802. The present data suggest that NPY- and CGRP-related peptides can indirectly interact in vivo with σ receptors to modulate cognitive processes associated with NMDA receptor function.     

Transferrin receptors in the normal human hippocampus and in Alzheimer's disease

Several lines of evidence suggest that aluminium may play a role in the pathogenesis of Alzheimer's disease (AD). The iron transport protein transferrin is the major transport protein for aluminium, and aluminium gains access to cells by means of a specific cell surface transferrin receptor. We have assessed the distribution of transferrin receptors in the normal and AD hippocampal formation using [³H]–transferrin ([³H]–Tf) binding and tritium film autoradiography, in order to assess the role of the transferrin receptor in AD. In normal brain, ³H]–Tf binding was highest in the pyramidal cell layers with CA2> dentate gyrus granule cell layer ≥Cal >CA3 ≥ CA4>subiculum>parahippocampal gyrus. In AD, significant reductions in [³H]–Tf binding were found in CA1, CA2 and CA4 pyramidal cell layers. The reduced [³H]–Tf binding in AD may, however, be due to poor pre–mortem agonal states which correlated with reduced [³H]–Tf binding. The discrepancy between the distribution of transferrin receptors in the hippocampus and those areas which are prone to the formation of senile plaques and neurofibrillary tangles suggests that if transferrin–mediated uptake of aluminium in AD/SDAT is significant in the pathogenesis of this disorder, it is not the only determinant of Alzheimer–type neuropathology.     

HIV-1 Envelope Protein gp120 Potentiates NMDA-evoked Noradrenaline Release by a Direct Action at Rat Hippocampal and Cortical Noradrenergic Nerve Endings

Exposure of rat or human neocortical or hippocampal tissue to glutamate receptor agonists elicits a Ca²⁺-dependent, exocytotic-like release of previously accumulated [³H]noradrenaline through activation of both N -methyl- d -aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors colocalized on the noradrenergic axon terminals. Here we show that the NMDA (100 μM)-evoked release of [³H]noradrenaline from superfused thin layers of isolated rat hippocampal or cortical nerve endings was potentiated when the human immunodeficiency virus type 1 coat protein gp120 was added to the superfusion medium concomitantly with NMDA. The effect of gp120 (10 pM to 3 nM) on the 100 μM NMDA-evoked release of [³H]noradrenaline was concentration-dependent; the maximal effect (-140% potentiation) was reached at 100 pM of gp120. The protein was inactive on its own. The [³H]noradrenaline release evoked by NMDA (100 μM) + gp120 (100 μM) was prevented by classical NMDA receptor antagonists, as well as by 10 μM memantine. Neither the release evoked by NMDA nor that elicited by NMDA + gp120 was sensitive to the nitric oxide synthase inhibitor N ^G -nitro- l -arginine, suggesting no involvement of nitric oxide. The [³H]noradrenaline release elicited by 100 nM AMPA was unaffected by gp120. The protein potentiated the release evoked by 100 nM glutamate; the effect of 100 pM gp120 was quantitatively identical to that of 1 μM glycine, with no apparent additivity between gp120 and glycine. The antagonism by 1 μM 7-chloro-kynurenic acid of the NMDA-induced [³H]noradrenaline release was reversed by glycine or gp120. The data are compatible with gp120 acting directly as a powerful positive allosteric modulator at a neuronal NMDA receptor.     

A Comparative Study of α2- and β-Adrenoceptor Distribution in Pigeon and Chick Brain

The pharmacological properties and anatomical distribution of α₂, β₁ and β₂-adrenoceptors in pigeon and chick brains were studied by both homogenate binding and tissue section autoradiography. [³H]Bromoxidine (α₂-adrenoceptor-), [³H]CGP 12177 (β-adrenoceptor) and [¹²⁵1]cyanopindolol (β-adrenoceptor) were used as radioligands. In both species, [³H]bromoxidine binding to avian brain tissue showed a pharmacological profile similar to that previously reported for α₂-adrenoceptors in mammals. Regarding the anatomical distribution, the areas with the highest densities of α₂-adrenoceptors in the pigeon brain included the hyperstriatum, nuclei septalis, tectum opticum and some brainstem nuclei. Most β-adrenoceptors found in tissue membranes and sections from chick and pigeon brain were of the β₂ subtype, in contrast to what has been reported in the mammalian brain, where the β₁ subtype is predominant. A striking difference was found between the two species regarding the densities of these receptors: while pigeon brain was extremely rich in [¹²⁵1]cyanopindolol binding throughout the brain (mainly cerebellum) in the pigeon, the levels of labelling in the chick brain were much lower; the exception was the cerebellum, which displayed a higher density than other parts of the brain in both species. Overall, our results support the proposed anatomical equivalences between a number of structures in the avian and mammalian encephalon.     

Distribution of Nicotinic Receptors in the Human Hippocampus and Thalamus

Neuronal nicotinic acetylcholine receptors consist of different subunits, α and β, with different subtype arrangement corresponding to distinct pharmacological and functional properties. The expression of α3, α7 and β2 mRNA in the human brain was studied by in situ hybridization and compared to [³H]nicotine, [³H]cytisine and [¹²⁵l]α-bungarotoxin binding in contiguous sections. The β2 probe showed a strong hybridization signal in the granular layer of the dentate gyrus and in the CA2/CA3 region of the hippocampus and in the insular cortex, and a signal of lower intensity in the subicular complex and entorhinal cortex. The α3 probe showed strong hybridization in the dorsomedial, lateral posterior, ventroposteromedial and reticular nuclei of the thalamus, and a weak signal in the hippocampal region and in the entorhinal, insular and cingular cortex. The amount of α7 mRNA was high at the level of the dentate granular layer and the CA2/CA3 region of the hippocampus, in the caudate nucleus and in the pulvinar and ventroposterolateral nuclei of the thalamus. [³H]Nicotine and [³H]cytisine binding appeared to be identical in anatomical distribution and relative intensity. It was high in the thalamic nuclei, the putamen and in the hippocampal formation in the subicular complex and the stratum lacunosum moleculare. The level of [¹²⁵l]α-bungarotoxin binding was particularly high in the hippocampus and in the pyramidal cells of the CA1 region, but was relatively low in the subicular complex. Our data indicate that in the human brain nicotinic receptor subtypes have discrete distributions, which are in part different from those of other species.     

Characterization of Excitatory Amino Acid Neurotoxicity in N-methyl-D-aspartate Receptor-deficient Mouse Cortical Neuronal Cells

Roles and mechanisms of N -methyl-D-aspartate (NMDA) receptors in glutamate neurotoxicity were investigated in cultures of NMDA receptor-deficient cortical neuronal cells. Mutant mice lacking a functional NMDA receptor were generated by gene targeting of the NR1 NMDA receptor subunit. Cortical neuronal cells prepared from wild-type NR1 ^+/+, heterozygous NR1 ^+/- and homozygous mutant NR1^-/- mice at 15–17 days of gestation grew indistinguishably from each other. Brief exposures (5 min) of both NR1 ^+/+ and NR1 ^+/- neuronal cells to glutamate or NMDA, but not kainate or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), resulted in widespread neuronal degeneration by the following day. In contrast, neither glutamate nor NMDA treatment caused neuronal degeneration in NR1 ^-/- cells, indicating that NMDA receptors are responsible for rapidly triggered glutamate neurotoxicity. The above four compounds were all effectivein inducing the death of NR1 ^+/+ and NR1 ^+/- neuronal cells after prolonged exposure (20–24 h). However, NMDA had no neurotoxic effects on NR1 -/- cells, although the other three compounds were neurotoxic with potencies comparable to those for NR1 ^+/+ and NR1 ^+/- cells. The AMPA and kainate receptors are thus sufficient for inducing slowly triggered glutamate neurotoxicity. Brief exposure of a mixed population of NR1 ^+/+ and NR1 ^-/- neuronal cells to NMDA selectively killed the NMDA receptor-expressing cells without any appreciable effects on neighbouring NMDA receptor-deficient cells. This finding further supports a direct and indispensable role for NMDA receptors in NMDA-evoked neuronal cell death.     

HIV-1 Envelope Proteins gp120 and gp160 Potentiate NMDA-induced [Ca2+]i Increase, Alter [Ca2+]i Homeostasis and Induce Neurotoxicity in Human Embryonic Neurons

The envelope glycoprotein gp120 of the human immunodeficiency virus HIV-1 has been proposed to cause neuron death in developing murine hippocampal cultures and rat retinal ganglion cells. In the present study, cultured human embryonic cerebral and spinal neurons from 8- to 10-week-old embryos were used to study the neurotoxic effect of gp120 and gp160. Electrophysiological properties as well as N -methyl- d -aspartate (NMDA)-induced currents were recorded from neurons maintained in culture for 10–30 days. Neither voltage-activated sodium or calcium currents nor NMDA-induced currents were affected by exposure of neurons to 250 pM gp120 or gp160. In contrast, when neurons were subjected to photometric measurements using the calcium dye indo-1 to monitor the intracellular free Ca²⁺ concentration ([Ca²⁺]_i), gp120 and gp160 (20–250 pM) potentiated the large rises in [Ca²⁺]_i induced by 50 μM NMDA. The potentiation of NMDA-induced Ca²⁺ responses required the presence of Ca²⁺ in the medium, and was abolished by the NMDA antagonist d -2-amino-5-phosphonovalerate (AP5) and the voltage-gated Ca²⁺ channel inhibitor nifedipine. Moreover, exposure of a subpopulation of spinal neurons (25% of the cells tested) to 20–250 pM gp120 or gp160 resulted in an increase in [Ca²⁺]_i that followed three patterns: fluctuations not affected by AP5, a single peak, and the progressive and irreversible rise of [Ca²⁺]_i. The neurotoxicity of picomolar doses of gp120 and gp160 cultures was estimated by immuno-fluorescence and colorimetric assay. Treatment of cultures with AP5 or nifedipine reduced gp120-induced toxicity by 70 and 100% respectively.     

A substantial number of Rasmussen syndrome patients have increased IgG, CD4+ T cells, TNFα, and Granzyme B in CSF

Purpose:   We studied the immunologic molecules in cerebrospinal fluid (CSF) and discussed their evolutional changes in pediatric patients with Rasmussen syndrome (RS).
Methods:   CSF samples collected from 27 patients with RS (average onset age, 7.5 ± 5.6 years) were studied. Cell count, protein, glucose, albumin, chloride, and immunoglobulin G (IgG) levels were measured by conventional methods. Surface markers of lymphocytes in CSF were examined by a cell sorter. Granzyme B, interferon γ (IFNγ), interleukin 4 (IL-4), tumor necrosis factor α (TNFα), and IL-12 in CSF were quantitated by enzyme-linked immunosorbent assay (ELISA). Autoantibodies against GluR ε2 (NR2B) were examined by immunoblot.
Results:   The data of the first CSF examination showed that IgG levels (Mann-Whitney U test, p < 0.01), CD4⁺ T cells (p = 0.02), TNFα levels (p < 0.01), and Granzyme B levels (p < 0.01) were elevated compared with disease controls. White blood cell count, IFNγ level, IL-12 level, and Granzyme B level were elevated, especially in the early stage of disease. CD4⁺ T cells, CD8⁺ cells, CD3⁺ T cells, IgG levels, and TNFα levels were elevated at all stages of disease evolution. Protein levels and albumin levels were elevated in the progressed stage. Autoantibodies against GluR ε2 (NR2B) (IgG) were found in 50% of patients in the early stage, and the positive rate was low at the progressed stage.
Discussion:   The present findings suggest that complex pathophysiologic mechanisms involving CD4⁺ T cells and CD8⁺ T cells change evolutionally during the progression of RS. A crucial cytotoxic process occurs in the early stage, and declines in the progressed stage.     

GX/Z Is Regulated by μ But Not δ Opioid Receptors in the Stimulation of the Low Km GTPase Activity in Mouse Periaqueductal Grey Matter

High affinity low K_m GTPase activity was measured in membrane preparations of adult mouse mesencephalic periaqueductal grey matter (PAG). Opioids displaying selectivity towards μ- or δ-opioid receptors (OR) activated the enzyme in a concentration-dependent manner. Antibodies to μ-OR greatly impaired the potential of μ-agonists, [D-Ala²,N-MePhe⁴,Gly-ol⁵]-enkephalin (DAMGO) and morphine, to increase hydrolysis of GTP. The same antibodies had little effect on [D-Pen^2,5]enkephalin (DPDPE) and [D-Ala²]deltorphin II, both agonists at δ-OR. Stimulation of GTPase by DPDPE and [D-Ala²]deltorphin II - but not by morphine or DAMGO - was diminished by antibodies to δ-OR. The blockade of G_i2α subunits by specific antibodies impaired the activation of Gα-related GTPase by all opioids. Antibodies in vitro, and oligodeoxynucleotides in vivo, prepared against Gx/zα subunits, reduced the release of Pi promoted by DAMGO and morphine. The impairment of Gx/z proteins also slightly reduced the effect of the δ2 agonist [D-Ala²]deltorphin II. At δ1 receptors, DPDPE fully expressed its activation of GTPase. These results indicate that in the PAG, μ-OR and δ-OR couple with Gi2 transducer proteins. Notably, μ-OR also regulates the pertussis toxin-insensitive G-protein Gx/z, an effect poorly exhibited by δ-OR in this tissue.     

Neurosteroids: Expression of Functional 3β-Hydroxysteroid Dehydrogenase by Rat Sensory Neurons and Schwann Cells

Steroids which are synthesized within the nervous system, such as progesterone, have been termed 'neurosteroids'. Levels of progesterone are much larger in peripheral nerves of rats and mice than in plasma, and persist after removal of the steroidogenic endocrine glands. Schwann cells are a source of progesterone: when isolated from embryonic dorsal root ganglia, they can synthesize progesterone from pregnenolone, the obligate precursor of all steroids. Locally produced progesterone has been shown to play an important role in myelination of peripheral nerve. We show here that sensory neurons from embryonic dorsal root ganglia also express 3β-hydroxysteroid dehydrogenase and can convert [³H]pregnenolone to [³H]progesterone. Moreover, when cultured under different conditions and incubated for 24 h in the presence of 100 nM [³H]pregnenolone, they produce 5–10 times more [³H]progesterone than Schwann cells. The conversion of pregnenolone to progesterone by neurons is further increased by a diffusible factor produced by Schwann cells. Sensory neurons can also metabolize progesterone to 5α-dihydroprogesterone, but unlike Schwann cells, they do not produce 3α,5α-tetrahydroprogesterone, a potent positive allosteric modulator of γ-aminobutyric acid type A receptors. We also show that cells isolated from the adult nervous system still have the capacity to convert [³H]pregnenolone to progesterone and its 5α-reduced metabolites: neurons and Schwann cells purified from dorsal root ganglia of 6 week old male rats show a similar pattern of pregnenolone metabolism to cells isolated from 18 day old embryos. These findings further support the important role of progesterone in the development and regeneration of the peripheral nervous system.     

β-Carbolines: Endogenous Ligands for the Benzodiazepine Receptor

Abstract: The interaction of benzodiazepines (BDZ) and β -carbolines with metal cations was investigated. Δmong the numerous transition metal cations, only four, Co²⁺, Ni², Cu²⁺ and Zn²⁺, specifically affected the receptor binding of [³H]diazepam and [³H] β -carboline-3-carboxylic acid ethyl ester ( β -CCE). The effects of these cations on [³H]diazepam binding were exactly opposite to those on [³H] β -CCE binding. An intraperitoneal injection of β -carboline-3-carboxylic acid methyl ester ( β -CCM) produced spike discharges and β-CCM induced spike discharges were prevented by an intramuscular injection of BDZ. These findings suggest that β -carbolines could be related to the proposed endogenous ligand and that BDZ might be antagonistic rather than agonistic of the naturally occurring ligand.     

Measurement of serum 7α-hydroxy-4-cholesten-3-one (or 7αC4), a surrogate test for bile acid malabsorption in health, ileal disease and irritable bowel syndrome using liquid chromatography-tandem mass spectrometry

Abstract  Bile acid malabsorption (BAM) is reported in up to 50% of patients with functional diarrhoea and irritable bowel syndrome with diarrhoea (IBS-D). Serum 7α-hydroxy-4-cholesten-3-one (7αHCO or 7αC4), an indirect measurement of hepatic bile acid synthesis, has been validated as a measurement of BAM relative to the ⁷⁵SeHCAT retention test. Our aim was to develop a serum 7αC4 assay, normal values, and compare results from healthy controls, patients with ileal Crohn's disease or resection, and patients with IBS-D or IBS with constipation (IBS-C). Stored serum samples were used from adult men and women in the following groups: 111 normal healthy controls, 15 IBS-D, 15 IBS-C, 24 with distal ileal Crohn's disease and 20 with distal ileal resection for Crohn's disease. We adapted a published high pressure liquid chromatography, tandem mass spectrometry (HPLC-MS/MS) assay. The HPLC-MS/MS assay showed good linearity in concentration range 0–200 ng mL⁻¹, sensitivity (lowest limit of detection 0.04 ng mL⁻¹), and high analytical recovery (average 99%, range 93–107%). The 5th to 95th percentile for 111 normal healthy controls was 6–60.7 ng mL⁻¹. There were significant overall group differences ( anova on ranks , P  < 0.001), with significantly higher values for terminal ileal disease or resection. There were significant differences between health and IBS ( anova , P  = 0.043) with higher mean values in IBS-D relative to controls (rank sum test, P  = 0.027). We have established a sensitive non-isotopic assay based on HPLC-MS/MS, determined normal 7αC4 values, and identified increased 7αC4 in IBS-D and in distal ileal resection and disease. This assay has potential as a non-invasive test for BAM in IBS.     

Functional Properties of Neuronal Nicotinic Acetylcholine Receptor Channels Expressed in Transfected Human Cells

To study how subunit composition affects the functional properties of neuronal nicotinic acetylcholine receptors (nAChRs), we examined the behaviour of acetylcholine (ACh)-induced single-channel currents in human BOSC 23 cells transiently transfected with various subunit cDNA combinations. For all nAChRs examined (chick and rat α₃β₄, chick α_<3/β₂, α₄β₂, α₄β₄, α₇and α₈, expression levels were high enough to allow measurements of acetylcholine-evoked whole-cell currents and nicotine-elicited Ca²⁺ transients as well as the functional characterization of nAChR channels. Unitary acetylcholine-evoked events of α₈ nAChR had a slope conductance of 23 pS, whereas two conductance classes (19–23 and 32–45 pS) were identified for all other nAChR channels. The mean channel open times were significantly longer for homomeric α₇ and α₈ nAChRs (6–7 ms) than for heteromeric nAChRs (1–3 ms), with the exception of α₃α₄nAChRs (8.4 ms for rat, 7 ms for chick). At least two species of heterologously expressed nAChRs (α₃α₄and α₃α₂) exhibited single-channel characteristics similar to those reported for native receptors. The variety of nAChRs channel conductance and kinetic properties encountered in human cells transfected with nAChR subunits contributes to the functional diversity of nAChRs in nerve cells.     

The Effect of Barium on [3H]Noradrenalin Release from the Human Neuroblastoma SH-SY5Y

Replacement of Ca²⁺ with Ba²⁺ in HEPES-buffered saline stimulated [³H]noradrenalin release in the human neuroblastoma clone SH-SY5Y by up to 20% of the cell content in the absence of other secretory stimuli. The Ba²⁺-evoked release was inhibited by 85% by 3 μM tetrodotoxin and 95% by 5 μM nifedipine. Ba²⁺ also increased the potency of K⁺-evoked release of [³H]noradrenalin, as maximal release was observed with 60 mM K⁺ compared with the 100 mM K⁺ necessary to achieve maximal release in the presence of Ca²⁺. In contrast, replacing Ca²⁺ with Ba²⁺ had little effect on carbachol- and bradykinin-evoked release of [³H]noradrenalin. No evidence was obtained from studies on changes in [Ca²⁺]_i (in response to 100 pM carbachol) using fura-2 that Ba²⁺ could enter intracellular stores in SH-SY5Y cells. Whole-cell patch-clamp studies showed that Ba²⁺ depolarizes SH-SY5Y cells as well as enhancing inward Ca²⁺ channel currents and shifting their voltage dependence to more negative values. These results are discussed in terms of the hypothesis that Ba²⁺ blocks K⁺ channels, leading to depolarization followed by opening of voltage-sensitive Na⁺ channels. This in turn opens voltage-sensitive L-type Ca²⁺ channels, which are coupled to the release of [³H]noradrenalin in SH-SY5Y cells.     

Effects of P-Aminophenyl Dichloroarsine on Reduced High-affinity [3H]Nicotine Binding Sites from Chick Brain: A Covalent,Yet Reversible,Agent for Neuronal Nicotinic Receptors

Neuronal nicotinic acetylcholine receptor (nAChR) α-subunits contain a conserved disulphide that is essential for function. Here, we have examined the effects of sulphydryl redox reagents on [³H]nicotine binding to chick brain nAChR immunoisolated with the monoclonal antibody mAb35. The disulphide reducing agent, dithiothreitol (DTT), inhibited [³H]nicotine binding [50% inhibitory concentration (IC₅₀) = 146 μM] but this effect was reversed (93±1.5%) by subsequent reoxidation with 1 mM dithio-bis(nitrobenzoic acid) (DTNB). The trivalent arsenical, p -aminophenyl dichloroarsine (APA), which reacts with pairs of spatially close sulphydryls, was a potent inhibitor of reoxidation by DTNB (IC₅₀= 35 nM). However, application of the 'anti-arsenical', 2,3-dimercaptopropane sulphonic acid (DMPS), restored agonist binding after APA treatment (50% effective concentration = 120 μM). Paradoxically, DMPS was also found to be a potent oxidizing agent of these receptors. Affinity alkylation of reduced nAChRs with bromoacetylcholine (BAC; 100 μM) irreversibly blocked nicotine binding (>90%). We propose (but have not proven) that APA interacts with the cysteines homologous to Cys192–193 in Torpedo AChRs, since APA pretreatment of reduced neuronal receptors protected against irreversible BAC alkylation, as shown by subsequent reversal of DMPS (2 mM; 20 min). This study illustrates the potent and reversible nature of the arsenical's covalent interaction with an isolated nAChR and suggests that modified arsenicals could be useful nAChR probes.     

Extracellular Magnesium and Anticonvulsant Effects of Valproate in Young Rat Hippocampus

Summary Extracellular field potential recordings were made in CA3 subfield of hippocampal slices from rats aged 11–22 days. In these experiments, we analyzed the effects induced by modifying [Mg²⁺] in the medium (1 or 2 mM) on (a) 4-aminopyridine (4-AP, 50 μM)-induced synchronous events (including ictal- and interictal-like epileptiform discharges as well as γ-aminobutyric acid (GABA)-mediated potentials), and (b) the changes induced by the antiepileptic drug (AED) valproate (VPA 2 mM) on such activities. Changing [Mg²⁺] from 1 to 2 mM induced age-dependent effects consisting of reduction in rate of occurrence of ictal-like discharges at 11–13 days (55% reduction, p <0.005) and 14–16 days (46% reduction, p <0.025) postpartum. At any age, the rate of occurrence and the amplitude of the GABA-mediated synchronous potentials tended to decrease in 1 mM [Mg²⁺] Similar effects were noted when changes in [Mg²⁺] were made during continuous application of the competitive antagonist of the N -methyl-D-aspartate (NMDA) receptor DL-2-amino-5-phosphonovalerate (APV 50 μM). As previously reported, interictal and ictal discharges were blocked by addition of VPA to medium containing 2 mM [Mg²⁺] Such an effect was not observed when [Mg²⁺] was decreased to 1 m M. In 1 m M , but not in 2 m M [Mg²⁺], VPA increased the amplitude of GABA-mediated synchronous potentials. Our results indicate that [Mg²⁺] plays a role in modulating occurrence of 4-AP-induced ictal activity and that it can influence the effects of VPA in this in vitro model of epileptogenesis. Because these findings are also observed in the presence of the NMDA receptor antagonist APV, we conclude that they are nof mediated by a mechanism linked to the NMDA receptor-ionophore.     

Modulation by substance P of synaptic transmission in the mouse hippocampal slice

The modulatory action of substance P on synaptic transmission of CA1 neurons was studied using intra‐ or extracellular recording from the mouse hippocampal slice preparation. Bath‐applied substance P (2–4 μ m ) or the selective NK₁ receptor agonist substance P methylester (SPME, 10 n m –5 μ m ) depressed field potentials (recorded from stratum pyramidale) evoked by focal stimulation of Schaffer collaterals. This effect was apparently mediated via NK₁ receptors since it was completely blocked by the selective NK₁ antagonist SR 140333. The field potential depression by SPME was significantly reduced in the presence of bicuculline. Intracellular recording from CA1 pyramidal neurons showed that evoked excitatory postsynaptic potentials (EPSPs) and evoked inhibitory postsynaptic potentials (IPSPs) were similarly depressed by SPME, which at the same time increased the frequency of spontaneous GABAergic events and reduced that of spontaneous glutamatergic events. The effects of SPME on spontaneous and evoked IPSPs were prevented by the ionotropic glutamate receptor blocker kynurenic acid. In tetrodotoxin (TTX) solution, no change in either the frequency of spontaneous GABAergic and glutamatergic events or in the amplitude of responses of pyramidal neurons to 4 μ m α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) or 10 μ m N ‐methyl‐ d ‐aspartate (NMDA) was observed. On the same cells, SPME produced minimal changes in passive membrane properties unable to account for the main effects on synaptic transmission. The present data indicate that SPME exerted its action on CA1 pyramidal neurons via a complex network mechanism, which is hypothesized to involve facilitation of a subset of GABAergic neurons with widely distributed connections to excitatory and inhibitory cells in the CA1 area.     

Release of Acetylcholine and Noradrenaline from the Cholinergic and Adrenergic Afferents in Rat Hippocampal CA1, CA3 and Dentate Gyrus Regions

An attempt was made to study the release of acetylcholine (ACh) and noradrenaline and their presynaptic modulation in isolated slice preparations dissected from different subfields of the hippocampus: CA1, CA3 and the dentate gyrus. The slices were perfused and loaded with [³H]choline or with [³H]noradrenaline. The release in response to field stimulation was determined radiochemically and the content of transmitters was assayed by a chemiluminescent method or by HPLC combined with electrochemical detection. After 30 min of loading with [³H]choline there were marked subregional differences in the specific activity of [³H]ACh content. The highest concentration was measured in the dentate gyrus and the lowest in CA3. Evidence was obtained that in all three subfields the cholinergic axon terminals are equipped with inhibitory muscarinic autoreceptors and the noradrenergic terminals with α₂-autoreceptors, as indicated by an increase in transmitter release when the tissue was exposed to selective muscarinic or α₂-adrenoceptor antagonists. In contrast, the cholinergic boutons are not equipped with α₂-adrenoceptors, and noradrenergic terminals do not possess inhibitory muscarinic receptors. It is therefore concluded that while the release of both ACh and noradrenaline is controlled by negative feedback modulation, there is no possibility of establishing a presynaptic inhibitory interaction between the two.     

NAD(P)H oxidase p22phox C242T polymorphism and ischemic stroke in Japan: the Fukuoka Stroke Registry and the Hisayama study

The C242T polymorphism of p22 ^phox , a component of NAD(P)H oxidase, may have an impact on cardiovascular diseases; however, the association between this polymorphism and brain infarction is not fully understood. Here, we investigate the relationship between the C242T polymorphism and brain infarction in Japan. We recruited 1055 patients with brain infarction and 1055 control subjects. A chi-squared test revealed that the T-allele frequency was lower in patients with cardioembolic infarction (5.6%) than in control subjects (11.0%, P  < 0.001); however, allele frequencies in patients with lacunar and atherothrombotic infarction (11.2%) were not significantly different from those in control subjects (11.0%). A multivariate-adjusted conditional logistic regression analysis also revealed no association between CT + TT genotype, and lacunar and atherothrombotic infarction (odds ratio = 0.97, 95% confidence interval: 0.72–1.32). To investigate the functional effects of the C242T polymorphism, we examined superoxide production in COS-7 cells cotransfected with Nox4 and p22 ^phox of each genotype. The superoxide-producing activity in those cells expressing p22 ^phox with the T allele was not significantly different from that in cells expressing p22 ^phox with the C allele. The present results suggest that the p22 ^phox C242T polymorphism may have a protective effect against cardioembolic infarction, but is not related to lacunar and atherothrombotic infarction in Japan.     

Induction and expression of GluA1 (GluR-A)-independent LTP in the hippocampus

Long-term potentiation (LTP) at hippocampal CA3–CA1 synapses is thought to be mediated, at least in part, by an increase in the postsynaptic surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptors induced by N -methyl- d -aspartate (NMDA) receptor activation. While this process was originally attributed to the regulated synaptic insertion of GluA1 (GluR-A) subunit-containing AMPA receptors, recent evidence suggests that regulated synaptic trafficking of GluA2 subunits might also contribute to one or several phases of potentiation. However, it has so far been difficult to separate these two mechanisms experimentally. Here we used genetically modified mice lacking the GluA1 subunit ( Gria1 ^−/− mice) to investigate GluA1-independent mechanisms of LTP at CA3–CA1 synapses in transverse hippocampal slices. An extracellular, paired theta-burst stimulation paradigm induced a robust GluA1-independent form of LTP lacking the early, rapidly decaying component characteristic of LTP in wild-type mice. This GluA1-independent form of LTP was attenuated by inhibitors of neuronal nitric oxide synthase and protein kinase C (PKC), two enzymes known to regulate GluA2 surface expression. Furthermore, the induction of GluA1-independent potentiation required the activation of GluN2B (NR2B) subunit-containing NMDA receptors. Our findings support and extend the evidence that LTP at hippocampal CA3–CA1 synapses comprises a rapidly decaying, GluA1-dependent component and a more sustained, GluA1-independent component, induced and expressed via a separate mechanism involving GluN2B-containing NMDA receptors, neuronal nitric oxide synthase and PKC.