Knockdown of AMPA receptor GluR2 expression causes delayed neurodegeneration and increases damage by sublethal ischemia in hippocampal CA1 and CA3 neurons.

Considerable evidence suggests that Ca(2+)-permeable AMPA receptors are critical mediators of the delayed, selective neuronal death associated with transient global ischemia and sustained seizures. Global ischemia suppresses mRNA and protein expression of the glutamate receptor subunit GluR2 and increases AMPA receptor-mediated Ca(2+) influx into vulnerable neurons of the hippocampal CA1 before the onset of neurodegeneration. Status epilepticus suppresses GluR2 mRNA and protein in CA3 before neurodegeneration in this region. To examine whether acute downregulation of the GluR2 subunit, even in the absence of a neurological insult, can cause neuronal cell death, we performed GluR2 "knockdown" experiments. Intracerebral injection of antisense oligodeoxynucleotides targeted to GluR2 mRNA induced delayed death of pyramidal neurons in CA1 and CA3. Antisense-induced neurodegeneration was preceded by a reduction in GluR2 mRNA, as indicated by in situ hybridization, and in GluR2 protein, as indicated by Western blot analysis. GluR2 antisense suppressed GluR2 mRNA in the dentate gyrus but did not cause cell death. The AMPA receptor antagonist 6-cyano-7-nitroquinoxiline-2,3-dione (CNQX) and the Ca(2+)-permeable AMPA receptor channel blocker 1-naphthyl acetyl spermine protected against antisense-induced cell death. This result indicates that antisense-induced cell death is mediated by Ca(2+)-permeable AMPA receptors. GluR2 antisense and brief sublethal global ischemia acted synergistically to cause degeneration of pyramidal neurons, consistent with action by a common mechanism. These findings demonstrate that downregulation of GluR2 is sufficient to induce delayed death of specific neuronal populations.

Global ischemia-induced increases in the gap junctional proteins connexin 32 (Cx32) and Cx36 in hippocampus and enhanced vulnerability of Cx32 knock-out mice.

Gap junctions are conductive channels that connect the interiors of coupled cells. In the hippocampus, GABA-containing hippocampal interneurons are interconnected by gap junctions, which mediate electrical coupling and synchronous firing and thereby promote inhibitory transmission. The present study was undertaken to examine the hypothesis that the gap junctional proteins connexin 32 (Cx32; expressed by oligodendrocytes, interneurons, or both), Cx36 (expressed by interneurons), and Cx43 (expressed by astrocytes) play a role in defining cell-specific patterns of neuronal death in hippocampus after global ischemia in mice. Global ischemia did not significantly alter Cx32 and Cx36 mRNA expression and slightly increased Cx43 mRNA expression in the vulnerable CA1, as assessed by Northern blot analysis and in situ hybridization. Global ischemia induced a selective increase in Cx32 and Cx36 but not Cx43 protein abundance in CA1 before onset of neuronal death, as assessed by Western blot analysis. The increase in Cx32 and Cx36 expression was intense and specific to parvalbumin-positive inhibitory interneurons of CA1, as assessed by double immunofluorescence. Protein abundance was unchanged in CA3 and dentate gyrus. The finding of increase in connexin protein without increase in mRNA suggests regulation of Cx32 and Cx36 expression at the translational or post-translational level. Cx32(Y/-) null mice exhibited enhanced vulnerability to brief ischemic insults, consistent with a role for Cx32 gap junctions in neuronal survival. These findings suggest that Cx32 and Cx36 gap junctions may contribute to the survival and resistance of GABAergic interneurons, thereby defining cell-specific patterns of global ischemia-induced neuronal death.

Effect of enzyme digestion on anionic sites and charge-selective permeability of dermo-epidermal junction.

To study components of anionic sites on the lamina densa of the dermo-epidermal junction (DEJ) and to assess the effect of removal of sialic acid or glycosaminoglycans on its charge-selective permeability, epidermal sheets, whose dermis had been removed by treatment with dithiothreitol, were digested with heparitinase, chondroitinase ABC, hyaluronidase, or neuraminidase. They were then stained with polyethyleneimine for demonstration of the anionic sites or incubated in a medium containing native anionic ferritin for tracer experiments. The anionic sites were completely removed after heparitinase digestion. Although the numerical density of the sites was not altered, their electron density was decreased after chondroitinase ABC digestion. The other enzymes had no effect on the sites. In the tracer experiments, heparitinase or neuraminidase increased the number of tracer molecules penetrating into the lamina lucida of the epidermal sheet, while the other enzymes had no effect on it. These data indicate that heparan sulfate, which is a main component of the anionic sites, plays an important role in the charge-selective permeability of the DEJ, whereas chondroitin sulfate, which seems to be contained in the sites, does not, probably because of its small amount. These data also indicate that sialic acid, which is not a main component of the anionic sites demonstrated with the cationic probe, has a role in the permeability function.

Immunohistochemical alterations in basement membrane components of squamous cell carcinoma.

To investigate alterations in the basement membrane (BM) in squamous cell carcinoma (SCC), we investigated 20 tumors. Four had the cytologic characteristics of Bowen's disease (SCC-BD) and 16 did not have them (SCC-NB). Tumors were studied immunohistochemically by double immunofluorescent staining by using mouse monoclonal antibodies to the core protein of heparan sulfate proteoglycan (HSPG) and chondroitin 6-sulfate glycosaminoglycan (Ch6S) as well as rabbit antiserum to laminin (LN) and type IV collagen (C-4). In well-differentiated and highly keratinized SCC-NB, LN, C-4, and HSPG could be detected in the tumor nest BM and showed no loss of continuity, but they were largely lost in poorly differentiated and poorly keratinized SCC-NB. This suggests that poorly differentiated SCC-NB cause greater enzymatic degradation of BM components than well-differentiated SCC-NB. Ch6S was detected in parts of the BM of SCC-BD, but it was absent in all SCC-NB examined. It appears that SCC-NB have lost the ability to synthesize Ch6S, and that SCC-BD degrade Ch6S although they continue to produce it. Thus, it appears that in SCC the BM is qualitatively different from that of normal epidermis, and that SCC-BD can be distinguished from SCC-NB by the Ch6S content of the BM.

Rapid turnover of tryptophan hydroxylase in serotonin producing cells: demonstration of ATP-dependent proteolytic degradation.

A rapid and continuous proteolysis of tryptophan hydroxylase was demonstrated with two mast cell lines derived from rat basophilic leukemia cells (RBL2H3) and mouse mastocytoma (FMA3). Under conditions in which protein biosynthesis was arrested by administration of cycloheximide, the decay profile of tryptophan hydroxylase protein was traced by Western blot analysis. Incorporation of [35S]methionine and the chase experiment performed without interfering with the metabolic stage also showed that tryptophan hydroxylase had been cleaved rapidly. The half life of the enzyme was 11-15 min in RBL2H3 cells and 40-60 min in FMA3 cells, and the process was demonstrated to be dependent on intracellular ATP.

Stimulation of tryptophan hydroxylase production in a serotonin-producing cell line (RBL2H3) by intracellular calcium mobilizing reagents.

RBL2H3 cells showed a remarkable increase in their level of tryptophan hydroxylase (up to 25-fold), the rate-limiting enzyme in serotonin biosynthesis, by stimulation with intracellular calcium mobilizers A23187, thapsigargin, and tBuBHQ as well as by stimulation with an antigen in the presence of IgE. The increase in the enzyme protein was visualized by Western blot analysis using anti-tryptophan hydroxylase antiserum. The enzyme turnover (Hasegawa et al., FEBS Lett., 368 (1995) 151-154) was not slowed down during the rise in tryptophan hydroxylase. Actinomycin D prevented the stimulation-induced elevation of the enzyme. These findings strongly suggest that this stimulation was achieved by the accelerated biosynthesis of tryptophan hydroxylase.

Low-dose ACTH therapy for West syndrome: initial effects and long-term outcome.

BACKGROUND: Most Japanese pediatric neurologists attempt other treatments before using adrenocorticotropic hormone (ACTH) therapy for West syndrome (WS), and even then, they use only a low-dose synthetic ACTH to avoid serious adverse effects. In this multi-institutional study, the authors analyzed the initial effects, adverse effects, and long-term outcome in patients treated with low-dose synthetic ACTH in Japan. METHODS: The medical records of 138 patients with WS, who were treated with low-dose synthetic ACTH therapy for the first time at the authors' institutions between 1989 and 1998, were analyzed. RESULTS: At the end of ACTH therapy, excellent effect on seizures was noted in 106 of 138 (76%) patients, good effect in 23 (17%), and poor effect in 9 (7%). Initial effects on EEG were excellent in 53 of 138 (38%) patients, good in 76 (55%), and poor in 9 (7%). As for seizure prognosis at the time of follow-up, 51 of 99 (52%) patients were seizure-free, whereas 48 (48%) patients had seizures. Mental outcome was normal in 6 of 98 (6%) patients, mild mental retardation in 16 (16%), moderate mental retardation in 26 (27%), and severe mental retardation in 50 (51%). The initial effects of ACTH on seizures and long-term outcome were not dose dependent (daily dosage 0.005 to 0.032 mg/kg, 0.2 to 1.28 IU/kg; total dosage 0.1 to 0.87 mg/kg, 4 to 34.8 IU/kg). The severity of adverse effects correlated with total dosage of ACTH, and the severity of brain volume loss due to ACTH correlated well with the daily dosage and total dosage of ACTH. CONCLUSION: Low-dose synthetic ACTH therapy is as effective for the treatment of WS as the higher doses used in previous studies. The dosage of synthetic ACTH used in the treatment of WS can be decreased as much as possible to avoid serious adverse effects.

Inositol 1,3,4,5-tetrakisphosphate as a mediator of neuronal death in ischemic hippocampus.

Selective death of CA1 pyramidal neurons after transient forebrain ischemia has attracted interest for its possible relation to the pathogenesis of memory deficits and dementia. Using whole cell patch-clamp recording from CA1 pyramidal neurons in hippocampal slices of gerbils after ischemia we studied the intracellular signaling mechanisms related to the phosphoinositide cycle. Intracellular application of an antibody against phosphatidylinositol 4,5-bisphosphate rescued ischemic neurons from stimulus-induced irreversible depolarization. Furthermore, application of inositol 1,3,4,5-tetrakisphosphate in normal cells caused an irreversible depolarization in response to synaptic input, which mimicked the deterioration of ischemic neurons. Depolarization of both ischemic and normal neurons in the presence of inositol 1,3,4,5-tetrakisphosphate was prevented by the addition of the Ca2+ chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetate. Application of antibody against inositol 1,4,5-triphosphate 3-kinase, which blocks formation of inositol 1,3,4,5-tetrakisphosphate, also protected against cell deterioration. Our results suggest that the vulnerability of hippocampal pyramidal neurons following ischemia is caused by a disturbed phosphoinositide cascade, with one metabolite, inositol 1,3,4,5-tetrakisphosphate, playing a key role in the induction of Ca2+ accumulation, which leads to neuronal death.

Abnormal Ca2+ homeostasis before cell death revealed by whole cell recording of ischemic CA1 hippocampal neurons.

Slices were made from the hippocampus of gerbils following transient ischemia achieved by clamping the carotid arteries for 5 min, and changes in the electrophysiology of CA1 pyramidal neurons were studied by whole cell patch-clamp recording as well as conventional intracellular recording. The great majority of CA1 neurons in slices made 2.5-3 days after ischemia showed reduced resting potentials and were easily depolarized by prolonged low-frequency stimulation or by tetanic stimulation of the Schaffer collateral/commissural input. This stimulus-induced depolarization was accelerated by intracellular injection of D-myo-inositol 1,4,5-triphosphate, which depolarized membrane potentials towards 0 mV without synaptic input stimulation. Intracellular application of BAPTA, a Ca2+ chelator, effectively blocked the stimulus-induced depolarization. When recording from ischemic neurons with patch pipettes containing both D-myo-inositol 1,4,5-triphosphate and BAPTA, excitatory postsynaptic currents were transiently potentiated by stimulation, but the membrane potential did not show stimulus-induced depolarization and remained steady for long periods. These results lend support to the view that the intracellular Ca2+ regulation system is severely disturbed following ischemia, and that input fiber stimulation leads to abnormal Ca2+ accumulation in ischemic neurons resulted in neuronal death. The reduction of free Ca2+ inside the ischemic neuron by BAPTA apparently saves neurons which are otherwise destined to delayed neuronal death.

Determination of transcriptional activities of typical gene promoters in HL-60 cells.

We developed a highly sensitive procedure for assaying chloramphenicol acetyltransferase (CAT) enzyme activity in extracts of eukaryotic cells transfected with the CAT gene expression vector, by modification of the partition extraction procedure described by Sleigh [Anal. Biochem. 156, 251-256 (1986)]. The sensitivity of the new method was improved 100-fold on commercial purified enzyme. In routine measurements with cell extracts a CAT activity as low as 1.3 x 10(-4) unit could be measured within an error of less than 30%. The CAT enzyme expressions in undifferentiated human promyelocytic leukemic cell line HL-60 from typical gene promoters could be measured by the new method and compared to select a stronger promoter. Similar measurements were made with more mature monocytic THP-1 cells to evaluate the change in the promoter activity with cell maturation. Differentiation induction with 12-O-tetradecanoylphorbol-13-acetate (TPA) activated transcription from the human immunodeficiency virus (HIV) promoter about 10-fold in HL-60 cells, as expected, but the level was less than that in untreated THP-1 cells. In addition, a similar activation was observed in THP-1 cells as well.

Rapid turnover of tryptophan hydroxylase is driven by proteasomes in RBL2H3 cells, a serotonin producing mast cell line.

Previously we demonstrated that tryptophan hydroxylase (TPH) undergoes very fast turnover driven by ATP-dependent proteolysis in serotonin producing mast cells [Hasegawa et al. (1995) FEBS Lett. 368, 151-154]. We searched for the major proteases involved in the rapid degradation of TPH in RBL2H3 cells. Among various protease inhibitors tested, proteasome inhibitors MG115, MG101, MG132, and lactacystin effectively inhibited the intracellular degradation of TPH. Administration of the proteasome inhibitors to cultured cells caused more than a 5-fold accumulation of TPH. Administration of the inhibitors together with cycloheximide stabilized the amount of TPH with no appreciable increase or decrease. Although MG-series proteasome inhibitors could inhibit calpain, the involvement of calpain was excluded since the cysteine protease inhibitor E-64-d, which acts on calpain, had no effect. Extracts of RBL2H3 cells were shown to contain a protease that digests TPH in an ATP-dependent manner and is sensitive to proteasome inhibitors. The ubiquitination of TPH could be visualized by Western blot analysis using both anti-TPH and anti-ubiquitin antibodies. Based on these results, we conclude that 26S proteasomes are mainly involved in the degradation of TPH. In the reported amino acid sequences of TPH from various sources including human, rabbit, rat, and mouse, a PEST sequence that is widely shared among short-lived proteins has been recognized. It was noted that the PEST sequence lies within the most conserved portion of the enzyme, the pteridine binding site.

Single glutamate channels in CA1 pyramidal neurones after transient ischaemia.

Patch clamp recordings were made from CA1 pyramidal neurones to study changes in the glutamate receptor subtypes in the gerbil hippocampus after transient ischaemia. In whole-cell recordings, the maximum chord conductances of AMPA currents in ischaemic neurones were increased over those of control neurones but NMDA-induced currents in the ischaemic neurones were smaller than the control. In AMPA-activated single channel currents, an open time histogram of the control neurones was well fitted by a single exponential function whereas in the ischaemic patches it was fitted by a double exponential function, indicating that currents consisted of at least two kinetically different types. These functional changes of the glutamate receptor channels may contribute to the abnormalities of the excitatory synaptic currents recorded in post-ischaemic CA1 neurones.

Reduced calcium elevation in hippocampal CA1 neurons of ischemia-tolerant gerbils.

Transient forebrain ischemia causes selective neuronal death in the hippocampal CA1 neurons. A short sublethal ischemic episode preceding ischemia of longer duration is known to increase tolerance against cell death. The mechanisms of this ischemic tolerance are still poorly understood. Here we show, using Ca2+ imaging, that intracellular calcium ([Ca2+]i) elevation in CA1 neurons after an anoxic-aglycemic episode is markedly inhibited in the ischemia-tolerant gerbil. The hippocampus of gerbils which did not acquire tolerance showed a high [Ca2+]i elevation during the anoxic-aglycemic episode, similar to controls. Since hypoxia/ischemia-induced neurodegeneration can be triggered by cytoplasmic Ca2+ overload, the tolerant gerbil may regulate calcium and keep [Ca2+]i below the critical level for initiating neuronal death.

Cyclic changes in NMDA receptor activation in hippocampal CA1 neurons after ischemia.

We studied N-methyl-D-aspartate (NMDA) receptor-mediated synaptic potentials in CA1 pyramidal neurons using hippocampal slices of gerbils after transient forebrain ischemia. In the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and bicuculline, stimulation of Schaffer collateral/commissural fibers induced field excitatory postsynaptic potentials (fEPSP) activated by NMDA receptors. We found that in many slices after ischemia, prolonged low-frequency stimulation (0.1-10 Hz) caused repeated depression and potentiation of the NMDA-mediated fEPSP. Changes in fEPSP amplitude were dependent on stimulus frequency and the cycle frequency ranged from 0.08 to 2.5 cycles/min. These cyclic changes were blocked by application of BAPTA-AM, a membrane-permeable Ca2+ chelator, but were little affected by application of verapamil or by lowering the Ca2+ in bathing solution. Intracellular recordings from CA1 neurons revealed that low-frequency stimulation caused periodic depolarizations of membrane potential accompanied by depression of the excitatory postsynaptic potentials. The cyclic changes of fEPSPs were blocked by inhibitors of protein kinase C (PKC) but were unaffected by inhibitors of Ca2+/calmodulin-dependent protein kinase II (CaMKII) or myosin light-chain kinase (MLCK). These results suggest that stimulus-dependent NMDA-receptor activation, mediated by PKC, takes place in the postischemic CA1 neurons and that the cyclic change may reflect abnormal intracellular Ca2+ signaling processes leading to neuronal degeneration.

Effects of perhexiline on contractile response of rabbit aorta to norepinephrine and potassium.

Perhexiline (3.2 X 10(-7)-6.4 X 10(-6) M) and verapamil (2 X 10(-8)-2 X 10(-7) M) inhibited the contraction of rabbit aorta induced with 40 mM K+ and with low concentrations (5.9 X 10(-9)-5.9 X 10(-8) M) of norepinephrine more effectively than the contraction induced by higher concentrations of norepinephrine. The tension induced by 1.2 X 10(-6) M norepinephrine was augmented with the above concentrations of perhexiline and verapamil. Higher concentrations (3.2 X 10(-5)-6.4 X 10(-5) M) of perhexiline significantly reduced the peak tension induced by norepinephrine. The results suggest that perhexiline is a Ca2+ antagonist and also has an unspecific spasmolytic action.

Involvement of the noradrenergic system in the seizures of epileptic El mice.

We studied the role of the noradrenergic system in the seizures of epileptic El mice. To this end, the anticonvulsant activity of adrenergic drugs was tested with a scoring method, and the binding of [3H]dihydroalprenolol, [3H]prazosin and [3H]yohimbine was evaluated in whole brains and various brain regions from stimulated and unstimulated El mice, and their maternal ddy mice. The seizures of El mice were inhibited by noradrenaline, phenylephrine, oxymetazoline, clonidine and yohimbine in a dose-dependent manner. These preventive effects of alpha-adrenoceptor agonists were antagonized by pretreatment with alpha-adrenoceptor antagonists. The preventive effect of yohimbine was reversed by pretreatment with clonidine or alpha-methyl-p-tyrosine, although the latter drug did not affect the anticonvulsant effect of clonidine. The binding of [3H]dihydroalprenolol was the same in the three groups of mice. More [3H]prazosin was bound in the cerebellum and striatum, and there were more [3H]yohimbine binding sites in the whole brain, cerebral cortex, hippocampus and brainstem of stimulated and unstimulated El mice than in the same areas of ddy mice. These findings suggest that up-regulated alpha 1- and alpha 2-adrenoceptors are involved in the inhibition of the seizures of El mice.

Inhibition of post-decapitation convulsions in the rat by dibenzothiepin neuroleptics via alpha 1-adrenoceptor blockade.

The mechanisms involved in inhibitory effects of isofloxythepin, a newly synthesized dibenzothiepin neuroleptic, on post-decapitation convulsions were studied in rats. Isofloxythepin (0.05-2.0 mg/kg s.c.) inhibited post-decapitation convulsions in a dose-dependent manner as shown by the decrease in the incidence and the shortening of the duration of convulsions. The convulsions were also inhibited by oxyprothepin, zotepine or chlorpromazine but not by haloperidol. Prazosin and bunazosin, both alpha 1-adrenoceptor antagonists, suppressed the post-decapitation convulsions but a non-selective alpha 2-adrenoceptor agonist, tolazoline, was without effect. The convulsions were inhibited dose dependently by clonidine, an alpha 2-adrenoceptor agonist, but were prolonged in duration by yohimbine, an alpha 2-adrenoceptor antagonist. Yohimbine antagonized the inhibitory effects of isofloxythepin, prazosin and clonidine. The noradrenaline-induced contraction of rat vas deferens was inhibited by isofloxythepin, prazosin or chlorpromazine. Isofloxythepin bound to alpha 1-receptors as did chlorpromazine in the rat brain cortex. The results imply that post-decapitation convulsions seem to be inhibited by a block of postsynaptic alpha 1-adrenoceptors, enhanced by a block of presynaptic alpha 2-adrenoceptors and reduced by isofloxythepin via the blocking of postsynaptic alpha 1-adrenoceptors. The convulsions thus could serve as a good model for studying the actions of drugs on the central nervous system alpha-adrenoceptors.

Different responses to auditory and somaesthetic stimulation in patients with an excessive startle: a report of pediatric experience.

OBJECTIVES: Children with cerebral injury often exhibit brief muscle contraction to a variety of stimuli. However, it remains to be determined whether or not the pattern of the reaction is stereotypical irrespective of the site stimulated. To answer this question, we studied electromyographic (EMG) responses to three types of stimuli in children. METHODS: The EMG responses of cranial and limb muscles were recorded after acoustic or somaesthetic stimulation in 6 patients and 23 control subjects. RESULTS: Acoustic stimuli evoked patterned motor activity with a rostrocaudal progression. Nose-tapping stimuli elicited reflex EMG activity in the VIIth cranial muscles that was similar to the R1 component of the electrical blink reflex. Sternum-tap stimuli evoked motor activity in the sternocleidomastoid and arm muscles, and this reflex was probably mediated through the cervical cord (H-reflex). Moreover, late reflexes were evoked following these early reflexes in the patients. In particular, atypical forms of myoclonic jerks were evoked on sternum-tap stimuli. CONCLUSIONS: Many types of primitive reflexes were evoked following three types of stimuli. These reflexes included startle reflex, trigeminomotor reflex, H-reflex and atypical forms of myoclonus, and they were enhanced in the patient group. There are many startle-mimicking reflexes.

Spontaneous excitatory postsynaptic currents in hippocampal CA1 pyramidal neurons of the gerbil after transient ischemia.

The changes in the spontaneous excitatory postsynaptic currents (sEPSCs) after transient cerebral ischemia were studied using whole-cell recording from CA1 pyramidal neurons in the gerbil. In neurons recorded 1-2 days after ischemia, sEPSCs had a slowed time course with the decay time constant fitted by a single exponential and it progressively increased after ischemia. Frequency and amplitude distribution of sEPSCs in ischemic neurons were not significantly different from those in the control neurons. The results support the view that abnormal non-N-methyl-D-aspartic acid currents originate at the degenerated postsynaptic site, unrelated to the presynaptic releasing mechanisms.

Association of NMDA receptor sites and seizures of El mice.

To investigate the possible role of N-methyl-D-aspartate (NMDA) receptors in the seizures of El mice, a genetic animal model of epilepsy, we measured [3H]3-[(+)-2-(carboxypiperazin-4-yl)][1,2-3H]- propyl-1-phosphonic acid (CPP) binding in several brain regions of El and ddY mice. At 22-24 weeks of age, the maximum number of binding sites (Bmax) of [3H]CPP was lower only in the cerebral cortex of both stimulated and unstimulated El mice (El(+) and El(-), respectively) than in that of ddY mice. A reduction in Bmax values of cortical [3H]CPP binding of El mice was detected after the age of 12 weeks. Cortical [3H]CPP binding in El(+) mice decreased further transiently after evoked seizures. No significant change was observed in El(-) mice after postural stimulation. These results suggest that El(+) and El(-) mice share seizure propensity and that activation of NMDA receptors is involved in the seizures of El mice.