Mortality risk factors and fulminant sub-phenotype in anaerobic bacteremia: a 10-year retrospective, multicenter, observational cohort study.

PURPOSE: During the last decade, the incidence of anaerobic bacteremia (AB) has been increasing. Patients with AB may develop complex underlying diseases, which can occasionally be accompanied by fatal or fulminant outcomes. However, the risk factors for AB-related mortality remain unclear. Herein, we sought to elucidate the risk factors for AB-related mortality. METHODS: In this multicenter, retrospective, observational study, we enrolled patients with culture-proven AB from six tertiary hospitals in Japan, between January 2012 and December 2021. Data on patient and infection characteristics, laboratory findings, treatment, and outcome were collected, and their associations with mortality were analyzed. RESULTS: A total of 520 participants were included. The 30-day mortality in the study cohort was 14.0% (73 patients), and malignant tumors were frequently observed comorbidities in 48% of the entire cohort. Multivariable logistic regression analysis showed a Charlson comorbidity score of > 6, serum creatinine level of > 1.17 mg/dL, and hypotension to be independent risk factors for 30-day mortality in AB (odds ratios [ORs] 2.12, 2.25, and 5.12, respectively; p < 0.05), whereas drainage significantly reduced this risk (OR, 0.28; p < 0.0001). Twelve patients (2.3% of the whole cohort and 16.4% of the deceased patients) presented with extremely rapid progression leading to fatal outcome, consistent with "fulminant AB." CONCLUSIONS: This study identified acute circulatory dysfunction and performance of drainage as independent predictive factors for 30-day AB-related mortality and revealed the existence of a fulminant AB sub-phenotype. Our findings could serve as a practical guide to predict the clinical outcomes of AB.

Association of subpleural ground-glass opacities with respiratory failure and RNAemia in COVID-19.

OBJECTIVES: To examine the radiological patterns specifically associated with hypoxemic respiratory failure in patients with coronavirus disease (COVID-19). METHODS: We enrolled patients with COVID-19 confirmed by qPCR in this prospective observational cohort study. We explored the association of clinical, radiological, and microbiological data with the development of hypoxemic respiratory failure after COVID-19 onset. Semi-quantitative CT scores and dominant CT patterns were retrospectively determined for each patient. The microbiological evaluation included checking the SARS-CoV-2 viral load by qPCR using nasal swab and serum specimens. RESULTS: Of the 214 eligible patients, 75 developed hypoxemic respiratory failure and 139 did not. The CT score was significantly higher in patients who developed hypoxemic respiratory failure than in those did not (median [interquartile range]: 9 [6-14] vs 0 [0-3]; p < 0.001). The dominant CT patterns were subpleural ground-glass opacities (GGOs) extending beyond the segmental area (n = 44); defined as "extended GGOs." Multivariable analysis showed that hypoxemic respiratory failure was significantly associated with extended GGOs (odds ratio [OR] 29.6; 95% confidence interval [CI], 9.3-120; p < 0.001), and a CT score > 4 (OR 12.7; 95% CI, 5.3-33; p < 0.001). The incidence of RNAemia was significantly higher in patients with extended GGOs (58.3%) than in those without any pulmonary lesion (14.7%; p < 0.001). CONCLUSIONS: Extended GGOs along the subpleural area were strongly associated with hypoxemia and viremia in patients with COVID-19. KEY POINTS: • Extended ground-glass opacities (GGOs) along the subpleural area and a CT score > 4, in the early phase of COVID-19, were independently associated with the development of hypoxemic respiratory failure. • The absence of pulmonary lesions on CT in the early phase of COVID-19 was associated with a lower risk of developing hypoxemic respiratory failure. • Compared to patients with other CT findings, the extended GGOs and a higher CT score were also associated with a higher incidence of RNAemia.

Fibronectin plays a major role in hypoxia-induced lenvatinib resistance in hepatocellular carcinoma PLC/PRF/5 cells.

Resistance to lenvatinib mesylate (LEN), a systemic chemotherapy that can be administered orally, has been a major issue for treatment of hepatocellular carcinoma (HCC). Although HCC is the tumor that most exhibits intratumoral hypoxia, which has been shown to be involved in the development of treatment resistance, there are no reports of LEN resistance in HCC treatment under hypoxia. The purpose of our study was to elucidate the mechanism of treatment resistance to LEN under hypoxia using HCC cell lines. We confirmed LEN resistance under hypoxic conditions in HCC cell lines. There was a significant increase in the IC50 value of PLC/PRF/5 cells from 13.0±0.8 µM in normoxia to 21.3±1.1 µM in hypoxia, but in HepG2 cells, the increase was not significant. To elucidate the LEN resistance mechanism of PLC/PRF/5 cells under hypoxia, we performed microarray analysis and extracted genes that are thought to be related to this mechanism. Furthermore, in-silico analysis confirmed significant changes in the extracellular matrix, and among them, FN1 encoding fibronectin was determined as the hub of the gene cluster. The expression of fibronectin in PLC/PRF/5 cells examined with immunofluorescence staining was significantly elevated in and outside of cells under hypoxia, and tended to decrease when cells were exposed to LEN under normoxia. Furthermore, the fibronectin concentration in the culture solution of PLC/PRF/5 cells examined by ELISA was 2.3 times higher under hypoxia than under normoxia under LEN(-) conditions, and 1.6 times higher under hypoxia than under normoxia under LEN(+) conditions. It is assumed that in PLC/PRF/5 cells, fibronectin is probably suppressed as an indirect effect of LEN under normoxia, but transcription factors such as HIF-1α are induced under hypoxia, thus enhancing the production of fibronectin and attenuating the effect of LEN, resulting in drug resistance. This behavior of fibronectin with LEN exposure under hypoxia is probably specific to PLC/PRF/5 cells. Further studies should verify the combined effective inhibition of fibronectin and the MAPK pathway as a promising therapeutic strategy to enhance the value of LEN in HCC treatment.

Predictive values of immune indicators on respiratory failure in the early phase of COVID-19 due to Delta and precedent variants.

Background: Immune response indicators in the early phase of COVID-19, including interferon and neutralizing responses against SARS-CoV-2, which predict hypoxemia remains unclear. Methods: This prospective observational study recruited patients hospitalized with COVID-19 (before emergence of omicron variant). As the immune indicators, we assessed the serum levels of IFN-I/III, IL-6, CXCL10 and VEGF, using an ELISA at within 5 days after the onset of symptoms, and serum neutralizing responses using a pseudovirus assay. We also assessed SARS-CoV-2 viral load by qPCR using nasal-swab specimens and serum, to assess the association of indicators and viral distribution. Results: The study enrolled 117 patients with COVID-19, of which 28 patients developed hypoxemia. None received vaccine before admission. Serum IFN-I levels (IFN-α and IFN-ß), IL-6, CXCL10, LDH and CRP were significantly higher in patients who developed hypoxemia. A significant association with nasopharyngeal viral load was observed only for IFN-I. The serum levels of IFN-α, IL-6, CXCL10 were significantly associated with the presence of RNAemia. Multivariable analysis showed higher odds ratio of IFN-α, with cut-off value of 107 pg/ml, in regard to hypoxemia (Odds ratio [OR]=17.5; 95% confidence interval [CI], 4.7-85; p<0.001), compared to those of IL-6, >17.9 pg/ml (OR=10.5; 95% CI, 2.9-46; p<0.001). Conclusions: This study demonstrated that serum IFN-α levels in the early phase of SARS-CoV-2 infection strongly predict hypoxemic respiratory failure in a manner different from that of the other indicators including IL-6 or humoral immune response, and instead sensitively reflect innate immune response against SARS-CoV-2 invasion.

Activation of protein kinase A and C prevents recovery from persistent depolarization produced by oxygen and glucose deprivation in rat hippocampal neurons.

Intracellular recordings were made from rat hippocampal CA1 neurons in rat brain slice preparations to investigate whether cAMP-dependent protein kinase (PKA) and calcium/phospholipid-dependent protein kinase C (PKC) contribute to the membrane dysfunction induced by oxygen and glucose deprivation (OGD). Superfusion of oxygen- and glucose-deprived medium produced a rapid depolarization ∼5 min after the onset of the superfusion. When oxygen and glucose were reintroduced immediately after the rapid depolarization, the membrane depolarized further (persistent depolarization) and reached 0 mV after 5 min from the reintroduction. The pretreatment of the slice preparation with PKA inhibitors, H-89 and Rp-cAMPS, and an adenylate cyclase inhibitor, SQ 22, 536, significantly restored the membrane toward the preexposure potential level after the reintroduction of oxygen and glucose in a concentration-dependent manner. On the other hand, a phospholipase C inhibitor, U73122, a PKC inhibitor, GF109203X, and a nonselective protein kinase inhibitor, staurosporine, also significantly restored the membrane after the reintroduction. Moreover, an inositol-1,4,5-triphosphate receptor antagonist, 2-aminoethyl diphenylborinate, and calmodulin inhibitors, trifluoperazine and W-7, significantly restored the membrane after the reintroduction, while neither an α-subunit-selective antagonist for stimulatory G protein, NF449, a Ca(2+)/calmodulin-dependent kinase II inhibitor, KN-62, nor a myosin light chain kinase inhibitor, ML-7, significantly restored the membrane after the reintroduction. These results suggest that the activation of PKA and/or PKC prevents the recovery from the persistent depolarization produced by OGD. The Ca(2+)/calmodulin-stimulated adenylate cyclase may contribute to the activation of PKA.

The validity of the simple methods of estimating chronic subdural hematoma volume.

OBJECTIVES: It was reported that the XYZ/2 technique (using length, width and height of hematoma) is a simple and reliable method of estimation of chronic subdural hematoma volume. Two subtypes of techniques enable to adequately estimate, it is unclear which is more accurate. Computer-assisted volumetric analysis is widely considered the gold standard for CSDH volumetric analysis. It is important to consider the stability of analyses between examiners, because individual, decision-making differences may be relevant to the analysis, as hematoma margin and length are hand-operated. In this study, we investigated potential measurement biases of three neurosurgeons and analyzed the validity of the XYZ/2 technique by comparing it to the gold standard method. METHOD: We retrospectively analyzed CT scans that indicated the need for an operation in 50 patients with CSDH in our department. Three neurosurgeons measured and calculated CSDH volumes independent of one another. We investigated potential measurement biases of three neurosurgeons and analyzed the validity of the XYZ/2 technique by comparing it to the gold standard method. The XYZ/2 technique includes the "maximal method" that uses the maximum length and maximum width of a slice to determine volume, and the "central method" that uses only the central slice to measure length and width. RESULTS: ICCs for the gold standard, central method, and maximal method were 0.945, 0.916, and 0.844, respectively, all of which indicated excellent reliability. For all examiners, the differences in calculation from gold standard and central method were not statistically significant (P>0.05). The estimations of CSDH volume calculated by the maximal method were significantly greater than the estimates calculated by the gold standard (P<0.05). CONCLUSIONS: This study proves that the XYZ/2 technique is a simple and reliable method of estimating CSDH volume. The "central method" in particular yielded similar results to that of the gold standard method.

Excitatory GABAergic synaptic potentials in the mesencephalic trigeminal nucleus of adult rat in vitro.

The mesencephalic trigeminal nucleus (MesV) contains the somata of primary afferent neurons innervating masticatory muscle spindles and the periodontal membrane. MesV afferent somata are unique in receiving synaptic inputs. Intracellular recordings in coronal pontine slices from adult rats were made from MesV neurons identified by having Cs-sensitive inward rectification and pseudounipolar morphology. Stimuli near the MesV evoked either a cluster of action potentials superimposed on a postsynaptic potential (PSP) or an antidromic spike at resting membrane potential (RMP). Membrane hyperpolarization revealed that each cluster of action potentials consisted of an antidromic spike and a subsequent PSP. Evoked PSPs in slices and miniature postsynaptic currents (mPSCs) recorded using whole-cell patch in dissociated MesV neurons were resistant to glutamate antagonists and strychnine but were reversibly abolished by 40 microM bicuculline. Superfusion of 1-10 mM GABA decreased input resistance and depolarized the membrane. Reversal potentials for evoked PSPs and GABA-induced depolarizations were similar and close to that for mPSCs which matched the Cl- equilibrium potential. Thus activation of synapses on MesV somata evokes GABAergic PSPs that generate action potentials at RMP in the adult. These data also indicate that primary afferent MesV neurons can act as interneurons in the central control of mastication.

Neuroprotective mechanisms of lidocaine against in vitro ischemic insult of the rat hippocampal CA1 pyramidal neurons.

To compare neuroprotective effects of lidocaine and procaine against ischemic insult, intracellular recordings were made from rat hippocampal CA1 pyramidal neurons in slice preparations. Superfusion of the slices with oxygen- and glucose-deprived medium (in vitro ischemia) produced a rapid depolarization 6 min from the onset. When oxygen and glucose were reintroduced, the membrane depolarized further until it reached 0 mV, and thereafter the membrane showed no functional recovery. Pretreatment with lidocaine (10 microM), but not procaine (50 microM), restored the membrane potential after the reintroduction of oxygen and glucose. Lidocaine, compared to procaine, significantly inhibited the reduction in both tissue ATP content and flavoprotein fluorescence during and after in vitro ischemia. Under electron microscopy, only lidocaine well preserved the structure of mitochondria in the CA1 pyramidal cell body. Extracellular recordings revealed that procaine reduced the field postsynaptic potential whereas lidocaine augmented it. Both drugs reduced the presynaptic volley dose-dependently. Neither lidocaine nor procaine significantly affected a rapid rise of the intracellular Ca2+ level produced by in vitro ischemia in the CA1 region. All the results suggest that the neuroprotective lidocaine action is due to the protection of the mitochondria to maintain the tissue ATP content during and after in vitro ischemia.

Monoclonal antibodies which differentiate high- and low-affinity binding sites of interleukin-2.

Five monoclonal antibodies (L15, L20, L23, L34, and L61) against human recombinant interleukin-2 were tested for their effects on the interleukin-2 bioactivity and binding. Four of these monoclonal antibodies, L15, L20, L34, and L61, which had neutralizing activity, completely blocked interleukin-2 binding to the high-affinity receptor. On the other hand, L23, which had a very weak neutralizing activity, blocked interleukin-2 binding to the low-affinity receptor. These results suggest that there are at least two distinct binding sites on the interleukin-2 molecule; those for the high-affinity receptor and those for the low-affinity receptor. These monoclonal antibodies should be useful tools in the study of the interaction between interleukin-2 and interleukin-2 receptor.

Evoked potential studies in mitochondrial encephalomyopathy.

Evoked potentials were studied in a patient with a mitochondrial encephalomyopathy revealing a defect of nicotinamideadenine dinucleotide dehydrogenase and cytochrome C oxidase in the mitochondria of a muscle biopsy specimen. The biopsy specimen showed myopathic changes with ragged-red fibers and markedly decreased cytochrome C oxidase in the muscle fibers. Subcortical somatosensory evoked potentials to median nerve stimulation were normal in the peak latencies of N9, N11, and N13. Cortical somatosensory evoked potentials to median nerve stimulation revealed significantly delayed peak latencies of N20, P20, P25, and N26, although N16 latency was normal. In particular, the interpeak latency between N16 and N20 was significantly delayed. In topographic maps, N20 and P20 were delayed in the peak latencies with normal scalp distributions. Dysfunction of somatosensory cortex indicated by the delay of cortical somatosensory evoked potentials may be related to a cortical mitochondrial abnormality. The absence of responses to auditory stimulation within 10 milliseconds could be related to the dysfunction of peripheral acoustic nerves.

Serum erythropoietin titers in the aged.

Erythropoietin (Epo) titers in the aged (from 70 to 89 years) were determined by a radioimmunoassay. Epo levels in normal elderly subjects were not different from levels in the young volunteers. The serum Epo levels in elderly patients with anemia were inversely related to hemoglobin levels; their regression coefficient was not worse than that found in young patients. It is suggested that the reactivity of Epo production to anemia in the aged may not be worse than that reactivity in the young.

Cortical somatosensory potentials evoked by magnetic stimulation of thoracic and lumbar roots.

We applied twin coil magnetic stimulation to thoracic and lumbar roots to evaluate the posterior column function in 50 normal subjects and 34 patients with neurologic disorders. In all nine patients with cervical myelopathy, there were abnormal somatosensory evoked potentials (SEPs) with stimulation of upper thoracic levels. In 14 patients with thoracic or lumbar myelopathy, there were normal SEPs with stimulation of spinal roots above the lesions and abnormal SEPs with stimulation below the lesions. In a patient with girdle sensation between T-3 and T-6, the peak latencies of P2 were significantly delayed with stimulation of T-2 and T-4, but peak latencies were normal with T-6, T-10, and L-3 stimulation. Ten patients with polyneuropathy had normal SEPs recorded with thoracic and lumbar root stimulation. SEPs by twin coil stimulation at thoracic and lumbar root levels are useful in detecting lesions of spinal cord or roots and for following their clinical course noninvasively.

Sudomotor potential evoked by magnetic stimulation of the neck.

We studied the sudomotor potential in the palm of the hand, evoked by magnetic stimulation of the neck, in 15 normal subjects and five patients with dysautonomia. The potentials consisted of two positive peaks (S1, S2), with mean (+/- SD) onset latencies of 408.5 +/- 31.8 and 619 +/- 61.4 msec. The sudomotor potential was clearly obtained only from the palm and was influenced by stimulus intensity, coil position, current direction in the coil, atropine or pilocarpine iontophoresis, and motor effort. With this method, we estimated the postganglionic sympathetic conduction time and the central reflex time, the mean values (+/- SD) of which were 408.5 +/- 31.8 and 323.3 +/- 45.2 msec. In three patients with severe dry skin, the sudomotor potential and the sympathetic skin response were not obtained. A patient with OPCA had a prolonged central reflex time, and a patient with alcoholic polyneuropathy showed a delayed sudomotor potential. Sudomotor potential is a noninvasive and useful method for evaluating sympathetic sudomotor function in patients with central and peripheral nervous system disorders.

Syndrome of continuous muscle-fiber activity: increased CSF GABA and effect of dantrolene.

In a patient with a syndrome of continuous muscle fiber activity, peripheral nerve block completely abolished the EMG discharges. Reduction of spontaneous discharges by epidural block and demonstration of a silent period after the H response suggested that the disorder may originate in the spinal cord or ventral roots, sparing inhibitory influences on the alpha-motoneuron to some extent. GABA (gamma-aminobutyric acid) content in CSF was remarkably increased. Therapeutically, dantrolene sodium was as effective as phenytoin or carbamazepine.

Denervation of eccrine glands in patients with familial amyloidotic polyneuropathy type I.

OBJECTIVE: To study the alterations in the structure and innervation of eccrine glands in familial amyloidotic polyneuropathy (FAP) type I with Val 30 Met transthyretin mutation. BACKGROUND: Anhidrosis of the distal lower limbs is a prominent feature of FAP type I. METHODS: Qualitative and morphometric study of amyloid deposition, eccrine glands, and their innervation in nine patients with FAP type I (duration of sensory symptoms, 8.4 +/- 3.9 years [mean +/- SD]; range, 3 to 15 years) and seven control subjects. RESULTS: On light microscopy, the endoneurium of cutaneous nerve fascicles had no definite amyloid deposition. Amyloid deposition was observed around eccrine glands in seven of nine patients. On electron microscopy, no focal destruction and degeneration of eccrine glands or ducts and of Schwann cell processes with or without nerve terminals or unmyelinated axons were observed in relation to adjacent amyloid deposition. Secretory vacuoles and granules of dark cells were markedly decreased in some secretory coils. Nerve terminals and unmyelinated axons of eccrine glands were considerably fewer in patients than in control subjects, and denervation was prominent in all patients. A few nerve terminals and unmyelinated axons of eccrine glands were present in patients who had experienced sensory symptoms for 3, 5, and 6 years, but were absent in patients with sensory symptoms for more than 7 years. CONCLUSIONS: Eccrine glands are markedly to totally denervated in patients with FAP type I and chronic sensory symptoms. The extent of denervation indicates the severity of autonomic denervation and therefore may suggest the timing of liver transplantation.

Neutralizing monoclonal antibodies against recombinant human interleukin-2.

A series of hybridoma cell lines which produce monoclonal antibodies (MAbs) against recombinant human interleukin-2 (rIL-2) have been established by fusion of murine myeloma cell line P3-NS1-1-AG4-1 and spleen cells of BALB/c mice which had been immunized with rIL-2. 48 hybridoma strains were selected by a solid-phase screening method which produced MAbs reacting with IL-2: four MAbs, L-15, L-20, L-34, and L-61, exhibited strong inhibition of the proliferating effect of rIL-2 on IL-2-dependent cell lines, NK7 and CTLL-2. L-61, the most potent MAb among them, also neutralized natural human IL-2, while the other three MAbs were unreactive. All the four MAbs were specific to human IL-2: they did not cross-react with mouse or rat IL-2. These MAbs are expected to be useful tools in the investigation of IL-2 function.

The prostaglandin E series modulates high-voltage-activated calcium channels probably through the EP3 receptor in rat paratracheal ganglia.

The modulation of high-voltage-activated (HVA) Ca2+ channels by the prostaglandin E series (PGE1 and PGE2) was studied in the paratracheal ganglion cells. Prostaglandin E1, E2, STA2 (a stable analogue of thromboxane A2), 17-phenyl-trinor-PGE2 (an EP1-selective agonist) and sulprostone (an EP3-selective agonist) inhibited the HVA Ca2+ current (HVA ICa) dose-dependently, and the rank order of potency to inhibit HVA Ca2+ channels was sulprostone>PGE2, PGE1>STA2>>17-phenyl-trinor-PGE2. SC-51089 (10(-5) M), a selective EP1-receptor antagonist, showed no effect on the PGE1- or PGE2-induced inhibition of the HVA ICa, thereby indicating that PGE1- and PGE2-induced inhibition of the HVA Ca2+ channels is possibly mediated by the EP3 receptor. The PGE1-sensitive component of the current was markedly reduced in the presence of omega-conotoxin-GVIA (3x10(-6) M), but not with nifedipine (3x10(-6) M). PGE1 and PGE2 also inhibited the remaining ICa in a saturating concentration of nifedipine, omega-conotoxin-GVIA and omega-conotoxin-MVIIC, suggesting that R-type Ca2+ channels are involved. The inhibitory effect of PGE1 or sulprostone was prevented by pretreatment with pertussis toxin [islet activating protein (IAP)] or phorbol-12-myristate-13-acetate (PMA), and the protein kinase C (PKC) inhibitor chelerythrine blocked the action of PMA. It was concluded that PGE1 selectively reduces both N- and R-type Ca2+ currents by activating a G-protein probably through the EP3 receptor in paratracheal ganglion cells.

Differential up-regulation of voltage-dependent Na+ channels induced by phenytoin in brains of genetically seizure-susceptible (E1) and control (ddY) mice.

We investigated the effect of in vivo administration of an antiepileptic drug, phenytoin, on the saxitoxin binding capacity of receptor site 1 of the Na+ channel alpha-subunit, and the expression activity of the channel messenger RNA in epileptic El mouse brains, as compared with parental ddY mice. Subchronic treatment with phenytoin (25 mg/kg per day) for 14 days increased the [3H]saxitoxin binding to brain-derived synaptic membranes of both El and control ddY mice in a time dependent manner. This increase plateaued at 21 +/- 4% in El mice and 28 +/- 3% in ddY control mice after administration of phenytoin for seven days. After cessation of treatment with phenytoin, [3H]saxitoxin binding capacity returned to the basal level within two weeks in both ddY and El brains. Scatchard plot analysis revealed that the phenytoin treatment caused a 20-30% increase in maximum binding capacity of [3H]saxitoxin binding without any change in equilibrium dissociation constant in the brain cortical synaptic membranes of both epileptic El and control ddY mice. A single injection of phenytoin (25 mg/kg) elevated the level of Na+ channel messenger RNA within 1 h in ddY mouse brains. The increase in Na+ channel messenger RNA reached a peak (about 80% increase) after 5 h of phenytoin administration in a concentration-dependent manner (6.25-50 mg/kg). On the other hand, in El mouse brains, Na+ channel messenger RNA was not elevated until more than 5 h after phenytoin injection, and was increased by only about 33%.(ABSTRACT TRUNCATED AT 250 WORDS)

Overproduction of voltage-dependent Na+ channels in the developing brain of genetically seizure-susceptible E1 mice.

We used E1 mice, a ddY mouse-derived, autosomal mutant strain and a model of hereditary sensory-precipitated epilepsy, to test the hypothesis that epileptic susceptibility may be associated with the activity of voltage-dependent ion channels. We examined the saxitoxin binding capacity of the receptor site 1 of the Na+ channel alpha-subunit, the expression activity of the Na+ channel mRNA, the veratridine-induced 22Na+ influx in the brain synaptosomes, and the regional distribution of Na+ channels in the brain. Compared with control ddY mice, in E1 mice which have not experienced seizures, the number of Na+ channels in the brain synaptosomes increased by approximately 20% starting at the fourth postnatal week through the adult stage as determined by [3H]saxitoxin binding assay. Northern blot hybridization analysis showed excess expression of Na+ channel mRNA (by 30-40%) coincidentally with Na+ channel increases. Regional analysis using the saxitoxin binding assay demonstrated approximately 1.3-fold denser distribution of Na+ channels in the cortex and cerebellum but not the hippocampus and midbrain including thalamus of E1 mice compared to ddY mice. Scatchard plot analysis for saxitoxin binding in the cortex of E1 mouse brains revealed higher maximum binding capacity (Bmax) values (ddY, 4.43 +/- 0.28 pmol/mg protein; E1, 5.43 +/- 0.25 pmol/mg protein) without a change in Kd (ddY, 1.05 +/- 0.03 nM; E1, 1.03 +/- 0.01 nM). Lastly, veratridine-evoked 22Na+ influx, sensitive to tetrodotoxin, was increased approximately 45% in the cortical synaptosomes in six-week-old E1 mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine modulation of high-voltage-activated calcium channels in the neurones acutely dissociated from rat paratracheal ganglia.

1. The modulation of high-voltage-activated (HVA) Ca2+ channels by acetylcholine (ACh) was studied in the paratracheal ganglion cells acutely dissociated from 2-week-old Wistar rats by use of the nystatin perforated patch recording configuration under voltage-clamp conditions. 2. ACh inhibited the HVA Ca2+ currents in a concentration- and voltage-dependent manner. 3. The inhibition was mimicked by a muscarinic agonist, oxotremorine. Pirenzepine and methoctramine produced parallel shifts to the right in the ACh concentration-response curves. Schild analysis of the ACh concentration-ratios yield pA2 values for pirenzepine and methoctramine of 6.85 and 8.57, respectively, suggesting the involvement of an M2 receptor. 4. Nifedipine, omega-conotoxin-GVIA and omega-conotoxin-MVIIC reduced the HVA I(Ca) by 16.8, 59.2 and 6.3%, respectively. A current insensitive to all of these Ca2+ antagonists, namely 'R-type', was also observed. The results indicated the existence of L-, N-, P/Q-, and R-type Ca2+ channels. 5. The ACh-sensitive current component was markedly reduced in the presence of omega-conotoxin-GVIA, but not with both nifedipine and omega-conotoxin-MVIIC. ACh also inhibited the R-type HVA I(Ca) remaining in saturating concentrations of nifedipine, omega-conotoxin-GVIA and omega-conotoxin-MVIIC. 6. The inhibitory effect of ACh was prevented by pretreatment with pertussis toxin. 7. It was concluded that ACh selectively reduces both the N- and R-type Ca2+ channels, by activating pertussis toxin sensitive G-protein through the M2 muscarinic receptor in paratracheal ganglion cells.