Brief Bursts Self-Inhibit and Correlate the Pyramidal Network

Inhibitory pathways are an essential component in the function of the neocortical microcircuitry. Despite the relatively small fraction of inhibitory neurons in the neocortex, these neurons are strongly activated due to their high connectivity rate and the intricate manner in which they interconnect with pyramidal cells (PCs). One prominent pathway is the frequency-dependent disynaptic inhibition (FDDI) formed between layer 5 PCs and mediated by Martinotti cells (MCs). Here, we show that simultaneous short bursts in four PCs are sufficient to exert FDDI in all neighboring PCs within the dimensions of a cortical column. This powerful inhibition is mediated by few interneurons, leading to strongly correlated membrane fluctuations and synchronous spiking between PCs simultaneously receiving FDDI. Somatic integration of such inhibition is independent and electrically isolated from monosynaptic excitation formed between the same PCs. FDDI is strongly shaped by I(h) in PC dendrites, which determines the effective integration time window for inhibitory and excitatory inputs. We propose a key disynaptic mechanism by which brief bursts generated by a few PCs can synchronize the activity in the pyramidal network.     

Human cholinesterase genes localized by hybridization to chromosomes 3 and 16

Summary A cloned human cDNA for cholinesterase (ChE) was used as a probe for in situ hybridization to spread lymphocyte chromosomes to map the structural human CHE genes to distinct chromosomal regions. The recent genetic linkage assignment of the CHE1 locus of the CHE gene to chromosome 3q was confirmed and further refined to 3q21-q26, close to the genes coding for transferrin (TF) and transferrin receptor (TFRC). The CHE1 allele localizes to a 3q region that is commonly mutated and then associated with abnormal megakaryocyte proliferation in acute myelodysplastic anomalies. In view of earlier findings that ChE inhibitors induce megakaryocytopoiesis in culture, this localization may indicate that ChEs are involved in regulating the differentiation of megakaryocytes. A second site for ChEcDNA hybridization was found on chromosome 16q11-q23, demonstrating that the CHE2 locus of the cholinesterase gene, which directs the production of the common C5 variant of serum ChE, also codes for a structural subunit of the enzyme and is localized on the same chromosome with the haptoglobin (HP) gene, both genes being found on the long arm of chromosome 16. The finding of two sites for ChEcDNA hybridization suggests that the two loci coding for human ChEs may include nonidentical sequences responsible for the biochemical differences between ChE variants.     

Human acetylcholinesterase and butyrylcholinesterase are encoded by two distinct genes

1. Various hybridization approaches were employed to investigate structural and chromosomal interrelationships between the human cholinesterase genes CHE and ACHE encoding the polymorphic, closely related, and coordinately regulated enzymes having butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) activities. 2. Homologous cosmid recombination with a 190-base pair 5' fragment from BuChEcDNA resulted in the isolation of four overlapping cosmid clones, apparently derived from a single gene with several introns. The Cosmid CHEDNA included a 700-base pair fragment known to be expressed at the 3' end of BuChEcDNA from nervous system tumors and which has been mapped by in situ hybridization to the unique 3q26-ter position. In contrast, cosmid CHEDNA did not hybridize with full-length AChEcDNA, proving that the complete CHE gene does not include AChE-encoding sequences either in exons or in its introns. 3. The chromosomal origin of BuChE-encoding sequences was further examined by two unrelated gene mapping approaches. Filter hybridization with DNA from human/hamster hybrid cell lines revealed BuChEcDNA-hybridizing sequences only in cell lines including human chromosome 3. However, three BuChEcDNA-homologous sequences were observed at chromosomal positions 3q21, 3q26-ter, and 16q21 by a highly stringent in situ hybridization protocol, including washes at high temperature and low salt. 4. These findings stress the selectivity of cosmid recombination and chromosome blots, raise the possibility of individual differences in BuChEcDNA-hybridizing sequences, and present an example for a family highly similar proteins encoded by distinct, nonhomologous genes.     

Calcium-Dependent Transglutaminase of Rat Sympathetic Ganglion in Development and After Nerve Injury

The activity of transglutaminase (TG) was examined in the rat superior cervical ganglion (SCG) during development and after postganglionic nerve crush. During postnatal development the enzyme activity is increased by sevenfold in parallel to protein content of the ganglion and reaches adult levels by day 35 after birth. The endogenous activity (enzyme activity assayed in the absence of the exogenous substrate) during development is transiently elevated with a peak at day 21 postnatal. In the adult ganglion the enzyme specific activity is evenly distributed in all subcellular compartments, but most of it is contained in the cytosol. Within the first hour after axotomy TG activity is rapidly and transiently elevated. The peak value, 80% above control levels, is attained by 30 min postoperative. At this time the activity is increased in all subcellular fractions, but the endogenous activity is selectively increased in the fraction containing nuclei. The enhanced TG activity after axotomy can be prevented by topical treatments with verapamil, an inhibitor of voltage-dependent calcium fluxes across excitable membranes, or with the calcium chelator EGTA. The results show that intracellular TG activity is present in the SCG and that it increases with postnatal growth of the ganglion. After axotomy the enzyme activity is rapidly and transiently increased in the ganglion and this elevation critically depends on calcium fluxes.     

Carbon dioxide-independent and -dependent components of light inhibition of the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene in oat leaves

Light inhibits while carbon dioxide enhances the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene in oat ( Avena sativa L. cv. Victory) leaf segments. The possibility that the light inhibition is mediated through changes of carbon dioxide has been investigated. The level of CO₂ increases or decreases in the sealed incubation vial in darkness or in light, respectively, which can apparently account for the differences in ACC-dependent ethylene production between the dark and light treatments. However, although the evolution of ethylene from ACC in the dark is reduced upon depletion of CO₂, the difference between light and dark is still very noticeable. Moreover, the production of the ethylene in CO₂-free air in the dark was still higher than in the light, where the concentration of CO₂ was 0.01%. It is proposed that the light effect on the conversion of ACC to ethylene is composed of two distinguishable components: one CO₂-mediated and the other CO₂-independent.     

Strain differences in rat adrenal biosynthetic enzymes and stress-induced increases in plasma catecholamines.

We have examined in two inbred rat strains basal and stress-induced increases in plasma levels of epinephrine (EPI) and norepinephrine (NE) and compared these with activities of the adrenal enzymes involved in the synthesis of catecholamines. There were no differences in basal levels of NE and EPI in plasma of adult male rats of the Wistar-Kyoto (WKY) and Brown-Norway (B-N) strains. However, following 5 min. of intermittent footshock, plasma levels of both catecholamines were twice as high in WKY rats as in B-N rats. In the adrenals of unstressed rats, activities of tyrosine hydroxylase and dopamine-beta-hydroxylase were significantly higher in B-N rats. In addition, the adrenal weights and the contents of NE but not EPI were greater in B-N rats. Thus, in these two rat strains, the capacity of the adrenal gland to synthesize and store catecholamines appeared to be inversely related to plasma levels of NE and EPI after stress. The differences between the strains appeared to be due to differences in the rates of removal of catecholamines from the peripheral circulation as well as to differences in the rate of release of catecholamines from the sympatho-adrenal medullary system. Thus biosynthetic enzyme activities need not be related directly to the capacity to release and elevate plasma levels of catecholamines following stressful stimulation.     

The T-Cell factor specific for poly(Tyr,Glu)-Poly(Pro)-Poly(Lys) is an I-Region gene product

The nature of a T-cell factor specific for poly(Tyr,Glu)-poly(Pro)-poly(Lys) [(T,G)-pro-L] was established in the present study. The activity of the (T,G)-Pro-L-specific factor was not removed by anti-mouse immunoglobulin Sepharose columns, suggesting that it is not a classical immunoglobulin. On the other hand, the factor lost its activity after passage through immunoadsorbents prepared with anti-H-2 sera raised against theH-2 haplotypes of the mouse strains in which the factor was prepared. Furthermore, this factor was adsorbed byI region-specific antisera but not by antisera directed against theI-J andI-C subregions as well as theK andD regions of theH-2 complex. Thus, the (T,G)-Pro-L-specific T-cell factor is most probably anI-A subregion gene product.     

Macrophage Specific Caspase-1/11 Deficiency Protects against Cholesterol Crystallization and Hepatic Inflammation in Hyperlipidemic Mice

Background & Aims

While non-alcoholic steatohepatitis (NASH) is characterized by hepatic steatosis combined with inflammation, the mechanisms triggering hepatic inflammation are unknown. In Ldlr^-/- mice, we have previously shown that lysosomal cholesterol accumulation in Kupffer cells (KCs) correlates with hepatic inflammation and cholesterol crystallization. Previously, cholesterol crystals have been shown to induce the activation of inflammasomes. Inflammasomes are protein complexes that induce the processing and release of pro-inflammatory cytokines IL-1b and IL-18 via caspase-1 activation. Whereas caspase-1 activation is independent of caspase-11 in the canonical pathway of inflammasome activation, caspase-11 was found to trigger caspase-1-dependent IL-1b and IL-18 in response to non-canonical inflammasome activators. So far, it has not been investigated whether inflammasome activation stimulates the formation of cholesterol crystals. We hypothesized that inflammasome activation in KCs stimulates cholesterol crystallization, thereby leading to hepatic inflammation.

Methods

Ldlr ^-/- mice were transplanted (tp) with wild-type (Wt) or caspase-1/11^-/- (dKO) bone marrow and fed either regular chow or a high-fat, high-cholesterol (HFC) diet for 12 weeks. In vitro, bone marrow derived macrophages (BMDM) from wt or caspase-1/11^-/- mice were incubated with oxLDL for 24h and autophagy was assessed.

Results

In line with our hypothesis, caspase-1/11^-/--tp mice had less severe hepatic inflammation than Wt-tp animals, as evident from liver histology and gene expression analysis in isolated KCs. Mechanistically, KCs from caspase-1/11^-/--tp mice showed less cholesterol crystals, enhanced cholesterol efflux and increased autophagy. In wt BMDM, oxLDL incubation led to disturbed autophagy activity whereas BMDM from caspase-1/11^-/- mice had normal autophagy activity.

Conclusion

Altogether, these data suggest a vicious cycle whereby disturbed autophagy and decreased cholesterol efflux leads to newly formed cholesterol crystals and thereby maintain hepatic inflammation during NASH by further activating the inflammasome.