Sympathetic Sprouting: Time Course of Changes of Noradrenergic, Cholinergic, and Serotonergic Markers in the Denervated Rat Hippocampus

Abstract: As a first step for experiments investigating the presynaptic characteristics of sympathetic fibers grown into the denervated hippocampus, we studied the time course of changes of neurochemical markers in the rat hippocampus, subsequent to aspiration lesions of the fimbria-fornix and the overlying callosal and cortical structures. At various postsurgical delays (1, 2, 8, 24, and 40 weeks), the activity of choline acetyltransferase, the high-affinity synaptosomal uptake of choline and noradrenaline, and the concentrations of noradrenaline, serotonin, and 5-hydroxyindoleacetic acid were measured in a dorsal, an intermediate, and a ventral part of the hippocampus. Levels of all markers were significantly reduced shortly (1–2 weeks) after the lesions. However, whereas the cholinergic (choline uptake and choline acetyltransferase activity) and the serotonergic (concentrations of serotonin and 5-hydroxyindoleacetic acid) markers remained significantly reduced for up to 40 weeks, both noradrenergic markers recovered to near-normal (noradrenaline uptake) or even supranormal (noradrenaline concentration) levels, although with clear-cut differences in the time course and the regional characteristics. The noradrenaline content reached control levels already 8 weeks after lesion surgery and was about two to three times higher 40 weeks later, with the most dramatic effects in the ventral hippocampus. In contrast, high-affinity noradrenaline uptake reached control values only 24 weeks after lesion and exceeded them only in the ventral hippocampus 40 weeks after surgery. It is concluded (a) that hippocampal noradrenaline concentration is a more sensitive marker for sympathetic sprouting than high-affinity noradrenaline uptake and (b) that functional in vitro studies on hippocampal sympathetic ingrowth appear to fit optimal conditions in the ventral hippocampus at a delay of at least 40 weeks after surgery.     

Cyclic AMP-Mediated Enhancement of High-Affinity Choline Transport and Acetylcholine Synthesis in Brain

Abstract: Intracerebroventricular administration of N⁶, 2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate (db-cyclic AMP) to mice increased high-affinity choline transport (HAChT) into synaptosomal preparations from the hippocampus, striatum, and frontal cortex in a time-dose-, and brain region-dependent manner. Similar observations were made when the cyclic AMP analogue 8-bromo-cyclic AMP, the adenylyl cyclase activator forskolin, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine were administered. Inhibition of phosphatase 1 and 2A, with okadaic acid, increased basal choline transport and enhanced the response to db-cyclic AMP. The early increase of HAChT activity induced by db-cyclic AMP was blocked by H-7 and H-89, protein kinase A inhibitors, but not by cycloheximide, a protein synthesis inhibitor. Kinetic analysis of the early changes of HAChT revealed an increase in the apparent V_max without a change of the K_m for choline. Hemicholinium-3 (HC-3) binding was not altered when studied 1 h after db-cyclic AMP administration. In contrast, HC-3 binding and HAChT activity were both elevated when estimated 3 h after the treatment, and pretreatment with cycloheximide partially prevented the db-cyclic AMP-induced HAChT rise. As evidence that enhanced HAChT is associated with a direct action of cyclic AMP-dependent pathways on the cholinergic nerve terminals, addition of 8-bromocyclic AMP to isolated hippocampal synaptosomes induced an increase of HAChT that was prevented by H-89. Choline acetyltransferase activity was not affected at any time during the studies. The synthesis of acetylcholine, however, was enhanced 1 h after db-cyclic AMP addition. Our studies show that cyclic AMP-mimetic compounds appear to modulate the choline carrier by a dual mode: an early increase of the maximal velocity without a change of the number of HC-3 binding sites and a late rise of transport that is accompanied by an increase of HC-3 binding. We postulate that HAChT and consequently acetylcholine synthesis in vivo is modulated, in part, by protein kinase A.     

Carnitine,carnitine acetyltransferase,and glutathione in Alzheimer brain

Glutathione and total carnitine (i.e., free carnitine plus acid-soluble carnitine esters) were measured in an affected (superior frontal gyrus; SFG) and unaffected (cerebellum: CBL) region of Alzheimer disease (AD) and control brains. Average glutathione content in AD SFG (n=13) and AD CBL (n=7) (7.9±2.1 and 11.9±4.0 nmol/mg protein, respectively (mean ±S.D.)) was similar to that in control SFG (n=13) and CBL (n=6) (7.7±2.0 and 11.6±2.6 nmol/mg protein, respectively). However, glutathione increased significantly with age in AD brain (p=0.003) but not in control brain. Average total carnitine in AD SFG (84±47 pmol/mg protein; n=10) and AD CBL (108±86 pmol/mg protein; n=7) was not significantly different from that in the corresponding regions of control brain (148±97 (n=10) and 144±107 (n=6) pmol/mg protein, respectively). However, a significant decline of total carnitine with age in both regions was noted for AD brain, but not for control brain. Carnitine acetyltransferase activity in the AD SFG (n=13) was not significantly different from that of control SFG (n=13) (1.83±1.05 and 2.04±0.82 nmol/min/mg protein, respectively). However, carnitine acetyltransferase activity of AD CBL (n=7) was significantly lower than that of control CBL (n=6) (1.33±0.88 versus 2.26±0.66 nmol/min/mg protein; p=0.05).     

Amino acid concentrations and selected enzyme activities in rat auditory,olfactory, and visual systems

Homogenates of specific brain regions of three sensory systems (auditory, olfactory, and visual) were prepared from pigmented Long-Evans Hooded rats and assayed for amino acid concentrations and activities of glutaminase, aspartate aminotransferase (total, cytosolic, and, by difference, mitochondrial), malate dehydrogenase, lactate dehydrogenase, and choline acetyltransferase. Comparing the quantitative distributions among regions revealed significant correlations between AAT and aspartate, between glutaminase and glutamate, between glutamate and glutamine, and between AAT plus glutaminase, or glutaminase alone, and the sum of aspartate, glutamate, and GABA, suggesting a metabolic pathway involving the synthesis of a glutamate pool as precursor to aspartate and GABA. Of the inhibitory transmitter amino acids, GABA concentrations routinely exceeded those of glycine, but glycine concentrations were relatively high in brainstem auditory structures.     

Choline acetyltransferase- and peptide immunoreactivity of submucous neurons in the small intestine of the guinea-pig

Summary The peptides cholecystokinin (CCK), neuropeptide Y (NPY), somatostatin (SOM), substance P (SP) and vasoactive intestinal peptide (VIP), and the synthesizing enzyme for acetylcholine, choline acetyltransferase (ChAT) were localized immunohistochemically in nerve cell bodies of the submucous ganglia in the small intestine of the guinea-pig. VIP-like immunoreactivity was found in 45% of submucous neurons. ChAT immunoreactivity was observed in a separate group of nerve cells, which made up 54% of the total population. There were three subsets of neurons immunoreactive for ChAT: (1) ChAT neurons that also contained immunoreactivity for each of the peptides CCK, SOM and NPY, representing 29% of all submucous neurons; (2) ChAT neurons that also contained SP-like immunoreactivity, representing 11% of all submucous neurons, and (3) ChAT cells that did not contain any detectable amount of the peptides that were localized in this study.     

Mitochondrial mutagenesis in Saccharomyces cerevisiae. III. Nitrous acid.

Nitrous acid (NA) induced mutations efficiently in mitDNA, conferring resistance to erythromycin and weakly induces mit- mutations. In some strains of yeast it also enhanced rho- mutations. The frequencies of nuclear and mitochondrial mutations induced with NA are compared.     

Survival and transfer in the human gut of poorly mobilizable (pBR322) and of transferable plasmids from the same carrier E. coli

We examined the survival of a host Escherichia coli K-12 bacterium containing two transferable plasmids (pLM2, pSL222-4) and one poorly mobilizable plasmid (pBR322), and the transfer of these three plasmids to endogenous bacteria in the human intestinal tract. The survival of this plasmid-carrying host organism in four human volunteers was 3.5 to 6 days at recovery rates of 10^?1 to 10^?4. This finding was similar to our previous survival data on the same organism bearing a single plasmid. The K-12 strain appeared to be under a strong selective disadvantage in the human gut, since, even when bearing a tetracycline-resistant plasmid, its titer did not increase despite the administration of tetracycline. Studies of transferability showed that, while the transfer-depressed incFII plasmid pSL222-4 transferred at a frequency of 10^?1 in culture, its transfer in the human gut was much less frequent. The number of new recipients per donor cell ingested was about 10^?5, which included new recipients arising by multiplication. The recovery of pSL222-4 transcipients was enhanced by the administration of tetracycline on day 6. Neither the transfer-repressed, broad host range incP plasmid pLM2, nor the plasmid pBR322, could be detected in any endogenous host bacteria. Using the transfer and mobilization frequencies obtained in culture and the number of new recipients of pSL222-4 in the intestinal tract, we estimated that any in vivo mobilization of pBR322 to a new recipient could not occur at a frequency higher than 10^?12.     

Novel shuttle plasmid vehicles for Escherichia-Streptococcus transgeneric cloning

A novel plasmid vector that is able to replicate both in Escherichia coli and in Streptococcus sanguis is described. This 9.2-kb plasmid, designated pVA856, carries Cm^r, Tc^r and Em^r determinants that are expressed in E. coli. Only the Em^r determinant is expressed in S. sanguis. Both the Cm^r and the Tc^r of pVA856 may be insertionally inactivated. This plasmid affords several different cleavage-ligation strategies for cloning in E. coli followed by subsequent introduction of chimeras in to S. sanguis. In addition, we have modified a previously described E. coli-S. sanguis shuttle plasmid [pVA838; Macrina et al., Gene 19 (1982) 345–353], so that it is unable to replicate in S. sanguis. The utility of such a plasmid for cloning and selecting sequences enabling autonomous replication in S. sanguis is demonstrated.