A Search for Discrete Cholinergic Nuclei in the Human Ventral Forebrain

Abstract: Slices cut from five frozen human brains were dissected into 2-mm cubes and assayed for choline acetyltransferase (ChAT) activity and protein content. A pattern of enrichment of ChAT activity was found ventral to the anterior commissure; this finding is consistent with the location of the enzyme in the cells of the nucleus basalis of Meynert. The region beneath the anterior commissure was the only place a discrete enrichment of activity could be found, and the precise topography of the enrichment was somewhat variable from brain to brain. The results are discussed in the light of recent knowledge concerning the source of the cortical cholinergic innervation.     

Heterotopic microvascular growth plate transplantation of the proximal fibula: an experimental canine model

The reconstructive potential of microvascular transplantation of skeletal growth plates was investigated through heterotopic transfers. The distal radius was resected in two series of puppies of a known large breed and substituted with a microsurgically revascularized transplant from the proximal fibula. Evaluation was conducted through serial roentgenograms, goniometric registration of joint mobility, volume measurements, histology, and fluorescent bone labeling. In the first series, development of neuropathic-like destruction of the weight-bearing graft ensued in the majority of the animals. In the second series, prolonged protection from weight bearing inhibited this destruction and resulted in hypertrophy of the revascularized epiphyseal end of the transplant but clearly reduced longitudinal growth, with only one transplant exhibiting longitudinal growth that exceeded 50 percent of the value for the control. This experiment demonstrates that skeletal growth plates possess a capacity for hypertrophy under the influence of increased loads. Whether this adaptability is sufficient to allow microvascular transplantation of growth plates to become a clinically useful procedure in children remains unclear. Further laboratory investigations are mandatory prior to clinical application of microvascular transfers of epiphyseal growth plates.     

The impact of ticks on pheasant territoriality

Pheasants are competent reservoir hosts for the Lyme disease spirochaete, Borrelia burgdorferi s.l., and carry large, but highly over-dispersed, infestations of the vector ticks, Ixodes ricinus . The effects of experimental reduction of tick infestation levels on the survival and territorial behaviour of male pheasants were studied. Over three years in two woodlands in southern England, birds were marked individually and half were fitted with a slow-release acaricide, which substantially reduced their tick burdens from March to August. Acaricide treatment affected reproductive success but had no discernible impact on the survival rates of male pheasants. The degree of wattle inflation by males, an indicator of territorial status and a correlate of harem acquisition, was significantly greater among treated males. In each year, a significantly higher proportion of treated (overall 44%) than control (22%) males acquired harems. Males that acquired females ranged over small areas on field edges. By contrast, those with no females ranged more widely in woods and the adjoining fields, increasing their exposure to questing ticks. The relative contribution of such roving males to tick-borne pathogen transmission may thus increase.     

Identification of highly reactive cysteinyl and methionyl residues of rabbit muscle phosphofructokinase

The reactivity of the 16 thiol groups of rabbit skeletal muscle phosphofructokinase has been studied extensively over the past 20 years. Several of these thiols show high reactivity with a variety of reagents, display differential reactivity in the presence of allosteric ligands and substrates, and appear to be important to function because their modification changes activity and regulatory properties. In the present study, the location in the primary structure of several highly reactive thiol groups has been established by reaction with [14C]iodoacetate. In the course of these studies, 2 methionyl residues that are located at or near proposed ligand-binding sites are readily carboxymethylated by iodoacetate. In addition to confirming the presence of the most reactive thiol group at sequence position 88, a thiol protected from reaction by the presence of fructose-6-P and cyclic AMP has been found at position 169. Cysteine 169 is close to a residue important to the binding of fructose-6-P in the homologous structure from Bacillus stearothermophilis phosphofructokinase. The modification of Cys-169 brings about extensive, but not total, loss of activity. Another cysteine, at position 232, was found to be highly reactive also. Substrate provided partial protection against carboxymethylation at this position. Carboxymethylation of enzyme restricted to methionines 74 and 173 brought about no changes in the total activity or in the ATP inhibition profile of the enzyme. This is significant since position 74 was projected on the basis of the homologous procaryotic structure to be important in the binding of nucleotide to the allosteric site.     

Muscarinic Agonists Evoke Neurotransmitter Release: Possible Roles for Phosphatidyl Inositol Bisphosphate Breakdown Products in Neuromodulation

Carbachol (CCh), a muscarinic agonist that elicits the formation of inositol trisphosphate (IP3) and diacylglycerol (DG), induces a calcium-dependent [3H]norepinephrine ([3H]NE) release [IC50 = (2.7 +/- 0.5) X 10(-4) M] in rat brain slices. Similarly, other muscarinic agonists evoke [3H]NE release which is specifically inhibited by muscarinic antagonists such as 3-quinuclidinyl benzilate, atropine, and N-methyl-4-piperidyl benzilate. The atropine-sensitive evoked release is effectively inhibited by neomycin (IC50 = 50 microM), a phospholipase C inhibitor that interferes with IP3-dependent cellular processes. In addition, polymyxin B, a rather selective inhibitor of protein kinase C (PK-C), abolishes the agonist-mediated release with a half-maximal effective concentration of 0.53 microM (750 ng/ml). These results have a significant implication for the mechanism by which agonists generating IP3 and DG act as inducers of neurotransmitter release in the CNS. However, since both neomycin and polymyxin B act also as N-calcium-channel blockers, other possible mechanisms are discussed. The CCh-induced release suggests that in the CNS an agonist-receptor interaction leads to a calcium-dependent neurotransmitter release, most likely via promoting the IP3/DG as second messengers followed by activation of PK-C.     

Vascularized bone allografts: in vitro assessment of cell-mediated and humoral responses

The immunologic consequences of transplantation of vascularized bone allografts have not been previously characterized. In this study, knee allografts, both vascularized and nonvascularized, were transplanted from Lewis rats to Brown Norway rats across a strong histocompatibility barrier. A total of 66 transplants and 8 control animals were evaluated. The vascularized knee grafts consisted of 1 cm of proximal tibia and distal femur with a minimal muscular cuff isolated on the femoral vessels, and these were transplanted to a heterotopic, subcutaneous position on the abdominal wall of the recipient rat. Nonvascularized allografts (identical but without anastomoses) were transplanted for comparison. The cell-mediated response was measured by lymphocytotoxicity assay, and the humoral response was measured by cytotoxic antibody assay, both employing 51Cr-labeled target cells. The timing and intensity of the immune response differed according to the type of graft. The vascularized bone allografts generated significant cell-mediated and humoral responses as early as 5 days posttransplant. A significant humoral response in nonvascularized bone allografts was not apparent until day 14, while cell-mediated response in these grafts was variable. These findings were correlated with the histologic appearance of the grafted tissue. Cyclosporine, which was administered to one group of vascularized bone allografts, resulted in the suppression of both types of immune responses. The histologic appearance of this group resembled that of isografts transplanted as controls. The clinical application of vascularized bone allografts may offer significant advantages over nonvascularized allografts in the reconstruction of massive bone defects. Complications such as nonunion, fracture, and collapse of articular segments seen in nonvascularized allograft transplantation may be avoided by preservation of the blood supply to the graft. Characterization of the immune response to vascularized bone allografts may subsequently allow the manipulation of the host and/or graft tissue and promote graft incorporation.     

Induction of methotrexate release from rat hepatocytes in suspension by alpha-adrenergic agents: involvement of calcium and metabolic energy

Hepatocytes in suspension which have accumulated [3H]methotrexate release the antifolate compound into the medium upon exposure to alpha-adrenergic agents. In the presence of metabolic poisons, such as sodium azide, dinitrophenol, or dicumarol, the release of methotrexate is attenuated, indicating that integrity of the cellular metabolic apparatus is required for response to the hormonal stimulus. In the presence of millimolar concentrations of the organic acid, probenecid, release of cellular methotrexate may be reduced (1 mM probenecid) or eliminated (2 mM probenecid), suggesting the involvement of a "membrane carrier." Microtubule poisons such as vincristine, vinblastine, and griseofulvin do not modify epinephrine + isobutyl methyl xanthine (IBMX)-induced release of methotrexate. The involvement of calcium in release of methotrexate from the hepatocyte is substantiated by a dose-dependent response to the calcium ionophore, A23187, in the presence of calcium, with a lack of response in the absence of calcium. These effects of A23187 are not related to inhibition of methotrexate influx. Other putative "calcium antagonists" such as tetracaine, neomycin sulfate, and 3,4,5-trimethoxybenzoic acid [8-(diethylamino)octyl ester], do not interfere with epinephrine + IBMX-induced release of [3H]methotrexate, suggesting that these agents may not be effective probes of calcium flux in the liver cell.     

Effects of Neuronal Activity on Inositol Phospholipid Metabolism in the Rat Autonomic Nervous System

The effect of nerve stimulation on inositol phospholipid hydrolysis in autonomic tissue was assessed by direct measurement of [3H]inositol phosphate production in ganglia that had been preincubated with [3H]inositol. Within minutes, stimulation of the preganglionic nerve increased the [3H]inositol phosphate content of the superior cervical sympathetic ganglion indicating increased hydrolysis of inositol phospholipids. This effect was blocked in a low Ca2+, high Mg2+ medium. It was also greatly reduced when nicotinic and muscarinic antagonists were present together in normal medium. However, neither the nicotinic antagonist nor the muscarinic antagonist alone appeared to be as effective as both in combination. In other experiments, stimulation of the vagus nerve caused dramatic increases in [3H]inositol phosphate in the nodose ganglion but did not increase [3H]inositol phosphate in the nerve itself. This effect was insensitive to the cholinergic antagonists. Thus, neuronal activity increased inositol phospholipid hydrolysis in a sympathetic ganglion rich in synapses, as well as in a sensory ganglion that contains few synapses. In the sympathetic ganglion, synaptic stimulation activated inositol phospholipid hydrolysis and this was primarily due to cholinergic transmission; both nicotinic and muscarinic pathways appeared to be involved.