Expression of human cationic trypsinogen (PRSS1) in murine acinar cells promotes pancreatitis and apoptotic cell death.

Hereditary pancreatitis (HP) is an autosomal dominant disease that displays the features of both acute and chronic pancreatitis. Mutations in human cationic trypsinogen (PRSS1) are associated with HP and have provided some insight into the pathogenesis of pancreatitis, but mechanisms responsible for the initiation of pancreatitis have not been elucidated and the role of apoptosis and necrosis has been much debated. However, it has been generally accepted that trypsinogen, prematurely activated within the pancreatic acinar cell, has a major role in the initiation process. Functional studies of HP have been limited by the absence of an experimental system that authentically mimics disease development. We therefore developed a novel transgenic murine model system using wild-type (WT) human PRSS1 or two HP-associated mutants (R122H and N29I) to determine whether expression of human cationic trypsinogen in murine acinar cells promotes pancreatitis. The rat elastase promoter was used to target transgene expression to pancreatic acinar cells in three transgenic strains that were generated: Tg(Ela-PRSS1)NV, Tg(Ela-PRSS1*R122H)NV and Tg(Ela-PRSS1*N29I)NV. Mice were analysed histologically, immunohistochemically and biochemically. We found that transgene expression is restricted to pancreatic acinar cells and transgenic PRSS1 proteins are targeted to the pancreatic secretory pathway. Animals from all transgenic strains developed pancreatitis characterised by acinar cell vacuolisation, inflammatory infiltrates and fibrosis. Transgenic animals also developed more severe pancreatitis upon treatment with low-dose cerulein than controls, displaying significantly higher scores for oedema, inflammation and overall histopathology. Expression of PRSS1, WT or mutant, in acinar cells increased apoptosis in pancreatic tissues and isolated acinar cells. Moreover, studies of isolated acinar cells demonstrated that transgene expression promotes apoptosis rather than necrosis. We therefore conclude that expression of WT or mutant human PRSS1 in murine acinar cells induces apoptosis and is sufficient to promote spontaneous pancreatitis, which is enhanced in response to cellular insult.

Termination of the geniculocortical projection in the striate cortex of macaque monkey: a quantitative immunoelectron microscopic study.

The goal of this present study was to derive a new estimate of the synaptic contribution of the dorsal lateral geniculate nucleus (dLGN) to the subdivisions of its main recipient layer, layer 4C, of striate cortex of macaque monkey. The projection from the dLGN and its terminal boutons within layer 4C were visualized by immunodetection of the calcium binding protein, parvalbumin (PV), which is expressed in relay cells of the dLGN. The proportion of asymmetric synapses formed by PV-positive boutons within the alpha and beta sublayers of 4C was estimated by using a nonbiased stereological counting method. The proportion of asymmetric synapses contributed by the PV-positive boutons to layer 4Calpha is 8.7%; to 4Cbeta is 6.9%. Assuming all the PV-positive asymmetric synapses derive from the dLGN relay cells, this gives a ratio of dLGN synapses per neuron of 192 in layer 4Calpha and 128 in layer 4Cbeta. Thus, the recurrent excitatory input from neighboring cortical neurons must play an important part in responses of the neurons lying at the input stage of the cortical circuit.

Synaptic and nonsynaptic localization of the GluR1 subunit of the AMPA-type excitatory amino acid receptor in the rat cerebellum.

The cellular and subcellular distribution of the GluR1 subunit of the AMPA-type excitatory amino acid receptor was determined in the cerebellar cortex of rat using immunocytochemistry. Two polyclonal antibodies were raised against the N- and C-terminal regions of the subunit. They both labeled a band in immunoblots of rat cerebellar membranes with a molecular weight corresponding to that predicted for this subunit of 105 kDa molecular mass. In light microscopy the distribution of immunoreactivity for the two antibodies was very similar. The molecular layer was strongly immunoreactive whereas no labeling was observed in the granular layer. Electron microscopy revealed that the antibody raised against the N-terminal part of the subunit recognizes an extracellular epitope(s), whereas the antibody against the C-terminal part recognizes an intracellular epitope(s) along the plasma membrane. In Bergmann glial cells the endoplasmic reticulum, Golgi apparatus, and multivesicular bodies were labeled, presumably demonstrating sites of synthesis and degradation for the GluR1 subunit, respectively. Immunoreactivity was associated with Bergmann glial processes surrounding Purkinje cell dendrites, spines, and the glutamate-releasing axon terminals of the parallel and climbing fibers. This suggests that the neurotransmitter glutamate and the AMPA-type glutamate receptors are involved in neuronal/glial communication. The GluR1 subunit was also found at glial membranes in contact with other glial cells. Purkinje cells showed immunoreactivity in the endoplasmic reticulum and multivesicular bodies. No immunoreaction was detected in basket and stellate cells. Immunoreactivity was observed at type 1 synaptic junctions, including the synaptic cleft. These synaptic junctions were between spines, often originating from Purkinje cell dendrites, and parallel or climbing fiber terminals. Our results demonstrate that the GluR1 subunit of the AMPA-type ionotropic excitatory amino acid receptor is present at both parallel and climbing fiber synapses, which are surrounded by glial processes containing the same receptor subunit.