Protein L-isoaspartyl methyltransferase (PIMT) is suggested to play a role in the repair of aged protein spontaneously incorporated with isoaspartyl residues. We generated PIMT-deficient mice by targeted disruption of the PIMT gene to elucidate the biological role of the gene in vivo. PIMT-deficient mice died from progressive epileptic seizures with grand mal and myoclonus between 4 and 12 weeks of age. An anticonvulsive drug, dipropylacetic acid (DPA), improved their survival but failed to cure the fatal outcome. L-Isoaspartatate, the putative substrate for PIMT, was increased ninefold in the brains of PIMT-deficient mice. The brains of PIMT-deficient mice started to enlarge after 4 weeks of age when the apical dendrites of pyramidal neurons in cerebral cortices showed aberrant arborizations with disorganized microtubules. We conclude that methylation of modified proteins with isoaspartyl residues is essential for the maintenance of a mature CNS and that a deficiency in PIMT results in fatal progressive epilepsy in mice.

We investigated some neurochemical changes that take place in the spinal cord dorsal horn in a mouse model of neuropathic cancer pain. The model was produced by inoculation of Meth-A sarcoma cells to the vicinity of the sciatic nerve, which resulted in growth of a tumor mass embedding the nerve. Hind paw-lifting, a behavioral sign of spontaneous pain, was at maximum on Day 18, but decreased thereafter. The decrease was likely caused by progression of motor paralysis. On Day 18, thermal and mechanical pain thresholds of the affected paw were significantly increased. Histologically, the sciatic nerve presented damages to both unmyelinated and myelinated fibers on Day 18, which were more pronounced on Day 25. In the spinal cord, c-Fos-positive cells were significantly increased in the superficial and deep layers on Day 18. The number of c-Fos-positive cells in the superficial layer correlated with the duration of paw-lifting. The increase in c-Fos-positive cells was still present on Day 25 despite decreased paw-lifting. Substance P and calcitonin gene-related peptide were up-regulated on Day 18 but down-regulated on Day 25. A marked up-regulation of dynorphin A (DynA) was present on Day 18 and persisted through Day 25. Our model caused progressive damage to the sciatic nerve and presented spontaneous pain-behavior while the paw became hyposensitive to mechanical and thermal stimuli. Since the up-regulation of DynA in the dorsal horn persisted and paralleled the increase in c-Fos-positive cells, the release of DynA may be associated with spontaneous pain in our model.

The rostral anteroventral cochlear nucleus (AVCN) of the chinchilla provides a preparation in which neuronal cell bodies and synapses in the mammalian central nervous system can be examined after direct freezing and freeze-substitution of rapidly excised brain stem slices. Cell bodies and synapses in the freeze-substituted AVCN differed from those in perfusion-fixed AVCN in several interesting respects. Despite of these differences, four types of synaptic terminal were distinguished in freeze-substituted AVCN and correlated with the four well-known types of perfusion-fixed terminal. Since the transmitter at each of the four types of terminal has been tentatively identified, the structure of synaptic vesicles and junctions in the freeze-substituted terminals could be related to transmitter type. Synaptic vesicles were uniformly round, but their diameters, deployment, and related cytoskeletal elements near the synaptic junction differed in each chemical type of synapse; the synapses thought to be cholinergic, for instance, had only a few vesicles clustered at their presynaptic junctions while the rest of the vesicles were separated from the junction by a network of fine filaments. Two types of filamentous components, short vertical projections from the postsynaptic membrane and thin filaments protruding from these projections, comprised the basic structure of the postsynaptic specialization, but their sizes and distribution differed at each chemical type of terminal. For instance, the postsynaptic specialization at the glycine terminal was distinguished by numerous thin filaments which curved sideways to run parallel to the plasmalemma. Thus, freeze-substitution gives new information about structural differences between chemically different types of synapses, which may reflect differences in their transmitter storage, release, and reception. In addition, the AVCN preparation is of general interest in making cell bodies in the mammalian central nervous system available to the various structural and analytical techniques which depend on direct, rapid freezing.