Disseminated encephalitozoonosis in captive, juvenile, cotton-top (Saguinus oedipus) and neonatal emperor (Saguinus imperator) tamarins in North America.

Disseminated encephalitozoonosis was diagnosed in 2 sibling, juvenile, cotton-top tamarins (Saguinus oedipus) and 3 sibling, neonatal, emperor tamarins (S. imperator) by use of histologic examination, histochemical analysis, electron microscopy, and polymerase chain reaction (PCR) analysis with nucleotide sequencing. All tamarins were captive born at zoos in North America and died with no premonitory signs of disease. The main pathologic findings were myocarditis (4/5), hepatitis (3/5), interstitial pneumonia (3/5), skeletal myositis (3/5), meningoencephalitis (2/5), adrenalitis (2/5), tubulointerstitial nephritis (1/5), myelitis (1/5), sympathetic ganglioneuritis (1/5), and retinitis (1/5). Central nervous system lesions were the most prominent findings in cotton-top tamarins. The inflammation was predominantly lymphocytic and suppurative in cotton-top tamarins, whereas emperor tamarins had granulomatous or lymphoplasmacytic lesions. Intralesional periodic acid-Schiff-, gram-, or acid-fast (or all 3)-positive, oval-to-elliptical shaped organisms were found in 1 cotton-top and the 3 emperor tamarins. By electron microscopy, these organisms were consistent with microsporidia of the genus Encephalitozoon. E. cuniculi genotype III was detected by PCR analysis and sequencing in paraffin-embedded brain, lung, and bone marrow specimens from the cotton-top tamarins. Although PCR results were negative for one of the emperor tamarins, their dam was seropositive for E. cuniculi by ELISA and Western blot immunodetection. These findings and recent reports of encephalitozoonosis in tamarins in Europe suggest that E. cuniculi infection may be an emerging disease in callitrichids, causing high neonatal and juvenile mortality in some colonies. The death of 2 less than 1-day-old emperor tamarins from a seropositive dam supports the likelihood of vertical transmission in some of the cases reported here.

Pharmacological modulation of Alzheimer's beta-amyloid precursor protein levels in the CSF of rats with forebrain cholinergic system lesions.

Abnormal deposition and accumulation of Alzheimer's amyloid beta-protein (A beta) and degeneration of forebrain cholinergic neurons are among the principal features of Alzheimer's disease. Studies in rat model systems have shown that forebrain cholinergic deficits are accompanied by induction of cortical beta-amyloid precursor protein (beta-APP) mRNAs and increased levels of secreted beta-APP in the CSF. The studies reported here determined whether the CSF levels of secreted beta-APP could be altered pharmacologically. In different experiments, rats with lesions of the forebrain cholinergic system received injections of vehicle, a muscarinic receptor antagonist scopolamine, or one of two cholinesterase inhibitors - diisopropyl phosphorofluoridate (DFP) or phenserine. Scopolamine was administered to determine whether the levels of beta-APP in the CSF could be increased by anticholinergic agents. The cholinesterase inhibitors were administered to determine whether the forebrain cholinergic system lesion-induced increases in CSF beta-APP could be reduced by cholinergic augmentation. Scopolamine administration led to a significant increase in the CSF levels of secreted beta-APP in sham-lesioned rats. Phenserine, a novel, reversible acetyl-selective cholinesterase inhibitor, significantly decreased the levels of secreted beta-APP in the CSF of forebrain cholinergic system-lesioned rats whereas DFP, a relatively non-specific cholinesterase inhibitor, failed to affect CSF levels of secreted beta-APP. These results suggest that the levels of secreted beta-APP in the CSF can be pharmacologically modulated but that this modulation is dependent upon the status of the forebrain cholinergic system and the pharmacological properties of the drugs used to influence it.

Application of stable isotope tracer combined with mass spectrometric detection for studying myo-inositol uptake by cultured neurons from fetal mouse: effect of trisomy 16.

A gas chromatographic (GC)/mass spectrometric method for studying myo-inositol uptake by neurons in vitro is described. Cultured cortical neurons from fetuses of diploid and trisomy 16 mouse (animal model for Down syndrome) were incubated with a physiological concentration of hexadeuterated myo-inositol for 2-40 min. Washed cells were lysed and scyllo-inositol (internal standard) was added to the intracellular material which contained labeled myo-inositol taken up by the cells as well as the endogenous, unlabeled myo-inositol. The samples were evaporated to dryness and the analytes were converted into acetate derivatives. The components were separated by capillary GC, and the m/z 379 ion for labeled myo-inositol and the m/z 373 ion for myo-inositol and scyllo-inositol generated by chemical ionization in an ion trap mass spectrometer were monitored. Quantitation of the deuterium-labeled myo-inositol taken up by the neuron along with endogenous myo-inositol was achieved for 2-40 min of incubation. The labeled myo-inositol uptake was linear for up to 20 min and was Na+ dependent in these neurons. This non-radioisotope method was used to demonstrate a significant (40%) increase in the rate of myo-inositol uptake by cortical neurons from the trisomy 16 mouse relative to control neurons. An increased myo-inositol uptake is consistent with evidence that the myo-inositol transporter gene is on both human chromosome 21 and mouse chromosome 16, and that myo-inositol concentrations are elevated in cerebrospinal fluid from adult Down syndrome individuals and brains from the fetal trisomy 16 mouse.

Quantification of acetylcholine in cell culture systems by semi-micro high-performance liquid chromatography and electrospray ionization mass spectrometry.

Electrospray ionization (ESI) was used to adapt the thermospray ionization liquid chromatographic/mass spectrometric (LC/MS) method of Liberato et al. to the determination of acetylcholine and choline released from cells in culture. A clonal cell line derived from a rat pheochromocytoma (PC12) was used and cultures depolarized in the presence of cholinesterase inhibitor to maximize the recovery of acetylcholine from the culture medium and protect against its hydrolysis. Concentration of acetylcholine and choline released into the incubation medium was accomplished with Sep-Pak C16 cartridges. The preformed analyte cations were resolved by reversed-phase high-performance liquid chromatography and analyzed without derivatization by electrospray ionization mass spectrometry with the use of acetyl[2H9]choline as internal standard. LC/MS allows for the direct determination of acetylcholine and choline, which is not possible with other methods.

Brain accumulation of myo-inositol in the trisomy 16 mouse, an animal model of Down's syndrome.

myo-Inositol and several other polyols were measured in the tissues of the trisomy 16 mouse (animal model of Down's Syndrome; human trisomy 21) and diploid controls. myo-Inositol was found to be selectively elevated in the brain of the trisomy 16 mouse. However, peripheral tissues showed no elevation. These results are consistent with the cerebrospinal fluid and plasma data reported previously on myo-inositol in Down's Syndrome subjects.

Increased inward current in septal neurons from the trisomy 16 mouse, a model for Down's syndrome.

We examined the electrophysiological properties of neurons cultured from the septum of the trisomy 16 mouse fetus, an animal model for Down's syndrome. The passive membrane properties were not different between trisomic and diploid septal neurons. We distinguished low-firing and high-firing populations of neurons based on differences in the firing rate evoked during current injection. Low-firing neurons fired three or fewer action potentials, high-firing neurons fired four or more. The membrane currents of low-firing trisomic neurons were not different from those of low-firing diploid neurons. However, high-firing trisomic neurons had an increased inward current and conductance, and a greater inward-to-outward conductance ratio. The increased current and conductance were independent of the passive electrical properties. The increased inward current in high-firing trisomic neurons was correlated with action potentials having faster depolarization rates. This greater excitability among this population of trisomic septal neurons, coupled with a reduced excitation in hippocampal neurons, may compromise septohippocampal and memory function.

Proctolin and excitation of the crayfish swimmeret system.

The ventral nerve cord of crayfish contains axons of five pairs of excitatory interneurons, each of which can activate the swimmeret system. Perfusion of the ventral nerve cord with the neuropeptide proctolin also activates the swimmeret system. The experiments reported here were conducted to test the hypothesis that one or more of these excitatory interneurons uses proctolin as a transmitter. Each of the five excitatory axons was located and stimulated separately in an individual crayfish, and similar motor activity was elicited by stimulating each of them. Quantitative comparison of spontaneous swimmeret motor patterns with activity caused by stimulating one of these excitatory axons, EC, or by perfusing with proctolin solutions showed that the motor patterns produced under these three conditions were not significantly different (P > 0.05). By using a new, affinity-purified proctolin antiserum, we labeled axons in the connective tissue between the last thoracic and first abdominal ganglion and compared the positions of labeled axons with the previously described positions of the excitatory axons. About 0.3% of the axons in these connective tissues showed proctolin-like immunoreactivity, but heavily labeled pairs of axons did occur bilaterally in the regions of excitatory swimmeret axons. The projections of these labeled axons into the abdominal ganglia were traced in serial plastic sections. Labeled processes were abundant in the lateral neuropils, the loci of the swimmeret pattern-generating circuitry. From this evidence, we propose that three of these excitatory swimmeret interneurons use proctolin as a transmitter, but that a fourth does not. The evidence for the fifth axon is ambiguous.

Modulation of the crayfish swimmeret rhythm by octopamine and the neuropeptide proctolin.

1. The swimmeret system can be excited by perfusing the neuropeptide proctolin through the isolated ventral nerve cord of the crayfish. Previously silent preparations begin to generate a characteristic motor pattern, the swimmeret rhythm, in the nerves that innervate the swimmerets. The response to proctolin is dose dependent and reversible. The threshold concentration of proctolin perfused through the ventral artery is approximately 10(-8) M. The EC50 is 1.6 X 10(-6) M. 2. Proctolin-induced motor patterns have periods and phases similar to those of spontaneously generated motor patterns. The durations of the bursts of impulses in power-stroke motor neurons generated in the presence of proctolin are, however, significantly longer than those that occur during spontaneous activity. 3. DL-Octopamine inhibits the swimmeret system, both when the system is spontaneously active and when it has been excited by proctolin. The inhibition by octopamine is dose dependent and reversible. The threshold for inhibition is approximately 10(-6) M, and the EC50 is approximately 5 X 10(-5) M. 4. Octopamine's effect is mimicked by its agonists, synephrine and norepinephrine. Synephrine has a lower threshold concentration than does octopamine, but norepinephrine is much less effective than octopamine. 5. Octopamine's inhibition is partially blocked by an antagonist, phentolamine. 6. Phentolamine also blocks inhibition of the swimmeret system by inhibitory command interneurons. This block is dose dependent and can be partially overcome by stimulating the command interneurons at higher frequencies. 7. Perfusion with 11 other suspected crustacean neurotransmitters and transmitter analogues did not similarly excite or inhibit the swimmeret system, so we suggest that proctolin and octopamine are transmitters used by the neurons that normally control expression of the swimmeret rhythm.