Dose-response characteristics of methylphenidate on locomotor behavior and on sensory evoked potentials recorded from the VTA, NAc, and PFC in freely behaving rats.

BACKGROUND: Methylphenidate (MPD) is a psychostimulant commonly prescribed for attention deficit/hyperactivity disorder. The mode of action of the brain circuitry responsible for initiating the animals' behavior in response to psychostimulants is not well understood. There is some evidence that psychostimulants activate the ventral tegmental area (VTA), nucleus accumbens (NAc), and prefrontal cortex (PFC). METHODS: The present study was designed to investigate the acute dose-response of MPD (0.6, 2.5, and 10.0 mg/kg) on locomotor behavior and sensory evoked potentials recorded from the VTA, NAc, and PFC in freely behaving rats previously implanted with permanent electrodes. For locomotor behavior, adult male Wistar-Kyoto (WKY; n = 39) rats were given saline on experimental day 1 and either saline or an acute injection of MPD (0.6, 2.5, or 10.0 mg/kg, i.p.) on experimental day 2. Locomotor activity was recorded for 2-h post injection on both days using an automated, computerized activity monitoring system. Electrophysiological recordings were also performed in the adult male WKY rats (n = 10). Five to seven days after the rats had recovered from the implantation of electrodes, each rat was placed in a sound-insulated, electrophysiological test chamber where its sensory evoked field potentials were recorded before and after saline and 0.6, 2.5, and 10.0 mg/kg MPD injection. Time interval between injections was 90 min. RESULTS: Results showed an increase in locomotion with dose-response characteristics, while a dose-response decrease in amplitude of the components of sensory evoked field responses of the VTA, NAc, and PFC neurons. For example, the P3 component of the sensory evoked field response of the VTA decreased by 19.8% +/- 7.4% from baseline after treatment of 0.6 mg/kg MPD, 37.8% +/- 5.9% after 2.5 mg/kg MPD, and 56.5% +/- 3.9% after 10 mg/kg MPD. Greater attenuation from baseline was observed in the NAc and PFC. Differences in the intensity of MPD-induced attenuation were also found among these brain areas. CONCLUSION: These results suggest that an acute treatment of MPD produces electrophysiologically detectable alterations at the neuronal level, as well as observable, behavioral responses. The present study is the first to investigate the acute dose-response effects of MPD on behavior in terms of locomotor activity and in the brain involving the sensory inputs of VTA, NAc, and PFC neurons in intact, non-anesthetized, freely behaving rats previously implanted with permanent electrodes.

Protein quaternary structure determination using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and chemical crosslinking

A means of analyzing protein quaternary structure using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI MS) and chemical crosslinking was evaluated. Proteins of known oligomeric structure, as well as monomeric proteins, were analyzed to evaluate the method. The quaternary structure of proteins of unknown or uncertain structure was investigated using this technique. The stoichiometry of recombinant E. coli carbamoyl phosphate synthetase and recombinant human farnesyl protein transferase were determined to be heterodimers using glutaraldehyde crosslinking, agreeing with the stoichiometry found for the wild type proteins. The stoichiometry of the gamma subunit of E. coli DNA polymerase III holoenzyme was determined in solution without the presence of other subunits to be a homotetramer using glutaraldehyde crosslinking and MALDI MS analysis. Chi and psi subunits of E. coli DNA polymerase III subunits appeared to form a heterodimer when crosslinked with heterobifunctional photoreactive crosslinkers.^ Comparison of relative % peak areas obtained from MALDI MS analysis of crosslinked proteins and densitometric scanning of silver stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels showed excellent qualitative agreement for the two techniques, but the quantitative analyses differed, sometimes significantly. This difference in quantitation could be due to SDS-PAGE conditions (differential staining, loss of sample) or to MALDI MS conditions (differences in ionization and/or detection). Investigation of pre-purified crosslinked monomers and dimers recombined in a specific ratio revealed the presence of mass discrimination in the MALDI MS process. The calculation of mass discrimination for two different MALDI time-of-flight instruments showed the loss of a factor of approximately 2.6 in relative peak area as the m/z value doubles over the m/z range from 30,000 to 145,000 daltons.^ Indirect symmetry was determined for tetramers using glutaraldehyde crosslinking with MALDI MS analysis. Mathematical modelling and simple graphing allowed the determination of the symmetry for several tetramers known to possess isologous D2 symmetry. These methods also distinguished tetramers that did not fit D2 symmetry such as apo-avidin. The gamma tetramer of E. coli DNA polymerase III appears to have isologous D2 symmetry. ^

COMPARISON OF MEASUREMENTS OF ABSORBED DOSE TO WATER USING A WATER CALORIMETER AND IONIZATION CHAMBERS FOR CLINICAL RADIOTHERAPY PHOTON AND ELECTRON BEAMS

With the development of the water calorimeter direct measurement of absorbed dose in water becomes possible. This could lead to the establishment of an absorbed dose rather than an exposure related standard for ionization chambers for high energy electrons and photons. In changing to an absorbed dose standard it is necessary to investigate the effect of different parameters, among which are the energy dependence, the air volume, wall thickness and material of the chamber. The effect of these parameters is experimentally studied and presented for several commercially available chambers and one experimental chamber, for photons up to 25 MV and electrons up to 20 MeV, using a water calorimeter as the absorbed dose standard and the most recent formalism to calculate the absorbed dose with ion chambers.^ For electron beams, the dose measured with the calorimeter was 1% lower than the dose calculated with the chambers, independent of beam energy and chamber.^ For photon beams, the absorbed dose measured with the calorimeter was 3.8% higher than the absorbed dose calculated from the chamber readings. Such differences were found to be chamber and energy independent.^ The results for the photons were found to be statistically different from the results with the electron beams. Such difference could not be attributed to a difference in the calorimeter response. ^