An improved metabolic calibrator for stress test equipment: protocol for its clinical utilization.

In previous articles, the technical specifications, validation and clinical applicability of a new metabolic calibrator for stress test apparatuses were described. However no indication was given of a basic protocol for the current clinical practice. Here we present an organised check system to satisfy this aim. A method for the exhaustive evaluation of the technical reliability of the metabolic instruments is presented together with the proposal of a uniform simulation approach to organise checks on-line of the apparatuses' ability to follow correctly the various test phases (protocol). Examples of recordings of checks using the standardised approach are shown. The method seems to be very effective. Every operation is computer directed.

Light microscopic and ultrastructural investigation of the dopaminergic innervation of the ventrolateral outgrowth of the rat inferior olive.

The ventrolateral outgrowth of the inferior olive is involved in the control of compensatory eye movement responses to optokinetic stimuli about the horizontal axis that is perpendicular to the ipsilateral anterior semicircular canal. Combining immunocytochemistry with retrograde tracing of WGA-BSA-gold, we demonstrated in the present study that this olivary subnucleus receives a substantial dopaminergic input, and that the prerubral parafascicular area and its surrounding regions form the sole source of this input. In addition, we investigated the postsynaptic distribution of the dopaminergic terminals in the inferior olive at the ultrastructural level. About a third (32%) of the dopaminergic terminals was found to make synaptic contacts in the olivary neuropil. The majority (81%) of these boutons terminated on cell bodies or extraglomerular dendrites, while the remaining terminals contacted dendritic spines inside glomeruli. In contrast, GABAergic terminals in the inferior olive formed more frequently (66%) synaptic contacts and they terminated more frequently (38%) in glomeruli. Thus, the ventrolateral outgrowth receives a dopaminergic input from the mesodiencephalic junction, and the postsynaptic distribution of this input reveals a characteristic pattern.

Gain adaptation and phase dynamics of compensatory eye movements in mice.

The vestibulocerebellum is involved in the control of compensatory eye movements. To investigate its role in the learning and timing of motor behaviour, eye movements in normal and mutant mice were investigated for the first time with the use of search coils. Wild-type mice showed the ability to increase the gain of their vestibulo-ocular reflex by visuo-vestibular training. This adaptation did not occur in lurcher mice, a natural mouse mutant that completely lacks Purkinje cells. During the optokinetic reflex the phase (timing) of the eye movements of lurchers lagged behind that of wild-type littermates, whereas during the vestibulo-ocular reflex it led that of the wild types. Ablations of different parts of the vestibulocerebellum indicated that the flocculus is necessary for the adaptation and the phase-leading effects of the cerebellum, whereas the nodulus might contribute to its phase-lagging effects. We conclude that Purkinje cells in the vestibulocerebellum are necessary for both learning and timing of compensatory eye movements in mice, and that the flocculus and nodulus may play antagonistic roles in these processes. The present description of the basic principles of cerebellar eye-movement control opens up the possibility to investigate the mechanisms of this motor behaviour at the molecular level in genetically manipulated mutant mice.

Detection of immunocytochemically stained rare events using image analysis.

The detection of rare-event cells circulating in peripheral blood using automated image analysis was evaluated using a model system consisting of cells from a breast cancer cell line (SKBR3) seeded in a mononuclear cell suspension. Slides of cells with optimal morphology were prepared according to an optimized preparation procedure based on centrifugal cytology in combination with formalin fixation. SKBR3 cells were immunocytochemically stained for cytokeratin using the cam 5.2 monoclonal antibody and labelled with alkaline phosphatase using CAS-red as substrate. Because, for optimal segmentation of cell images, plain differences in absorption wavelength are required, the red immunostaining was combined with a green nuclear counter-staining based on ethyl green. Slides were automatically screened for cytokeratin-positive SKBR3 cells resulting in a lowest detectable frequency of one positive cell per 1.87 x 10(6) negative cells. A comparison between manual screening and automated screening for cytokeratin-positive cells showed a high level of correlation (0.9998). For the definition of the total number of objects per slide, two counting procedures were evaluated. Results were close to the visual score with a coefficient of variation of 0.47% for the counting procedure used in this study. It is concluded that optimization of preparation and staining procedures for the detection of rare-event cells using automated image analysis results in optimal image contrast and, consequently, in an increase in sensitivity for detecting rare events.