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Notes: It has been an earnest desire of protein crystallographers to collect fast, accurate, high resolution diffraction data from protein crystals, preferably with exposure time as short as possible. In order to meet this challenge, a new type of Weissenberg camera has been developed for the recording of diffraction intensity from protein crystals using synchrotron radiation. The BL6A2 line has a plane-bending mirror designed by Y. Sato. The optical bench with triangular tilt-cut Si crystal monochromator was designed by N. Kamiya and was installed in the BL6A2 hutch. The Weissenberg camera was set on the 2θ arm of the optical bench. This camera can be used with Fuji Imaging Plate (IP) as an x-ray detector, and the reading out of the image from the IP is carried out by using BA100. The characterization of this system was carried out using the native crystal of chicken gizzard G-actin DNase I complex and its Yb3+, PCMB, indium, and FMA derivatives. Since these crystals are very sensitive for x rays, the resolution limit of the diffraction was 5 A(ring) with a 4-circle diffractometer on a rotating anode x-ray generator (N. Sakabe et al., J. Biochem. 95, 887. This complex was crystallized in space group P2,2,2, with a=42.0, b=225.3, and c=77.4 A(ring). The data were collected with this system with the 430-mm radius cassette when Photon Factory was operated at 2.5 GeV and 270 mA and the wavelength λ=1.004 A(ring) was chosen. In order to avoid overlapping of diffraction spots, oscillation angle range and coupling constant (degree/mm) were settled on the basis of simulation patterns of diffraction spots up to the maximum resolution to be measured considering the direction of the crystal axes, wavelength, radius of the camera, and mosaicness of the crystal. When the oscillation axis was a axis, the oscillation angle range was selected at either 10° (1°/mm) or 5° (0.5°/mm) depending on the density of reciprocal lattice points along the incident beam, and typical exposure time in each IP was 50 and 25 s, respectively. The exposure was stopped after 10 times oscillation. The total range of 117.5° was recorded on 16 sheets of IP with an overlapping range 0.5°.The data processing was carried out using program weis coded by T. Higashi. Two complete data sets along the a and c axes were collected using two crystals, independently, and the merge R(F2) for native crystal was 0.068. In order to know the feasibility of the data (F+−F−), Patterson maps were calculated with data of each derivative, and heavy atom vectors clearly appeared as prominent peaks in the Harker sections of the Patterson maps of both Yb3+ and PCMB derivatives. The heavy atom positions were obtained from the combinations of different Patterson and different Fourier maps, and were refined by using least-squares techniques. The final figure of merit up to 2.5 A(ring) resolution was 0.61 with 22 700 reflections. The assignment of DNase I part on the electron density maps is progressing using the Nicholson molecular model referring to its structure reported by C. Oefner and D. Suck [J. Mol. Biol. 192, 605 (1986)]. In addition to promising results of ω-aminoacid pyruvate aminotransferase [N. Watanabe et al., Book of abstracts of second Japan-China Bilateral Symposium on Biophysics, p. 83 (1988)], these results support that this data collection system consisting of a new type of Weissenberg camera using SR, Fuji Imaging Plate, BA100, and program weis is one of the fastest and most accurate systems for protein crystallography in use today. We thank the Education Ministry and the Foundations of Yamada, Naito and Toray for the financial support of this project. We are grateful to Dr. K. Namba of ERATO for our use of BA100.

Notes: The spatial and temporal organization of climbing fibre activation of Purkinje cells, the so-called complex spikes, were studied in the rat cerebellar Crus II folium utilizing a multiple microeletrode recording technique. As many as 32 Purkinje cells could be simultaneously recorded by using a custom-built electronic amplifier system and a special data storage device. Analysis of the auto-correlation activity of complex spikes in any given group of Purkinje cells indicated that activation occurs with a particular rhythmicity having a base firing of 10 Hz. Cross-correlation of spontaneous complex spikes demonstrated, in addition to a particular rhythmicity, an extraordinarily high degree of synchronicity within a particular spatial distribution of Purkinje cells. Thus, Purkinje cells organized in rostra-caudal rows tend to fire within 1 ms of each other for distances as far as 800 μm (the width of a folium) from the ‘master’ neuron. By contrast, Purkinje cells located medial or lateral to the master neuron showed almost no cross-correlation. Administration of harmaline to the animal increased the degree of auto- and cross-correlation but did not change the spatial order of the distribution of the cross-correlation. The results indicate that the olivo-cerebellar system is organized in such a way that climbing fibre afferents may be activated in a close-to-synchronous and rhythmic fashion. The spatial distribution of these afferents over the cortex is such as to activate rostro-caudal bands of Purkinje cells which tend to fire in a close-to-synchronous manner.

Notes: Multiple recordings from Purkinje cells in the rat cerebellum allowed the mechanism responsible for the activation of rows of synchronous complex spikes to be investigated. By determining the spatial distribution of the climbing fibre reflex that follows electrical microstimulation of the cerebellar cortex, it was shown that the mechanism for the simultaneity of firing was the electrotonic interactions between neurons in the inferior olive (IO). The spatial organization of the complex spike activity was shown to be regulated by GABAergic inhibitory input into the IO, probably arising from the cerebellar nuclear neurons. The rostro-caudal organizion of the complex spike activity following physiological stimulation (tactile stimulation of the upper and lower lip) demonstrated the same spatial distribution of synchronous activity in the cerebellar cortex as did the spontaneous activity and this was also disrupted by GABA blockers. Finally, complex spike responses to physiological stimulation indicate that the IO is capable of gating sensory inputs in accordance with its intrinsic autorhythmicity and that strong peripheral stimuli reset the oscillatory properties of the IO. The functional implications of the synchronicity and of the temporo-spatial organizion of complex spikes in the cerebellar cortex are discussed in the context of motor coordination and timing.

Notes: Summary An injection/circulation-timing switch was developed to simply and precisely initate the timing unit of the fundus camera for angiographic documentation. After starting the timer, the switch, mounted on the syringe used for injection of the tracer material, subsequently provides documentation of the total elapsed time of injection. Such quantitative data can enhance the evaluation of angiographic results and contribute to their universal comparability when combined with such qualitative data as injection location and total injectate.

Notes: Summary Laser coagulation using 50 and 100 mW of power, a spot of 50 microns and an exposure time of 0,2 seconds were performed on rat eye fundi. We observed the effects which were produced in the retina and choroid utilizing both fluorescence photomicroscopy with incident excitation light and fluorescence angiography. No distinct differences were noticed between the effects produced by 50 and by 100 mW. Both energies damaged choroid and retina, especially the outer part of the retina. Moreover, fluorescein angiography within 24 hours gave us effective information for estimating the applied laser effect.

Notes: Summary Argon laser coagulations with a power of 100 mW and 300 mW, exposure time 0.02 and 0.2 sec, and constant spot size of 100 microns were applied on normal cat fundi. From 1 and 2 hrs to 7 days after the coagulation, the treated eyes were observed angiographically and by fluorescence microscopy after dye injection; freeze dried eyes were examined under a fluorescence microscope. Abnormal dye leakages originated from the choroid and from retinal vessels were detected in the specimens examined within 72 hrs after the coagulation.

Notes: Summary Stimulation of the cerebellar dentate nucleus in monkeys elicited responses in the frontal association cortex (area 9) on the contralateral side to the stimulation, in addition to those in the motor (area 4) and premotor (area 6) cortices which were reported previously. The responses in the frontal association cortex were characterized by surface positive-deep negative field potentials in the cortex. They contrasted with surface negative-deep positive potentials in the motor and premotor cortices on the same dentate nucleus stimulation. In the rostral part of the premotor cortex (area 6) on the border of area 9, both types of responses were induced and admixed. The relay nucleus of the thalamus was suggested for the dentate-induced responses in the frontal association cortex.

Notes: Summary 1. In lightly nembutalized cats, effects of high frequency (60–100/sec) repetitive stimulation of the midbrain reticular formation (RF) were tested upon the thalamo-cortical (T-C) neurones which project from the anterior ventral (VA) nucleus of the thalamus and its vicinities to the parietal association cortex and convey impulses responsible for cortical recruiting and spindling-like responses. 2. Tonic maintained or rhythmic grouped firings of the T-C neurones recorded as extracellular units with microelectrode in the thalamus were in the majority suppressed by high frequency RF stimulation during and often for a short time after the stimulation, and they showed later tonic discharges for many seconds or minutes. Intracellular recording from the T-C neurones revealed hyperpolarizing potential changes corresponding to the suppression on high frequency RF stimulation. Field potential analysis in the VA nucleus indicated that the hyperpolarization is ascribed at least in part to IPSPs elicited in the T-C neurones. Responses in some other types of thalamic neurones to the RF stimulation were exemplified to be compared and related with those of the T-C neurones. 3. Desynchronization of the parietal electrocorticogram coincided with the suppression of the T-C neurones and lasted for the time of the later tonic discharges, which contrasted with the rhythmic grouped discharges of the T-C neurones in association with recruiting and spindling-like responses of the cortex. Relations between the thalamo-cortical recruiting system and the ascending reticular activating system were discussed.