Single Light Flashes Research Articles

Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes: I. In vitro studies with haemophilus influenzae transforming DNA and yeast photoreactivating enzyme

The photoenzymatic repair of ultraviolet (UV) lesions in DNA is a photochemical reaction which occurs in an enzyme-substrate complex between these lesions and photoreactivating enzyme (PRE). It is shown here that high intensity flash illumination (duration of the order of 1 millisecond) causes this repair in essentially all enzyme-substrate complexes present at the time. This fact allows several kinds of studies. (1) By applying the flash at timed intervals after mixing PRE and UV-irradiated bacterial transforming DNA, the formation of enzyme-substrate complex can be observed directly. The process takes of the order of minutes for its completion at the usual reaction concentrations, and the effect of changing concentration shows that most of this time is required for the extremely dilute reactants (∼ 10 −9 M) to encounter each other in solution. (2) When a sequence of flashes is applied with UV lesions in excess, the resulting stepwise repair permits complex formation to be studied at each level of recovery, from the first set of lesions erased to the last. The result shows that the lesions are heterogeneous with respect to rate of complex formation. (3) The rate of complex formation depends on temperature, but once a comples has been formed at 37° it does not rapidly dissociate on shifting the temperature to 2°. Repeating the experiments at lower light intensity, where not all the complexes present are repaired at each flash, then shows that the actual photochemical process in the complex is temperature-independent over the 2–37° range—at least for the most rapidly complexing lesions. (4) When PRE is allowed to complex with irradiated transforming DNA in the dark, and irradiated non-transforming DNA is subsequently added flashes applied at various times after this addition allow the dark dissociation of enzyme-substrate complexes to be followed. Comparison with the converse experiment, in which PRE is first complexed with lesions on non-transforming DNA, shows that some complexes are extremely stable, the enzyme preferentially remaining on the first DNA with which it forms a complex even after 2 h. Complexes with UV lesions on the synthetic polydeoxynucleotides dA:dT and dG:dC are more stable than most of complexes with natural DNA. (5) Measurement of complex formation under conditions of DNA excess, where essentially all the enzyme is bound, permits determination of the number of enzyme molecules relative to the number of UV lesions. The reasonable assumption that the number of lesions equals the number of pyrimidine dimers recoverable from the DNA then gives an absolute count. Provided the molecular weight of 3·10 4 given by Muhammed is approximately correct, the proportion of pure enzyme to totat protein in crude yeast extract is about 2·10 −6.

Preference for different visual stimulus sequences in children, adolescents, and young adults

Using a cross-sectional approach male Ss ranging in age from 8 to 20 years were asked to make preference judgments with regard to all possible pairs of eight different visual stimulus sequences. The stimulus sequences consisted of a different number (3, 5, 8, 13, 22, 37, 61, 100) of single light flashes spaced equally within a 10 sec interval. With increasing age Ss preferred slower stimulus sequences. These results are in close agreement with earlier results on auditory stimulus material.

Alpha Blocking “Conditioning,” Sensitization and Interstimulus Interval

5 groups of Ss trained with ISIs of 0, 0.5, 1, 2, and 5 sec. were used in a study of trace alpha blocking “conditioning.” CS was a 0.5-sec. tone, and US was a single light flash. 20 female college students showing at least 50%-time alpha rhythm in their resting EEGs served as Ss. Alpha blocking Rs were defined as a 25% reduction in alpha amplitude during the 0.5 sec. of the CS (initial R) and the 0.5 sec. immediately preceding the US (trace R), as compared to the 0.5 sec. immediately preceding the CS. Analysis of variance of the Rs showed no effect from ISI, a highly significant response effect, with trace Rs more numerous than initial Rs, a highly significant trials effect, and a significant response × trials interaction. Trend analysis of the trials and the interaction confirmed and extended these results by showing significant over-all linear, cubic and quintic components. Initial Rs consistently decrease (habituation), while trace Rs showed significant decreases and increases. Initial Rs were attributed to sensitization, while two types of pre-US or trace Rs were identified: one, at the beginning of “conditioning”, possibly related to initial Rs and undergoing fast habituation, and “true” trace Rs which developed after 20 trials and subsequently habituated slower than initial Rs. Differences between trace and delayed alpha blocking Rs were also discussed.

Short-latency electrodermal reaction and traffic accidents.

For 7 of 24 Ss, a single light flash evoked an unanticipated short-latency (100–200 msec.) increase in voltage between 2 fingertip electrodes passing a 2 μa. of direct current. The occurrence of this electrodermal phenomenon and the occurrence of a traffic accident over a 3-yr. period were related at the .059 level. This finding is discussed in relation to simple reaction time and driving GSR. In addition, consideration is given to possible reasons why this short-latency phenomenon has not been reported in some 1000 electrodermal studies covering a century.

Photosensitive epilepsy: Relationships between the visual evoked responses and the epileptiform discharges induced by intermittent photic stimulation

In fifteen patients with photosensitive epilepsy the characteristics of the EEG epileptiform discharges induced by intermittent photic stimulation (IPS) were investigated. In all the patients generalized EEG epileptiform discharges were induced by IPS. In eight of them (1st group) the discharges appeared first in the occipital area, whereas in the remaining seven patients (2nd group) they occured simultaneously over all head regions or appeared earlier over the anterior areas of both sides. Averaged visual evoked responses (VERs) to single flashes of light in the patients were similar morphologically to those of normal subjects. In these, the VER recorded from the mid-occipital area consisted of 4–6 rhythmic wavelets occurring within 100 msec from the flash and of larger deflexions following such wavelets. The initial component of the response is positive and has a peak latency of 20–27 msec. In six patients of the 1st group certain components of the VER were extraordinarily augmented. The earliest spikes of the EEG epileptiform discharges appearing first over the occipital regions were interpreted as unusually augmented VERs. The photosensitivity of the patients decreased remarkably during drowsiness. No epileptiform discharges were induced during slow wave sleep. In the REM stage of sleep the discharges were on the contrary as promptly induced as in the waking state. This phenomenon did not seem to be due to pupillary miosis. Monocular stimulation markedly reduced the epileptogenic effect of IPS as compared to binocular stimulation. Background illumination appeared to modify the epileptogenic effects of IPS. Ophthalmological examination revealed incomplete red-green color blindness in one patient and quadrantic hemianopsia in another. In the remaining subjects no pathological conditions of the retina were found. Some considerations on the neural mechanisms involved in epileptic photosensitivity are presented.

Recovery of Retinal Function From Suppression Caused by Flashes of Light

When the dark-adapted, totally color-blind eye is stimulated with pairs of single flashes of light of 0.15 millilambert (mL) luminance and of 10 msec duration the difference in size of the two electroretinograms evoked, as related to the dark interval between the stimuli, indicates exponential decay of suppression caused by the first stimulus. In these conditions the main component of the ERG is the scotopic b-wave. Using pairs of light flashes of much higher luminance (150 mL, 20,000 photopic troland, 10 msec duration) for stimulation, the recovery of retinal function from suppression caused by the first stimulus is not only delayed by several hundred milliseconds, but also the time constant of the process is longer. In these conditions the ERG consists of both a negative and a positive component.

Photomyoclonic response of epileltic and non-epileptic subjects during wakefulness, sleep and arousal

1. 1. The clinical photomyoclonic response (PMR) of eleven photosensitive epileptic patients and two non-epileptic subjects was studied by polygraphic EEG and ultraviolet apparatus recording of single responses and by computer summation. The responses spread after single light flashes over the deltoid, biceps, forearm flexors, quadriceps and gastrocnemius muscles with latencies of about: 61, 64, 65, 83 and 95 msec respectively. Agonists and antagonists contracted almost simultaneously. 2. 2. These latencies are virtually identical to those of the sub-clinical physiological reflex described by Bickford as the “photo-motor response”. The PMR is considered as an enhancement of it. 3. 3. In NREM sleep, following arousal from NREM sleep and during REM sleep, the PMR and the photoconvulsive response were diminished. Following arousal from REM sleep they were less diminished and quickly became those of wakefulness. 4. 4. In patients with petit mal absences and petit mal myoclonus, arousal from NREM sleep, unlike that from REM sleep, provoked prolonged myoclonus associated with EEG poly-spike and wave discharges while the PMR and PCR remained suppressed.

Molecules and memories.

MOLECULES AND MEMORIES BERNARD W. AGRANOFF* We believe we are studying the molecular basis of behavior. Perhaps this presentation will add strength to the thesis that neurochemistry is at the threshold of membership in that new sect, "molecular biology." Among our credentials are the following: (a) We use biochemical tools to study fundamental biological phenomena, (b) We find it exciting, (c) We cannot isolate reaction products. Ofcourse, the term "molecular" has already been used freely. There is, for example, ajournai concerned with "Molecular Pathology." Soon we may see on our bookshelves the "Molecular Psychopathology of Everyday Life," and perhaps "The Molecular Anatomy ofMelancholy." In this essay, the behavioral response will be considered as follows: An external stimulus is converted to electrical impulses in a sense organ. Interposed between this encoding process and the output, or "read out"— i.e., the electrical impulses leaving a central nerve apparatus to the muscles involved in the observed response—lies the associative mechanism. By a selection or instruction process, the brain makes decisions and records, in some as yet unknown form, information for a particular response which becomes, with time, preferred. We call this information memory. It has been compared to the biological information storage seen in immune body formation, enzyme induction, and the genetic process [i]. Most of our present knowledge of the nature of nerve impulses and interactions is based on electrophysiological observations. A single light flash can be detected electrically along the optic radiation and, by averaging techniques, evoked responses from repetitive external stimuli can be * Mental Health Research Institute and Department ofBiological Chemistry, The University of Michigan, Ann Arbor, Michigan 48104. My co-workers on the work with goldfish are Dr. Roger E. Davis and Dr.JohnJ. Brink (Interdisciplinary Training Fellows under USPHS training grant No. 5T7-MH-7417), Miss Patty Bright, and Mr. Paul Klinger. The research is sponsored by a National Science Foundation grant. 13 detected from the scalp ofan intact animal [2]. Experiments on the eye of limulus [3] andthefrog [4] indicatethat encoding ofstimuli can behighly selective. Whether learning can occur in sensory organs is not yet known. By comparison, chemical correlates ofnerve activity are crude. Increased temperature over the occipital cortex has been observed only after repeated massive visual stimulation [5]. Under similar conditions, increased local diffusion ofinert solutes has also been observed autoradiographically [6, 7]. Recently, increased incorporation of amino acids in the motor neurons ofthe rat spinal cord after prolonged exercise has been observed by autoradiography [8]. By means of microchemical measurements, changes in the RNA ofDeiter cells and the surrounding glia have been reported in rats following vestibular stimulation. Specific changes in the composition of RNA are also reported following training [9]. The chemical or electrical nature ofmemory has eluded clarification. It may not reside exclusively in the brain. Studies with transected planarians [10] suggest the existence ofa diffuse memory. Surgical lesions [11] as well as local electrical stimulation [12] and injections (see below) indicate some degree oflocalization in the temporal and hippocampal regions of higher animals. In what special way might chemical approaches be ofvalue in studies on memory? The answer lies in the hypothesis that memoryformation (the fixation ofexperience) involves theformation ofcovalent chemical bonds. It is supported by the following: i. The evolutionary experience ofa species is storedin DNA, the genetic "memory" molecule. DNA is assembled by formation of covalent chemical linkages and it does not appear to undergo metabolic turnover in the decoding process. The behavioral model would by analogy require molecules or structures which do not turn over and would thus accrete in the brain with time [13]. Some brain lipids fulfil these requirements. (It is known that brain cells do not contain on the average more DNA than other somatic cells [14].) Evidence for changes in the size ofthe cortex [15] is compatible with such an accretion process. We could classify the formation ofnew synapses as a type ofaccretion. A diminution process is also conceivable, in which irrelevant interneuronal connections are destroyedasafunctionofexperience . Suchapossibilityisratherdiscouraging from the standpoint ofchemical detection. It is easier to find the difference between 0 and 1 than between 100 and 101. 14 Bernard W. Agranoff · Molecules and Memories Perspectives in Biology and Medicine · Autumn 196...

A study of the mode of action of 3-(4-chlorophenyl)-1,1-dimethylurea on photosynthesis.

Abstract The inhibition of steady-state photosynthetic oxygen evolution by 3-(4-chlorophenyl)- i,i -dimethylurea was studied in Chlorella as a function of light intensity, wavelength and temperature. The results of this study were taken to indicate that the inhibition is located very close to photochemical System II. The site of inhibition was determined more accurately by making use of the phenomenon of activation by a background light of the oxygen evolution produced by a single brief flash of white light. This activation was interpreted as being the result of oxidation of the primary substrate of photochemical Reaction II. The inhibitor appears to block oxygen evolution by inactivating this primary substrate.

Phase of alpha brain waves, reaction time and visually evoked potentials

A reaction time study was conducted in which twenty subjects were asked to respond to single flashes of light by closing a response switch as quickly as possible. The flashes were presented during six different phase intervals of alpha waves to determine whether reaction times would be related to alpha phase, thus reflecting changes in cortical excitability. Reaction times were found to be reliably faster when the stimulus light was flashed during certain portions of the alpha wave, thus supporting the hypothesis of an excitability cycle being related to the alpha wave. Inked plots of the resulting averaged visually evoked potentials yielded a complex wave consisting of eight distinct components in the first 300 msec of the response. Only two of these components correlated positively and significantly with reaction time. The peak delay of the earliest and most highly correlated of the two components was used as a measure for the interval of time required for the volley initiated by the flash to have reached the cortex and the neural integration necessary for “perception” to have occurred. When the alpha phase during which the light was flashed was corrected by this amount (57 msec), fastest mean reaction times were found to fall on a surface negative phase of the wave while the slowest fell on a positive phase.

Effect of electrocutaneous digital stimulation on the detection of single and double flashes of light.

Effect of electrocutaneous digital stimulation on the detection of single and double flashes of light.

Neural limitations of visual excitability V. Cerebral after-activity evoked by photic stimulation

In locally anesthetized and in lightly anesthetized cats, a 5-msec flash of light to the eye evokes from the visual cortex a “primary” potential, followed by a train of after-discharges which may last 300 msec or longer. The duration, frequency and amplitude of after-discharges are variable, but it is clear that the total duration becomes longer with increasing intensity or duration of the photic stimulus. These after-discharges can be disrupted by the response to a subsequent longer flash when the onset of the second flash occurs before the total response to the first flash is over (i.e. within 300 msec). If one plots, on the ordinate, the reduction in the duration of the after-discharges elicited by a preceding test flash and, on the abscissa, the conditioning flash-test flash interval, the curve is similar to those obtained in previous studies of the psychophysical threshold in the human subject. These findings suggest that the perception of a single flash of light is related to the whole complex of waves elicited by a light flash and not just to the “primary” response.

DECAY OF SUPPRESSION OF RETINAL FUNCTION AFTER SHORT FLASHES OF LIGHT--ELECTRORETINOGRAPHIC MEASUREMENTS IN THE DARK ADAPTED HUMAN EYE.

When the dark adapted human eye is stimulated with pairs of single flashes of light of 5 lux intensity and 125 msec duration, the difference in size of the b-waves of the two ERGs evoked, as related to the dark interval between the stimuli, indicates exponential decay of the suppression caused by the first stimulus.

Electrographic patterns of the occipital lobe in man: A topographic study based on use of implanted electrodes

An attempt was made to determine the topographic distribution of electrographic patterns recorded from deep in the occipital lobe of 26 patients by means of 31 implanted leads with 167 electrodes. The occipital patterns were divided into two groups: One group is widely distributed throughout the whole occipital lobe and extends beyond its limits to the parietal and temporal lobes. These include the alpha rhythms (8–16 c/sec) and the fast rhythms (25–50 c/sec). The alpha rhythms appear to originate from multiple occipital and extra-occipital cortical generators variously overlapping and influencing each other, probably under the relative control of a central pacemaker. The other group is recorded from discrete areas of the occipital lobe in or near the calcarine region. The responses to steady illumination of the eyes (slow on-responses and off-responses and fast disharge), to single flashes of light, and to repetitive flashes (on-responses, off-responses, and driving responses) belong to this group. The small foci, from which responses to patterned visual stimulation (lambda waves) were recorded, were located not only in the medial portion but also in the inferolateral and occasionally in the superolateral portion of the occipital lobe.

An electrophysiological study of the superior colliculus and visual cortex

In cats anesthetized with Nembutal, simultaneous recording of evoked potentials from superior colliculus and visual cortex to single light flashes gave mean onset latencies of 36.5 msec in the former and 27 msec in the latter. Onset latency to electric stimulation of optic nerve was 8 msec in colliculus and over 1 msec in cortex. Since similar latency differences were obtained from colliculus and cortex.to both retinal (photic) and postretinal (electric) stimulation, latency differences were attributed to slower conduction in the retinocollicular pathway. The thresholds were higher in superior colliculus than visual cortex to light flashes of short duration or low intensity, or electric pulses applied to the optic nerves. Simultaneously recorded collicular and cortical evoked potentials differed from each other in shape, duration, and several other respects. With microelectrodes, three functionally differentiated layers of the superior colliculus were distinguished: a dorsal layer in which evoked potentials and single units responding to optic stimuli were obtained; an intermediate layer in which evoked potentials of unchanged amplitude, but no driven units, were recorded; and a ventral layer in which evoked potentials were greatly reduced, and where only “spontaneously” discharging units were found. The optically driven units were classified as those firing with short latency, with long latency, to light-on only, to light-on and light-off, and those inhibited by photic or optic nerve stimuli.

Multiple response of visual cortex of the rat to photic stimulation

The primary and the later multiple responses of the visual cortex of the anesthesized (nembutal) rat to flash do not differ significantly from those recorded from the cat and rabbit under similar conditions. In the rat the multiple response consists of surface-positive potentials recurring at 160–240 msec intervals following the primary response. Intracortical recordings indicate a close correspondence in potential changes between the primary and the multiple response. Local application of strychnine sulphate did not abolish the multiple response. In the unanesthetized rat with permanently implanted cortical electrodes the late response to a single flash of light is characterized by a series of high-amplitude negative waves with sharp initially-positive activity interposed between waves. This potential complex, which has a wave-and-spike form, also recurs at 160–240 msec intervals. This response is labile and sensitive to the “arousal” level of the animal. It occurs only after the animal has “habituated” to the flash and is suppressed by novel sensory stimuli as well as high-frequency electrical stimulation of the brain stem. Control procedures show that the response is not a cortical injury potential or other artifact. The negative potentials are attenuated by nembutal anesthesia to a greater degree than the sharp positive component, and this residual activity is the multiple response recorded from the anesthetized preparation.

Hypothalamic and anteroventral mesencephalic photic responses in the cat

Recent anatomical studies have demonstrated the existence of an afferent optic-diencephalic tract, arising in the retina and terminating throughout the hypothalamus. Experiments were designed to determine whether or not this pathway responded to light stimulation. By means of recording electrodes stereotaxically inserted into the diencephalon and mesencephalon, responses to single intense light flashes and “photic drive” at rates of 6 to 20 flashes per second were observed in mid-line structures of the hypothalamus and anteroventral mesencephalon in cats. The characteristic potential recorded on the electroencephalograph did not resemble the typical “on-off” response commonly found along the classical optic pathway. The hypothalamus response consisted of a single slow-wave response with a duration of 30 msec. Hypothalamic structures responding to single light flashes also responded to photic stimulation at rates of 6 to 20 flashes per second. Pentobarbital sodium, removal of the eyes, severing the optic nerves, and covering the cat's eyes abolished these responses. Sparine, a tranquilizer, failed to alter them. This investigation indicates the existence of an extraoptic tract to the hypothalamus and anteroventral mesencephalon which reacts to light. Anatomical evidence for the existence of such a tract has been described previously; however, precise determination of the retinal origin of the hypothalamic responses could not be determined.

Cortical electrical responses to visual stimulation in the human infant

Full-term and premature babies have been stimulated on one or more occasions by single and repetitive flashes of light. The responses recorded from the occipital cortex (scalp) differ from those of adults in being of ( a) more variable wave-form, ( b) more variable (and often higher) amplitude, ( c) longer latency, and ( d) greater “fatigability”. Developmental changes with increasing age have been demonstrated; the most notable of these are ( a) change from a response with initial negative phase in the earliest records of some subjects to the classical response with initial positive phase, ( b) decreasing response latency, and ( c) increasing ability to respond to more and more rapidly repeated stimuli. Response latency is also inversely correlated with body weight up to about 12 lb. ( r = −.80). Curves of response latency vs. age and of response latency vs. body weight are 2-legged rather than monotonic, the breaks in the curves occurring at 4 weeks post-term and 9 lb., respectively. This phenomenon may reflect either a growth spurt in the visual system or different developmental rates in the two parts (scotopic and photopic ?) of the visual system. The physiological evidence appears to be related to anatomical evidence of immaturity and developmental change, but available anatomical data are not detailed enough for close correlation. The human data described are in most respects similar to those reported for cats and rabbits. The visual system of the human infant appears to be physiologically more mature at birth than that of the cat and the rabbit, but the latter develop more rapidly postnatally. Individual differences are as striking in the human as they are in the other two species.

Electroencephalographic Patterns of the Goldfish (Carassius Auratus L.)*

ABSTRACT Bipolar surface electrodes were used to record electrical potentials from the brain of the goldfish, Carassius auratus L. Characteristic patterns of spontaneous electrical activity were described for telencephalon, mesencephalon, cerebellum and medulla oblongata. Changes in these patterns under deepening urethane narcosis were noted. Rapid repetitive flashing light caused a change in the normal pattern of the mesencephalon which resembled the mammalian arousal. High-frequency (18–24 cyc./sec.) low-amplitude activity replaced the characteristic low-frequency waves (7–14 cyc./sec.); the lower-frequency activity returned 1–5 min. after cessation of stimulation. Responses to single light flashes were described from the mesencephalon and cerebellum. An ‘A complex’ from the mesencephalon consisted of two to four rapid diphasic spikes, with a latency of 30–40 msec., duration of 40–50 msec., and amplitude of about 100 μ.V. A ‘B complex’ consisted of a large negative wave, duration 90–110 msec, and amplitude 100–200 μ,V., followed by a small positive deflexion, and a slow negative after-potential. The cerebellar response had a latency of 60–80 msec., duration of 100–120 msec., amplitude of 80–125 μV. The nature of these two components was discussed. Monocular blinding abolished the response to light in the contralateral half of the optic tectum and in the cerebellum. The amplitude of the low-frequency spontaneous activity characteristic of the mesencephalon was reduced in the contralateral tectum. Regional differences in the tectal response to single light flashes were described. Microelectrodes were used to record the photic response from deeper tectal layers. After destruction of parts of the retina by electrocautery, a disappearance of the A complex and 40–50% reduction of the B complex was observed in restricted tectal areas. The distribution of the areas of changed response was shown to correspond to a coarse overlapping projection of retinal quadrants of the fish eye on to the contralateral optic tectum. A possible basis for the localization of this projection, in agreement with anatomical and electrophysiological findings, was described.

The duration of human electroencephalographic arousal responses elicited by photic stimulation

Observations on the duration of arousal responses elicited by single stroboscopic light flashes of constant intensity and duration have been made in 80 normal adults. Normal values have been established for the response duration curves for periods up to 30 min. The response duration in the first 30 sec. tends to be almost two times as long as the duration seen after 30 sec. The values then stabilize and remain stable for periods as long as 30 min. Recovery of the prolonged duration of the early responses is incomplete in 20 min. but complete after 24 hours. Extraneous sound stimuli and attention have no effect on the duration of the arousal response. The validity of using arousal response duration in assessing the influence of drugs and disease on central nervous system activity is established.