Action Spectrum of Delayed Luminescence in Rat Brain Tissue at Room Temperature

G.K. Smirnov

Bioanalytical Laboratory, Ecological Safety Research Center,

Russian Academy of Sciences, St.-Petersburg

Summary

Action spectrum of delayed luminescence of rat brain tissue was obtained for the wavelength of excitation light in the range of 250 to 615 nm at the temperatures of about 24°C. This spectrum has peaks at approximately 490, 480, 420, 365, 330 and 280 nm.

Keywords: rat brain tissue, delayed luminescence, action spectrum

Introduction

It is known that in some biological objects such as green plant leaves, it is possible to observe very weak luminescence in the time range of milliseconds and even seconds after exposure to light (delayed luminescence) [1]. The existence of delayed luminescence (DL) in plants is connected with conversion of light energy into chemical energy via photosynthesis. Earlier the author of this communication supposed that the same property of delayed luminescence may be exhibited by brain tissue if, as it was shown in some works [2-4], a brain tissue can react to light directly, without the participation of retina. Indeed, we have found the photoluminescence of rat brain tissue in the time range of milliseconds [5]. Later we had measured the temperature dependence of this photoluminescence and found that its intensity increases as the temperature increases [6]. Relying on this experiments we concluded that this photoluminscence is a delayed luminescence. In the present communication we attempt to elucidate the nature of rat brain tissue DL with the aid of its action spectrum obtained for the wavelength of excitation light in the range of 250 to 615 nm.

Experimental Details

For the experiments were used male Vistar rats weighing 200-250 g. The rats were anaesthetized, then, following decapitation the brains were removed and immediately placed in a freezer. Once frozen, the brain tissue specimens were cut from the brain's outer surface located between two frontal sections, spaced at about 6 mm so that the crossing of the optic nerves (optic chiasm) was between them. Specimens had dimensions of 6x6 mm and thickness of 1-2 mm (perpendicular to the brain's outer surface). A total of four brains were used and 3-4 tissue specimens were taken from each.

For further specimen manipulation copper plates were used. One side of each plate was sooted in order to decrease the noise level when measuring the DL. Each plate had a hole of 4 mm in diameter. The brain tissue specimen was laid on the unsooted side of the copper plate immediately after cutting from frozen brain in such a manner that the hole in the plate was completely closed by the specimen. Then the specimen on the plate was placed in a lightproof box and dried at 39°-40°C for 24 hours. As we found, the drying of specimen ensured that the intensity of DL may be increased about ten times. All procedures with brains and brain tissue specimens before measurements were made under a dim red light.

The experimental arrangement consists of fluorimeter ÔË (FL)-2006 (Moscow State University and Krasnoyarsk Agricultural Institute), monochromator with diffraction grating ÌÄÐ (MDR)-2 (Leningrad Optical-Mechanical Association) and light source with mercury lamp ÄÐØ (DRSh)-250-3 (250 W) or mercury quartz lamp ÑÂÄ (SVD)-120 (120 W).

The direction of illumination in experimental arrangement was perpendicular to the direction of measurement of luminescence. A plate with specimen was located at 45° to both of this directions and the sooted side was presented to the light. When measuring the noise level, the sooted plate without hole was used. For measurements of intensity of illuminance a calibrated photodiod (A.F. Ioffe Physical-Technical Institute) was used. The temperature of the specimen was controlled with the use of a thermocouple placed at 1 mm from the specimen, and a flat heater under the copper plate. These measurements were conducted before placement and also after removal of the specimen plate for each wavelength of exciting light. The value of illuminance was different for different wavelengths and lay in the range of 7*10-5 to 1*10-3 W/cm2 (of 2*1012 to 3*1013photon/cm2s).

The sequence of wavelengths of illuminance light was randomised for each experiment and chosen from the constant set of wavelengths from linear spectrum of mercury.

The working cycle of fluorimeter consisted of an illuminance period of 9.1 ms, a darkness period of 5.5 ms and a registration period of 2.4 ms within the darkness period. The time interval between the end of the illuminance period and the beginning of the registration period equalled to 1.5 ms. During the registration period one measurement of DL intensity was made. On each wavelength of excitation light about 600 such measurements were made during a span of about 10 s. While calculating the value of DL intensity, the first 50 and last 100 measurements were rejected and the average of the remaining measurements was taken as the value of DL intensity for its wavelength of illumination.

Results and Discussion

The results obtained are presented in the following figure and table.

Each spectrum is an average of spectra for all specimens from the corresponding area. Relative intensities of DL were obtained by dividing measured intensities by the value of exciting light intensity (photon/ cm2s) for the corresponding wavelength. The locations of areas for which corresponding average spectrum was obtained are shown with solid segments on the circles which schematically represent the frontal section of the brain.

The relative quantity of brain tissue specimens whose action spectra of DL had peaks at wavelengths of exciting light which are indicated in the table.

The relative quantity of specimens for each wavelength indicated in the table was calculated by dividing the quantity of specimens whose action spectra of DL had peak at this wavelength, by the total quantity of specimens from the respective area. In so doing, it was assumed that peaks at 365 nm for side area were taken up by peaks at 330 nm. It is necessary to emphasise that in each specimen each of its spectrum peaks was one of those indicated in the table and there were no peaks at any other wavelengths.

When interpreting our data we took as the starting point the work [3] where it was demonstrated that the action spectrum of secretion of luteinizing hormone in quails whose brains were illuminated with light of different wavelengths, was close to the rhodopsin absorbance spectrum which had a maximum at about 500 nm. If it is granted that in our experiments peak of spectrum observed at about 490 nm is also due to rhodopsin, then other peaks can probably also be ascribed to the products of rhodopsin photolysis which consists in separation of the retinal chromophore from opsin in the molecule of rhodopsin followed by conversions of retinal [7]. The absorption spectra shows that these processes are accompanied by gradual disappearance of peak at 500 nm (rhodopsin) and the rise of peak at 365-380 nm (free retinal) [8,9]. One can find the peaks near both of these wavelengts in our action spectra of DL. The retinal may be further converted to vitamin A, whose absorption maximum lies at 328 nm [9]. Among the products of rhodopsin photolysis are metarhodopsin I, II and III with adsorbance maximums at 478, 380 and 460 nm, respectively [7]. In our spectra one can find peaks apparently corresponds to metarhodopsin I and II. Unfortunately, we had not made measurements in the region of 380 nm. Furthermore, it is known that opsin gives an absorbance maximum at about 280 nm [7] and the peak near this wavelength is represented in the action spectra for specimens taken from the side and top surface of the brain. The only peak which is not due to rhodopsin photolysis products or rhodopsin itself, is the peak at 420 nm but this wavelength coincides with the maximum of adsorbance of the blue-sensitive photopigment (of human retina cones) which is chemically related to rhodopsin [7], and coincides with the maximum of adsorbance of blood too. But we havn't found the DL signal from blood in our experiments.

Unfortunately, as far as is known, there are no data for the existence of luminescence of rhodopsin or its derivatives in the millisecond range at room temperature, except tryptophan phosphorescence. The excitation spectrum of tryptophan phosphorescence (in solution) lies in the range from 250 to 300 nm and has the maximum near 280 nm [10] (compare it with spectra for "top" and "side" on the figure).

So, relying on these results we have no possibility to conclude confidently that rhodopsin is the source of DL in the rat brain tissue. But these results shows that this unknown substance may bear chemical similarities with rhodopsin.

A comparison of spectra for specimens taken from top, side and bottom areas of the brain as well as data in the table, show that along with obvious similarity of spectra there are some differences between them.

These differences are probably caused by

i) differences in functioning of hypothetitcal photoreceptors in these areas; and

ii) differences in sequence of wavelengths of illuminance.

The assumption that hypothetical photoreceptors of different areas can function differently is in general agreement with the results of experiments on ducks [2] and rats [11] which have shown that physiological reactions studied in these investigations after direct illumination of brain were possible to observe only when the region of hypothalamus was illuminated (in our experiments this region corresponds to the bottom area). In our previous article [5] it was shown that the intensity of DL of undried brain tissue depends of the location of the area from which the specimen was cut.

The relationship between spectra and sequence of wavelengths of illumination is possible because the result of later illuminance by light of any wavelength may depends on what the result of illuminance of specimen by light of previous wavelength was.

The existence of DL in the rat brain tissue as well as known data on penetration of rather considerable amount of light into the brains of sheep, dogs, rabbits and rats [12] make much more plausible the existence of photoreception of rat brain tissue. Data on DL action spectrum are a good reference points for further investigations in this area.

References

1. Strehler, B.L., Arnold, W. J. Gen. Physiology, 34 (1951) 809-820.

2. Benoit, J. Yale J. Biol. Med., 34 (1961) 97-116.

3. Foster, R.G., Follett, B.K. J. Comp. Physiol., 157 (1985) 519-528.

4. Wade, P.D., Taylor, J., Siekevitz, P. Proc. Natl. Acad. Sci. USA, 85 (1988) 9322-9326.

5. Smirnov, G.K., Komarov, V.Ya. Biofizika, 41 (1996) 744-748.

6. Smirnov, G.K., Komarov, V.Ya. Biofizika (in review).

7. Ostrovsky, M.A., Govardovskii, V.I. in Physiology of vision (in Russian) (ed. Byzov, A.L.) 5-58 ("Nauka" Publishing House, 1992).

8. Wolken, J.J. Vision. Biophysics and Biochemistry of the Retinal Photoreceptors (Thomas, Springfield, Illinois, 1966).

9. Dowling, J.E. The Retina. An Approachable Part of the Brain (The Belknap University Press, Cambridge, Massachusetts, 1987).

10. Vanderkooi J.M., Calhoun D.B., Englander S.W. Science, 236 (1987) 568-569.

11. Lisk, R.D., Kannwischer, L.R. Science, 146 (1964) 272-273.

12. Ganong, W.F., Shepherd, M.D., Wall, J.R., Van Brunt, E.E., Clegg, M.T. Endocrinology, 72 (1963) 962-963.

G.K. Smirnov,

Bioanalytical Laboratory, Ecological Safety Research Center,

Russian Academy of Sciences,

Korpusnaya 18, Saint-Petersburg, 197042 Russia.

Phone: (812)-230-78-97.

Fax: (812) 235-43-61

E-mail:

kyuya@srces.samson.spb.su

or:

lsmirnov@ban.spb.ru