Use of a Simplified Method of Optical Recording to Identify Foci of Maximal Neuron Activity in the Somatosensory Cortex of White Rats

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1 Neuroscience and Behavioral Physiology, Vol. 31, No. 2, 2001 Use of a Simplified Method of Optical Recording to Identify Foci of Maximal Neuron Activity in the Somatosensory Cortex of White Rats M. Yu. Inyushin, A. B. Vol nova, and D. N. Lenkov Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 85, No. 11, pp , November, Original article submitted March 31, Eight mongrel white male rats were studied under urethane anesthesia, and neuron activity evoked by mechanical and/or electrical stimulation of the contralateral whiskers was recorded in the primary somatosensory cortex. Recordings were made using a digital USB chamber attached to the printer port of a Pentium 200MMX computer running standard programs. Optical images were obtained in the barrel-field zone using a differential signal, i.e., the difference signal for cortex images in control and experimental animals. The results obtained here showed that subtraction of averaged sequences of frames yielded images consisting of spots reflecting the probable position of activated groups of neurons. The most effective stimulation consisted of natural low-frequency stimulation of the whiskers. The method can be used for preliminary mapping of cortical zones, as it provides for rapid and reproducible testing of the activity of neuron ensembles over large areas of the cortex. KEY WORDS: Optical recording, somatosensory cortex, whiskers. Recording of intrinsic optical signals from neurons was first proposed in 1986 by Grinvald et al. [5], using potential-dependent dyes; the method was then used for recording the intrinsic electrical signals of nerve cells both in vivo [4] and in brain slices [11]. The optical properties of neurons were found to change in accordance with their activity, allowing their locations to be determined. Experiments of this type generally measure the difference in brightness between active and inactive zones of nerve tissue in reflected light, though whether this change occurs because of changes in the absorption of light, in the fluorescent properties of neurons, or for other reasons thus far remains unclear. The reflective properties of nerve cells can change, for example because of changes in neuron volume [2]. Some authors [11] ascribe these changes to the uptake of Cl ions by astroglial cells, as experimental studies have shown that extracellular chloride blocks the optical signal. The authors believed that chloride uptake leads to increases in the volume of Developmental Neurobiology Laboratory, A. A. Ukhtomskii Physiological Science Research Institute, St. Petersburg State University, 7/9 Universitetsksya Bank, St. Petersburg, Russia. astroglial cells and decreases in their uptake of natural chromophores, such as hemoglobin, cytochromes, and even reduced NAD [13]. Increases in neuron activity are accompanied by increases in their oxygen requirement, and changes in light reflection correspond to the transition between oxyhemoglobin and deoxyhemoglobin, because the latter absorbs more red light. Thus, an activated zone of cortex is darker than the neighboring area. There is one further possible explanation for the observed optical signal that it reflects changes in blood flow in the microzone of the brain surface [12]. However, experiments based on blockade of vasodilatation had no effect on the intrinsic signal of the cortex [6]. This hypothesis also does not correspond completely to the fact that optical signals can be obtained in brain slices [11]. Thus, the question of the mechanisms of this phenomenon remains open and it appears that intrinsic optical signal, which is dependent on nerve cell activity, arises from several simultaneous origins. A complex specialized apparatus is used for recording intrinsic optical signals in the infrared region of the spectrum in nerve tissues. However, as first shown by Peterson and Goldreich [13], simpler methods can also be used in the visible part of the spectrum, /01/ $ Plenum Publishing Corporation

2 202 using standard digital CCD cameras, microscopes, and programming developed for biological experiments and freely available across the internet (for example, at ftp://maxrad6.uthscsa.edu). The technique of digital filtration allows optical recording of neuron activity to be applied (for example, for recording neuron responses in the somatosensory cortex of rats during mechanical stimulation of the whiskers) by recording changes in the reflectivity of the cortex through the dura mater and even through a thinned skull [9]. As shown by Dowling et al. [3], simplified and accelerated optical methods, which make no physical contact and involve little trauma, are especially useful for general mapping of large surface areas of the cerebral cortex. This method, for example, has been used for surgical operations in humans for pre-operative mapping of the cortex [7]. The aim of the present work was to develop a method for optical recording of the activity of neuron ensembles in the primary somatosensory cortex (C1) of rats during mechanical stimulation and/or electrical stimulation of a small group of contralateral whiskers. Since our studies in recent years have been directed to studying functional changes in neuron activity in various areas of the rat cerebral cortex, these being associated with the whisker system [1, 8], we believe that mapping using optical recording could significantly facilitate the search for foci of maximum activity and for express analysis of receptive fields in adult and developing rats. METHODS Experiments were conducted on eight white mongrel rats weighing g. After induction of general anesthesia with urethane (1 g/kg, i.p.), standard craniotomies were performed, starting with a small opening being made in the bone with a dental drill without damaging the dura mater, which was not removed during the experiments. Skin and bone around the opening were covered with black matte paper to reduce parasitic light reflections. The surface of the cortex was coated with a thin layer of Vaseline oil and was covered with a microscope cover slip in such a way as to avoid forming an air bubble between the glass and the oil. The cortex was illuminated with a variety of light sources; since studies reported by other authors recording intrinsic optical signals in the visible range involved measuring changes in absorption of light by the cortex of no more than 1 2% [3, 13], the light source had to have a level of stability an order of magnitude greater than this. Another requirement was to provide a high enough level of illumination (around 1,000 lux) in order to take advantage of the maximum sensitivity of the digital video camera. A third requirement was a narrow Inyushin, Vol nova, and Lenkov range of emitted light (corresponding to the range at which the greatest changes in neuron optical signals occurs). The use of a semiconductor laser (wavelength 660 nm), which we were the first to try, failed to give stable results because of fluctuation between different modes on reflection from the cortex, which led to the appearance of excessive background optical noise. A halogen lamp (20 W, 12 V) was subsequently used with a current stabilizer based on an operational amplifier built in our laboratory. This provided a direct current flowing through the lamp with a stability of the order of 0.001% with control over the level of illumination. A narrow-pass filter type 3S2 was also used, with a central bandpass wavelength of 520 nm. Recordings were made using a CCD camera (charge-coupled device) produced by QuickCam attached to the input of a 455 CCD microsystem produced by Texas Instruments; this yielded a signal corresponding to a matrix of µm square pixels. This camera was connected to the printer port of the computer and could be used as an external peripheral device attached to the personal computer with digital control, this allowing regulation of the brightness level (using direct digitization, the camera gave an eight-bit brightness measure, i.e., expressing 256 shades), the levels of black and white, the color saturation, the frame frequency, etc. A standard Windows interface was used, running the free image processing program UTHSCSA ImageTool 2.0 (a program for optical biological studies produced by the Initiative Group, University of Texas, UTHSCSA) (ftp://maxrad6.uthscsa.edu). Using the built-in functions of the ImageTool 2.0 program, monochrome images of the surface of the somatosensory cortex in the whisker representation area were collected at a rate of 25 frames/sec using half-frames, where the duration of each frame was 40 msec. Establishment of the black and white levels was by hand with calibration at the beginning of each experiment. This calibration of the camera allowed about 250 gradations of black to be obtained for the level of illumination obtaining in each experiment. Totals of frames were collected in each experiment (films were recorded in the avi format), and ImageTool 2.0 was then used to obtain a single frame with a reduced noise level using the built-in averaging function. This removed the effects of irrelevant mechanical oscillations in the cortical zone being studied (pulsatile movements of vessels, vibration) and noise in the recording apparatus. The video camera was connected to an MBS-2 binocular magnifier via an optical adapter. Recording was performed using a 10 ocular; the objective was notched to the 2 position. Focusing was adjusted to the surface of the cortex, and the objective was then brought to within µm of the cortex, thus determining the depth of optical recording.

3 Use of a Simplified Method of Optical Recording 203 Fig. 1. Sequences of frames showing the results of two experiments using optical recording of neuron activity in the rat somatosensory cortex after mechanical stimulation of the contralateral whiskers. A) Accumulation of images in controls; B) after deviation of the contralateral whiskers (caudal or rostral group). C) Difference image. The scale bar corresponds to 1 mm. RESULTS AND DISCUSSION Optical images in the cortical barrel-field zone of the C1 were obtained using a differential optical signal the difference in cortical images before and after subthreshold electrical or mechanical stimulation of the whiskers. Two digital methods could be used to obtain the differential optical signal. In one, frames were subtracted (values for each pixel were subtracted from each other) and the result was multiplied by the contrast coefficient. In the other method (used, for example, in the NIH-Image program), frames are divided by each other, the result then being processed as in the first method. We used frame subtraction, the control image being subtracted from the experimental image using the quantitative subtraction function in ImageTool 2.0. Recordings were made using a computer containing a Pentium 200MMX processor and 64 Mbytes of operating memory. All frames were stored on the hard disk and processed during experiments to identify areas of increased activity in the C1 cortex. Before each series of pictures, frames were accumulated in each experiment to use, after averaging, as controls. After stimulation of one or several neighboring whiskers, further series of frames were accumulated; after averaging, these provided an image of the cortex reflecting stimulation-induced changes. The results showed that subtraction of one sequence of images from another yielded an image consisting of spots in the area of the somatosensory cortex, apparently indicating the locations of barrels corresponding to the activated group of whiskers. This

4 204 Inyushin, Vol nova, and Lenkov Fig. 2. Diagram showing the distribution of optical densities of images from the rat somatosensory cortex during mechanical stimulation of the contralateral whiskers. The Y axis shows image density in arbitrary units; the X and Z axes show distances across the cortical surfaces, mm. observation was reliably reproduced in different experiments; the orientation marker for positioning was provided by the distribution of blood vessels on the surface of the cortex. Figure 1 shows results from two experiments. The image on the left (A) shows an accumulated image from the control; the central image (B) is after stimulation of the contralateral whiskers (caudal or rostral group); the image on the right (C) is the difference between the images, and the dark spots reflect the presumptive position of the focus of excitation in the animal s somatosensory cortex. When the group of whiskers being deviated was changed, the position of the dark spots on the surface of the cortex changed correspondingly. Analysis of the brightness of the image obtained in one of the two images is shown in Fig. 2. It should be noted that the dark spot recorded on the cortical surface is a gradient whose peak is essentially in the center of the spot, and the amplitude of the peak amounted to 4 5 pixel values (the increase after contrasting was six-fold). This apparently represents the distribution of the focus of peak neuron activity on the surface of the cortex [10]. The sizes of the optically active areas of the cortex (of the order of µm) also agree with published data on the sizes of barrels in the right somatosensory cortex [9, 13]. When more sensitive instrumentation (a cooled multidischarge CCD camera) was used, the microzones of the cortical surface undergoing changes in brightness could increase to 100 µm, which is significantly larger than the size of barrels as defined by a cytochrome-peroxidase method [5]. In our experiments, it seems likely that the low sensitivity of the apparatus should reveal only the center of the area of evoked changes in cortical neuron ensembles (Fig. 2). The size of the active zones recorded here also depended on the degree of whisker activation [5]. The most intense optical signal in our observations were recorded in conditions of natural subthreshold stimuli of the whiskers (deviation), while electrical stimulation did not produce any result. Electrocutaneous stimulation of the whisker pad, which simultaneously activates a large group of close-lying whiskers, led to masking of neuron activity in the corresponding microzones of C1 because of mutual inhibition, while natural stimulation revealed neuron responses of a separate pool of neurons in the barrel field. Data published in recent years indicate that the optical recording method is very sensitive and allows the

5 Use of a Simplified Method of Optical Recording 205 consequences of moving the whiskers to be detected even when this is through small angles [14]. Thus, the optical method of recording neuron activity has great potential for studying the receptive fields of cortical neurons, their plastic properties, the consequences of sensory deprivation [1], and, perhaps, transplantation of cortical tissue. There is special potential for the preliminary mapping of cortical zones, allowing rapid and reliable testing of the activity of neuron ensembles over large areas of the cortical surface and to identify plastic changes in its physiological characteristics. This study was supported by the Russian Fund for Basic Research (Code No ). REFERENCES 1. A. B. Vol nova, T. V. Golikova, A. Yu. Ignashchenkova, and D. N. Lenkov, Long-term changes in the latent periods of afferent and efferent responses of neurons in the vibrissal motor cortex after unilateral section of the infraorbital nerve in neonates, Zh. Évolyuts. Biokhim. Fiziol. (in press). 2. L. B. Cohen, Changes in neuron structure during action potential propagation and synaptic transmission, Physiol. Rev., 53, (1973). 3. J. L. Dowling, M. M. Henegar, D. Liu, C. M. Rovainen, and T. A. Woolsey, Rapid optical imaging of whisker responses in the rat barrel cortex, J. Neurosci. Meth., 66, No. 2, (1996). 4. R. D. Frostig, E. E. Lieke, D. Y. Ts o, and A. Grinvald, Cortical functional architecture and local coupling between neuronal activity and the microcirculation revealed by in vivo high-resolution optical imaging of intrinsic signals, Proc. Natl. Acad. Sci. USA, 87, (1990). 5. A. Grinvald, E. Lieke, R. D. Frostig, C. D. Gilbert, and T. N. Wiesel, Functional architecture of cortex revealed by optical imaging of intrinsic signal, Nature, 324, (1986). 6. M. M. Haglund, D. W. Hochman, J. R. Meno, A. G. Ngai, and H. R. Winn, Mechanisms underlying the intrinsic signal during optical imaging of rat somatosensory cortex, Soc. Neurosci. Abstr., 20, 1264 (1994). 7. M. M. Haglund, G. A. Ojemann, and D. W. Hochman, Optical imaging of epileptiform and functional activity in human cerebral cortex, Nature, 358, (1992). 8. D. N. Lenkov, A. B. Vol nova, and T. V. Golikov, The physiological characteristics of neurons of the rat s primary motor cortex after neonatal vibrissae sensory deprivation, in: Proceedings of the 12th Meeting of the International Society for Developmental Neuroscience, Canada (1998). 9. S. A. Masino, M. Kwon, Y. Dory, and R. D. Frostig, Characterization of functional organization within rat barrel cortex using intrinsic optical imaging through a thinned skull, Proc. Natl. Acad. Sci. USA, 90, (1993). 10. S. A. Masino and R. D. Frostig, Quantitative long-term imaging of the functional representation of a whisker in rat barrel cortex, Proc. Natl. Acad. Sci. USA, 93, (1996). 11. B. A. McVicar, T. W. Watson, F. E. LeBlanc, S. G. Borg, and P. Federico, Mapping of neural activity patterns using intrinsic optical signals: from isolated brain preparations to the intact human brain, in: Optical Imaging of Brain Functions and Metabolism, Plenum Press (1993), pp S. M. Narayan, E. M. Santori, A. J. Blood, L. Sikkens, and A. W. Toga, Functional increases in blood volume over somatosensory cortex, J. Cereb. Blood Flow Metab., 15, (1995). 13. B. E. Peterson and D. Goldreich, A new approach to optical imaging applied to a rat barrel cortex, J. Neurosci. Meth., 54, (1994). 14. B. E. Peterson, D. Goldreich, and M. M. Merzenich, Optical imaging and electrophysiology of rat barrel cortex. I. Responses to small single-vibrissa deflections, Cerebral Cortex, 8, No. 2, (1998). 15. B. R. Sheth, C. I. Moore, and M. Sur, Temporal modulation of spatial borders in rat barrel cortex, J. Neurophysiol., 79, No. 1, (1998).