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Changes in intrinsic optical signal of rat neocortical slices following afferent stimulation
Neuroscience Letters 180 (1994) 227~30
ELSEVIER
H[UROSCIENCE LETTERS
Changes in intrinsic optical signal of rat neocortical slices following afferent stimulation K n u t H o l t h o f f a, Hans-Ulrich D o d t b, Otto W. Witte a'* "Neurologische Klinik der Heinrich-Heine-Universitiit, Moorenstr. 5, D-40225 Diisseldorf German), bMax-Planck-Institut fiir Psychiatrie, Klinisches lnstitut, Klinische Neuropharmakologie, Kraeplinstr 2, D-80804 Miinchen, Germany Received 29 April 1994; Revised version received 2 September 1994; Accepted 2 September 1994
Abstract Changes in intrinsic optical signal of rat neocortical slices following afferent stimulation were recorded using darkfield infraredvideomicroscopy. Response amplitude was linearly related to stimulation intensity. The intensity of the optical signal reached its maximum 3 s after onset of stimulation and redecayed with a mean time constant of 23 _+7.1 s. The optical signal had a columnar shape. The size of the column was independent from stimulation intensity with stimuli of medium amplitudes. The extent of the optical signal corresponded to the extent of the electrical activation. Changes in intrinsic optical properties may be a useful tool for the study of spread of excitation in neuronal tissue in vitro. Key words'." Intrinsic optical signal; Darkfield infrared videomicroscopy; Neocortical slice; Afferent stimulation; Rat
Various optical methods have been developed to investigate the neuronal activity with 2-dimensional spatial resolution. Using fluorescence ratio imaging with the intracellular Ca2*-indicator fura-2 [20], activity-dependent changes in intracellular Ca 2+ in single cells [16,18], presynaptic terminals, or postsynaptic dendrites [17] could be demonstrated in vitro. Extrinsic optical signals caused by voltage sensitive dyes allow to detect changes in membrane potential of single neurons in culture [8] and neuronal populations with high resolution in time and space [1,2,6]. Optical detection of neuronal activity using intrinsic signals was first described by Hill and Keynes [10] who measured changes in light scattering of active nerves. Using darkfield microscopy it was possible to measure electrical activity of cultured neurons [19]. Maps of functional activity of mammalian cortex in vivo have been obtained by measuring the change of light reflected from animal [3,5,7] and human brain [9]. Since the intrinsic optical signals have a time course several orders of mag-
*Corresponding author. Fax: (49) (211) 3118485. 0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(94)00683-0
nitude slower than the electrical signals, they cannot substitute for recordings with extrinsic optical dyes. Changes of intrinsic optical signal can also be recorded in brain slices using a combination of darkfield technique and infrared-videomicroscopy [4]. The aim of the present study was to characterize this intrinsic optical signal. 400/lm thick neocortical coronal slices were prepared from juvenile male Wistar rats (14 days old) using a vibratome. The slices were stored at room temperature in artificial cerebrospinal fluid containing (in mM): NaCI 124, NaHCO3 26, KCI 3, CaC12 2, MgSO4 2, NaH2PO4 1.25, and glucose 10, equilibrated with 95% O2 and 5% CO2 to pH 7.4. Experiments were done on 20 slices of 13 animals. In the recording chamber the slices were stored submerged at 32°C. To avoid changing reflections on the boundary between water and air, we covered the recording chamber with a small cover slip. An upright microscope (Axioskop FS, Zeiss) with an x 2.5 Neofluar objective (0.075 n.A.) and a darkfield central stop was used. The illumination light was filtered with a bandpass interference filter (750 + 50 nm, Oriel). The video imaging system consisted of a CCD (charge coupled device) camera (C 2400-77 type 01, Hamamatsu), a camera control
228
K. ttollhotf cl a/./.\euro.science Lelters' l,YO l1994) 227 230
Fig. I. Change of intrinsic optical signal in rat ncocortical slice IkHlovxing affcrcnt stimulation. The stimulation electrode is marked by, a vvhitc boundar 5. I:~ar 300 lira.
unit with a shading correction system, a video processing unit (DVS 3000, Hamamatsu) and a S-VHS Videorecorder (RTV 950 PC, Blaupunkt). The experiments were analyzed off-line using a Macintosh f:X personal computer equipped with a fi'ame grabber card and the Image N I H software. If not mentioned otherwise number of experiments refers to number of slices. For stimulation (pulses of 200 #s/2- 10 V in a train of 50 Hz tk~r 2 s) a concentric bipolar electrode (tip diameter 0.1 mm) was used. 11 was placed perpendicular to the surface of the slice at the border of layer VI and the white matter. Field potentials were measured in layer II/I11 with glass microelectrodes filled with 0.5 M NaC1. To detect stimulation-induced changes of the intrinsic signal, analog contrast enhancement, background subtraction, and an 8-fold digital enhancement were applied on-line to the video image obtained from the CCD-camera. Ten images per second were ewduated off-line. To exclude artefacts, we determined the difference between optical intensity in a region of interest and a not involved reference region. Full-width half-maximum of the columnar intrinsic signals were determined at peak of signal after end of stimulation. For characterisation of the changes of intrinsic optical signal, the time course and the spatial extent were determined. Stimulation at the border of layer VI to the white matter caused a change of the intrinsic signal in a columnar region tlnoughout the cortical layers II Vl perpendicular to the orientation of the cortex (Fig. 1). Layer I was never involved. The time course of the change of the intrinsic optical signal was rather slow. The intensity of the optical signal reached its maximum amplitude 3 s alter onset of stimulation, and redecayed with a mean time constant of 23 _+ 7.1 s (n = 48 stimulations of 7 slices) to baseline level (Fig. 2A). The anaplitude of optical signal showed a linear relationship to stimulation strength (Fig. 2B) with a mean correlation coefficient r = 0 . 9 9 + 0 . 0 1 (n = 7). With meditnn stimulation intensity yielding re-
sponses in the plateau range of lull-width half-maximum as discribed below, signal was at least one order of magnitude greater than noise. The time constant of decay was related to stimulation strength with stimuli of small amplitude, but remained unaltered with stimuli o1" medium and high amplitude (Fig. 3A). In 52% of the experiments, a biexponential time course was observed. The two time constants (20 + 3.6 s vs. 27 + 4.6 s; n = 31 stimulations of 7 slices) were significant different (P < 0.01, Student's t-test). The intrinsic optical signal shows a good reproducibility. In a series of 10 subsequent stimulations with 5 min intervalls the amplitude of the tenth optical signal was 100 + 2.2% (n = 6) of the first. To determine the width of the stimulation induced columnar intrinsic signal, we chose the full-width halfmaximum ( F W H M ) measured tangentially in cortical layer I1/III. The relation between F W H M and stimulation strength followed an inverted sigmoid curve
A
loo
50 .c_
m lOs
B
//
loo
.=z, 5o .E
O-
• I
I
I
I
I
2
4
6
8
10
stimulation strength (V)
Fig. 2. Time course and peak amplitude of intrinsic optical signal in relation to stimulation strength. A: time courses of change of intrinsic optical signal in layer lI/III after stimulation with different strength at the border of layer VI and the white matter. Arrow marks onset of stimulation. B: relation between peak amplitude of intrinsic optical signal and stimtflation strength. Line indicates linear regression analysis,,
229
K. Holthoff et al./ Neuroscience Letters 180 (1994) 227-230
A
40 35
t
3O
0
o
20
E 15 ¸ 105I
I
I
I
I
2
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4
5
6
stimulation strength (V)
B
500-
/
400E E
'~ 300 E (" J= t-.
200
in rat hippocampal slices by measuring changes of light transmission. The origin of changes of intrinsic optical signal described here remains to be investigated. The reflected intrinsic optical signal of mammalian cortex in vivo was suggested to be composed of changes of blood volume, oxygen delivery and light scattering [5]. Lipton [14] concluded that the reflectance changes of guinea pig cerebral cortical slices were due to alterations of cell volume and extracellular space, respectively. The changes of light transmission in rat hippocampal brain slices following afferent stimulation were attributed to cell swelling [15]. In a range with stimuli of medium strength the columnar shape of intrinsic optical signal was constant. Columnar patterns of cortical activity have been described previously [11-13] by [14C]deoxyglucose technique in monkey striate cortex and rat barrel cortex. The shape of activity in rat barrel cortex induced by stroking contralateral C3 vibrissa was similar to the optical signal described here. The present study shows that intrinsic optical signals in brain slices have a high signal-to-noise ratio, show a good reproducibility and may therefore be a useful tool for the study of spread of excitation in neuronal tissue.
"10
"~
This work was supported by Grants Hess DFG 830/6 to O.W.W. and by grant from BMFT to Dr. W. Zieglg~nsberger.
100
0
=
--
I
[
I
I
I
1
I
2
4
6
8
10
12
14
stimulation strength (V)
Fig. 3. Time constant of decay and full-width half-maximum (FWHM) of intrinsic optical signal. A: relation between time constant of optical signal decay and stimulation strength. Values are means of three experiments. B: relation between FWHM of the optical signal measured in layer II/III and stimulation strength.
140 120
100
(Fig. 3B). With increasing amplitude of the stimulation pulses, the FWHM first increased, then remained unchanged for a range of stimuli with medium amplitude, and finally increased again with very strong stimuli. The mean signal FWHM obtained with stimuli of medium strength was 230 + 9.9/zm (n = 6). For investigation of the correlation between changes of intrinsic optical signal and electrical activity, field potentials in layer II/III at different distances to longitudinal axis of optical signal were recorded. Such experiments showed that the extent of the optical signal corresponded to the extent of the electrical activation (Fig. 4). The mean correlation coefficient yielded from a regression analysis was 0.97 _+0.02 (n = 6). The time course of changes in intrinsic optical signal in rat neocortical slices was very slow and exceeded the time course of electrical activation by tens of seconds. MacVicar and Hochman [15] found similar time courses of intrinsic optical signals following afferent stimulation
C
80
2O 0
j
-20 l
I
I
I
I
-1000
-500
0
500
1000
distance in p.m Fig. 4. Correlation between intrinsic optical signal and electrical activity. Intensity profile measured tangentially in cortical layer lull/ at peak of optical signal. Squares indicate normalized amplitude of field potentials (ampl/amplm,0, measured in layer II/III. Insets show field potential recordings of baseline, maxima/, and half-maximal amplitude. Height and width of insets correspond to 4 mV and 60 ms, respectively.
230
K. Holthq/f et al./Neuroscience Letter,~ 180 (1994) 227 230
[1] Albowitz, B., Kuhnt, U. and Ehrenreich, L., Optical recording of epileptiform voltage changes in the neocortical slice, Exp. Brain Res., 81 (1990) 241 256. [2] Blasdel, G.G. and Salama, G., Voltage-sensitive dyes reveal a modular organization in monkey striate cortex, Nature, 321 (1986) 579 585. [3] Bonhoeffer, T. and Grinvald, A., Iso-orientation domains in cat visual cortex are arranged in pinwheel-like patterns, Nature, 353 (1991) 429 431. [4] Dodt+ H.-U. and Zieglgansberger W., Visualization of the spread of excitation in brainslices with infrared-darkfield microscopy, Eur. J. Physiol., Suppl. 442 (1993). [5] Frostig, R.D., Lieke, E.E., Ts'o, D.Y. and Grinvald, A., 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) 6082 6086. [6] Grinvald+ A., Frostig, R.D., Lieke, E. and Hildesheim, R., Optical imaging of neuronal activity, Physiol. Rev., 68 (1988) 1285 1366. [7] Grinvald, A.. Frostig, R.D., Siegel, R.M. and Bartfeld+ E., Highresolution optical imaging of functional brain architecture in the awake monkey, Proc. Natl. Acad. Sci. LISA, 88 (1991) 11559 11563. [8] Grinvald. A., Ross, W.N. and Farber+ 1., Simultaneous optical measurements of electrical activity from multiple sites on processes of cultured neurons, Proc. Natl. Acad. Sci. USA. 78 (1981) 3245 3249. [9] Haglund, M.M., Ojemann, G.A. and Hochman, D.W., Optical imaging of epileptiform and functional activity in human cerebral cortex, Nature, 358 (1992) 668 671. [10] Hill, D.K. and Keynes, R.D., Opacity changes in stimulated nerve, J. Physiol.. 108 (1949) 278 281. [11] Kennedy, C , Des Rosiers, M.H.. Sakurada, O., Shinohara, M.,
Reivich, M.+ Jehle, J.W. and Sokoloff, L., Metabolic mapping of the primary visual system of the monkey by means of the autoradiographic [HC]deoxyglucose technique, Proc. Natl. Acad. Sci. USA, 73 (1976) 4230M234. [12] Kossut, M. and Hand, E, Early development of changes in cortical representation of C3 vibrissa following neonatal denervation of surrounding vibrissa receptors: a 2-deoxyglucose study in the rut, Neurosci. Lctt., 46 (1984) 7 12. [13] K ossut. M. and Hand, R, The development of the vibrissal cortical colurnn: a 2-deoxyglucose study in the rat, Neurosci. Lett., 46 (1984) 1 6. [14] Lipton, P.. Effects of membrane depolarization on light scattering by cerebral cortical slices, J. Physiol., 231 (1973) 365 383. [15] MacVicar. B.A. and Hochman. D.+ Imaging of synaptically ew>ked intrinsic optical signals in hippocampal slices, J. Neurosci.. 11 (1991) 1458 1469. [16] Regehr, W.G., Connor, J.A. and Tank, D.W., Optical imaging of calcium accumulation in hippocampal pyramidal cells during synaptic activation, Nature, 341 (1989) 533 536. [I 7] Regehr, W.G. and Tank, D.W.. Selective fura-2 loading of presynaptic terminals and nerve cell processes by local perfusion in mammalian brain slice, J. Neurosci. Methods, 37 (1991) 111 119. [18] Ross, W.N., Lasser-Ross, N. and Werman, R., Spatial and temporul analysis of calcium-dependent electrical activity in guinea pig Purkinje cell dendrites, Proc. R. Lond. B, 240 (1990) 173 185. [19] Stepnoski. R.A., LaPorta, A., Raccuia-Behling, F., Blonder, G.E., Slusher, R.E. and Kleinfeld, D., Noninvasive detection of changes in membrane potential in cultured neurons by light scattering, Proc. Natl, Acad. Sci. USA, 88 (1991) 9382 9386. [2(I] Tsien, R.Y. and Poenie, M., Fluorescence ratio imaging: a new window into intracellular ionic signaling, Trends Biochem+ Sci., I I (1986) 450 455.
ELSEVIER
H[UROSCIENCE LETTERS
Changes in intrinsic optical signal of rat neocortical slices following afferent stimulation K n u t H o l t h o f f a, Hans-Ulrich D o d t b, Otto W. Witte a'* "Neurologische Klinik der Heinrich-Heine-Universitiit, Moorenstr. 5, D-40225 Diisseldorf German), bMax-Planck-Institut fiir Psychiatrie, Klinisches lnstitut, Klinische Neuropharmakologie, Kraeplinstr 2, D-80804 Miinchen, Germany Received 29 April 1994; Revised version received 2 September 1994; Accepted 2 September 1994
Abstract Changes in intrinsic optical signal of rat neocortical slices following afferent stimulation were recorded using darkfield infraredvideomicroscopy. Response amplitude was linearly related to stimulation intensity. The intensity of the optical signal reached its maximum 3 s after onset of stimulation and redecayed with a mean time constant of 23 _+7.1 s. The optical signal had a columnar shape. The size of the column was independent from stimulation intensity with stimuli of medium amplitudes. The extent of the optical signal corresponded to the extent of the electrical activation. Changes in intrinsic optical properties may be a useful tool for the study of spread of excitation in neuronal tissue in vitro. Key words'." Intrinsic optical signal; Darkfield infrared videomicroscopy; Neocortical slice; Afferent stimulation; Rat
Various optical methods have been developed to investigate the neuronal activity with 2-dimensional spatial resolution. Using fluorescence ratio imaging with the intracellular Ca2*-indicator fura-2 [20], activity-dependent changes in intracellular Ca 2+ in single cells [16,18], presynaptic terminals, or postsynaptic dendrites [17] could be demonstrated in vitro. Extrinsic optical signals caused by voltage sensitive dyes allow to detect changes in membrane potential of single neurons in culture [8] and neuronal populations with high resolution in time and space [1,2,6]. Optical detection of neuronal activity using intrinsic signals was first described by Hill and Keynes [10] who measured changes in light scattering of active nerves. Using darkfield microscopy it was possible to measure electrical activity of cultured neurons [19]. Maps of functional activity of mammalian cortex in vivo have been obtained by measuring the change of light reflected from animal [3,5,7] and human brain [9]. Since the intrinsic optical signals have a time course several orders of mag-
*Corresponding author. Fax: (49) (211) 3118485. 0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(94)00683-0
nitude slower than the electrical signals, they cannot substitute for recordings with extrinsic optical dyes. Changes of intrinsic optical signal can also be recorded in brain slices using a combination of darkfield technique and infrared-videomicroscopy [4]. The aim of the present study was to characterize this intrinsic optical signal. 400/lm thick neocortical coronal slices were prepared from juvenile male Wistar rats (14 days old) using a vibratome. The slices were stored at room temperature in artificial cerebrospinal fluid containing (in mM): NaCI 124, NaHCO3 26, KCI 3, CaC12 2, MgSO4 2, NaH2PO4 1.25, and glucose 10, equilibrated with 95% O2 and 5% CO2 to pH 7.4. Experiments were done on 20 slices of 13 animals. In the recording chamber the slices were stored submerged at 32°C. To avoid changing reflections on the boundary between water and air, we covered the recording chamber with a small cover slip. An upright microscope (Axioskop FS, Zeiss) with an x 2.5 Neofluar objective (0.075 n.A.) and a darkfield central stop was used. The illumination light was filtered with a bandpass interference filter (750 + 50 nm, Oriel). The video imaging system consisted of a CCD (charge coupled device) camera (C 2400-77 type 01, Hamamatsu), a camera control
228
K. ttollhotf cl a/./.\euro.science Lelters' l,YO l1994) 227 230
Fig. I. Change of intrinsic optical signal in rat ncocortical slice IkHlovxing affcrcnt stimulation. The stimulation electrode is marked by, a vvhitc boundar 5. I:~ar 300 lira.
unit with a shading correction system, a video processing unit (DVS 3000, Hamamatsu) and a S-VHS Videorecorder (RTV 950 PC, Blaupunkt). The experiments were analyzed off-line using a Macintosh f:X personal computer equipped with a fi'ame grabber card and the Image N I H software. If not mentioned otherwise number of experiments refers to number of slices. For stimulation (pulses of 200 #s/2- 10 V in a train of 50 Hz tk~r 2 s) a concentric bipolar electrode (tip diameter 0.1 mm) was used. 11 was placed perpendicular to the surface of the slice at the border of layer VI and the white matter. Field potentials were measured in layer II/I11 with glass microelectrodes filled with 0.5 M NaC1. To detect stimulation-induced changes of the intrinsic signal, analog contrast enhancement, background subtraction, and an 8-fold digital enhancement were applied on-line to the video image obtained from the CCD-camera. Ten images per second were ewduated off-line. To exclude artefacts, we determined the difference between optical intensity in a region of interest and a not involved reference region. Full-width half-maximum of the columnar intrinsic signals were determined at peak of signal after end of stimulation. For characterisation of the changes of intrinsic optical signal, the time course and the spatial extent were determined. Stimulation at the border of layer VI to the white matter caused a change of the intrinsic signal in a columnar region tlnoughout the cortical layers II Vl perpendicular to the orientation of the cortex (Fig. 1). Layer I was never involved. The time course of the change of the intrinsic optical signal was rather slow. The intensity of the optical signal reached its maximum amplitude 3 s alter onset of stimulation, and redecayed with a mean time constant of 23 _+ 7.1 s (n = 48 stimulations of 7 slices) to baseline level (Fig. 2A). The anaplitude of optical signal showed a linear relationship to stimulation strength (Fig. 2B) with a mean correlation coefficient r = 0 . 9 9 + 0 . 0 1 (n = 7). With meditnn stimulation intensity yielding re-
sponses in the plateau range of lull-width half-maximum as discribed below, signal was at least one order of magnitude greater than noise. The time constant of decay was related to stimulation strength with stimuli of small amplitude, but remained unaltered with stimuli o1" medium and high amplitude (Fig. 3A). In 52% of the experiments, a biexponential time course was observed. The two time constants (20 + 3.6 s vs. 27 + 4.6 s; n = 31 stimulations of 7 slices) were significant different (P < 0.01, Student's t-test). The intrinsic optical signal shows a good reproducibility. In a series of 10 subsequent stimulations with 5 min intervalls the amplitude of the tenth optical signal was 100 + 2.2% (n = 6) of the first. To determine the width of the stimulation induced columnar intrinsic signal, we chose the full-width halfmaximum ( F W H M ) measured tangentially in cortical layer I1/III. The relation between F W H M and stimulation strength followed an inverted sigmoid curve
A
loo
50 .c_
m lOs
B
//
loo
.=z, 5o .E
O-
• I
I
I
I
I
2
4
6
8
10
stimulation strength (V)
Fig. 2. Time course and peak amplitude of intrinsic optical signal in relation to stimulation strength. A: time courses of change of intrinsic optical signal in layer lI/III after stimulation with different strength at the border of layer VI and the white matter. Arrow marks onset of stimulation. B: relation between peak amplitude of intrinsic optical signal and stimtflation strength. Line indicates linear regression analysis,,
229
K. Holthoff et al./ Neuroscience Letters 180 (1994) 227-230
A
40 35
t
3O
0
o
20
E 15 ¸ 105I
I
I
I
I
2
3
4
5
6
stimulation strength (V)
B
500-
/
400E E
'~ 300 E (" J= t-.
200
in rat hippocampal slices by measuring changes of light transmission. The origin of changes of intrinsic optical signal described here remains to be investigated. The reflected intrinsic optical signal of mammalian cortex in vivo was suggested to be composed of changes of blood volume, oxygen delivery and light scattering [5]. Lipton [14] concluded that the reflectance changes of guinea pig cerebral cortical slices were due to alterations of cell volume and extracellular space, respectively. The changes of light transmission in rat hippocampal brain slices following afferent stimulation were attributed to cell swelling [15]. In a range with stimuli of medium strength the columnar shape of intrinsic optical signal was constant. Columnar patterns of cortical activity have been described previously [11-13] by [14C]deoxyglucose technique in monkey striate cortex and rat barrel cortex. The shape of activity in rat barrel cortex induced by stroking contralateral C3 vibrissa was similar to the optical signal described here. The present study shows that intrinsic optical signals in brain slices have a high signal-to-noise ratio, show a good reproducibility and may therefore be a useful tool for the study of spread of excitation in neuronal tissue.
"10
"~
This work was supported by Grants Hess DFG 830/6 to O.W.W. and by grant from BMFT to Dr. W. Zieglg~nsberger.
100
0
=
--
I
[
I
I
I
1
I
2
4
6
8
10
12
14
stimulation strength (V)
Fig. 3. Time constant of decay and full-width half-maximum (FWHM) of intrinsic optical signal. A: relation between time constant of optical signal decay and stimulation strength. Values are means of three experiments. B: relation between FWHM of the optical signal measured in layer II/III and stimulation strength.
140 120
100
(Fig. 3B). With increasing amplitude of the stimulation pulses, the FWHM first increased, then remained unchanged for a range of stimuli with medium amplitude, and finally increased again with very strong stimuli. The mean signal FWHM obtained with stimuli of medium strength was 230 + 9.9/zm (n = 6). For investigation of the correlation between changes of intrinsic optical signal and electrical activity, field potentials in layer II/III at different distances to longitudinal axis of optical signal were recorded. Such experiments showed that the extent of the optical signal corresponded to the extent of the electrical activation (Fig. 4). The mean correlation coefficient yielded from a regression analysis was 0.97 _+0.02 (n = 6). The time course of changes in intrinsic optical signal in rat neocortical slices was very slow and exceeded the time course of electrical activation by tens of seconds. MacVicar and Hochman [15] found similar time courses of intrinsic optical signals following afferent stimulation
C
80
2O 0
j
-20 l
I
I
I
I
-1000
-500
0
500
1000
distance in p.m Fig. 4. Correlation between intrinsic optical signal and electrical activity. Intensity profile measured tangentially in cortical layer lull/ at peak of optical signal. Squares indicate normalized amplitude of field potentials (ampl/amplm,0, measured in layer II/III. Insets show field potential recordings of baseline, maxima/, and half-maximal amplitude. Height and width of insets correspond to 4 mV and 60 ms, respectively.
230
K. Holthq/f et al./Neuroscience Letter,~ 180 (1994) 227 230
[1] Albowitz, B., Kuhnt, U. and Ehrenreich, L., Optical recording of epileptiform voltage changes in the neocortical slice, Exp. Brain Res., 81 (1990) 241 256. [2] Blasdel, G.G. and Salama, G., Voltage-sensitive dyes reveal a modular organization in monkey striate cortex, Nature, 321 (1986) 579 585. [3] Bonhoeffer, T. and Grinvald, A., Iso-orientation domains in cat visual cortex are arranged in pinwheel-like patterns, Nature, 353 (1991) 429 431. [4] Dodt+ H.-U. and Zieglgansberger W., Visualization of the spread of excitation in brainslices with infrared-darkfield microscopy, Eur. J. Physiol., Suppl. 442 (1993). [5] Frostig, R.D., Lieke, E.E., Ts'o, D.Y. and Grinvald, A., 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) 6082 6086. [6] Grinvald+ A., Frostig, R.D., Lieke, E. and Hildesheim, R., Optical imaging of neuronal activity, Physiol. Rev., 68 (1988) 1285 1366. [7] Grinvald, A.. Frostig, R.D., Siegel, R.M. and Bartfeld+ E., Highresolution optical imaging of functional brain architecture in the awake monkey, Proc. Natl. Acad. Sci. LISA, 88 (1991) 11559 11563. [8] Grinvald. A., Ross, W.N. and Farber+ 1., Simultaneous optical measurements of electrical activity from multiple sites on processes of cultured neurons, Proc. Natl. Acad. Sci. USA. 78 (1981) 3245 3249. [9] Haglund, M.M., Ojemann, G.A. and Hochman, D.W., Optical imaging of epileptiform and functional activity in human cerebral cortex, Nature, 358 (1992) 668 671. [10] Hill, D.K. and Keynes, R.D., Opacity changes in stimulated nerve, J. Physiol.. 108 (1949) 278 281. [11] Kennedy, C , Des Rosiers, M.H.. Sakurada, O., Shinohara, M.,
Reivich, M.+ Jehle, J.W. and Sokoloff, L., Metabolic mapping of the primary visual system of the monkey by means of the autoradiographic [HC]deoxyglucose technique, Proc. Natl. Acad. Sci. USA, 73 (1976) 4230M234. [12] Kossut, M. and Hand, E, Early development of changes in cortical representation of C3 vibrissa following neonatal denervation of surrounding vibrissa receptors: a 2-deoxyglucose study in the rut, Neurosci. Lctt., 46 (1984) 7 12. [13] K ossut. M. and Hand, R, The development of the vibrissal cortical colurnn: a 2-deoxyglucose study in the rat, Neurosci. Lett., 46 (1984) 1 6. [14] Lipton, P.. Effects of membrane depolarization on light scattering by cerebral cortical slices, J. Physiol., 231 (1973) 365 383. [15] MacVicar. B.A. and Hochman. D.+ Imaging of synaptically ew>ked intrinsic optical signals in hippocampal slices, J. Neurosci.. 11 (1991) 1458 1469. [16] Regehr, W.G., Connor, J.A. and Tank, D.W., Optical imaging of calcium accumulation in hippocampal pyramidal cells during synaptic activation, Nature, 341 (1989) 533 536. [I 7] Regehr, W.G. and Tank, D.W.. Selective fura-2 loading of presynaptic terminals and nerve cell processes by local perfusion in mammalian brain slice, J. Neurosci. Methods, 37 (1991) 111 119. [18] Ross, W.N., Lasser-Ross, N. and Werman, R., Spatial and temporul analysis of calcium-dependent electrical activity in guinea pig Purkinje cell dendrites, Proc. R. Lond. B, 240 (1990) 173 185. [19] Stepnoski. R.A., LaPorta, A., Raccuia-Behling, F., Blonder, G.E., Slusher, R.E. and Kleinfeld, D., Noninvasive detection of changes in membrane potential in cultured neurons by light scattering, Proc. Natl, Acad. Sci. USA, 88 (1991) 9382 9386. [2(I] Tsien, R.Y. and Poenie, M., Fluorescence ratio imaging: a new window into intracellular ionic signaling, Trends Biochem+ Sci., I I (1986) 450 455.