Lasers in Surgery and Medicine 12329-337 (1992)
He-Ne Laser Irradiation of Single Identified Neurons Pave1 Balaban, PhD, Rinat Esenaliev, MS, Tiina Karu, PhD, Elena Kutomkina, MS, Vladilen Letokhov, PhD, Alexander Oraevsky, PhD, and Nikolay Ovcharenko, BS lnstitute of Higher Nervous Activity and Neurophysiology (i? 64,lnstitute of Spectroscopy (R.E., V.L., A.O., N.O.), and Laser Technology Center (T.K., E.K.), Academy of Sciences, 7 42092 Troitzk, Moscow Region, Russia
Silent (LPa2 and LPa3) and spontaneously active (V3, V5, V17) neurons of subesophageal ganglia of Helix pomatia were irradiated via a 125-mm fiber probe with a 10-mW He-Ne laser (A = 632.8 nm), and the rate of membrane depolarization, duration of latent period, and probability of spike activity were measured as the functions of light intensity. It was found that silent neurons can not be activated by He-Ne laser irradiation. When the spontaneously active neurons generating spikes every 7-10 min were irradiated in between their spontaneous spikes, the depolarization of membrane and generation of action potentials occurred as a function of light intensity, I. The probability of spike generation increased until the intensity reached 1 W/cm2,and when I = 4 W/cm2was equal to 1. The depolarization of the membrane had a threshold at I = 0.1 W/cm2, then increased with increasing the intensity, and reached a plateau at I = 0.7 W/cm2(depolarization rate 0.18 mV/s). Duration of the latent period decreased from 28 s to 17 s when the intensity was increased from 0.05 to 0.3 W/cm2. Further increase of the light intensity, from 0.3 to 1.5 W/cm2, caused a less pronounced change in the duration of the latent period (e.g., latent period equal to 11 s at I = 1.5 W/cm2). 0 1992 Wiley-Liss,
Inc.
Key words: He-Ne laser, silent neurons, spike activity, spontaneously active neurons, depolarization of membrane, Helix pomatia
area has been performed with He-Ne laser (see, for example, [18-211). In spite of that, detailed Starting with the classical paper of Arvani- investigation of red light action on neural activity taki and Chalazonitis 111, a big body of experi- is practically absent. He-Ne laser radiation (16mental data gathered during past decades gives mW laser power, 4-mm beam diameter, 8-J/cm2 the evidence that excitable cells like neurons [2- irradiation dose) applied to the skin overlying the 4,6-12,261 and myocardial cells [1,5,13-15,243 sciatic nerve in rats increased the action potencan be stimulated by various wavelengths of UV tials of the sciatic nerve 181. The irradiation of the [11,24] and visible 12-10,12-17,261 light. skin (4-mm area) overlying the median nerve of On the other hand, successful (laser) light humans with a l-mW He-Ne laser (400 flashes at treatments of a variety of painful rheumatologic the repetition rate of 3.1 Hz, dose and intensity and neurologic conditions have been described not shown) via a fiberoptic catheter produced a [18-211. If the neurons have photosensitivity [2- somatosensory evoked potential obtained at Erb's 121 and irradiation alters neural firing in human beings [16,18,22], it is reasonable to assume that Accepted for publication November 26, 1991. these two phenomena could be connected with Address reprint requests to Pave1 Balaban, Inst. of Higher pain therapy by laser light. Nervous Activity and Neurophysiology, Academy ofSciences, A great deal of clinical work in pain-relief 142092 Troitzk, Moscow, Russia. INTRODUCTION
0 1992 Wiley-Liss, Inc.
330
Balaban et al.
Fig. 1. Schematic representation of neuronal localization on the dorsal surface of the subesophageal ganglia complex. LPlG and RPlG, left and right plural ganglia; VG, visceral ganglion; CPlC, cerebropedal connective (adapted from [231).Identifiable neurons are numbered.
point. Prolonged exposure (4,800 flashes under the same experimental conditions) resulted in a decrease of the amplitude of electrically evoked potential [61. The He-Ne laser radiation had no effect on the pulsation frequency of the neuroreceptor cells of Astacus leptodactilus under conditions of both irradiation of the whole cell (intensity 5.1OP2W/cm2, and irradiation time up to 40 min) and microirradiation of its individual parts (laser intensity %loe4W/cm2,irradiation time up to 40 min) [lo]. The irradiation with a He-Ne laser (8 mW/cm2, irradiation time 2 s, repetition rate 10 Hz, irradiated area 2 mm2)resulted in the propagation of a variable potential along the human basal complex [161. The irradiation with a 4-mW He-Ne laser was found to decrease the duration of synaptic depression after the spike, accomplished by the intensification of the nuclear plasmic exchange of the exposed spinal cord neurons of cats. The threshold dose was found to be 1-3 J/cm2 [17]. The goal of our experiments was an investigation into possibility of stimulation of identified
single silent and spontaneously active neurons by He-Ne laser radiation and determination of parameters of neuronal responses (membrane depolarization rate, duration of latent period, probability of spikes) as the functions of light parameters. MATERIALS AND METHODS Chemicals
All chemicals were finest available grade purchased from Reanal (Budapest, Hungary) and Chemapol (Praha, Checoslovakia). Object of Investigation and Its Preparation
Experiments were carried out on giant identified neurons (soma diameter range was from 50 to 100 pm>of the edible snail Helix pomatia. The location of the neuron on the dorsal surface of the subesophageal ganglia complex is shown in Figure 1.The identification of neurons is described in detail in [23]. In our experiments we studied the reaction of silent (LPa2, LPa3, RPa5, RPa6, Fig.
He-Ne Laser Irradiation of Neurons
331
[ n I I \
LASER BEAM
REFERENCE ELECTRODE
\
-
Fig. 2. Principal scheme of the experimental arrangement.
1)and spontaneously active neurons 0.73, V5, V8, V9, V17, Fig. 1.)to the He-Ne laser CW irradiation. The isolation of ganglia is described in detail in [23]. The isolated central nervous system (CNS) was pinned to the wax floor of a rectangular chamber (20 mm x 30 mm), filled with snail saline (80 mM NaCl, 4 mM KC1, 7 mM CaCl, 5 mM MgC1, 10 mM HEPES, pH = 7.55) at 21°C stabilized temperature. The freshly isolated CNS was kept in the dark for 2-3 h before the beginning of irradiation experiment. During that time the electric activity of the neurons under study was recorded. For every experiment, a newly prepared CNS was used. In all experiments, the location of the CNS in the chamber was identical.
period after the penetration of the electrode into the cell. These characteristics then remained constant for tens of hours. A referent Ag-AgC1 electrode was placed in the chamber containing the isolated CNS. A conventional technique was used for recording the electrical activity monitored with a storhge oscilloscope (model M42, Medicor, Budapest, Hungary) and on a chart-recorder H30-30/4 (Krasnodar, USSR). Irradiation
Radiation of a He-Ne laser LG-79/2 (Lvov, USSR, h = 632.8 nm) with output power 10 mW was focused with the aid of an objective on input end of a fused silica fiber with 125-pm core diameter. The output tip of the fiber was fixed on a Recording of the Electrical Activity micromanipulator and positioned at a distance of The membrane potential was recorded with about 100-p,m from the soma of the recorded neuintracellular glass microelectrode filled by 2 M ron. potassium chloride (Fig. 2). Threshold potential, Measurement of the laser radiation power, resting potential, and neuronal spontaneous ac- P, at the output end of the fiber was carried out tivity were constant starting after a 30-40-min before every experiment with the aid of a cali-
Balaban et al.
332
I
fiz
ON
OFF
Fig. 3. A scheme of the neuronal response to irradiation and the measured parameters. AU, a change in membrane potential; At, duration of latent period.
brated photodiode. The power was varied by calibrated filters located before the objective. The area of the irradiated neuronal soma surface, S,, was equal to 2.9-10-4 cm2 and estimated taking into account the energy distribution at the fiber output and the divergence of laser light. The diameter of the laser beam was determined as the width where the intensity drops down to a l/e value. Intensity of the irradiation, I, was calculated as I = P/S1. Measured Parameters Figure 3 represents a schematic response of a neuron to the irradiation and indicates the parameters of the neuronal response. The rectangular trace at the bottom shows the time when the light was switched on and off. Membrane potential shift, AU, was measured from the chart-recorder. Rate of depolarization, V, was calculated as the ratio of membrane potential shift, AU, to the time interval, At, required for the occurrence of this shift. Real-time recording of the membrane potential during laser irradiation allowed to investigate the latency, L (a period of time from the start of light influence on the membrane potential t o its plateau value, Fig. 3.), as a function of the irradiation parameters, and to estimate the dependence of spike probability on laser irradiation intensity. Spike probability, Psp, was calculated by the expression Psp = (NsdNirrad)I, where N,, is the number of neurons responding to light wlth spikes, and Nimadis the total number of neurons irradiated with light with intensity, I. RESULTS
Irradiation of silent parietal neurons LPa2, LPa3, RPa5, and RPa6 (Fig. 1) was carried out using various intensities (up t o 4 W/cm2)of light
during a 1min time. After 30-40 min of resting time the same irradiation procedure was repeated. Neuronal sensibility was estimated in 6 preparations of CNS using AU as a criterion. Resting potentials of mentioned identified silent neurons varied from -65 mV to -74 mV, and no significant correlation between these changes and irradiation was observed. These results suggest that under our experimental conditions the silent neurons are not responsive to irradiation. In contrast, spontaneously active neurons in visceral ganglion (V3, V5, V8, V9, and V17, Fig. 1)were sensitive to irradiation. An example of the spontaneous activity of a bursting cell V5 is shown in Figure 4. As a response to the irradiation with low intensity light, this cell usually responded with a reversible depolarizing shift of membrane potential (Fig. 5). This response was highly reproducible in every experiment when the light was applied repeatedly 8 times at 510-min intervals. Similar results were obtained in 10 preparations. Typical examples of the neuron V5 responses are shown in Figure 5. The irradiation with intensity 0.065 W/cm2 (Fig. 5, curve 1) elicited a depolarizing shift of about 5 mV, which reached a plateau during the stimulation period (60 s). Restoration of membrane potential was observed in 30 s after the end of the irradiation. No spikes were elicited by this intensity of light stimulus. The irradiation with the light of intensity 0.156 W/cm2evoked not only a shift of membrane potential, but also the action potentials (Fig. 5, curve 2) with a latency about 25 s. The increase of irradiation intensity up to 0.96 W/cm2 (Fig. 5, trace 3) elicited the response with a great number of spikes. It is necessary to note that the threshold intensity varied in different preparations, but in all cases probability of spike appearance, P, was dependent on the light intensity (Fig. 6). The behavior of this dependence is almost linear up to 1 W/cm2(Fig. 6). Further increase of irradiation intensity leads to slow rise of spike probability. When the light intensity exceeded 4 W/cm2, the probability, P, reaches its maximum (P = 1).Statistical analysis shows that the absence of spike activity can be expected in 85% cases when the neural cells were irradiated with light intensity of 0.065 W/cm2 (Fig. 5, curve 1).At the intensity 0.156 W/cm2, the spike activity (Fig. 5, curve 2) was recorded for 35% of irradiated neurons, and at the intensity 0.96 W/cm2,80% of the irradiated neurons will respond. At the intensities higher
He-Ne Laser Irradiation of Neurons
333
L
5 MUW
I
Fig. 4. Spontaneous activity of the neuron V5.
- - .
I
I
-
I
3 Fig. 5. Typical responses of the neuron V5 to the irradiation with light X = 632.8 nm at various intensities. Below every trace, the moment when the light was switched on and off is shown.
than 4 W/cm2,all irradiated neurons V5 produced spikes. The depolarization rate, AU/At (Fig. 7), and duration of latent period, L (Fig. 81, were esti-
mated as a function of laser intensity. The depolarization rate was very low when the light intensity was less than 0.1 W/cm2.The increase of the intensity up to 0.7 Wkm’ led to a significant in-
Balaban et al.
334
04 0
0.5
1.0
1.5
I N T E N S I T Y , W/crn2 o !
:
::tl+q 0.1
t:
I
1.0
:::
I
10 IN TE N S I T Y
W,cm2
Fig. 6. Probability of the spike activity of the neuron V5 as a function on the He-Ne laser radiation intensity.
1
1
2
o_ i l
n
I
IN TE N SI T Y,
W/crn2
Fig. 7. Dependence of the depolarization rate, AUlAt, for the V5 neuron upon the intensity of He-Ne laser radiation (open circles, results of different experiments; filled circles, the mean value of all experiments).
crease of the depolarization rate, but when the intensity exceeded 0.7 W/cm2, the depolarization rate reached the plateau (Fig. 7) on the level about 0.18 mV/s. Duration of the latent period L decreased from 28 s t o 17 s with the increase of the light intensity from 0.05 to 0.3 W/cm2(Fig. 8). Further increase of the light intensity led to less rapid decrease of the latency (Fig. 8). DISCUSSION
The extraretinal photoreception (i.e. biological response initiated through photoreceptors others than the eye) is a wide-spread biological phenomenon occurring in a variety of excitable as
Fig. 8. Dependence of the latent period, At (L), of the neuron V5 upon the intensity of He-Ne laser radiation (open circles, results of different experiments;filled circles, the mean value of all experiments).
well as nonexcitable cells (see [351 for review). In case of nonexcitable cells and microorganisms, the respiratory chain components are believed t o be primary chromophores [27,281.The respiratory chain components are also found to be responsible for photoresponses of excitable cells [1,4,5,7,10, 15,261. In the case of Aplysia neurons the laser light is believed can be preferentially absorbed by cytoplasmic lipochondria. These yellow-orange pigmented granules underwent structural changes accompanied by the release of calcium under action of short flashes of irradiation [25,33,341. In our experiments, the effects of the He-Ne laser irradiation with the intensity range from 0.05 to 4.0 Wkm2 were investigated. It was found that only spontaneously active cells of the snail nervous systems were sensitive to the light stimulation. The neuronal responses were demonstrated as a function of light intensity (Figs. 6-8). In the present study the effect of laser irradiation on identified silent and active neural cells is described. The previous He-Ne laser irradiation studies [6,8,10,16,171 dealt with the nerve cells acting as conductors of neural spikes. Therefore, the comparison of our data with published results of neural cell irradiation can not be made directly. No difference in the laser effect on spontaneously active and silent neuron elements can be expected in cases when the nerve cells act only as conductors of neural spikes. For instance, the effect of the He-Ne laser radiation was found only after the preceding stimulation of neural spontaneous activity [6,8], while no effect was seen in
He-Ne Laser Irradiation of Neurons 335 experiments on neuroreceptor cells of the crayfish for biological tissue irradiation in the visible specin which no prestimulation was used [lo]. tral range), the heat diffusion time is given by the It was shown that blue light can produce expression stimulation (activation) of silent identified neurons in Aplysia californica (A = 488 nm in [2], and A = 490 nm in [251), but irradiation by the Nd: YAG laser (near infrared spectral region, A = where x = 1.3-10-3cm2/sis thermal diffusivity of 1060 nm [21) was not effective. The irradiation by soft biological tissue with a high water content, the He-Cd laser (A = 446.1 nm, I = 10-104 W/ and lmin is the minimal linear dimension of the cm2)has been shown to produce the excitation of volume being irradiated. The length of the cylinnonidentified neurons in crayfish, while He-Ne der heated is equal to the effective penetration laser irradiation with I = 2-103-2.104 W/cm2was depth of laser radiation, leff.For a simple estimanot effective [lo]. These data suggest that silent tion we may assume that diameter, d, of this cylneurons can be activated by blue light, while red inder is equal to the diameter of the laser beam. It light and near infrared radiation are not effective. is easy to see that the model of elongated cylinder For now, an effective spectral range for molluscan is suitable for our experimental conditions, if one silent neurons activation is thought to be 470- takes leffequal t o 0.1 cm (as in the case of a hu510 nm (maximum at 490 nm [25]),but more pre- man blood vessel wall at A = 633 nm 1291) and dl cise investigation on different neurons is clearly = (4S,/.rr)= 4" 2.9.10-4 cm2/3.14 = 0.019 cm
He-Ne Laser Irradiation of Single Identified Neurons Pave1 Balaban, PhD, Rinat Esenaliev, MS, Tiina Karu, PhD, Elena Kutomkina, MS, Vladilen Letokhov, PhD, Alexander Oraevsky, PhD, and Nikolay Ovcharenko, BS lnstitute of Higher Nervous Activity and Neurophysiology (i? 64,lnstitute of Spectroscopy (R.E., V.L., A.O., N.O.), and Laser Technology Center (T.K., E.K.), Academy of Sciences, 7 42092 Troitzk, Moscow Region, Russia
Silent (LPa2 and LPa3) and spontaneously active (V3, V5, V17) neurons of subesophageal ganglia of Helix pomatia were irradiated via a 125-mm fiber probe with a 10-mW He-Ne laser (A = 632.8 nm), and the rate of membrane depolarization, duration of latent period, and probability of spike activity were measured as the functions of light intensity. It was found that silent neurons can not be activated by He-Ne laser irradiation. When the spontaneously active neurons generating spikes every 7-10 min were irradiated in between their spontaneous spikes, the depolarization of membrane and generation of action potentials occurred as a function of light intensity, I. The probability of spike generation increased until the intensity reached 1 W/cm2,and when I = 4 W/cm2was equal to 1. The depolarization of the membrane had a threshold at I = 0.1 W/cm2, then increased with increasing the intensity, and reached a plateau at I = 0.7 W/cm2(depolarization rate 0.18 mV/s). Duration of the latent period decreased from 28 s to 17 s when the intensity was increased from 0.05 to 0.3 W/cm2. Further increase of the light intensity, from 0.3 to 1.5 W/cm2, caused a less pronounced change in the duration of the latent period (e.g., latent period equal to 11 s at I = 1.5 W/cm2). 0 1992 Wiley-Liss,
Inc.
Key words: He-Ne laser, silent neurons, spike activity, spontaneously active neurons, depolarization of membrane, Helix pomatia
area has been performed with He-Ne laser (see, for example, [18-211). In spite of that, detailed Starting with the classical paper of Arvani- investigation of red light action on neural activity taki and Chalazonitis 111, a big body of experi- is practically absent. He-Ne laser radiation (16mental data gathered during past decades gives mW laser power, 4-mm beam diameter, 8-J/cm2 the evidence that excitable cells like neurons [2- irradiation dose) applied to the skin overlying the 4,6-12,261 and myocardial cells [1,5,13-15,243 sciatic nerve in rats increased the action potencan be stimulated by various wavelengths of UV tials of the sciatic nerve 181. The irradiation of the [11,24] and visible 12-10,12-17,261 light. skin (4-mm area) overlying the median nerve of On the other hand, successful (laser) light humans with a l-mW He-Ne laser (400 flashes at treatments of a variety of painful rheumatologic the repetition rate of 3.1 Hz, dose and intensity and neurologic conditions have been described not shown) via a fiberoptic catheter produced a [18-211. If the neurons have photosensitivity [2- somatosensory evoked potential obtained at Erb's 121 and irradiation alters neural firing in human beings [16,18,22], it is reasonable to assume that Accepted for publication November 26, 1991. these two phenomena could be connected with Address reprint requests to Pave1 Balaban, Inst. of Higher pain therapy by laser light. Nervous Activity and Neurophysiology, Academy ofSciences, A great deal of clinical work in pain-relief 142092 Troitzk, Moscow, Russia. INTRODUCTION
0 1992 Wiley-Liss, Inc.
330
Balaban et al.
Fig. 1. Schematic representation of neuronal localization on the dorsal surface of the subesophageal ganglia complex. LPlG and RPlG, left and right plural ganglia; VG, visceral ganglion; CPlC, cerebropedal connective (adapted from [231).Identifiable neurons are numbered.
point. Prolonged exposure (4,800 flashes under the same experimental conditions) resulted in a decrease of the amplitude of electrically evoked potential [61. The He-Ne laser radiation had no effect on the pulsation frequency of the neuroreceptor cells of Astacus leptodactilus under conditions of both irradiation of the whole cell (intensity 5.1OP2W/cm2, and irradiation time up to 40 min) and microirradiation of its individual parts (laser intensity %loe4W/cm2,irradiation time up to 40 min) [lo]. The irradiation with a He-Ne laser (8 mW/cm2, irradiation time 2 s, repetition rate 10 Hz, irradiated area 2 mm2)resulted in the propagation of a variable potential along the human basal complex [161. The irradiation with a 4-mW He-Ne laser was found to decrease the duration of synaptic depression after the spike, accomplished by the intensification of the nuclear plasmic exchange of the exposed spinal cord neurons of cats. The threshold dose was found to be 1-3 J/cm2 [17]. The goal of our experiments was an investigation into possibility of stimulation of identified
single silent and spontaneously active neurons by He-Ne laser radiation and determination of parameters of neuronal responses (membrane depolarization rate, duration of latent period, probability of spikes) as the functions of light parameters. MATERIALS AND METHODS Chemicals
All chemicals were finest available grade purchased from Reanal (Budapest, Hungary) and Chemapol (Praha, Checoslovakia). Object of Investigation and Its Preparation
Experiments were carried out on giant identified neurons (soma diameter range was from 50 to 100 pm>of the edible snail Helix pomatia. The location of the neuron on the dorsal surface of the subesophageal ganglia complex is shown in Figure 1.The identification of neurons is described in detail in [23]. In our experiments we studied the reaction of silent (LPa2, LPa3, RPa5, RPa6, Fig.
He-Ne Laser Irradiation of Neurons
331
[ n I I \
LASER BEAM
REFERENCE ELECTRODE
\
-
Fig. 2. Principal scheme of the experimental arrangement.
1)and spontaneously active neurons 0.73, V5, V8, V9, V17, Fig. 1.)to the He-Ne laser CW irradiation. The isolation of ganglia is described in detail in [23]. The isolated central nervous system (CNS) was pinned to the wax floor of a rectangular chamber (20 mm x 30 mm), filled with snail saline (80 mM NaCl, 4 mM KC1, 7 mM CaCl, 5 mM MgC1, 10 mM HEPES, pH = 7.55) at 21°C stabilized temperature. The freshly isolated CNS was kept in the dark for 2-3 h before the beginning of irradiation experiment. During that time the electric activity of the neurons under study was recorded. For every experiment, a newly prepared CNS was used. In all experiments, the location of the CNS in the chamber was identical.
period after the penetration of the electrode into the cell. These characteristics then remained constant for tens of hours. A referent Ag-AgC1 electrode was placed in the chamber containing the isolated CNS. A conventional technique was used for recording the electrical activity monitored with a storhge oscilloscope (model M42, Medicor, Budapest, Hungary) and on a chart-recorder H30-30/4 (Krasnodar, USSR). Irradiation
Radiation of a He-Ne laser LG-79/2 (Lvov, USSR, h = 632.8 nm) with output power 10 mW was focused with the aid of an objective on input end of a fused silica fiber with 125-pm core diameter. The output tip of the fiber was fixed on a Recording of the Electrical Activity micromanipulator and positioned at a distance of The membrane potential was recorded with about 100-p,m from the soma of the recorded neuintracellular glass microelectrode filled by 2 M ron. potassium chloride (Fig. 2). Threshold potential, Measurement of the laser radiation power, resting potential, and neuronal spontaneous ac- P, at the output end of the fiber was carried out tivity were constant starting after a 30-40-min before every experiment with the aid of a cali-
Balaban et al.
332
I
fiz
ON
OFF
Fig. 3. A scheme of the neuronal response to irradiation and the measured parameters. AU, a change in membrane potential; At, duration of latent period.
brated photodiode. The power was varied by calibrated filters located before the objective. The area of the irradiated neuronal soma surface, S,, was equal to 2.9-10-4 cm2 and estimated taking into account the energy distribution at the fiber output and the divergence of laser light. The diameter of the laser beam was determined as the width where the intensity drops down to a l/e value. Intensity of the irradiation, I, was calculated as I = P/S1. Measured Parameters Figure 3 represents a schematic response of a neuron to the irradiation and indicates the parameters of the neuronal response. The rectangular trace at the bottom shows the time when the light was switched on and off. Membrane potential shift, AU, was measured from the chart-recorder. Rate of depolarization, V, was calculated as the ratio of membrane potential shift, AU, to the time interval, At, required for the occurrence of this shift. Real-time recording of the membrane potential during laser irradiation allowed to investigate the latency, L (a period of time from the start of light influence on the membrane potential t o its plateau value, Fig. 3.), as a function of the irradiation parameters, and to estimate the dependence of spike probability on laser irradiation intensity. Spike probability, Psp, was calculated by the expression Psp = (NsdNirrad)I, where N,, is the number of neurons responding to light wlth spikes, and Nimadis the total number of neurons irradiated with light with intensity, I. RESULTS
Irradiation of silent parietal neurons LPa2, LPa3, RPa5, and RPa6 (Fig. 1) was carried out using various intensities (up t o 4 W/cm2)of light
during a 1min time. After 30-40 min of resting time the same irradiation procedure was repeated. Neuronal sensibility was estimated in 6 preparations of CNS using AU as a criterion. Resting potentials of mentioned identified silent neurons varied from -65 mV to -74 mV, and no significant correlation between these changes and irradiation was observed. These results suggest that under our experimental conditions the silent neurons are not responsive to irradiation. In contrast, spontaneously active neurons in visceral ganglion (V3, V5, V8, V9, and V17, Fig. 1)were sensitive to irradiation. An example of the spontaneous activity of a bursting cell V5 is shown in Figure 4. As a response to the irradiation with low intensity light, this cell usually responded with a reversible depolarizing shift of membrane potential (Fig. 5). This response was highly reproducible in every experiment when the light was applied repeatedly 8 times at 510-min intervals. Similar results were obtained in 10 preparations. Typical examples of the neuron V5 responses are shown in Figure 5. The irradiation with intensity 0.065 W/cm2 (Fig. 5, curve 1) elicited a depolarizing shift of about 5 mV, which reached a plateau during the stimulation period (60 s). Restoration of membrane potential was observed in 30 s after the end of the irradiation. No spikes were elicited by this intensity of light stimulus. The irradiation with the light of intensity 0.156 W/cm2evoked not only a shift of membrane potential, but also the action potentials (Fig. 5, curve 2) with a latency about 25 s. The increase of irradiation intensity up to 0.96 W/cm2 (Fig. 5, trace 3) elicited the response with a great number of spikes. It is necessary to note that the threshold intensity varied in different preparations, but in all cases probability of spike appearance, P, was dependent on the light intensity (Fig. 6). The behavior of this dependence is almost linear up to 1 W/cm2(Fig. 6). Further increase of irradiation intensity leads to slow rise of spike probability. When the light intensity exceeded 4 W/cm2, the probability, P, reaches its maximum (P = 1).Statistical analysis shows that the absence of spike activity can be expected in 85% cases when the neural cells were irradiated with light intensity of 0.065 W/cm2 (Fig. 5, curve 1).At the intensity 0.156 W/cm2, the spike activity (Fig. 5, curve 2) was recorded for 35% of irradiated neurons, and at the intensity 0.96 W/cm2,80% of the irradiated neurons will respond. At the intensities higher
He-Ne Laser Irradiation of Neurons
333
L
5 MUW
I
Fig. 4. Spontaneous activity of the neuron V5.
- - .
I
I
-
I
3 Fig. 5. Typical responses of the neuron V5 to the irradiation with light X = 632.8 nm at various intensities. Below every trace, the moment when the light was switched on and off is shown.
than 4 W/cm2,all irradiated neurons V5 produced spikes. The depolarization rate, AU/At (Fig. 7), and duration of latent period, L (Fig. 81, were esti-
mated as a function of laser intensity. The depolarization rate was very low when the light intensity was less than 0.1 W/cm2.The increase of the intensity up to 0.7 Wkm’ led to a significant in-
Balaban et al.
334
04 0
0.5
1.0
1.5
I N T E N S I T Y , W/crn2 o !
:
::tl+q 0.1
t:
I
1.0
:::
I
10 IN TE N S I T Y
W,cm2
Fig. 6. Probability of the spike activity of the neuron V5 as a function on the He-Ne laser radiation intensity.
1
1
2
o_ i l
n
I
IN TE N SI T Y,
W/crn2
Fig. 7. Dependence of the depolarization rate, AUlAt, for the V5 neuron upon the intensity of He-Ne laser radiation (open circles, results of different experiments; filled circles, the mean value of all experiments).
crease of the depolarization rate, but when the intensity exceeded 0.7 W/cm2, the depolarization rate reached the plateau (Fig. 7) on the level about 0.18 mV/s. Duration of the latent period L decreased from 28 s t o 17 s with the increase of the light intensity from 0.05 to 0.3 W/cm2(Fig. 8). Further increase of the light intensity led to less rapid decrease of the latency (Fig. 8). DISCUSSION
The extraretinal photoreception (i.e. biological response initiated through photoreceptors others than the eye) is a wide-spread biological phenomenon occurring in a variety of excitable as
Fig. 8. Dependence of the latent period, At (L), of the neuron V5 upon the intensity of He-Ne laser radiation (open circles, results of different experiments;filled circles, the mean value of all experiments).
well as nonexcitable cells (see [351 for review). In case of nonexcitable cells and microorganisms, the respiratory chain components are believed t o be primary chromophores [27,281.The respiratory chain components are also found to be responsible for photoresponses of excitable cells [1,4,5,7,10, 15,261. In the case of Aplysia neurons the laser light is believed can be preferentially absorbed by cytoplasmic lipochondria. These yellow-orange pigmented granules underwent structural changes accompanied by the release of calcium under action of short flashes of irradiation [25,33,341. In our experiments, the effects of the He-Ne laser irradiation with the intensity range from 0.05 to 4.0 Wkm2 were investigated. It was found that only spontaneously active cells of the snail nervous systems were sensitive to the light stimulation. The neuronal responses were demonstrated as a function of light intensity (Figs. 6-8). In the present study the effect of laser irradiation on identified silent and active neural cells is described. The previous He-Ne laser irradiation studies [6,8,10,16,171 dealt with the nerve cells acting as conductors of neural spikes. Therefore, the comparison of our data with published results of neural cell irradiation can not be made directly. No difference in the laser effect on spontaneously active and silent neuron elements can be expected in cases when the nerve cells act only as conductors of neural spikes. For instance, the effect of the He-Ne laser radiation was found only after the preceding stimulation of neural spontaneous activity [6,8], while no effect was seen in
He-Ne Laser Irradiation of Neurons 335 experiments on neuroreceptor cells of the crayfish for biological tissue irradiation in the visible specin which no prestimulation was used [lo]. tral range), the heat diffusion time is given by the It was shown that blue light can produce expression stimulation (activation) of silent identified neurons in Aplysia californica (A = 488 nm in [2], and A = 490 nm in [251), but irradiation by the Nd: YAG laser (near infrared spectral region, A = where x = 1.3-10-3cm2/sis thermal diffusivity of 1060 nm [21) was not effective. The irradiation by soft biological tissue with a high water content, the He-Cd laser (A = 446.1 nm, I = 10-104 W/ and lmin is the minimal linear dimension of the cm2)has been shown to produce the excitation of volume being irradiated. The length of the cylinnonidentified neurons in crayfish, while He-Ne der heated is equal to the effective penetration laser irradiation with I = 2-103-2.104 W/cm2was depth of laser radiation, leff.For a simple estimanot effective [lo]. These data suggest that silent tion we may assume that diameter, d, of this cylneurons can be activated by blue light, while red inder is equal to the diameter of the laser beam. It light and near infrared radiation are not effective. is easy to see that the model of elongated cylinder For now, an effective spectral range for molluscan is suitable for our experimental conditions, if one silent neurons activation is thought to be 470- takes leffequal t o 0.1 cm (as in the case of a hu510 nm (maximum at 490 nm [25]),but more pre- man blood vessel wall at A = 633 nm 1291) and dl cise investigation on different neurons is clearly = (4S,/.rr)= 4" 2.9.10-4 cm2/3.14 = 0.019 cm
