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Intracellular recording and staining of directionally selective motion detecting neurons in fly optic lobe
RESEARCH
NOTE
INTRACELLULAR RECORDING AND STAINING OF DIRECTIONALLY SELECTIVE MOTION DETECHNG NEURONS IN FLY OPTIC LOBE DAZEDR. DVORAK,LEWISG. BISHOPand HENDRK E. ECKERT Department of Biological Sciences University of Southern California. University Park. Los Angeles California 9ooO7. U.S.A. (Receiced
25 June 1974: in rerised form
The optomotor response. that is. the tendency of an animal to turn in the direction of a moving object in its visual field. has been used to study movement perception in insects. A specific response. such as the turning torque. the thrust. or the lift produced in tethered flight or by the walking animal. is measured as a function of the parameters of a moving striped pattern. From such measurements several workers have described the functional properties of the neural network mediating the optomotor response (e.g. review Reichar& 1969:Goetz 1972; Kirschfeld 1972.1973; Eckert 1973; Reichardt. 1973). Extensive anatomical studies have been made upon the dipteran and hymenopteran visual systems (e.g. Trujillo-Cenoz and Melamed 1966; Braitenberg. 1967, 1972; Strausfeld. 1971; Ribi 1973). Direct correlation of the behavioral results to the neurological substrate could be made by electrophysiological experiments if: (1) the organization of the system is such that one can find in it single neurons whose electrical activity encodes recognizable portions of the behavioral response; and (2) the neurons from which recordings are taken can be identified and traced morphologically. With regard to point (1) neurons have been isolated that contain in their responses much of the information prerequisite to the optomotor response. In the lobula of blowflies and houseflies (Bishop and Keehn, 1966. 1967) and in the homologous area in the optic lobe of the bee (Kaiser and Bishop, 1970) extracellular recordings have been taken from directionally selective motion detecting cells. These cells are excited by pattern movement in a specific direction (preferred direction) and inhibited by pattern movement opposite to the preferred direction (null direction). These cells are thought to be part of the neural system underlying the optomotor response because their responses (average steady state spike frequency) either encode information necessary to the optomotor response or correlate with it. For example. these units exhibit a strong dependence upon the direction of object movement. and correlate with the optomotor response (torque produced in tethered flight) in their dependence upon the spatial
I5 July 1971)
wavelength. angular velocity and mean brightness of a rotating striped pattern. Two distinct groups of motion units were found (Bishop. Keehn and McCann. 1968): those excited by pattern movement in the horizontal direction and those excited by movement in the vertical direction. Goetz (review. 1972) concluded that optomotor responses in Drosophila are driven by distinct horizontal and vertical directionally Selective motion detecting systems. We have recorded intracellularly from the directionally selective motion units in flies; we have stained ten of them well by iontophoretic injection of the fluorescent dye procion yellow M4R.S. Hence we have demonstrated that the need expressed in point (2) for recordings from identified cells is possible, even though the diameters of the cells involved are as small as 3 pm. Adult male and female greenbottle flies. Pltaeniciu sericaru and white-eyed (chalky mutant) blowflies. Calliphora erythrocephala. were used in this study. The animals were intact and not anesthetized. They were secured in soft wax (Surgident Co.) thus preventing any movements of the animal. A suitable portion of the exoskeleton was removed from the back of the head to allow access to the optic lobe. A silver-silver chloride indifferent electrode was placed in the body fluid near the recording site. The animal was placed in front of a screen onto which a movable pattern of black and white stripes was projected. The spatial wavelength of the pattern was 20”. the velocity approximately 20’/ sec. The cells were impaled with microelectrodes filled with a 4”/, (w/v) solution of procion yellow M4RS or with 3 M KCl. The resistances of the two types of electrodes was 200-6OOMn and 20-50MR. respectively. Intracellular potentials were amplified by conventional means (Bioelectric Instruments PAD 2a preamplifier), and recorded on an FM tape recorder (Hewlett Packard 3960). Iontophoresis of the marker dye was accomplished by injecting lO-30nA of oscillating current (frequency 1 Hz) into the neuron for 30-90 sec. The dye was allowed to diffuse for 6- 18 hr at 4°C. The head was fixed in a 4% formaldehyde solution overnight. dehydrated cleared embedded in paraplast, and sectioned
451
Research Note
Fig. I. Intracellular recording from a directionally selective motion detecting cell in the lobulus of the blowfly (preferred direction: dorsal to ventral). (a) Spontaneous activity, pattern illuminated. (b) and (d) Response to movement of a striped pattern (bars) in the preferred direction. (c) Bar indicates movement of the striped pattern in the null direction. Markers: 60 mV. 05 sec.
ventral (a down unit). The axon has an average diaat I2 pm. The histological material was examined unmeter ofapproximately 45 pm. The cell can be traced der a Zeiss transmission fluorescence microscope. from the peripheral extent of the lobulus to the midThe responses of the motion detecting cells recorded intracellularly are similar to those obtained from brain over a distance of approximately 300 pm. The extracellular recordings. The activity in the presence of approximate location of this cell is shown schematian illuminated but stationary pattern. varied from 3 to cally in Fig. 2(a) (arrow). Although several hours were 25 spike&z [Fig. l(a)]. Movement of the pattern in allowed for the procion dye to diffuse throughout the the preferred direction evoked a response of up to 100 cell. it is not certain that the entire cell was filled. spikes/set [Figs. l(b). I(d)], while movements in the Motion detecting neurons with contralateral visual null direction typically inhibited spontaneous activity fields and horizontal preferred directions were located [Fig. I(c)]. Overshooting action potentials (30-90 mV). near the anterior surface of the lobulus. Cells with contralateral visual fields and vertical preferred directions monophasic action potentials (5-25 mV) and EPSP’s (I-10 mV) were routinely recorded. This variation in were found near the posterior surface of the lobulus. waveform and amplitude of observed potentials indicates that recording sites were located in axons, neurites and somata (see Murphy, 1973). Resting membrane potentials varied from 20-6OmV. The most stable recordings (usually associated with over-shooting spikes) were as long as 30 min. In a previous publication it was reported that in the lobula region extracellular recordings had been obtained from several different types of directionally selective motion detecting units (Bishop er al., 1968). These types were distinguished by location of visual field, contralateral or ipsilateral. and by direction of preferred pattern movemenf that is: (1) horizontal. back to front or front to back; and (2) vertical. up or down. We have succeeded in recording intracellularly from each of these types of units. Fig. 2. (a) Schematic diagram of a cross-sectional view of Figure 2(b) shows a stained neuron which had a conthe head of a fly from the midbrain to the periphery. The tralateral visual held and responded maximally to patarrow indicates the approximate location of the neuron tern movement in the vertical direction from dorsal to shown in Fig. 2(b).
Fig. 2(b). Directionally
selective motion detecting cell stained by intracellular 10 pm. (Horizontal section.)
yellow M4RS. Marker:
injection OSprocion
453
Research Note Both types of neurons projected to the periesophageal
region. Those cells with ipsilateral visual fields were located near the anterior surface of the lobulus and extended ventro-posteriorly to the circumesophageal connective. In no case did a neuron with contralateral visual field extend beyond the midline of the midbrain. Accurate mapping of these cells by comparison with Golgi stained material is our immediate goal.
Acknowledgements-Supported by AFOSR grant number 71-21 I2 and NSF grant number GB 30733. We thank Mr. Mark Mayo for valuable technical assistance.
REFERENCES
Bishop L. G. and Keehn D. G. (1966) Two types of motion sensitive neurons in the fly optic lobe. Snture. Lond. 212. 1373-1376. Bishop L. G. and Keehn D. G. (1967) Neural correlates of the optomotor response in the fly. Kyberneric 3. 288-295. Bishop L. G.. Keehn D. G. and McCann G. D. (1968) Studies of motion detection by interneurons of the optic lobes and brain of the flies. Calliphora phaenicia and &fusca domestico.J. Neuroph!aioL 31. 509-525. Braitenberg V. (1967) Patterns of projection in the visual system of the fly. Expl Brain Res. 3. 271-298. Braitenberg V. (1972) Periodic structures and structural gradients in the visual ganglia of the fly. In Information Processing in the Visua/ Srsrents of Arthropods (edited by Wehner R.J. p. 3- I5 Springer. Berlin.
Eckert H. (1973) Optomotorrsche Untersuchungen am visuellen System der StubenRiege .Il~t.sra tlonresricn L. Kjherneric 14. l-13. Goetz K. G. (1972) Processing ol cues from the moving environment in the Drosophila navigation system. In I&rmotion
Processing
in
rhe
Lisual
S.Wevr
01’ Arrbrop0d.v
(edited by Wehner R.I. p. 225-263. Springer. Berlin. Kaiser W. and Bishop L. G. (1970) Directionally selective motion detecting units in the optic lobe of the honeybee. Z. rergl. Physiol. 67. 40.3-413. Kirschfeld K. (1972) The visual system of .Lfuscu: studies in optics. structure and function. In I&vrnarim Procc~ssinq in the k’isua/ S.rstetnsof Amhropods (edited by Wehner R.). p. 61-74. Springer. Berlin. Kirschfeld K. (1973) Optomotorische Reaktionen der Biene auf bewegte “Polarizations-Muster” Z. ,Vaturfbrsch. 2&z. 329-338. Murphy R. K. (1973) Characterization of an insect neuron which cannot be visualized in SIIU. In Ierraccllular Stairrinq in k’rurohioloqy (edited by Kater S. B. and Nicholson C.), p. 135 150. Springer. Berlin. Reichardt W. (1969) Movement perception in insects. In ProcessingofOptical Data by Organisms and by Machines (edited by Reichardt W.). Reichardt W. (1973) Musterinduziertc Flugorientierung. Verhaltensversuche an der Fliege Musco dmmicu. Naturwiss 60. IX- I38 Ribi W. (1973) Doctoral dissertation. Zurich. Strausfeld N. J. (197 I I The organization of the insect visual system. Z. Zelf/iirsch. 121. M-454. Trujillo-Cenoz 0. and Melamed J. (1966) Electron microscope observations on the peripheral and intermediate retinas ofdipterans. In The Functional Organkation qfrhe Compound Eye (edited by Bernard C. G.J. pp. 339-361. Pergamon Press Oxford.
NOTE
INTRACELLULAR RECORDING AND STAINING OF DIRECTIONALLY SELECTIVE MOTION DETECHNG NEURONS IN FLY OPTIC LOBE DAZEDR. DVORAK,LEWISG. BISHOPand HENDRK E. ECKERT Department of Biological Sciences University of Southern California. University Park. Los Angeles California 9ooO7. U.S.A. (Receiced
25 June 1974: in rerised form
The optomotor response. that is. the tendency of an animal to turn in the direction of a moving object in its visual field. has been used to study movement perception in insects. A specific response. such as the turning torque. the thrust. or the lift produced in tethered flight or by the walking animal. is measured as a function of the parameters of a moving striped pattern. From such measurements several workers have described the functional properties of the neural network mediating the optomotor response (e.g. review Reichar& 1969:Goetz 1972; Kirschfeld 1972.1973; Eckert 1973; Reichardt. 1973). Extensive anatomical studies have been made upon the dipteran and hymenopteran visual systems (e.g. Trujillo-Cenoz and Melamed 1966; Braitenberg. 1967, 1972; Strausfeld. 1971; Ribi 1973). Direct correlation of the behavioral results to the neurological substrate could be made by electrophysiological experiments if: (1) the organization of the system is such that one can find in it single neurons whose electrical activity encodes recognizable portions of the behavioral response; and (2) the neurons from which recordings are taken can be identified and traced morphologically. With regard to point (1) neurons have been isolated that contain in their responses much of the information prerequisite to the optomotor response. In the lobula of blowflies and houseflies (Bishop and Keehn, 1966. 1967) and in the homologous area in the optic lobe of the bee (Kaiser and Bishop, 1970) extracellular recordings have been taken from directionally selective motion detecting cells. These cells are excited by pattern movement in a specific direction (preferred direction) and inhibited by pattern movement opposite to the preferred direction (null direction). These cells are thought to be part of the neural system underlying the optomotor response because their responses (average steady state spike frequency) either encode information necessary to the optomotor response or correlate with it. For example. these units exhibit a strong dependence upon the direction of object movement. and correlate with the optomotor response (torque produced in tethered flight) in their dependence upon the spatial
I5 July 1971)
wavelength. angular velocity and mean brightness of a rotating striped pattern. Two distinct groups of motion units were found (Bishop. Keehn and McCann. 1968): those excited by pattern movement in the horizontal direction and those excited by movement in the vertical direction. Goetz (review. 1972) concluded that optomotor responses in Drosophila are driven by distinct horizontal and vertical directionally Selective motion detecting systems. We have recorded intracellularly from the directionally selective motion units in flies; we have stained ten of them well by iontophoretic injection of the fluorescent dye procion yellow M4R.S. Hence we have demonstrated that the need expressed in point (2) for recordings from identified cells is possible, even though the diameters of the cells involved are as small as 3 pm. Adult male and female greenbottle flies. Pltaeniciu sericaru and white-eyed (chalky mutant) blowflies. Calliphora erythrocephala. were used in this study. The animals were intact and not anesthetized. They were secured in soft wax (Surgident Co.) thus preventing any movements of the animal. A suitable portion of the exoskeleton was removed from the back of the head to allow access to the optic lobe. A silver-silver chloride indifferent electrode was placed in the body fluid near the recording site. The animal was placed in front of a screen onto which a movable pattern of black and white stripes was projected. The spatial wavelength of the pattern was 20”. the velocity approximately 20’/ sec. The cells were impaled with microelectrodes filled with a 4”/, (w/v) solution of procion yellow M4RS or with 3 M KCl. The resistances of the two types of electrodes was 200-6OOMn and 20-50MR. respectively. Intracellular potentials were amplified by conventional means (Bioelectric Instruments PAD 2a preamplifier), and recorded on an FM tape recorder (Hewlett Packard 3960). Iontophoresis of the marker dye was accomplished by injecting lO-30nA of oscillating current (frequency 1 Hz) into the neuron for 30-90 sec. The dye was allowed to diffuse for 6- 18 hr at 4°C. The head was fixed in a 4% formaldehyde solution overnight. dehydrated cleared embedded in paraplast, and sectioned
451
Research Note
Fig. I. Intracellular recording from a directionally selective motion detecting cell in the lobulus of the blowfly (preferred direction: dorsal to ventral). (a) Spontaneous activity, pattern illuminated. (b) and (d) Response to movement of a striped pattern (bars) in the preferred direction. (c) Bar indicates movement of the striped pattern in the null direction. Markers: 60 mV. 05 sec.
ventral (a down unit). The axon has an average diaat I2 pm. The histological material was examined unmeter ofapproximately 45 pm. The cell can be traced der a Zeiss transmission fluorescence microscope. from the peripheral extent of the lobulus to the midThe responses of the motion detecting cells recorded intracellularly are similar to those obtained from brain over a distance of approximately 300 pm. The extracellular recordings. The activity in the presence of approximate location of this cell is shown schematian illuminated but stationary pattern. varied from 3 to cally in Fig. 2(a) (arrow). Although several hours were 25 spike&z [Fig. l(a)]. Movement of the pattern in allowed for the procion dye to diffuse throughout the the preferred direction evoked a response of up to 100 cell. it is not certain that the entire cell was filled. spikes/set [Figs. l(b). I(d)], while movements in the Motion detecting neurons with contralateral visual null direction typically inhibited spontaneous activity fields and horizontal preferred directions were located [Fig. I(c)]. Overshooting action potentials (30-90 mV). near the anterior surface of the lobulus. Cells with contralateral visual fields and vertical preferred directions monophasic action potentials (5-25 mV) and EPSP’s (I-10 mV) were routinely recorded. This variation in were found near the posterior surface of the lobulus. waveform and amplitude of observed potentials indicates that recording sites were located in axons, neurites and somata (see Murphy, 1973). Resting membrane potentials varied from 20-6OmV. The most stable recordings (usually associated with over-shooting spikes) were as long as 30 min. In a previous publication it was reported that in the lobula region extracellular recordings had been obtained from several different types of directionally selective motion detecting units (Bishop er al., 1968). These types were distinguished by location of visual field, contralateral or ipsilateral. and by direction of preferred pattern movemenf that is: (1) horizontal. back to front or front to back; and (2) vertical. up or down. We have succeeded in recording intracellularly from each of these types of units. Fig. 2. (a) Schematic diagram of a cross-sectional view of Figure 2(b) shows a stained neuron which had a conthe head of a fly from the midbrain to the periphery. The tralateral visual held and responded maximally to patarrow indicates the approximate location of the neuron tern movement in the vertical direction from dorsal to shown in Fig. 2(b).
Fig. 2(b). Directionally
selective motion detecting cell stained by intracellular 10 pm. (Horizontal section.)
yellow M4RS. Marker:
injection OSprocion
453
Research Note Both types of neurons projected to the periesophageal
region. Those cells with ipsilateral visual fields were located near the anterior surface of the lobulus and extended ventro-posteriorly to the circumesophageal connective. In no case did a neuron with contralateral visual field extend beyond the midline of the midbrain. Accurate mapping of these cells by comparison with Golgi stained material is our immediate goal.
Acknowledgements-Supported by AFOSR grant number 71-21 I2 and NSF grant number GB 30733. We thank Mr. Mark Mayo for valuable technical assistance.
REFERENCES
Bishop L. G. and Keehn D. G. (1966) Two types of motion sensitive neurons in the fly optic lobe. Snture. Lond. 212. 1373-1376. Bishop L. G. and Keehn D. G. (1967) Neural correlates of the optomotor response in the fly. Kyberneric 3. 288-295. Bishop L. G.. Keehn D. G. and McCann G. D. (1968) Studies of motion detection by interneurons of the optic lobes and brain of the flies. Calliphora phaenicia and &fusca domestico.J. Neuroph!aioL 31. 509-525. Braitenberg V. (1967) Patterns of projection in the visual system of the fly. Expl Brain Res. 3. 271-298. Braitenberg V. (1972) Periodic structures and structural gradients in the visual ganglia of the fly. In Information Processing in the Visua/ Srsrents of Arthropods (edited by Wehner R.J. p. 3- I5 Springer. Berlin.
Eckert H. (1973) Optomotorrsche Untersuchungen am visuellen System der StubenRiege .Il~t.sra tlonresricn L. Kjherneric 14. l-13. Goetz K. G. (1972) Processing ol cues from the moving environment in the Drosophila navigation system. In I&rmotion
Processing
in
rhe
Lisual
S.Wevr
01’ Arrbrop0d.v
(edited by Wehner R.I. p. 225-263. Springer. Berlin. Kaiser W. and Bishop L. G. (1970) Directionally selective motion detecting units in the optic lobe of the honeybee. Z. rergl. Physiol. 67. 40.3-413. Kirschfeld K. (1972) The visual system of .Lfuscu: studies in optics. structure and function. In I&vrnarim Procc~ssinq in the k’isua/ S.rstetnsof Amhropods (edited by Wehner R.). p. 61-74. Springer. Berlin. Kirschfeld K. (1973) Optomotorische Reaktionen der Biene auf bewegte “Polarizations-Muster” Z. ,Vaturfbrsch. 2&z. 329-338. Murphy R. K. (1973) Characterization of an insect neuron which cannot be visualized in SIIU. In Ierraccllular Stairrinq in k’rurohioloqy (edited by Kater S. B. and Nicholson C.), p. 135 150. Springer. Berlin. Reichardt W. (1969) Movement perception in insects. In ProcessingofOptical Data by Organisms and by Machines (edited by Reichardt W.). Reichardt W. (1973) Musterinduziertc Flugorientierung. Verhaltensversuche an der Fliege Musco dmmicu. Naturwiss 60. IX- I38 Ribi W. (1973) Doctoral dissertation. Zurich. Strausfeld N. J. (197 I I The organization of the insect visual system. Z. Zelf/iirsch. 121. M-454. Trujillo-Cenoz 0. and Melamed J. (1966) Electron microscope observations on the peripheral and intermediate retinas ofdipterans. In The Functional Organkation qfrhe Compound Eye (edited by Bernard C. G.J. pp. 339-361. Pergamon Press Oxford.