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Use of microelectrophoresis in the autoradiographic demonstration of fiber projections
274
SHORT COMMUNICATIONS
Use of microelectrophoresis in the autoradiographic demonstration of fiber projections Utilizing axonal transport of radioactive substances as a tool in tracing neuronal connections has been considered but infrequently exploited2,4-6,8,12,14. Surprisingly few neuroanatomists have made use of this elegant technique. Only recently Cowan et al. critically analyzed this method; they found it useful and even superior to other tracing techniques, particularly those based on terminal or anterograde degenerationL The fact that axonal flow can be used as a physiological vehicle rules out many of the uncertainties of the degeneration process. Moreover, undesirable 'impure lesions', in which fibers of the white matter are destroyed, can be avoided. The only traumatic event still affecting the brain region under study with this new tracing technique is the injection of a radiochemical from a syringe. Therefore, even less disruption would be expected by applying the radiochemical iontophoretically by means of a microelectrode. This paper reports on the elaboration of such a microinjection technique. Double-barreled microelectrodes were prepared using glass capillaries of 2.5 m m diameter subdivided by an inner glass wall. The tip was gently broken while viewed through a microscope with a resulting tip size of 3-4/zm. The shank diameter was about 15 #m at a 200/zm distance from the tip. One of the barrels was filled with a radioactive amino acid, e.g., [3H]glycine. One ml of the glycine solution (1 mCi/ml, spec. act. 2.3 Ci/mmole, Radiochemical Centre Amersham) was dried under nitrogen. The sediment was dissolved in 20 #1 of 0.1 M acetic acid. Two/~1 of this solution were injected directly into one of the electrode barrels from a syringe without air space (Hamilton 7105 N), the needle being inserted up to the electrode shoulder. Thus, filling the electrodes to the tip was easily carried out within 5-10 min. A silver wire was then inserted into the shank and fixed to the barrel with sealing wax. The second barrel can be used as an extracellular recording electrode. The efficiency of the [3H]glycine electrophoresis was checked in vitro 15. The electrode tip was immersed into a drop of 0.9 M NaCI contained in a small metal vessel (airgun pellet) which had been connected to ground. The tracer barrel was connected to a battery-type floating amplifier configuration which provided a controlled current3,1°,13. A current of 2 × 10-7 m was applied from barrel to ground for 5 min. The amount of radioactive material moved by the current into the NaC1 solution was then measured in a Beckman scintillationWcounter. Electrodes providing at least 4000 counts/rain in vitro were found to give satisfactory results in tissue labeling experiments in vivo. These investigations were carried out on Long Evans hooded rats, 8 weeks old. Under Nembutal anesthesia the head of the rat was firmly fastened to a stereotactic holder. A 2 m m opening was made into the occipital skull and the visual cortex exposed. The electrode was inserted ca. 0.5 m m into the cortex by means of a micromanipulator, and an iontophoretic current of 1-2 × 10-7 A was applied for 10-15 min. After injection, the opening was covered securely with lyophilized dura. The rats were killed 5 days later by formalin perfusion and the brain embedded in paraBrain Research, 39 (1972) 274-277
SHORT COMMUNICATIONS
275
Fig. 1. Spread of radioactive material in the visual cortex of the rat following microelectrophoresis of [3H]glycine 5 days before sacrifice. Autoradiography, counterstained by toluidine blue. × 75.
Fig. 2. A, Within the center of the injected area radioactive material is accumulated in the nerve cell bodies, the site of protein synthesis. B, Radioactive material transported subcortically in a distinct fiber bundle. Autoradiography, counterstained by toluidine blue. × 460. plast. A u t o r a d i o g r a p h s were made on 6 # m serial sections coated with NTB-3 photoemulsion. After 3 weeks exposure, the autoradiographs were developed and counterstained by toluidine blue. The iontophoretic application o f the radiochemical resulted in labeling o f a confined area o f the occipital cortex (area 17/18) (Fig. 1). The perikarya o f the nerve cells were heavily labeled a r o u n d the center o f the area injected with [3H]glycine (Fig. 2A). This labeled region measured approximately 400/~m in diameter. The density o f the silver grains decreased with increasing distance f r o m the center o f the focus. The area o f the radioactively labeled nerve cell bodies measured about 1000 ± 200 # m in diameter. Brain Research, 39 (1972) 274--277
276
SHORT COMMUNICATIONS
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m Fig. 3. Target area of labeled cortical neurons within the geniculate body. Radioactivity is confined to the neuropil, the presumable site of their terminals. Note the absence of silver grains over the neighboring area (right) and over the non-terminal fiber tracts. Autoradiography, counterstained by toluidine blue. × 740.
N o significant tissue damage was observed in the area of electrode penetration. N o t even an electrode track could be detected. The high rate of amino acid incorporation in the center o f the injected field led to the assumption that the neurons were not impaired in their metabolic functions. The high amount o f radioactivity in the neuropil was attributed to cellulifugal transport in the dendrites and axons o f the labeled neurons and to glial cell uptake and incorporation. Radioactive material was also found in areas bordering the injection site. In contrast with the immediate site of iontophoresis where radioactivity was greatest in the perikarya, the neighboring areas showed activity predominantly in the neuropil. This suggested that the radioactive material had been transported, e.g., from area 17 by way of the U-fibers to areas 18 and 18a, and was now located in the terminals o f axons originating from neurons of area 17. A transport of radioactive material could definitely be observed in fibers of the subcortical white matter (Fig. 2B). These were probably fibers projecting from the site of injection to the thalamus. A circumscribed area of the lateral geniculate body was clearly labeled in the thalamus (Fig. 3). The label was located exclusively in the neuropil where terminals of incoming cortical axons would be expected 11. Radioactive materials were also detected in a fiber bundle o f the corpus callosum. It is likely that they represent fibers projecting from the injection site to the contralateral visual cortex. Besides a low general~background, there were no deposits o f radioactive material in any other part of the brain. This finding stresses the specificity and the accuracy of our method. Brain Research, 39 ('1972) 274-277
SHORT COMMUNICATIONS
277
I o n t o p h o r e t i c injection o f r a d i o a c t i v e m a t e r i a l s p r o v i d e s a m e a n s o f tracing fiber c a n n e c t i o n s a n d d e m o n s t r a t i n g t e r m i n a l s in the target n u c l e u s o f a given n e u r o nal p o p u l a t i o n . The i o n t o p h o r e t i c a p p l i c a t i o n by means o f a m i c r o e l e c t r o d e d i d not cause tissue d a m a g e because no v o l u m e was injected into the tissue as w o u l d o c c u r with injection b y syringe. Instead, electrophoresis enables the r a d i o c h e m i c a l to enter the tissue in exchange for o t h e r electrically c h a r g e d particles. By m o d i f y i n g the m i c r o electrophoresis c u r r e n t it is possible to a p p l y very small a m o u n t s o f r a d i o a c t i v e precursors resulting in a labeling o f limited areas. P a r a m e t e r s o f such a m o d i f i c a t i o n have been defined by Herz et al. 7. Lastly, m i c r o e l e c t r o d e s m a k e it possible simultaneously to collect m o r p h o l o g ical a n d e l e c t r o p h y s i o l o g i c a l d a t a L T h e electrode used in this study p e r m i t s extracellular recording. This is o f great a d v a n t a g e for the identification o f a target area, p a r t i c u l a r l y if the a r e a is l o c a t e d subcortically o r in the b r a i n stem. R e c o r d i n g o f p o t e n t i a l s e v o k e d by s t i m u l a t i o n o f defined afferents c o u l d o p e n a new d i m e n s i o n in the precision o f identification o f fiber p r o j e c t i o n s in the C N S . Max-Planck-lnstitut fiir Psychiatrie, 8000 Munich 23 (G.F.R.)
PETER SCHUBERT GEORG W. KREUTZBERG HANS D. LUX
1 COWAN, W. M., GOTTLIEB, D. 1., HENDRICKSON, A., PRICE, J. L., AND WOOLSEY, T.A., The autoradiographic demonstration of axonal connections in the central nervous system, Brain Research, 37 (1972) 21-51.
2 DROZ, B., AND BARONDES,S. M., Nerve endings: rapid appearance of labeled protein shown by electron microscope radioautography, Science, 165 (1969) 1131-1133. 3 GLOBUS, A., Lux, H. D., AND SCHUBERT, P., Somadendritic spread of intracellularly injected tritiated glycine in cat spinal motoneurons, Brain Research, 11 (1969) 440-445. 4 GOLDBERG,S., ANDKOTANI,M., The projection of optic nerve fibers in the frog, Rana catesbeiana, as studied by radioautography, Anat. Rec., 158 (1967) 325-332. 5 GRAFSTEIN,B., Transport of protein by goldfish optic nerve fibers, Science, 157 (1967) 196-198. 6 HENDRICKSON, A., Electron microscopic radioautography: Identification of origin of synaptic terminals in normal nervous tissue, Science, 165 (1969) 194-196. 7 HERZ, A., ZIEGLG*NSBERGER,W., AND F~.RBER, G., Microelectrophoretic studies concerning spread of substances in brain tissue, Exp. Brain Res., 9 (1969) 221-235. 8 LASEK,R., JOSEPH, B. S., ANDWHITLOCK,D. G., Evaluation of a radioautographic neuroanatomical tracing method, Brain Research, 8 (1968) 319-336. 9 Lux, H.D., SCHUBERT, P., AND KREUTZBERG,G. W., Direct matching of morphological and electrophysiologicaldata in cat spinal motoneurones. In P. ANDERSONANDJ. K. S. JANSEN(Eds.), Excitatory Synaptic Mechanisms, Universitetsforlaget, Oslo, 1970, pp. 189-198. 10 Lux, H. D., SCHUBERT,P., KREUTZBERG,G. W., AND GLOBUS, A., Excitation and axonal flow: Autoradiographic study on motoneurons intracellularly injected with a all-amino acid, Exp. Brain Res., 10 (1970) 197-204. l l NAUTA, W. J. H., AND BUCHER, V. M., Efferent connections of the striate cortex in the albino rat, J. comp. Neurol., 100 (1954) 257-295. 12 SCHONBACH,J., AND CUI~NOD, M., Axoplasmic migration of protein. A light microscopic autoradiographic study in the avian retinotectal pathway, Exp. Brain Res., 12 (1971) 275-282. 13 SCHUBERT,P., LUX, H. D., AND KREUTZBERG,G. W., Single cell isotope injection technique, a tool for studying axonal and dendritic transport, Acta neuropath. (Berl.), Suppl. 5 (1971) 179-186. 14 WEISS, P., AND HOLLAND, Y., Neuronal dynamics and axonal flow. II. The olfactory nerve as model test object, Proc. nat. Acad. Sci. (Wash.), 57 (1967) 259-264. 15 ZIEGLG.~NSBERGER,W., HERZ, A., AND TESCHEMACHER,HJ., Electrophoretic release of tritiumlabelled glutamic acid from micropipettes in vitro, Brain Research, 15 (1969) 298-300. (Accepted December 21st, 1971) Brain Research, 39 (1972) 274-277
SHORT COMMUNICATIONS
Use of microelectrophoresis in the autoradiographic demonstration of fiber projections Utilizing axonal transport of radioactive substances as a tool in tracing neuronal connections has been considered but infrequently exploited2,4-6,8,12,14. Surprisingly few neuroanatomists have made use of this elegant technique. Only recently Cowan et al. critically analyzed this method; they found it useful and even superior to other tracing techniques, particularly those based on terminal or anterograde degenerationL The fact that axonal flow can be used as a physiological vehicle rules out many of the uncertainties of the degeneration process. Moreover, undesirable 'impure lesions', in which fibers of the white matter are destroyed, can be avoided. The only traumatic event still affecting the brain region under study with this new tracing technique is the injection of a radiochemical from a syringe. Therefore, even less disruption would be expected by applying the radiochemical iontophoretically by means of a microelectrode. This paper reports on the elaboration of such a microinjection technique. Double-barreled microelectrodes were prepared using glass capillaries of 2.5 m m diameter subdivided by an inner glass wall. The tip was gently broken while viewed through a microscope with a resulting tip size of 3-4/zm. The shank diameter was about 15 #m at a 200/zm distance from the tip. One of the barrels was filled with a radioactive amino acid, e.g., [3H]glycine. One ml of the glycine solution (1 mCi/ml, spec. act. 2.3 Ci/mmole, Radiochemical Centre Amersham) was dried under nitrogen. The sediment was dissolved in 20 #1 of 0.1 M acetic acid. Two/~1 of this solution were injected directly into one of the electrode barrels from a syringe without air space (Hamilton 7105 N), the needle being inserted up to the electrode shoulder. Thus, filling the electrodes to the tip was easily carried out within 5-10 min. A silver wire was then inserted into the shank and fixed to the barrel with sealing wax. The second barrel can be used as an extracellular recording electrode. The efficiency of the [3H]glycine electrophoresis was checked in vitro 15. The electrode tip was immersed into a drop of 0.9 M NaCI contained in a small metal vessel (airgun pellet) which had been connected to ground. The tracer barrel was connected to a battery-type floating amplifier configuration which provided a controlled current3,1°,13. A current of 2 × 10-7 m was applied from barrel to ground for 5 min. The amount of radioactive material moved by the current into the NaC1 solution was then measured in a Beckman scintillationWcounter. Electrodes providing at least 4000 counts/rain in vitro were found to give satisfactory results in tissue labeling experiments in vivo. These investigations were carried out on Long Evans hooded rats, 8 weeks old. Under Nembutal anesthesia the head of the rat was firmly fastened to a stereotactic holder. A 2 m m opening was made into the occipital skull and the visual cortex exposed. The electrode was inserted ca. 0.5 m m into the cortex by means of a micromanipulator, and an iontophoretic current of 1-2 × 10-7 A was applied for 10-15 min. After injection, the opening was covered securely with lyophilized dura. The rats were killed 5 days later by formalin perfusion and the brain embedded in paraBrain Research, 39 (1972) 274-277
SHORT COMMUNICATIONS
275
Fig. 1. Spread of radioactive material in the visual cortex of the rat following microelectrophoresis of [3H]glycine 5 days before sacrifice. Autoradiography, counterstained by toluidine blue. × 75.
Fig. 2. A, Within the center of the injected area radioactive material is accumulated in the nerve cell bodies, the site of protein synthesis. B, Radioactive material transported subcortically in a distinct fiber bundle. Autoradiography, counterstained by toluidine blue. × 460. plast. A u t o r a d i o g r a p h s were made on 6 # m serial sections coated with NTB-3 photoemulsion. After 3 weeks exposure, the autoradiographs were developed and counterstained by toluidine blue. The iontophoretic application o f the radiochemical resulted in labeling o f a confined area o f the occipital cortex (area 17/18) (Fig. 1). The perikarya o f the nerve cells were heavily labeled a r o u n d the center o f the area injected with [3H]glycine (Fig. 2A). This labeled region measured approximately 400/~m in diameter. The density o f the silver grains decreased with increasing distance f r o m the center o f the focus. The area o f the radioactively labeled nerve cell bodies measured about 1000 ± 200 # m in diameter. Brain Research, 39 (1972) 274--277
276
SHORT COMMUNICATIONS
"o
• It
• ~ e Jo
e~
•
qb •
Q w
I
3
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m Fig. 3. Target area of labeled cortical neurons within the geniculate body. Radioactivity is confined to the neuropil, the presumable site of their terminals. Note the absence of silver grains over the neighboring area (right) and over the non-terminal fiber tracts. Autoradiography, counterstained by toluidine blue. × 740.
N o significant tissue damage was observed in the area of electrode penetration. N o t even an electrode track could be detected. The high rate of amino acid incorporation in the center o f the injected field led to the assumption that the neurons were not impaired in their metabolic functions. The high amount o f radioactivity in the neuropil was attributed to cellulifugal transport in the dendrites and axons o f the labeled neurons and to glial cell uptake and incorporation. Radioactive material was also found in areas bordering the injection site. In contrast with the immediate site of iontophoresis where radioactivity was greatest in the perikarya, the neighboring areas showed activity predominantly in the neuropil. This suggested that the radioactive material had been transported, e.g., from area 17 by way of the U-fibers to areas 18 and 18a, and was now located in the terminals o f axons originating from neurons of area 17. A transport of radioactive material could definitely be observed in fibers of the subcortical white matter (Fig. 2B). These were probably fibers projecting from the site of injection to the thalamus. A circumscribed area of the lateral geniculate body was clearly labeled in the thalamus (Fig. 3). The label was located exclusively in the neuropil where terminals of incoming cortical axons would be expected 11. Radioactive materials were also detected in a fiber bundle o f the corpus callosum. It is likely that they represent fibers projecting from the injection site to the contralateral visual cortex. Besides a low general~background, there were no deposits o f radioactive material in any other part of the brain. This finding stresses the specificity and the accuracy of our method. Brain Research, 39 ('1972) 274-277
SHORT COMMUNICATIONS
277
I o n t o p h o r e t i c injection o f r a d i o a c t i v e m a t e r i a l s p r o v i d e s a m e a n s o f tracing fiber c a n n e c t i o n s a n d d e m o n s t r a t i n g t e r m i n a l s in the target n u c l e u s o f a given n e u r o nal p o p u l a t i o n . The i o n t o p h o r e t i c a p p l i c a t i o n by means o f a m i c r o e l e c t r o d e d i d not cause tissue d a m a g e because no v o l u m e was injected into the tissue as w o u l d o c c u r with injection b y syringe. Instead, electrophoresis enables the r a d i o c h e m i c a l to enter the tissue in exchange for o t h e r electrically c h a r g e d particles. By m o d i f y i n g the m i c r o electrophoresis c u r r e n t it is possible to a p p l y very small a m o u n t s o f r a d i o a c t i v e precursors resulting in a labeling o f limited areas. P a r a m e t e r s o f such a m o d i f i c a t i o n have been defined by Herz et al. 7. Lastly, m i c r o e l e c t r o d e s m a k e it possible simultaneously to collect m o r p h o l o g ical a n d e l e c t r o p h y s i o l o g i c a l d a t a L T h e electrode used in this study p e r m i t s extracellular recording. This is o f great a d v a n t a g e for the identification o f a target area, p a r t i c u l a r l y if the a r e a is l o c a t e d subcortically o r in the b r a i n stem. R e c o r d i n g o f p o t e n t i a l s e v o k e d by s t i m u l a t i o n o f defined afferents c o u l d o p e n a new d i m e n s i o n in the precision o f identification o f fiber p r o j e c t i o n s in the C N S . Max-Planck-lnstitut fiir Psychiatrie, 8000 Munich 23 (G.F.R.)
PETER SCHUBERT GEORG W. KREUTZBERG HANS D. LUX
1 COWAN, W. M., GOTTLIEB, D. 1., HENDRICKSON, A., PRICE, J. L., AND WOOLSEY, T.A., The autoradiographic demonstration of axonal connections in the central nervous system, Brain Research, 37 (1972) 21-51.
2 DROZ, B., AND BARONDES,S. M., Nerve endings: rapid appearance of labeled protein shown by electron microscope radioautography, Science, 165 (1969) 1131-1133. 3 GLOBUS, A., Lux, H. D., AND SCHUBERT, P., Somadendritic spread of intracellularly injected tritiated glycine in cat spinal motoneurons, Brain Research, 11 (1969) 440-445. 4 GOLDBERG,S., ANDKOTANI,M., The projection of optic nerve fibers in the frog, Rana catesbeiana, as studied by radioautography, Anat. Rec., 158 (1967) 325-332. 5 GRAFSTEIN,B., Transport of protein by goldfish optic nerve fibers, Science, 157 (1967) 196-198. 6 HENDRICKSON, A., Electron microscopic radioautography: Identification of origin of synaptic terminals in normal nervous tissue, Science, 165 (1969) 194-196. 7 HERZ, A., ZIEGLG*NSBERGER,W., AND F~.RBER, G., Microelectrophoretic studies concerning spread of substances in brain tissue, Exp. Brain Res., 9 (1969) 221-235. 8 LASEK,R., JOSEPH, B. S., ANDWHITLOCK,D. G., Evaluation of a radioautographic neuroanatomical tracing method, Brain Research, 8 (1968) 319-336. 9 Lux, H.D., SCHUBERT, P., AND KREUTZBERG,G. W., Direct matching of morphological and electrophysiologicaldata in cat spinal motoneurones. In P. ANDERSONANDJ. K. S. JANSEN(Eds.), Excitatory Synaptic Mechanisms, Universitetsforlaget, Oslo, 1970, pp. 189-198. 10 Lux, H. D., SCHUBERT,P., KREUTZBERG,G. W., AND GLOBUS, A., Excitation and axonal flow: Autoradiographic study on motoneurons intracellularly injected with a all-amino acid, Exp. Brain Res., 10 (1970) 197-204. l l NAUTA, W. J. H., AND BUCHER, V. M., Efferent connections of the striate cortex in the albino rat, J. comp. Neurol., 100 (1954) 257-295. 12 SCHONBACH,J., AND CUI~NOD, M., Axoplasmic migration of protein. A light microscopic autoradiographic study in the avian retinotectal pathway, Exp. Brain Res., 12 (1971) 275-282. 13 SCHUBERT,P., LUX, H. D., AND KREUTZBERG,G. W., Single cell isotope injection technique, a tool for studying axonal and dendritic transport, Acta neuropath. (Berl.), Suppl. 5 (1971) 179-186. 14 WEISS, P., AND HOLLAND, Y., Neuronal dynamics and axonal flow. II. The olfactory nerve as model test object, Proc. nat. Acad. Sci. (Wash.), 57 (1967) 259-264. 15 ZIEGLG.~NSBERGER,W., HERZ, A., AND TESCHEMACHER,HJ., Electrophoretic release of tritiumlabelled glutamic acid from micropipettes in vitro, Brain Research, 15 (1969) 298-300. (Accepted December 21st, 1971) Brain Research, 39 (1972) 274-277