Brain Research, 108 (1976) 175-179
175
© ElsevierScientificPublishingCompany,Amsterdam- Printed in The Netherlands
An improved technique for the microinjection of horseradish peroxidase
ALVIN J. BEITZANDGARY W. KING Departments of Anatomy and Physiology, Universityof Minnesota, Minneapolis, Minn. 54555 (U.S.A.)
(Accepted February 9th, 1976)
Since the introduction of the method of retrograde neuroanatomical tracing by LaVail et alP, using horseradish peroxidase (HRP) as a marker, this technique has become an important tool for determining the projections of neurons in the central nervous system. The method is based on the retrograde movement of HRP from the region of axon terminals to the parent cell bodies. It has been suggested that HRP is transported along the axon to the cell body through tubules of the endoplasmic reticulumT,10,is, multivesicular bodies and membrane-bound vesiclesT,10. Once in the cell body, the HRP is concentrated into dense bodies large enough to be resolved with the light microscope and localized mainly in the perinuclear Golgi region. These dense brown cytoplasmic granules therefore characterize peroxidase-positive neurons whose axons have taken up the marker. In exploring the usefulness of HRP as a retrograde tracer, it was noted that HRP injected into brain tissue in aqueous solutions tends to diffuse widely from the injection site s, particularly in non-cortical gray matter. One of the possible complications arising from the diffusion of peroxidase from the injection site is the spread of HRP backward along the path of the pipette through the brain 1,5,11,12. This spread of HRP may be a serious problem for two reasons. (1) When injected into non-cortical gray matter, the HRP may spread back along the pipette path to the overlying cortex, thus giving rise to a potential source of error in the interpretation of afferents projecting to the area in question. (2) Injured axons along the pipette track may take up HRP 1-'%6 and transport it back to their parent cell bodies, yielding erroneous results. To prevent the diffusion of HRP back along the pipette track, we developed a technique in which sterile mineral oil is slowly injected into the brain as the pipette is withdrawn. The experiments were performed on 35 adult cats anesthetized with ketamine hydrochloride (Ketalar, Parke-Davis, 50 mg i.m.). A 35 ~o solution of HRP (0.030.30/zl) in sterile saline (0.85 ~o) was injected into the dentate nucleus of the cerebellum of each cat through a stereotaxically placed glass pipette. The pipettes were approximately 3-4 cm long and were prepared by drawing out glass capillary tubes of 1 mm external diameter (Friedrich and Dimmock, Inc.) to internal tip diameters of 50-100 /zm. The pipette was connected to a short segment of polyethylene tubing, and both
176
were filled with sterile mineral oil. The tubing was connected to a I #1 syringe (Hamilton 700-1N), and the desired amount of H R P was drawn into the tip of the pipette. In addition, a second oil-filled syringe (10 #1) was kept in readiness to be used during the withdrawal process. The cats were divided into two groups: group I, consisting of 20 cats injected with H R P ; group I1, consisting of 15 cats in which sterile mineral oil followed the injection of HRP. In both groups 1 and I1 the H R P solution was injected over a period of 1-5 min, after which the system was allowed to equilibrate for a further 1-15 rain before withdrawing the pipette. However, only in group II was sterile mineral oil injected into the track as the pipette was withdrawn. To accomplish this, the pipette was first withdrawn I mm from the site of injection of HRP. The polyethylene tubing was then disconnected from the 1 /~1 syringe and connected to the 10/~l syringe situated adjacent to it. As the pipette was slowly withdrawn over a period of 30 sec, about 15 ¢tl of sterile mineral oil was injected into the brain approximating the volume originally occupied by the pipette. The quantity of oil injected was monitored by watching the advance of an air bubble introduced into the polyethylene tubing when the syringes were switched.
Fig. 1. A transverse section through the cerebellum and medulla of a cat from group I which was sacrificed 36 h after injection, illustrating the injection site and spread of HRP along the pipette track.
177 Twenty-four to 48 h after injection the animals were anesthetized and perfused transcardially with isotonic saline followed by a mixture of 1.25 % glutaraldehyde and 1% paraformaldehyde. The brains were promptly removed, placed in fresh fixative for 12 h, and then rinsed for 20-24 h in 0.1 M phosphate buffer (pH 7.4) containing 20% sucrose. Sections were cut at 50/~m on a freezing microtome and reacted with 3,Y-diaminobenzidine 9 for 15 min. The sections were transferred through two rinsing solutions of 0.1 M phosphate buffer and then individually mounted on gelatinized slides. Once dry, alternate slides were stained with cresylecht violet. In most cases in both groups I and II, the injection site was centered in the region of the dentate nucleus. The size of the injection site was defined under the light microscope as the extent of visible HRP reaction product. The size was found to be dependent on the volume of HRP injected with larger volumes of HRP resulting in larger injection sites. The sites were often roughly spherical and measured 0.8-3.0 mm in diameter. In a number of cases, however, small brown spurs were observed to protrude from the sphere along fibers that passed to or from the site of injection. There was no consistent difference in the shape of injection sites between groups I and II except for the region along the pipette track.
Fig. 2. A transverse section through the cerebellum and medulla of a cat from group II which was sacrificed 40 h after injection, depicting the injection site and the absence of spread of HRP along the pipette track.
178 The results of a typical microinjection of HRP from group I are illustrated in Fig. 1. The injection site, approximately 2 mm in diameter, is seen as a sphere in the region of the dentate nucleus. In addition, HRP has spread back along the path of the pipette through the cerebellar white matter into the cerebellar cortex. This backward diffusion of HRP was a consistent finding and was observed in 18 of the 20 cats included in group I. Fig. 2 illustrates the results of a typical injection obtained from a cat in group |l. In this case the injection site is also approximately 2 mm in diameter, but the spread of HRP backward along the pipette path has been eliminated. No spread was observed in any of the cats receiving the mineral oil injection as the pipette was withdrawn*. This technique caused a small amount of visible damage to the brain tissue by producing a cylindrical hole about 1 mm in diameter along the path of the pipette. Since this damage was confined to the area above the injection site, and the visible H R P reaction product was confined to the injection site, it seems unlikely that injured axons or cells above the injection site could have taken up the peroxidase. The importance of preventing the spread of H R P back along the pipette track was demonstrated in our own work when comparing peroxidase-positive cells found in the brains from cats in groups I and II. Cats in group I, for example, showed positive cells in the lateral reticular nucleus (LRN) indicating a projection from the L R N to the dentate nucleus and overlying cerebellar cortex. No positive cells, however, could be demonstrated in the L R N from cats in group lI. The latter finding agress well with the work of Kfinzle 4 who was unable to find direct fiber connections between the L R N and the dentate using the autoradiographic tracing technique. The present results indicate that the injection of mineral oil following the injection of H R P can eliminate one of the present problems associated with the peroxidase technique. The authors thank Dr. Morris Smithberg, Dr. James R. Bloedel, and Dr. Charles K. Knox for their careful reading and comments on this paper, and Diane Jacoby for help in preparing the manuscript. This study was supported in part by USPHS Grants G M I I 4 , GM572, and HL16430. 1 BUNT, A. H., HENDRICKSON,A. E., LUND, J. S., LUND, R. D., AND FucrtS, A. F., Monkey retinal ganglion cells: morphometric analysis and tracing of axonal projections, with a consideration of the peroxidase technique, J. comp. NeuroL, 164 (1975) 265-285. 2 DE VITO, J. L., CLAUSING,K. W., AND SMITH, O. A., Uptake and transport of horseradish peroxidase by cut end of the vagus nerve, Brain Research, 82 (1974) 269-271. 3 HALPERIN,J. J., AND LAVAIL,J. H., A study of the dynamics of retrograde transport and accumulation of horseradish peroxidase in injured neurons, Brain Research, 100 (1975) 253-269. 4 KONZLE, H., Autoradiographic tracing of the cerebellar projections from the lateral reticular nucleus in the cat, Exp. Brain Res., 22 (1975) 255-266. * Preliminary results from injections into areas of the brain stem, using a rostral approach of the pipette, also indicate an absence of spreading of the HRP along the pipette track.
179 5 LAVAIL,J. H., personal communication. 6 LAVA]L,J. H., The retrograde transport method, Fed. Proc., 34 (1975) 1618-1624. 7 LAVA]L,J. H., ANDLAVA]L,M. M., The retrograde intraaxonal transport of horseradish peroxidase in the chick visual system: a light and electron microscopic study, J. comp. NeuroL, 157 (1974) 303-358. 8 LAVA]L,J. H., AND LAVAIL,M. M., Retrograde axonal transport in the central nervous system, Science, 176 (1972) 1416-1417. 9 LAVA]L,J. H., WINSTON,K. R., AND TlSH, A., A method based on retrograde intraaxonal transport of protein for identification of cell bodies of origin of axons terminating within the CNS, Brain Research, 58 (1973) 470-477. 10 NAUTA,H. J. W., KAISERMAN-ABRAMOF,I. R., AND LASEK,R. J., Electron microscopic observations of horseradish peroxidase transported from the caudoputamen to the substantia nigra in the rat: possible involvement of the agranular reticulum, Brain Research, 85 0975) 373-384. 11 ROMA~NANO,M. A., AND MACmWICZ,R. J., Peroxidase labeling of motor cortex neurons projecting to the ventrolateral nucleus in the cat, Brain Research, 83 (1975) 469-473. 12 SI~RLOCK,D. A., AND RA]SMAN,G., A comparison of anterograde and retrograde axonal transport of horseradish peroxidase in the connections of the mammillary nuclei in the rat, Brain Research, 85 (1975) 321-324. 13 SOTELO, C., AND RtCH~, D., The smooth endoplasmic reticulum and the retrograde and fast orthograde transport of horseradish peroxidase in the nigro-striato-nigral loop, Anat. EmbryoL, 146 (1974) 209-218.
175
© ElsevierScientificPublishingCompany,Amsterdam- Printed in The Netherlands
An improved technique for the microinjection of horseradish peroxidase
ALVIN J. BEITZANDGARY W. KING Departments of Anatomy and Physiology, Universityof Minnesota, Minneapolis, Minn. 54555 (U.S.A.)
(Accepted February 9th, 1976)
Since the introduction of the method of retrograde neuroanatomical tracing by LaVail et alP, using horseradish peroxidase (HRP) as a marker, this technique has become an important tool for determining the projections of neurons in the central nervous system. The method is based on the retrograde movement of HRP from the region of axon terminals to the parent cell bodies. It has been suggested that HRP is transported along the axon to the cell body through tubules of the endoplasmic reticulumT,10,is, multivesicular bodies and membrane-bound vesiclesT,10. Once in the cell body, the HRP is concentrated into dense bodies large enough to be resolved with the light microscope and localized mainly in the perinuclear Golgi region. These dense brown cytoplasmic granules therefore characterize peroxidase-positive neurons whose axons have taken up the marker. In exploring the usefulness of HRP as a retrograde tracer, it was noted that HRP injected into brain tissue in aqueous solutions tends to diffuse widely from the injection site s, particularly in non-cortical gray matter. One of the possible complications arising from the diffusion of peroxidase from the injection site is the spread of HRP backward along the path of the pipette through the brain 1,5,11,12. This spread of HRP may be a serious problem for two reasons. (1) When injected into non-cortical gray matter, the HRP may spread back along the pipette path to the overlying cortex, thus giving rise to a potential source of error in the interpretation of afferents projecting to the area in question. (2) Injured axons along the pipette track may take up HRP 1-'%6 and transport it back to their parent cell bodies, yielding erroneous results. To prevent the diffusion of HRP back along the pipette track, we developed a technique in which sterile mineral oil is slowly injected into the brain as the pipette is withdrawn. The experiments were performed on 35 adult cats anesthetized with ketamine hydrochloride (Ketalar, Parke-Davis, 50 mg i.m.). A 35 ~o solution of HRP (0.030.30/zl) in sterile saline (0.85 ~o) was injected into the dentate nucleus of the cerebellum of each cat through a stereotaxically placed glass pipette. The pipettes were approximately 3-4 cm long and were prepared by drawing out glass capillary tubes of 1 mm external diameter (Friedrich and Dimmock, Inc.) to internal tip diameters of 50-100 /zm. The pipette was connected to a short segment of polyethylene tubing, and both
176
were filled with sterile mineral oil. The tubing was connected to a I #1 syringe (Hamilton 700-1N), and the desired amount of H R P was drawn into the tip of the pipette. In addition, a second oil-filled syringe (10 #1) was kept in readiness to be used during the withdrawal process. The cats were divided into two groups: group I, consisting of 20 cats injected with H R P ; group I1, consisting of 15 cats in which sterile mineral oil followed the injection of HRP. In both groups 1 and I1 the H R P solution was injected over a period of 1-5 min, after which the system was allowed to equilibrate for a further 1-15 rain before withdrawing the pipette. However, only in group II was sterile mineral oil injected into the track as the pipette was withdrawn. To accomplish this, the pipette was first withdrawn I mm from the site of injection of HRP. The polyethylene tubing was then disconnected from the 1 /~1 syringe and connected to the 10/~l syringe situated adjacent to it. As the pipette was slowly withdrawn over a period of 30 sec, about 15 ¢tl of sterile mineral oil was injected into the brain approximating the volume originally occupied by the pipette. The quantity of oil injected was monitored by watching the advance of an air bubble introduced into the polyethylene tubing when the syringes were switched.
Fig. 1. A transverse section through the cerebellum and medulla of a cat from group I which was sacrificed 36 h after injection, illustrating the injection site and spread of HRP along the pipette track.
177 Twenty-four to 48 h after injection the animals were anesthetized and perfused transcardially with isotonic saline followed by a mixture of 1.25 % glutaraldehyde and 1% paraformaldehyde. The brains were promptly removed, placed in fresh fixative for 12 h, and then rinsed for 20-24 h in 0.1 M phosphate buffer (pH 7.4) containing 20% sucrose. Sections were cut at 50/~m on a freezing microtome and reacted with 3,Y-diaminobenzidine 9 for 15 min. The sections were transferred through two rinsing solutions of 0.1 M phosphate buffer and then individually mounted on gelatinized slides. Once dry, alternate slides were stained with cresylecht violet. In most cases in both groups I and II, the injection site was centered in the region of the dentate nucleus. The size of the injection site was defined under the light microscope as the extent of visible HRP reaction product. The size was found to be dependent on the volume of HRP injected with larger volumes of HRP resulting in larger injection sites. The sites were often roughly spherical and measured 0.8-3.0 mm in diameter. In a number of cases, however, small brown spurs were observed to protrude from the sphere along fibers that passed to or from the site of injection. There was no consistent difference in the shape of injection sites between groups I and II except for the region along the pipette track.
Fig. 2. A transverse section through the cerebellum and medulla of a cat from group II which was sacrificed 40 h after injection, depicting the injection site and the absence of spread of HRP along the pipette track.
178 The results of a typical microinjection of HRP from group I are illustrated in Fig. 1. The injection site, approximately 2 mm in diameter, is seen as a sphere in the region of the dentate nucleus. In addition, HRP has spread back along the path of the pipette through the cerebellar white matter into the cerebellar cortex. This backward diffusion of HRP was a consistent finding and was observed in 18 of the 20 cats included in group I. Fig. 2 illustrates the results of a typical injection obtained from a cat in group |l. In this case the injection site is also approximately 2 mm in diameter, but the spread of HRP backward along the pipette path has been eliminated. No spread was observed in any of the cats receiving the mineral oil injection as the pipette was withdrawn*. This technique caused a small amount of visible damage to the brain tissue by producing a cylindrical hole about 1 mm in diameter along the path of the pipette. Since this damage was confined to the area above the injection site, and the visible H R P reaction product was confined to the injection site, it seems unlikely that injured axons or cells above the injection site could have taken up the peroxidase. The importance of preventing the spread of H R P back along the pipette track was demonstrated in our own work when comparing peroxidase-positive cells found in the brains from cats in groups I and II. Cats in group I, for example, showed positive cells in the lateral reticular nucleus (LRN) indicating a projection from the L R N to the dentate nucleus and overlying cerebellar cortex. No positive cells, however, could be demonstrated in the L R N from cats in group lI. The latter finding agress well with the work of Kfinzle 4 who was unable to find direct fiber connections between the L R N and the dentate using the autoradiographic tracing technique. The present results indicate that the injection of mineral oil following the injection of H R P can eliminate one of the present problems associated with the peroxidase technique. The authors thank Dr. Morris Smithberg, Dr. James R. Bloedel, and Dr. Charles K. Knox for their careful reading and comments on this paper, and Diane Jacoby for help in preparing the manuscript. This study was supported in part by USPHS Grants G M I I 4 , GM572, and HL16430. 1 BUNT, A. H., HENDRICKSON,A. E., LUND, J. S., LUND, R. D., AND FucrtS, A. F., Monkey retinal ganglion cells: morphometric analysis and tracing of axonal projections, with a consideration of the peroxidase technique, J. comp. NeuroL, 164 (1975) 265-285. 2 DE VITO, J. L., CLAUSING,K. W., AND SMITH, O. A., Uptake and transport of horseradish peroxidase by cut end of the vagus nerve, Brain Research, 82 (1974) 269-271. 3 HALPERIN,J. J., AND LAVAIL,J. H., A study of the dynamics of retrograde transport and accumulation of horseradish peroxidase in injured neurons, Brain Research, 100 (1975) 253-269. 4 KONZLE, H., Autoradiographic tracing of the cerebellar projections from the lateral reticular nucleus in the cat, Exp. Brain Res., 22 (1975) 255-266. * Preliminary results from injections into areas of the brain stem, using a rostral approach of the pipette, also indicate an absence of spreading of the HRP along the pipette track.
179 5 LAVAIL,J. H., personal communication. 6 LAVA]L,J. H., The retrograde transport method, Fed. Proc., 34 (1975) 1618-1624. 7 LAVA]L,J. H., ANDLAVA]L,M. M., The retrograde intraaxonal transport of horseradish peroxidase in the chick visual system: a light and electron microscopic study, J. comp. NeuroL, 157 (1974) 303-358. 8 LAVA]L,J. H., AND LAVAIL,M. M., Retrograde axonal transport in the central nervous system, Science, 176 (1972) 1416-1417. 9 LAVA]L,J. H., WINSTON,K. R., AND TlSH, A., A method based on retrograde intraaxonal transport of protein for identification of cell bodies of origin of axons terminating within the CNS, Brain Research, 58 (1973) 470-477. 10 NAUTA,H. J. W., KAISERMAN-ABRAMOF,I. R., AND LASEK,R. J., Electron microscopic observations of horseradish peroxidase transported from the caudoputamen to the substantia nigra in the rat: possible involvement of the agranular reticulum, Brain Research, 85 0975) 373-384. 11 ROMA~NANO,M. A., AND MACmWICZ,R. J., Peroxidase labeling of motor cortex neurons projecting to the ventrolateral nucleus in the cat, Brain Research, 83 (1975) 469-473. 12 SI~RLOCK,D. A., AND RA]SMAN,G., A comparison of anterograde and retrograde axonal transport of horseradish peroxidase in the connections of the mammillary nuclei in the rat, Brain Research, 85 (1975) 321-324. 13 SOTELO, C., AND RtCH~, D., The smooth endoplasmic reticulum and the retrograde and fast orthograde transport of horseradish peroxidase in the nigro-striato-nigral loop, Anat. EmbryoL, 146 (1974) 209-218.
