Intracellular staining of cortical neurons by pressure microinjection of horseradish peroxidase and recovery by core biopsy

EXPERIMENTAL

NEUROLOGY

58,

138-144 (1978)

RESEARCH

NOTE

Intracellular Staining of Cortical Microinjection of Horseradish Recovery by Core MASAKI

SAKAI,

HIROKO

SAKAI,

AND

Neurons by Pressure Peroxidase and Biopsy CHARLES

D.

WOODY

3

Brain Research Institute, Departments of Anatomy and Psychiatry, and Laboratory of Newophysiology, Mental RetardationResearch Center, University of California, Los Angeles, California 90024 Received

July

5, 1977

Intracellular iontophoresis of Procion dye (IS), cobalt (14), radioactive amino acids (5), and horseradish peroxidase (HRP) (3, 6, 10, 12, 13, 17) has been used for the morphological identification of electrophysiologically studied neurons. Limitations in this approach include the requirement of passageof large, polarizing currents over a long period of time. The currents can alter cell activity, and the electrode may be dislodged before the iontophoresis is completed. In addition there is considerable variability in the amount of material released. The use of pressure injection may avoid or reduce these problems (1, 4, 9, 15). It was possible to inject as little as 1 nl [3H] choline chloride solution reliably into visible ganglion cells of Aplysia (11). However, it has not been possible to pressure inject through micropipets with sufficiently small tips to penetrate and hold comparatively smaller neurons of mammals. We developed a technique for pressure injection of small mammalian cortical neurons using an electrode treated with silicone; preliminary results were reported elsewhere (16). After injecting HRP intracellularly in an awake animal, the neuron is recovered by tissue core biopsy. This method permits Abbreviation : HRP-horseradish peroxidase. 1 We thank Ehud Gruen for technical assistance and H. Koike, S. T. Kitai, and B. Wong for helpful suggestions. This project was supported by the Air Force Office of Scientific Research Grant 76-3074 and also, in part, by NSF BNS 76-06886. Dr. Masaki Sakai is on leave from the Primate Research Institute, Kyoto University, Japan. 138 0014-4886/78/0581-0138$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

INTRACELLULAR

PRESSURE

MICROIN

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JECTION

rapid retrieval of an identifiable neuron from the chronically prepared animal. Intracellular pressure microinjection of HRP was carried out in unanesthetized cats that had been previously prepared, under anesthesia, for subsequent intracellular recording as described elsewhere (19). The same glass microelectrode was used for recording and pressure injection. Theta-type 2 capillaries (external diameter 1.5 to 2.0 mm) were washed in boiled methanol, rinsed in acetone, immersed a few minutes in 1% Siliclad (ClayAdams, 1950), and filled with fresh, filtered 4% HRP [3: 1 Sigma Types II and VI in 0.2 M KC1 (more recently, 0.6 to 1.0 M KCI) with Tris buffer, pH S.61. After a platinum-iridium or stainless-steel wire was inserted, the shank of the electrode was placed in a rigid plastic tube (Pharmaseal, Inc., K-SOL) and sealed with Pyseal (Fisher Scientific Co. C-228). The tube was connected to a regulated (Unimetrics, Inc., 10078) source of air pressure (60 to 90 lb/in. ‘) through a solenoid valve (Clippard Model 2013) (Fig. 1A). Then, under a dissecting microscope, the tip was placed under mineral oil and gently broken against a piece of glass to a diameter of about 1 pm while applying air pressure. After satisfactory breaking (< 170 Ma), exuded droplets were visible under the microscope with an output rate of approximately 1 pm3/s. Detailed descriptions of the apparatus for introducing the micropipet into the cortex and of the recording system have appeared elsewhere (19). Electrodes were inserted into the brain via a guide tube through the incised dura in a small burr hole (diameter 3 mm) in the skull. The intracellular activity was led through a Mentor amplifier to a Hewlett-Packard FM tape recorder. After recording intracellular activity, pressure was applied for 1 s or longer, depending on the desired extent of injection. In more than half of the cells, injection time was less than 10 s. Whenever the cell was lost, pressure was turned off manually, as quickly as possible. After a successful injection of a cell, blood clots and other debris were removed from the burr hole, the incision through the dura was enlarged, and a modified 13-gauge needle inserted. A core of brain tissue was aspirated by applying back pressure through a syringe filled with isotonic saline. The core (diameter 2.0 mm) was rinsed in isotonic saline to remove blood, cut into 6-mm or other desired length, and placed in 20 ml fixative (1.25% glutaraldehyde, 1% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3). All the above processes for core recovery were normally carried out within 5 min after injecting the cell. Fig. 1B illustrates the core biopsy procedures. As many as five cores could be taken from the pericruciate cortex of each hemisphere in the cats (N = 16). 2 Theta tubing was used for ease of filling and for more than one injectable solution per electrode.

the possibility

of ultimately

using

SAKAI,

SAKAI,

AND

WOODY

FIG. 1. Schematic illustration of techniques for pressure microinjection (A) and core biopsy (B). A-The microelectrode (m) is connected to a holder with Pyseal (c). A pressure meter (p) and solenoid valve (s) are inserted between the electrode and the air source. The electrode is introduced through the incised dura into the brain via a guide tube (g). B-A tissue core is aspirated through a modified Id-gauge needle connected to a syringe filled with saline. This picture shows the core expressed into isotonic saline. The

method

hydrochloride) Leavitt

(8),

DAB (3,3’-diaminobenzidine tetraand histological processing was similar to thaz of Jones and

for

development

but the fixation

with

time was shortened

because

of the small

size

of the tissue core. The core was refrigerated at least 4 h in fixative, trans-

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141

ferred to 0.1 M phosphate buffer containing 30% sucrose for a minimum of 1 h, cut in frozen serial sections of 30 to 40-pm thickness, and placed 20 min in 0.05% DAB in Tris buffer (pH 7.3),; then 3% Ha02 was added and incubated another 20 min. Sections were mounted on gelatin-coated slides and counterstained with cresyl violet. Thirty-two neurons were penetrated, studied electrophysiologically, and then injected intracellularly. Soma, dendrites, and axons were densely

FIG. 2. A pyramidal cell of layer V stained by intracellularly injected HRP. All calibration bars are 10 pm. A-A long apical dendrite can be seen. The arrow indicates an axon. B-Intracellular activity of the cell shown in A : (I) penetration; (II) single action potential; note the smoothly rising phase that is characteristic of cortical neurons; (III) application of lo-ms, OS-nA, depolarizing current pulse. The artifacts from these pulses, delivered every 100 ms, can be seen above in (I). All vertical calibrations are 40 mV. Arrow shows beginning of spiking after penetration. CSpines are seen on the apical dendrite of this cell. D-Two axon collaterals branch off from the main axon 100 Frn from the soma (arrows). Red blood cells also react to DAB.

142

SAKAI,

SAKAI,

AND

WOODY

stained. Some axons and dendrites could be traced for several hundred micrometers. Spines were stained in sufficient detail to distinguish differences in their distribution and morphology. Axon collaterals having a diameter of less than 1 w were also observed. In 27 of 32 cells, the locations of their soma in the cortical layer were identified, 1 cell in layer I, 4 in II, 8 in III, 13 in V, and 1 in VI. Five cells were not precisely located due to an inappropriate angle of transection. Several cell types were identified including 18 pyramidal and 1 inverted pyramidal cell. All but 4 cells fit Colonnier’s classification system (2). The largest cell found was a pyramidal cell in layer V, having a soma of SO-pm length and 30-pm width.3 The smallest cell found was a stellate cell in layer II having a 10-w diameter. The amplitude of action potentials was 20 to 62 mV ; baseline potential shift on penetration was 20 to 72 mV. Figure 2 shows a pyramidal cell which was located in layer V of the pericruciate cortex. Electrical activity of the cell is shown in B. For this cell, after a 4-s pressure microinjection, the tissue core was removed and placed in fixative within 2 min. Nonetheless, HRP spread surprisingly far (A). The apical shaft of the dendrite was traced a distance of 540 pm in the serial sections. The axon was traced 360 pm, to near the white matter, and four axon collaterals were observed (D). Three of the collaterals ran parallel to the main axon, back to near the soma. Numerous stubby, mushroom-shape, and thin spines (7) were identified at the middle and distal regions of the dendrites (C) . There was no systematic relation between injection time (1 to 15 min) and color density of the stained cell. As many as 78% of cells in which injections were attempted could be successfully recovered. Successful injections were invariably accompanied by sudden decreases in electrode resistance, measured by a Wheatstone bridge. In 5 of 32 cells, some slight, diffuse extracellular spread of HRP was seen around the injected cell. This was thought to be caused by a delay in releasing the pressure after losing the cell. These results indicate that pressure microinjection of HRP can be successfully used to mark intracellularly recorded neurons in the cortex of awake cats. Recovery of the cells by a biopsy technique permits rapid retrieval of multiple cores from a chronically prepared animal. The pressure technique is superior to iontophoretic injection in permitting extensive staiming in a very short time. It also permits continuous electrical recording during the marking procedure and avoids the passage of large current as well as the need for polar iontophoretic agents. Cortical cells with somas \of lo-pm diameter as well as larger cells with somas of 50 ysn were stained. B All measurements are not corrected

for

shrinkage

during

histology.

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There was no indication that brief pressure microinjection caused progressive depolarization, irreversibly increased frequencies of discharge, or other obvious injury. REFERENCES T. K. 1969. Techniques of intracellular microinjection. Pages 404423 in M. LAVALL~E, 0. F. SCHANNE, AND N. C. H&BERT, Eds., G&s Microelectrodes. John Wiley and Sons, New York/London/Sydney/Toronto. COLONNIER, M. L. 1966. The structural design of the neocortex. Pages 1-23 in J. C. ECCLES, Ed., Bruiti and Con.scious Experience. Springer-Verlag, New York/Heidelberg/Berlin. CULLHEIM, S., AND J-O. KELLERTH. 1976. Combined light and electron microscopic tracing of neurons, including axons and synaptic terminals, after intracellular injection of horseradish peroxidase. Neurosci. Lett. 2 : 307-313. CURTIS, D. R. 1955. Microelectrophoresis. Phys. Tech. Biol. Res. 5: 144-190. GLOBUS, A., H. D. Lux, AND P. SCHUBERT. 1968. Soma dendritic spread of intracellularly injected tritiated glycine in cat spinal motoneurons. Brain Res. 11: 440-44s. JANKOWSBA, E., J. RESTAD, AND J. WESTMAN. 1976. Intracellular application of horseradish peroxidase and its light and electron microscopical appearance in spinocervical tract cells. Brain Res. 105 : 557-562. JONES, E. G., AND T. P. S. POWELL. 1969. Morphological variations in the dendritic spines of the neocortex. J. Cell. Sci. 5: 509-529. JONES, E. G., AND R. Y. LEAVITT. 1974. Retrograde axonal transport and the demonstration of non-specific projections to the cerebral cortex and striatum from thalamic intralaminar nuclei in the rat, cat and monkey. J. Co*rcp. Neural. 154: 349-378. KATER, S. B., C. NICHOLSON, AND W. J. DAVIS. 1973. A guide to intracellular staining techniques. Pages 307-326 in S. B. KATER AND C. NICHOLSON, Eds., Intracellular Staining in Neurobiology. Springer-Verlag, New York/Heidelberg/Berlin. KITAI, S. T., J. D. KOCSIS, R. J. PRESTON, AND M. SUGIMORI. 1976. Monosynaptic inputs to caudate neurons identified by intracellular injection of horseradish peroxidase. Bra& Res. 109 : 601-606. KOIKE, H., M. EISENSTADT, AND J. H. SCHWARTZ. 1972. Axonal transport of newly synthesized acetylcholine in an identified neuron of Aplysia. Bra& Res. 37 : 152-159. LIGHT, A. R., AND R. G. DURKOVIC. 1976. Horseradish peroxidase: An improvement in intracellular staining of single, electrophysiologically characterized neurons. Exp. Newel. 53 : 847-853. MCCREA, R. A., G. A. BISHOP, AND S. T. KITAI. 1976. Intracellular staining of Purkinje cells and their axons with horseradish peroxidase. Brain Res. 118: 132-136. PITMAN, R. M., C. D. TWEEDLE, AND M. J. COHEN. 1972. Branching of central neurons; intracellular cobalt injection for light and electron microscopy. Science 176: 412-414. REMLER, M., A. SELVERSTON, AND D. KENNEDY. 1968. Lateral giant fibers of crayfish ; Location of somata by dye injection. Scieme 162 : 281-283. SAKAI, M., H. SAKAI, AND C. WOODY. 1977. Identification of intracellularly re-

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