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A reliable technique for marking the location of extracellular recording sites using glass micropipettes
?~eur~'icncc Letters, 81 (1987) I00 I[~4 Elsevier Scientific Publishers Ireland Lid
100
NSL 04881
A reliable technique for marking the location of extracellular recording sites using glass micropipettes Daniel J. Simons 1 and Peter W. Land 2 Departments qf IPhysiology and of : Neurobiology, Anatomy and ('ell Science, University q["Pittsburgh School ¢JfMedieine, Pittsburgh, PA 15261 (U.S.A.) (Received 6 April 1987; Revised version received and accepted 2 June 1987)
Key words: Microelectrode; Horseradish peroxidase (HRP); Extracellular recording A simple and highly reliable technique is described for marking the locations of extracellularly recorded neurons using double-barreled glass micropipettes. One barrel contains 3 M NaC1 for recording; the other contains horseradish peroxidase (HRP) that is iontophoretically ejected using low currents. With appropriate processing of the tissue, small well-localized spots are produced that contain a small number of HRP-filled neurons. The technique is effective in marking sequential recording sites within individual electrode tracks and in multiple penetrations using the same microelectrode.
The use of fluid-filled glass micropipettes for recording the extracellular signs of neuron action potentials offers a number of advantages for single-unit analyses of nervous tissue. First, glass micropipettes are inexpensive and their fabrication can be precisely controlled, permitting considerable flexibility in their overall shapes and tip diameters. Second, their smooth and graually tapering shafts facilitate tissue penetration and reduce dimpling at the tissue's surface. Tissue disruption in the immediate vicinity of the recording tip and along electrode tracks of several millimeters in length is considerably less severe than that produced by most metal electrodes. This is especially desirable when numerous, closely-spaced penetrations are required for sampling neuronal activity within a small area of tissue. Third, glass micropipettes provide excellent resolution in single-unit recordings, particularly when recording tip diameters are on the order of I /tm. Although fluid-filled microelectrodes with such small recording surfaces have relatively high resistances, good signal-to-noise ratio is presumably facilitated by the ability to position the recording tip close to the neuronal element being sampled, i.e. cell soma. The last two features, minimal tissue disruption and high resolution, are particularly desirable when attempting to study regions containing many small, densely-packed neurons, such as in granular layers of the cerebral cortex. Correspondence." D.J. Simons, Department of Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261~ U.S.A. 0304-3940/87/$ 03.50 (~) 1987 Elsevier Scientific Publishers Ireland Ltd.
101
A serious and persistent drawback to using glass micropipettes is the difficulty in producing discrete markers which aid the identification of recording sites in histological material. Following an early report by Thomas and Wilson [8] micropipettes have been filled with a variety of dyes that can be ejected iontophoretically or by pressure. Some of these dyes, such as Fast green [8], diffuse rapidly from the injection site and either disappear or fail to remain as localized spots. Other dyes such as Pontamine blue [3], Methyl blue [9] and Alcian blue [5] are effective only when suspended in electrolyte solutions of low salt content. This results in undesirably large internal resistances which exacerbate problems of high noise inherent to the glass micropipette [2]. In addition, relatively large current densities are required for iontophoretic passage of these dyes into the tissue. This frequently leads to blockage of the recording electrode or actual melting of the tip and consequent failure of dye deposition; moreover, the recording quality of the electrode may deteriorate and multiple injections, for example at different recording sites along an electrode track, often cannot be madc reliably. We have circumvented these problems by (1) employing a double-barreled electrode in which recording and dye solutions are loaded into separate compartments [7] and (2) using a marker, horseradish peroxidase (HRP), that can be iontophoretically deposited using low current and that is actively taken up by only a small number of neurons in the immediate vicinity of the electrode tip (see also ref. 6). With this technique we are able to reliably produce discrete, long-lasting marks that can be readily identified in a variety of histologically processed specimens. Electrodes are made from microcapillary-filled double-barreled glass capillary tubes having outside diameters of 1.2 or 1.5 ram. In order to conveniently position the glass in a standard electrode puller (Narishige or Kopf), one of the barrels is shortened by incrementally breaking it from both ends. The double-barreled glass is placed flat on a hard surface, and the corner of a razor blade is forced down between the two glass capillaries while applying slight pressure towards the capillary that is being shortened. The glass is broken off in small increments by starting at one end of the tube and working towards the center. The breaking procedure is controlled by clamping the two capillaries with the index finger, which is moved centrally in steps of approximately 5 ram. The glass fractures along the junction of the fused capillaries up to a point underneath the finger which acts as a shock absorber to prevent further spread of mechanical stress down the length of the fused glass tubes. To ensure that the same capillary tube is broken off at the other end, the outsidc corner of that capillary is first nipped with a pair of small wire cutters. This small fracture of the outside corner weakens the structure of the glass sufficiently to promote breakage of only that capillary when the razor blade is first inserted there to initiate the breaking procedure. The finished product is a single long capillary tube with a double-barreled segment centered in the middle 40 50 mm (see Fig. I). Thc two ends can then be placed in the microelectrode puller as if a single-barreled electrode was being pulled, the only modification required being adjustment of the heating element to fit concentrically around the double-barreled central region. Once the glass is pulled (in our configuration to a total tip diameter of approximately 1.5 2.0/Lm), one barrel is filled with 3 M NaCI and the other with 10% w/v HRP
102
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in 0.05 M Tris-HC1 (pH 6.8). The short barrel is backfilled using a 30-gauge syringe needle whereas the longer barrel can be filled by backfilling or by immersion of the back end in the NaCI or H R P solution. Separate contact wires are inserted into each barrel and are led to the recording amplifier or to a constant current generator. Resistances of the 3 M NaCl-filled recording electrode are 8-12 M,(2. For iontophoresis of HRP from the other barrel pulsed current (7 s on, 14 s off) of 0.6/IA (electrode positive) is applied for 60--90 s. The reference lead from the preparation is connected only to the negative terminal of the current source and not to earth ground. Animals can be perfused with a variety of aldehyde fixatives suitable for H RP histochemistry. Our experiments dictate that we mark the position of recording sites with respect to metabolic subdivisions of the cortex revealed by cytochrome oxidase (CO) activity. We therefore typically use a fixative developed for CO histochemistry composed of 2% paraformaldehyde and 1.5% glutaraldehyde in 0.1 M phosphate buffer [4]. Brains are postfixed for 8- 24h at 4'C and then transferred to 30% sucrose buffer until sectioning. Forty to 80 izm frozen sections are reacted according to the method of Adams [1]. An important parameter in obtaining well-localized spots of reaction product is the surviwfl time following the last H R P deposition. Diffusion of the enzyme from the deposition site is rapid, and with survival times of 2 4 h a halo of reaction product can be observed which may extend as much as 500 lira from the iniection site. With longer survival times, i.e. 6 h or more, this halo dissipates so that reaction product is restricted to the immediate vicinity of the intended spot. Since long survival times are sometimes impractical in acute experiments, the reaction product halo can be significantly reduced by postfixing the sectioned tissue in 2% glutaraldehyde for 1 2 h prior to the histochemical reaction. This additional postlixation blocks the reactivity of the diffusion halo, presumably by further denaturing the enzyme. The comparatively higher concentration of enzyme taken up by cells in the immediate vicinity of the electrode tip makes it possible to stain them histochemically in spite of the additional postfixation. Moreover, even ifa light halo persists, the spot is readily identifiable by the presence of several highly reactive somata at its center. An additional consideration is the potential cross-reactivity of the ejected H RP in histological procedures, such as CO staining, that utilize diaminobenzidine as a chromagen. This can be avoided by processing alternate sections for HRP and for CO. Most of the cross-reactivity in the latter sections can be avoided by including 10 mg catalase (Sigma) per 100 ml of the CO reaction mixture. Using the described injection parameters and histological processing, H RP spots of 50- 100 lira diameter are reliably produced (Fig. 1B, D). The spots typically consist of 2 6 heavily labeled adjacent cell bodies surrounded by scattered bits of reaction product debris. Labeling often extends into proximal dendritic segments and axons frequently are observed emanating from the side of HRP deposition. Occasionally labeled cells are observed at some distance from the spot; these have presumably been ! filled by retrograde transport of the enzyme and are readily distinguished from the cells at the spot's actual location by their considerably less dense labeling. The larger reaction product halo, which may be evident with short survival times, has not in
104 any instance precluded u n a m b i g u o u s identification o f the H R P marks. In addition, there is no observable tissue d a m a g e such as a lesion in the vicinity o f the H R P spots. Reaction p r o d u c t frequently can be seen outlining the electrode track and appears to be associated with red blood cells that enter the track as the shaft o f the electrode penetrates the tissue (e.g. Fig. I C). Reconstruction is also aided by a Nissl counterstain which reveals small gila-like cells that populate the electrode track. To date we have attempted to produce 73 spots during 27 recording experiments involving the rodent s o m a t o s e n s o r y cortex and have recovered 71 o f them. In at least half o f the attempts, more than one spot was m a d e using the same microelectrode to record and then deposit H R P in a single electrode penetration or in different penetrations. The electrodes yielded g o o d quality recordings, even in layer IV where cell bodies are comparatively small and packed closely together. Recorded activity there typically consists o f 2 or 3 different size spikes, with the largest amplitude unit (~> 150/tV) being easily discriminated from the smaller spikes a n d / o r b a c k g r o u n d activity. Recordings are stable and single units can be studied for periods as long as several hours. Moreover, the electrodes are quite sturdy, and provided that the dura is removed they can be used for several penetrations without severe deterioration in either the quality o f the recordings or the ability to reliably produce H R P spots. We have also been using these electrodes to successfully record and mark sites in the rat ventrobasal thalamus. In s u m m a r y , we describe a simple and reliable technique for accurately marking the location o f extracellularly recorded neurons using H R P . The technique is suitable for m a r k i n g sequential recording sites in individual electrode tracks and in multiple tracks using the same electrode. The histochemical proceduce used for visualizing the H R P spots is straightforward and inexpensive, and it produces a permanent record o f electrode position. Finally, the m e t h o d is compatible with m a n y standard fixatives. Therefore a variety o f counterstains may be employed, including other histochemical procedures. Supported by N I H G r a n t NS 19950: We thank J. Griffin for help with the illustrations and Alice Diven for typing the manuscript. 1 Adams, J.C., Heavy metal intensification of DAB-based HRP reaction product, J. Histochem. Cytochem., 29 (1981) 775. 2 Geddes, L.A., Electrodes and the Measurement of Bioelectric Events, Wiley, New York, 1972. 3 Kaneko, A. and Hashimoto, H., Recording site of the single cone response determined by an electrode marking technique. Vision Res., 7 (1967) 847 851. 4 Land, P.W. and Simons, D.J., Cytochrome oxidase staining in the rat Sml barrel cortex, J. Comp. Neurol., 238 (1985) 225 235. 5 Lee, B.B., Mandl, G. and Stean, J.P.B., Microelectrode tip position marking in nervous tissue: a new dye method, Electroencephalogr. Clin. Neurophysiol., 27 (1969) 610-613. 6 Lynch, G., Deadwyler, S. and Gall, C., Labeling of central nervous system neurons with extracellular recording microelectrodes, Brain Res., 66 (1974) 337 341. 7 Simons, D.J., Durham; D. and Woolsey, T.A., Functional organization of mouse and rat Sml barrel cortex following vibrissal damage on different postnatal days, Somatosens. Res., 1 (1984) 207 245. 8 Thomas, R.C. and Wilson, V.J., Precise localization of Renshaw cells with a new marking technique. Nature (London), 206 (1965) 211 213. 9 Thomas, R.C. and Wilson, V.J., Marking single neurons by staining with intracellular recording microelectrodes, Science, 151 (1966) 1538 1539.
100
NSL 04881
A reliable technique for marking the location of extracellular recording sites using glass micropipettes Daniel J. Simons 1 and Peter W. Land 2 Departments qf IPhysiology and of : Neurobiology, Anatomy and ('ell Science, University q["Pittsburgh School ¢JfMedieine, Pittsburgh, PA 15261 (U.S.A.) (Received 6 April 1987; Revised version received and accepted 2 June 1987)
Key words: Microelectrode; Horseradish peroxidase (HRP); Extracellular recording A simple and highly reliable technique is described for marking the locations of extracellularly recorded neurons using double-barreled glass micropipettes. One barrel contains 3 M NaC1 for recording; the other contains horseradish peroxidase (HRP) that is iontophoretically ejected using low currents. With appropriate processing of the tissue, small well-localized spots are produced that contain a small number of HRP-filled neurons. The technique is effective in marking sequential recording sites within individual electrode tracks and in multiple penetrations using the same microelectrode.
The use of fluid-filled glass micropipettes for recording the extracellular signs of neuron action potentials offers a number of advantages for single-unit analyses of nervous tissue. First, glass micropipettes are inexpensive and their fabrication can be precisely controlled, permitting considerable flexibility in their overall shapes and tip diameters. Second, their smooth and graually tapering shafts facilitate tissue penetration and reduce dimpling at the tissue's surface. Tissue disruption in the immediate vicinity of the recording tip and along electrode tracks of several millimeters in length is considerably less severe than that produced by most metal electrodes. This is especially desirable when numerous, closely-spaced penetrations are required for sampling neuronal activity within a small area of tissue. Third, glass micropipettes provide excellent resolution in single-unit recordings, particularly when recording tip diameters are on the order of I /tm. Although fluid-filled microelectrodes with such small recording surfaces have relatively high resistances, good signal-to-noise ratio is presumably facilitated by the ability to position the recording tip close to the neuronal element being sampled, i.e. cell soma. The last two features, minimal tissue disruption and high resolution, are particularly desirable when attempting to study regions containing many small, densely-packed neurons, such as in granular layers of the cerebral cortex. Correspondence." D.J. Simons, Department of Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261~ U.S.A. 0304-3940/87/$ 03.50 (~) 1987 Elsevier Scientific Publishers Ireland Ltd.
101
A serious and persistent drawback to using glass micropipettes is the difficulty in producing discrete markers which aid the identification of recording sites in histological material. Following an early report by Thomas and Wilson [8] micropipettes have been filled with a variety of dyes that can be ejected iontophoretically or by pressure. Some of these dyes, such as Fast green [8], diffuse rapidly from the injection site and either disappear or fail to remain as localized spots. Other dyes such as Pontamine blue [3], Methyl blue [9] and Alcian blue [5] are effective only when suspended in electrolyte solutions of low salt content. This results in undesirably large internal resistances which exacerbate problems of high noise inherent to the glass micropipette [2]. In addition, relatively large current densities are required for iontophoretic passage of these dyes into the tissue. This frequently leads to blockage of the recording electrode or actual melting of the tip and consequent failure of dye deposition; moreover, the recording quality of the electrode may deteriorate and multiple injections, for example at different recording sites along an electrode track, often cannot be madc reliably. We have circumvented these problems by (1) employing a double-barreled electrode in which recording and dye solutions are loaded into separate compartments [7] and (2) using a marker, horseradish peroxidase (HRP), that can be iontophoretically deposited using low current and that is actively taken up by only a small number of neurons in the immediate vicinity of the electrode tip (see also ref. 6). With this technique we are able to reliably produce discrete, long-lasting marks that can be readily identified in a variety of histologically processed specimens. Electrodes are made from microcapillary-filled double-barreled glass capillary tubes having outside diameters of 1.2 or 1.5 ram. In order to conveniently position the glass in a standard electrode puller (Narishige or Kopf), one of the barrels is shortened by incrementally breaking it from both ends. The double-barreled glass is placed flat on a hard surface, and the corner of a razor blade is forced down between the two glass capillaries while applying slight pressure towards the capillary that is being shortened. The glass is broken off in small increments by starting at one end of the tube and working towards the center. The breaking procedure is controlled by clamping the two capillaries with the index finger, which is moved centrally in steps of approximately 5 ram. The glass fractures along the junction of the fused capillaries up to a point underneath the finger which acts as a shock absorber to prevent further spread of mechanical stress down the length of the fused glass tubes. To ensure that the same capillary tube is broken off at the other end, the outsidc corner of that capillary is first nipped with a pair of small wire cutters. This small fracture of the outside corner weakens the structure of the glass sufficiently to promote breakage of only that capillary when the razor blade is first inserted there to initiate the breaking procedure. The finished product is a single long capillary tube with a double-barreled segment centered in the middle 40 50 mm (see Fig. I). Thc two ends can then be placed in the microelectrode puller as if a single-barreled electrode was being pulled, the only modification required being adjustment of the heating element to fit concentrically around the double-barreled central region. Once the glass is pulled (in our configuration to a total tip diameter of approximately 1.5 2.0/Lm), one barrel is filled with 3 M NaCI and the other with 10% w/v HRP
102
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t
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=
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+ •
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~
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~
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,,<
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~-.~ ~ ~.-= ._= 2 --
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103
in 0.05 M Tris-HC1 (pH 6.8). The short barrel is backfilled using a 30-gauge syringe needle whereas the longer barrel can be filled by backfilling or by immersion of the back end in the NaCI or H R P solution. Separate contact wires are inserted into each barrel and are led to the recording amplifier or to a constant current generator. Resistances of the 3 M NaCl-filled recording electrode are 8-12 M,(2. For iontophoresis of HRP from the other barrel pulsed current (7 s on, 14 s off) of 0.6/IA (electrode positive) is applied for 60--90 s. The reference lead from the preparation is connected only to the negative terminal of the current source and not to earth ground. Animals can be perfused with a variety of aldehyde fixatives suitable for H RP histochemistry. Our experiments dictate that we mark the position of recording sites with respect to metabolic subdivisions of the cortex revealed by cytochrome oxidase (CO) activity. We therefore typically use a fixative developed for CO histochemistry composed of 2% paraformaldehyde and 1.5% glutaraldehyde in 0.1 M phosphate buffer [4]. Brains are postfixed for 8- 24h at 4'C and then transferred to 30% sucrose buffer until sectioning. Forty to 80 izm frozen sections are reacted according to the method of Adams [1]. An important parameter in obtaining well-localized spots of reaction product is the surviwfl time following the last H R P deposition. Diffusion of the enzyme from the deposition site is rapid, and with survival times of 2 4 h a halo of reaction product can be observed which may extend as much as 500 lira from the iniection site. With longer survival times, i.e. 6 h or more, this halo dissipates so that reaction product is restricted to the immediate vicinity of the intended spot. Since long survival times are sometimes impractical in acute experiments, the reaction product halo can be significantly reduced by postfixing the sectioned tissue in 2% glutaraldehyde for 1 2 h prior to the histochemical reaction. This additional postlixation blocks the reactivity of the diffusion halo, presumably by further denaturing the enzyme. The comparatively higher concentration of enzyme taken up by cells in the immediate vicinity of the electrode tip makes it possible to stain them histochemically in spite of the additional postfixation. Moreover, even ifa light halo persists, the spot is readily identifiable by the presence of several highly reactive somata at its center. An additional consideration is the potential cross-reactivity of the ejected H RP in histological procedures, such as CO staining, that utilize diaminobenzidine as a chromagen. This can be avoided by processing alternate sections for HRP and for CO. Most of the cross-reactivity in the latter sections can be avoided by including 10 mg catalase (Sigma) per 100 ml of the CO reaction mixture. Using the described injection parameters and histological processing, H RP spots of 50- 100 lira diameter are reliably produced (Fig. 1B, D). The spots typically consist of 2 6 heavily labeled adjacent cell bodies surrounded by scattered bits of reaction product debris. Labeling often extends into proximal dendritic segments and axons frequently are observed emanating from the side of HRP deposition. Occasionally labeled cells are observed at some distance from the spot; these have presumably been ! filled by retrograde transport of the enzyme and are readily distinguished from the cells at the spot's actual location by their considerably less dense labeling. The larger reaction product halo, which may be evident with short survival times, has not in
104 any instance precluded u n a m b i g u o u s identification o f the H R P marks. In addition, there is no observable tissue d a m a g e such as a lesion in the vicinity o f the H R P spots. Reaction p r o d u c t frequently can be seen outlining the electrode track and appears to be associated with red blood cells that enter the track as the shaft o f the electrode penetrates the tissue (e.g. Fig. I C). Reconstruction is also aided by a Nissl counterstain which reveals small gila-like cells that populate the electrode track. To date we have attempted to produce 73 spots during 27 recording experiments involving the rodent s o m a t o s e n s o r y cortex and have recovered 71 o f them. In at least half o f the attempts, more than one spot was m a d e using the same microelectrode to record and then deposit H R P in a single electrode penetration or in different penetrations. The electrodes yielded g o o d quality recordings, even in layer IV where cell bodies are comparatively small and packed closely together. Recorded activity there typically consists o f 2 or 3 different size spikes, with the largest amplitude unit (~> 150/tV) being easily discriminated from the smaller spikes a n d / o r b a c k g r o u n d activity. Recordings are stable and single units can be studied for periods as long as several hours. Moreover, the electrodes are quite sturdy, and provided that the dura is removed they can be used for several penetrations without severe deterioration in either the quality o f the recordings or the ability to reliably produce H R P spots. We have also been using these electrodes to successfully record and mark sites in the rat ventrobasal thalamus. In s u m m a r y , we describe a simple and reliable technique for accurately marking the location o f extracellularly recorded neurons using H R P . The technique is suitable for m a r k i n g sequential recording sites in individual electrode tracks and in multiple tracks using the same electrode. The histochemical proceduce used for visualizing the H R P spots is straightforward and inexpensive, and it produces a permanent record o f electrode position. Finally, the m e t h o d is compatible with m a n y standard fixatives. Therefore a variety o f counterstains may be employed, including other histochemical procedures. Supported by N I H G r a n t NS 19950: We thank J. Griffin for help with the illustrations and Alice Diven for typing the manuscript. 1 Adams, J.C., Heavy metal intensification of DAB-based HRP reaction product, J. Histochem. Cytochem., 29 (1981) 775. 2 Geddes, L.A., Electrodes and the Measurement of Bioelectric Events, Wiley, New York, 1972. 3 Kaneko, A. and Hashimoto, H., Recording site of the single cone response determined by an electrode marking technique. Vision Res., 7 (1967) 847 851. 4 Land, P.W. and Simons, D.J., Cytochrome oxidase staining in the rat Sml barrel cortex, J. Comp. Neurol., 238 (1985) 225 235. 5 Lee, B.B., Mandl, G. and Stean, J.P.B., Microelectrode tip position marking in nervous tissue: a new dye method, Electroencephalogr. Clin. Neurophysiol., 27 (1969) 610-613. 6 Lynch, G., Deadwyler, S. and Gall, C., Labeling of central nervous system neurons with extracellular recording microelectrodes, Brain Res., 66 (1974) 337 341. 7 Simons, D.J., Durham; D. and Woolsey, T.A., Functional organization of mouse and rat Sml barrel cortex following vibrissal damage on different postnatal days, Somatosens. Res., 1 (1984) 207 245. 8 Thomas, R.C. and Wilson, V.J., Precise localization of Renshaw cells with a new marking technique. Nature (London), 206 (1965) 211 213. 9 Thomas, R.C. and Wilson, V.J., Marking single neurons by staining with intracellular recording microelectrodes, Science, 151 (1966) 1538 1539.