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Interference of biocytin with opioid-evoked hyperpolarization and membrane properties of rat spinal substantia gelatinosa neurons
Neuroscience Letters 297 (2001) 117±120
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Interference of biocytin with opioid-evoked hyperpolarization and membrane properties of rat spinal substantia gelatinosa neurons William A. Eckert III 1, Helen H. Willcockson, Alan R. Light* Department of Cell and Molecular Physiology, CB#7545 Medical Sciences Research Building, University of North Carolina, Chapel Hill, NC 27599±7545, USA Received 18 August 2000; received in revised form 15 November 2000; accepted 15 November 2000
Abstract In our laboratory, preliminary whole-cell, tight seal recordings of rat spinal substantia gelatinosa neurons including biocytin in the patch pipette yielded a signi®cantly smaller proportion of neurons hyperpolarized by selective opioid agonists compared with recordings without biocytin. Therefore, we investigated the effects of biocytin inclusion on opioid responses and other membrane properties during whole-cell, tight seal recordings of these neurons. The percentage of neurons hyperpolarized by m-, d1-, and k-selective opioids was signi®cantly reduced when 1% but not #0.2% biocytin was included in the recording pipette, compared with neurons recorded without biocytin. However, a signi®cantly higher proportion of neurons ®red spontaneous action potentials with either 0.05±0.2 or 1% biocytin compared to no biocytin. Resting membrane potential, input impedance and the proportion of neurons displaying transient outward recti®cation were each signi®cantly altered for neurons recorded with 1% but not 0.05±0.2% biocytin. These effects may be due to a relatively speci®c blockade of diverse potassium channel types. Because ef®cient labeling can be achieved with 0.1% biocytin with whole-cell recording, higher concentrations are contraindicated. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Techniques; Methods; Lamina II; Opioid receptors; Patch clamp; In vitro slice; Spinal cord
The biotin molecule intracellular tracers, neurobiotin and biocytin are widely used to label cells and identify their morphological features, usually in conjunction with electrophysiological characterization of the same cell. In these studies, it is crucial that the tracer does not alter any of the electrophysiological properties under investigation [5]. Previously, recordings of rat neostriatal neurons recorded with sharp intracellular electrodes demonstrated little to no alteration in electrophysiological properties when 1±3% neurobiotin was used [6,8]. By contrast, SchloÈsser and colleagues [6] also found that including 1% neurobiotin in the patch pipette during whole-cell, tight seal recordings of these same neurons profoundly increased action potential duration in a manner resembling the effects observed from potassium channel blockers. Despite the similarities in molecular structure between biocytin and neurobiotin [3], * Corresponding author. Tel.: 11-919-966-1177; fax 11-919966-6927. E-mail address: [email protected] (A.R. Light). 1 Present address: Department of Anatomy, Keck Center for Integrative Neuroscience, University of California San Francisco, CA 94143-0452, USA
to our knowledge there have been no reports of altered electrophysiological properties with the use of biocytin during whole-cell, tight seal or intracellular recordings. Here we report that inclusion of biocytin in the patch pipette during whole-cell, tight-seal recordings of rat substantia gelatinosa (SG) neurons can signi®cantly reduce the proportion of neurons hyperpolarized by selective opioid agonists and may alter other membrane properties, depending upon the biocytin concentration used. Blind whole-cell, tight seal recordings of rat SG neurons from spinal cord slices were performed as described previously [7]. Brie¯y, spinal cords were removed and 500±600 mm transverse slices were prepared by sectioning with a vibratome. Slices were superfused with roomtemperature, arti®cial cerebral spinal ¯uid (ACSF) containing (in mM): 120 NaCl, 2.5 KCl, 2.5 CaCl2, 26 NaHCO3, 1.5 MgSO4, 1.25 NaH2PO4, and 10 glucose, pH 7.40±7.45, continuously gassed with 95% O2/5% CO2. Recording pipettes were fabricated as in Light and Willcockson [4] and had a DC resistance of 5±10 MV when ®lled with potassium gluconate internal solution containing (in mM): 130 K-gluconate, 5 NaCl, 1 CaCl2, 1 MgCl2, 11 ethylene
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 68 4- 0
118
W.A. Eckert III et al. / Neuroscience Letters 297 (2001) 117±120
glycol-bis(b 0aminoethyl ether)-N,N,N 0 ,N 0 -tetraacetic acid (EGTA), 10 N-2-hydroxyethylpiperazine-N 0 -2-ethanesulfonic acid (HEPES), 0.1 guanosine triphosphate (GTP) (lithium salt, Sigma Chemical, St. Louis, MO), and 2 Mgadenosine triphosphate (ATP) (magnesium salt, equine, Sigma) (pH 7.3). Patch pipette tips were ®lled with internal solution without biocytin and pipettes were back®lled with 0, 0.05, 0.1, 0.2 or 1.0% biocytin (free base; Sigma) in the same internal solution. Current clamp (bridge mode) recordings were made with an AxoClamp 2A electrometer (Axon Instruments, Foster City, CA). Electrode signals were immediately displayed on a storage oscilloscope and strip chart recorder, stored using Axotape software (Axon Instruments) and saved on magnetic tape for subsequent analyses. One to four of the following selective opioid agonists (Sigma) (m): [d-Ala 2, N-Me-Phe 4, Gly 5-ol]-enkephalin (DAMGO, acetate salt), (k): trans-(^)-3,4-dichloro-Nmethyl-n-(2-[1-pyrrolidinyl]cyclohexyl) benzeneacetamide (U50488H, methanesulfonate salt) or d-(5a,7a,8b)-(1)-Nmethyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-8-yl]benzeneacetamide (U69593), (d1): [d-Pen 2, Pen 5]-enkephalin (DPDPE), and (d2): [d-Ala 2,Glu 4]deltorphin (Deltorphin II) were applied to individual SG neurons via bath perfusate. Most opioid-responsive neurons were hyperpolarized only by one subtype-selective agonist. After recording biocytin®lled neurons, slices were prepared for histological processing and visualization of biocytin as described in Eckert et al. [1]. Whole-cell, tight seal recordings were obtained from a total of 143 SG neurons. Ninety-two of these were recorded with biocytin included in the patch pipette internal solution and 51 were recorded without biocytin. Forty-seven of the biocytin-containing pipettes were ®lled with 1% biocytin and 45 were ®lled with 0.05±0.2% biocytin. In preliminary whole-cell, tight seal experiments, we observed a reduction in the proportions of SG neurons hyperpolarized by selective m-, k-, and d opioids when 1% biocytin was included in the patch pipette. Eleven of 30 neurons (37%) recorded without biocytin in the electrode were hyperpolarized by one or more opioids, whereas only ®ve of 40 (13%) were hyperpolarized when 1% biocytin was included. To directly address the hypothesis that biocytin diffusion into the cytoplasm of SG neurons during whole-cell recordings reduces the proportion of SG neurons hyperpolarized by selective opioid agonists, we alternated recordings between pipettes with (n 7) and without 1% biocytin (n 21) in potassium gluconate internal solution. We found that a lower proportion of SG neurons was hyperpolarized when 1% biocytin was included (two of seven; 29%) relative to those hyperpolarized without biocytin inclusion (nine of 21; 43%). The proportions of neurons hyperpolarized by opioids with 0.05±0.2% biocytin were nearly identical with those recorded without biocytin (Fig. 1). Overall, 13/24 SG neurons (54%) were hyperpolarized by the m-opioid agonist, DAMGO, without biocytin included and 22/43 (51%) with 0.05±0.2% biocytin vs. only 6/29 (21%)
Fig. 1. Histogram of effects of patch pipette biocytin concentration on the percentage of SG neurons hyperpolarized by selective opioid agonists: m- (DAMGO), d1- (DPDPE), d2- (deltorphin II), and k- (U69593 or U50488H).
when 1% biocytin was included (P , 0:05, x 2-test). The d1opioid agonist, DPDPE, hyperpolarized 5/27 neurons (19%) when biocytin was not included and 4/28 (14%) with 0.05± 0.2% biocytin vs. 0/32 (0%) with 1% biocytin included (P , 0:05, x 2-test). The k-opioids, U69593 and U50488H, together hyperpolarized 4/27 (15%) without biocytin included and 4/32 (13%) with 0.05±0.2% biocytin vs. 0/29 (0%) with 1% biocytin included (P , 0:05, x 2-test). Because d2-selective agonists hyperpolarized few neurons, we could not attribute a reduction in proportion of responders to biocytin when using any concentration of biocytin (Fig. 1). There were no signi®cant differences in magnitudes of opioid-evoked hyperpolarizations or conductance increases with different concentrations of biocytin (Student's t-test). When neurons were randomly assigned to two different groups independent of biocytin concentration, there were no signi®cant differences between groups in the proportions of neurons hyperpolarized by opioids (x 2test). Other membrane properties were also signi®cantly altered with inclusion of biocytin in the whole-cell electrode. Mean resting membrane potential was 249.4 ^ 1.0 mV without biocytin, 246.8 ^ 0.9 mV with 0.05±0.2% and 246.5 ^ 1.0 mV with 1% biocytin (P , 0:05, Student's t-test). Input impedance was 784 ^ 37 MV without biocytin, 882 ^ 50 MV with 0.05±0.2% biocytin and 653 ^ 35 MV with 1% biocytin (P , 0:05, Student's t-test). Evoked action potential heights and widths were not signi®cantly altered with inclusion of biocytin. However, these parameters were measured with only nine neurons recorded with 1% biocytin included. Seven of 51 neurons (14%) recorded without biocytin ®red spontaneous action potentials; whereas, 17 of 45 (38%) recorded with 0.05±0.2% biocytin (P , 0:01, x 2-test) and 21 of 47 (45%) recorded
W.A. Eckert III et al. / Neuroscience Letters 297 (2001) 117±120
119
Fig. 2. Negative confocal image (grayscale) of a `stalked' SG neuron from the border of rat spinal lamina II-inner and II-outer ®lled with 0.1% biocytin visualized with streptavidin-Texas Red (1:150) (Vector, Burligname, CA). Dorsal is up, arrows indicate biocytin-®lled axons, scale bar 20 mm.
with 1% biocytin (P , 0:01, x 2-test) ®red spontaneous action potentials. Twenty-one of 51 neurons (39%) recorded without biocytin and 18 of 45 (40%) recorded with 0.05± 0.2% biocytin demonstrated a transient outward recti®cation upon offset of hyperpolarizing current, as described by Yoshimura and Jessell [9]. By contrast, only eight of 41 (20%) showed similar recti®cation when 1% biocytin was included (P , 0:05, x 2-test). There were no signi®cant differences in any of the membrane properties investigated between neurons randomly assigned to two groups independent of biocytin concentrations (Student's t-test; x 2-test). The intracellular targets of biocytin remain to be determined. Given the diversity of membrane properties affected by inclusion of biocytin, it is dif®cult to postulate a single speci®c mechanism. Nevertheless, opioid-evoked hyperpolarizations [7], resting membrane potential [2], and transient outward recti®cation [9] are each mediated by different types of potassium channels, e.g. G-protein-coupled inward rectifying potassium channels, inward rectifying potassium channels and A-type potassium channels, respectively. Also, one of the candidate targets for the effects of neurobiotin suggested by SchloÈsser and colleagues [6] was a potassium channel (activated by depolarization). Thus, if any speci®c mechanism exists for these biocytin effects, it may be a global blockade of potassium channels. However, the lower mean input impedance we observed for neurons recorded with 1% biocytin seems suggestive of an increase in overall membrane conductance rather than a decrease.
Therefore, the potential effects of biocytin on membrane properties are likely diverse and multidirectional. These results demonstrate that the intracellular tracer biocytin, which is widely used to label recorded neurons, can signi®cantly affect activation of opioid-evoked hyperpolarizations as well as other membrane properties of neurons when recorded in the whole-cell, tight-seal con®guration. Although the effects on opioid responses, resting potential, input impedance and transient outward recti®cation were only found at the highest biocytin concentration tested (1%), neurons recorded with low (0.05±0.2%) and high (1%) biocytin concentrations differed from those recorded without biocytin in the proportions of neurons ®ring spontaneous action potentials. Given these effects of 1% biocytin and the fact that Golgi-like labeling of somata, dendrites and axons can be ef®ciently achieved with 0.1% biocytin within 10 min of whole-cell recordings (Fig. 2), there is little reason to use higher than 0.1% biocytin. Alternatively, if higher biocytin concentration is for some reason necessary for desired labeling, one may consider using sharp electrodes rather whole-cell recording methods because there would be little to no diffusion of biocytin during recording and thus little to no concern about biocytinmediated alteration of membrane properties. The authors wish to thank Dr Michael Chua, Curtis Connor, Kirk McNaughton, Dr Steve Schneider, Bonnie Taylor-Blake, and Mel Roberts for their technical assis-
120
W.A. Eckert III et al. / Neuroscience Letters 297 (2001) 117±120
tance, and Dr Allan Basbaum for helpful comments during the writing of this manuscript. This work was supported by NIH grant R01-NS16433. [1] Eckert, W.A. III, McNaughton, K.K. and Light, A.R., Morphology and axonal arborization of inner lamina II neurons of rat spinal cord hyperpolarized by m-opioid selective agonists, in preparation. [2] Isomoto, S., Kondo, C. and Kurachi, Y., Inwardly rectifying potassium channels: Their molecular heterogeneity and function, Jpn. J. Physiol., 47 (1997) 11±39. [3] Kita, H. and Armstrong, W., A biotin-containing compound N-(2-aminoethyl)biotinamide for intracellular labeling and neuronal tracing studies: comparison with biocytin, J. Neurosci. Methods, 37 (1991) 141±150. [4] Light, A.R. and Willcockson, H.H., Spinal laminae I and II neurons in rat recorded in vivo in the whole-cell, tight seal
[5] [6] [7]
[8] [9]
con®guration: Properties and opioid responses, J. Neurophysiol., 82 (1999) 3316±3326. Nicholson, C. and Kater, S.B., The development of intracellular staining, In Kater, S.B., Nicholson, C. (Eds.), Intracellular Staining in Neurobiology, Springer, New York, 1973, pp. 1±19. SchloÈsser, B., Bruggencate, G. and Sutor, B., The intracellular tracer neurobiotin alters electrophysiological properties of rat neostriatal neurons, Neurosci. Lett., 249 (1998) 13±16. Schneider, S.P., Eckert III, W.A. and Light, A.R., Opioid-activated postsynaptic, inward rectifying potassium currents in whole cell recordings in substantia gelatinosa neurons, J. Neurophysiol., 80 (1998) 2954±2962. Xi, X.Z. and Xu, Z.C., The effect of neurobiotin on membrane properties and morphology of intracellularly labeled neurons, J. Neurosci. Methods, 65 (1996) 27±32. Yoshimura, M. and Jessell, T.M., Membrane properties of rat substantia gelatinosa neurons in vitro, J. Neurophysiol., 62 (1989) 109±118.
www.elsevier.com/locate/neulet
Interference of biocytin with opioid-evoked hyperpolarization and membrane properties of rat spinal substantia gelatinosa neurons William A. Eckert III 1, Helen H. Willcockson, Alan R. Light* Department of Cell and Molecular Physiology, CB#7545 Medical Sciences Research Building, University of North Carolina, Chapel Hill, NC 27599±7545, USA Received 18 August 2000; received in revised form 15 November 2000; accepted 15 November 2000
Abstract In our laboratory, preliminary whole-cell, tight seal recordings of rat spinal substantia gelatinosa neurons including biocytin in the patch pipette yielded a signi®cantly smaller proportion of neurons hyperpolarized by selective opioid agonists compared with recordings without biocytin. Therefore, we investigated the effects of biocytin inclusion on opioid responses and other membrane properties during whole-cell, tight seal recordings of these neurons. The percentage of neurons hyperpolarized by m-, d1-, and k-selective opioids was signi®cantly reduced when 1% but not #0.2% biocytin was included in the recording pipette, compared with neurons recorded without biocytin. However, a signi®cantly higher proportion of neurons ®red spontaneous action potentials with either 0.05±0.2 or 1% biocytin compared to no biocytin. Resting membrane potential, input impedance and the proportion of neurons displaying transient outward recti®cation were each signi®cantly altered for neurons recorded with 1% but not 0.05±0.2% biocytin. These effects may be due to a relatively speci®c blockade of diverse potassium channel types. Because ef®cient labeling can be achieved with 0.1% biocytin with whole-cell recording, higher concentrations are contraindicated. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Techniques; Methods; Lamina II; Opioid receptors; Patch clamp; In vitro slice; Spinal cord
The biotin molecule intracellular tracers, neurobiotin and biocytin are widely used to label cells and identify their morphological features, usually in conjunction with electrophysiological characterization of the same cell. In these studies, it is crucial that the tracer does not alter any of the electrophysiological properties under investigation [5]. Previously, recordings of rat neostriatal neurons recorded with sharp intracellular electrodes demonstrated little to no alteration in electrophysiological properties when 1±3% neurobiotin was used [6,8]. By contrast, SchloÈsser and colleagues [6] also found that including 1% neurobiotin in the patch pipette during whole-cell, tight seal recordings of these same neurons profoundly increased action potential duration in a manner resembling the effects observed from potassium channel blockers. Despite the similarities in molecular structure between biocytin and neurobiotin [3], * Corresponding author. Tel.: 11-919-966-1177; fax 11-919966-6927. E-mail address: [email protected] (A.R. Light). 1 Present address: Department of Anatomy, Keck Center for Integrative Neuroscience, University of California San Francisco, CA 94143-0452, USA
to our knowledge there have been no reports of altered electrophysiological properties with the use of biocytin during whole-cell, tight seal or intracellular recordings. Here we report that inclusion of biocytin in the patch pipette during whole-cell, tight-seal recordings of rat substantia gelatinosa (SG) neurons can signi®cantly reduce the proportion of neurons hyperpolarized by selective opioid agonists and may alter other membrane properties, depending upon the biocytin concentration used. Blind whole-cell, tight seal recordings of rat SG neurons from spinal cord slices were performed as described previously [7]. Brie¯y, spinal cords were removed and 500±600 mm transverse slices were prepared by sectioning with a vibratome. Slices were superfused with roomtemperature, arti®cial cerebral spinal ¯uid (ACSF) containing (in mM): 120 NaCl, 2.5 KCl, 2.5 CaCl2, 26 NaHCO3, 1.5 MgSO4, 1.25 NaH2PO4, and 10 glucose, pH 7.40±7.45, continuously gassed with 95% O2/5% CO2. Recording pipettes were fabricated as in Light and Willcockson [4] and had a DC resistance of 5±10 MV when ®lled with potassium gluconate internal solution containing (in mM): 130 K-gluconate, 5 NaCl, 1 CaCl2, 1 MgCl2, 11 ethylene
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 68 4- 0
118
W.A. Eckert III et al. / Neuroscience Letters 297 (2001) 117±120
glycol-bis(b 0aminoethyl ether)-N,N,N 0 ,N 0 -tetraacetic acid (EGTA), 10 N-2-hydroxyethylpiperazine-N 0 -2-ethanesulfonic acid (HEPES), 0.1 guanosine triphosphate (GTP) (lithium salt, Sigma Chemical, St. Louis, MO), and 2 Mgadenosine triphosphate (ATP) (magnesium salt, equine, Sigma) (pH 7.3). Patch pipette tips were ®lled with internal solution without biocytin and pipettes were back®lled with 0, 0.05, 0.1, 0.2 or 1.0% biocytin (free base; Sigma) in the same internal solution. Current clamp (bridge mode) recordings were made with an AxoClamp 2A electrometer (Axon Instruments, Foster City, CA). Electrode signals were immediately displayed on a storage oscilloscope and strip chart recorder, stored using Axotape software (Axon Instruments) and saved on magnetic tape for subsequent analyses. One to four of the following selective opioid agonists (Sigma) (m): [d-Ala 2, N-Me-Phe 4, Gly 5-ol]-enkephalin (DAMGO, acetate salt), (k): trans-(^)-3,4-dichloro-Nmethyl-n-(2-[1-pyrrolidinyl]cyclohexyl) benzeneacetamide (U50488H, methanesulfonate salt) or d-(5a,7a,8b)-(1)-Nmethyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-8-yl]benzeneacetamide (U69593), (d1): [d-Pen 2, Pen 5]-enkephalin (DPDPE), and (d2): [d-Ala 2,Glu 4]deltorphin (Deltorphin II) were applied to individual SG neurons via bath perfusate. Most opioid-responsive neurons were hyperpolarized only by one subtype-selective agonist. After recording biocytin®lled neurons, slices were prepared for histological processing and visualization of biocytin as described in Eckert et al. [1]. Whole-cell, tight seal recordings were obtained from a total of 143 SG neurons. Ninety-two of these were recorded with biocytin included in the patch pipette internal solution and 51 were recorded without biocytin. Forty-seven of the biocytin-containing pipettes were ®lled with 1% biocytin and 45 were ®lled with 0.05±0.2% biocytin. In preliminary whole-cell, tight seal experiments, we observed a reduction in the proportions of SG neurons hyperpolarized by selective m-, k-, and d opioids when 1% biocytin was included in the patch pipette. Eleven of 30 neurons (37%) recorded without biocytin in the electrode were hyperpolarized by one or more opioids, whereas only ®ve of 40 (13%) were hyperpolarized when 1% biocytin was included. To directly address the hypothesis that biocytin diffusion into the cytoplasm of SG neurons during whole-cell recordings reduces the proportion of SG neurons hyperpolarized by selective opioid agonists, we alternated recordings between pipettes with (n 7) and without 1% biocytin (n 21) in potassium gluconate internal solution. We found that a lower proportion of SG neurons was hyperpolarized when 1% biocytin was included (two of seven; 29%) relative to those hyperpolarized without biocytin inclusion (nine of 21; 43%). The proportions of neurons hyperpolarized by opioids with 0.05±0.2% biocytin were nearly identical with those recorded without biocytin (Fig. 1). Overall, 13/24 SG neurons (54%) were hyperpolarized by the m-opioid agonist, DAMGO, without biocytin included and 22/43 (51%) with 0.05±0.2% biocytin vs. only 6/29 (21%)
Fig. 1. Histogram of effects of patch pipette biocytin concentration on the percentage of SG neurons hyperpolarized by selective opioid agonists: m- (DAMGO), d1- (DPDPE), d2- (deltorphin II), and k- (U69593 or U50488H).
when 1% biocytin was included (P , 0:05, x 2-test). The d1opioid agonist, DPDPE, hyperpolarized 5/27 neurons (19%) when biocytin was not included and 4/28 (14%) with 0.05± 0.2% biocytin vs. 0/32 (0%) with 1% biocytin included (P , 0:05, x 2-test). The k-opioids, U69593 and U50488H, together hyperpolarized 4/27 (15%) without biocytin included and 4/32 (13%) with 0.05±0.2% biocytin vs. 0/29 (0%) with 1% biocytin included (P , 0:05, x 2-test). Because d2-selective agonists hyperpolarized few neurons, we could not attribute a reduction in proportion of responders to biocytin when using any concentration of biocytin (Fig. 1). There were no signi®cant differences in magnitudes of opioid-evoked hyperpolarizations or conductance increases with different concentrations of biocytin (Student's t-test). When neurons were randomly assigned to two different groups independent of biocytin concentration, there were no signi®cant differences between groups in the proportions of neurons hyperpolarized by opioids (x 2test). Other membrane properties were also signi®cantly altered with inclusion of biocytin in the whole-cell electrode. Mean resting membrane potential was 249.4 ^ 1.0 mV without biocytin, 246.8 ^ 0.9 mV with 0.05±0.2% and 246.5 ^ 1.0 mV with 1% biocytin (P , 0:05, Student's t-test). Input impedance was 784 ^ 37 MV without biocytin, 882 ^ 50 MV with 0.05±0.2% biocytin and 653 ^ 35 MV with 1% biocytin (P , 0:05, Student's t-test). Evoked action potential heights and widths were not signi®cantly altered with inclusion of biocytin. However, these parameters were measured with only nine neurons recorded with 1% biocytin included. Seven of 51 neurons (14%) recorded without biocytin ®red spontaneous action potentials; whereas, 17 of 45 (38%) recorded with 0.05±0.2% biocytin (P , 0:01, x 2-test) and 21 of 47 (45%) recorded
W.A. Eckert III et al. / Neuroscience Letters 297 (2001) 117±120
119
Fig. 2. Negative confocal image (grayscale) of a `stalked' SG neuron from the border of rat spinal lamina II-inner and II-outer ®lled with 0.1% biocytin visualized with streptavidin-Texas Red (1:150) (Vector, Burligname, CA). Dorsal is up, arrows indicate biocytin-®lled axons, scale bar 20 mm.
with 1% biocytin (P , 0:01, x 2-test) ®red spontaneous action potentials. Twenty-one of 51 neurons (39%) recorded without biocytin and 18 of 45 (40%) recorded with 0.05± 0.2% biocytin demonstrated a transient outward recti®cation upon offset of hyperpolarizing current, as described by Yoshimura and Jessell [9]. By contrast, only eight of 41 (20%) showed similar recti®cation when 1% biocytin was included (P , 0:05, x 2-test). There were no signi®cant differences in any of the membrane properties investigated between neurons randomly assigned to two groups independent of biocytin concentrations (Student's t-test; x 2-test). The intracellular targets of biocytin remain to be determined. Given the diversity of membrane properties affected by inclusion of biocytin, it is dif®cult to postulate a single speci®c mechanism. Nevertheless, opioid-evoked hyperpolarizations [7], resting membrane potential [2], and transient outward recti®cation [9] are each mediated by different types of potassium channels, e.g. G-protein-coupled inward rectifying potassium channels, inward rectifying potassium channels and A-type potassium channels, respectively. Also, one of the candidate targets for the effects of neurobiotin suggested by SchloÈsser and colleagues [6] was a potassium channel (activated by depolarization). Thus, if any speci®c mechanism exists for these biocytin effects, it may be a global blockade of potassium channels. However, the lower mean input impedance we observed for neurons recorded with 1% biocytin seems suggestive of an increase in overall membrane conductance rather than a decrease.
Therefore, the potential effects of biocytin on membrane properties are likely diverse and multidirectional. These results demonstrate that the intracellular tracer biocytin, which is widely used to label recorded neurons, can signi®cantly affect activation of opioid-evoked hyperpolarizations as well as other membrane properties of neurons when recorded in the whole-cell, tight-seal con®guration. Although the effects on opioid responses, resting potential, input impedance and transient outward recti®cation were only found at the highest biocytin concentration tested (1%), neurons recorded with low (0.05±0.2%) and high (1%) biocytin concentrations differed from those recorded without biocytin in the proportions of neurons ®ring spontaneous action potentials. Given these effects of 1% biocytin and the fact that Golgi-like labeling of somata, dendrites and axons can be ef®ciently achieved with 0.1% biocytin within 10 min of whole-cell recordings (Fig. 2), there is little reason to use higher than 0.1% biocytin. Alternatively, if higher biocytin concentration is for some reason necessary for desired labeling, one may consider using sharp electrodes rather whole-cell recording methods because there would be little to no diffusion of biocytin during recording and thus little to no concern about biocytinmediated alteration of membrane properties. The authors wish to thank Dr Michael Chua, Curtis Connor, Kirk McNaughton, Dr Steve Schneider, Bonnie Taylor-Blake, and Mel Roberts for their technical assis-
120
W.A. Eckert III et al. / Neuroscience Letters 297 (2001) 117±120
tance, and Dr Allan Basbaum for helpful comments during the writing of this manuscript. This work was supported by NIH grant R01-NS16433. [1] Eckert, W.A. III, McNaughton, K.K. and Light, A.R., Morphology and axonal arborization of inner lamina II neurons of rat spinal cord hyperpolarized by m-opioid selective agonists, in preparation. [2] Isomoto, S., Kondo, C. and Kurachi, Y., Inwardly rectifying potassium channels: Their molecular heterogeneity and function, Jpn. J. Physiol., 47 (1997) 11±39. [3] Kita, H. and Armstrong, W., A biotin-containing compound N-(2-aminoethyl)biotinamide for intracellular labeling and neuronal tracing studies: comparison with biocytin, J. Neurosci. Methods, 37 (1991) 141±150. [4] Light, A.R. and Willcockson, H.H., Spinal laminae I and II neurons in rat recorded in vivo in the whole-cell, tight seal
[5] [6] [7]
[8] [9]
con®guration: Properties and opioid responses, J. Neurophysiol., 82 (1999) 3316±3326. Nicholson, C. and Kater, S.B., The development of intracellular staining, In Kater, S.B., Nicholson, C. (Eds.), Intracellular Staining in Neurobiology, Springer, New York, 1973, pp. 1±19. SchloÈsser, B., Bruggencate, G. and Sutor, B., The intracellular tracer neurobiotin alters electrophysiological properties of rat neostriatal neurons, Neurosci. Lett., 249 (1998) 13±16. Schneider, S.P., Eckert III, W.A. and Light, A.R., Opioid-activated postsynaptic, inward rectifying potassium currents in whole cell recordings in substantia gelatinosa neurons, J. Neurophysiol., 80 (1998) 2954±2962. Xi, X.Z. and Xu, Z.C., The effect of neurobiotin on membrane properties and morphology of intracellularly labeled neurons, J. Neurosci. Methods, 65 (1996) 27±32. Yoshimura, M. and Jessell, T.M., Membrane properties of rat substantia gelatinosa neurons in vitro, J. Neurophysiol., 62 (1989) 109±118.