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Intracellular labeling of dentate granule cells in fixed tissue permits quantitative analysis of dendritic morphology
Neuroscience Letters, 118 (1990) 249-251
249
Elsevier Scientific Publishers Ireland Ltd. NSL 07229
Intracellular labeling of dentate granule cells in fixed tissue permits quantitative analysis of dendritic morphology Alicia M . F e l t h a u s e r a n d B r e n d a J. C l a i b o r n e Division of Life Sciences, The University of Texas at San Antonio, San Antonio, TX 78285 (U.S.A.)
(Received 18 October 1989; Revised version received22 June 1990; Accepted 27 June 1990) Key words: Dendrite; Dentate gyrus; Fixed tissue; Fluorescent dye; Hippocampus; Rat
Although cortical neurons in fixed tissue can be labeled by intracellular dye injection, it is not yet known whether the dye diffuses to the most distal tips of the dendrites, or whether all dendritic branches of a neuron are labeled. In this study we addressed these questions by injecting granule neurons from the rat dentate gyrus in fixed, 400-gm-thick hippocampal slices with the fluorescent dye Lucifer yellow and comparing the resultant dendritic morphologies with those of neurons labeled in slices maintained in vitro. Results indicated that each dendritic branch was filled to its most distal tip and that the average number of branches per neuron in the fixed tissue was the same as that seen after in vitro labeling. Thus, intracellular labeling of neurons in fixed tissue permits quantitative studies of dendritic tree structure.
Recently, neurons in fixed slices of the m a m m a l i a n brain have been labeled intracellularly with the fluorescent dye Lucifer yellow [1, 2, 6, 8, 9, 10, 12]. This technique has a number of advantages over injections in vivo or in vitro, including the large number of neurons that can be labeled in a single experiment, the ability to inject neurons that have small cell bodies and the use of human autopsy material for cellular studies [1, 2, 6, 10]. Although the dendritic trees of neurons injected in fixed tissue appear qualitatively similar to those stained by other methods [1, 9, 10], it is not yet known if the entire tree is filled with the dye. Specific questions include whether each branch is filled to its most distal tip and whether all dendritic branches of a neuron are labeled. These issues must be addressed before neurons labeled intracellularly in fixed tissue can be used for quantitative studies. The present work was undertaken, therefore, to examine the dendritic morphologies of granule neurons from the rat dentate gyrus labeled by intracellular injection in fixed hippocampal slices. The granule cells are particularly advantageous for such a study because virtually all of their dendrites reach the top of the molecular layer [4, 5, l 1], thereby providing a reference point to assess whether dendrites fill completely in fixed tissue. In addition, the number of branches per neuron is known from previous quantitative studies of granule cells labeled by Correspondence: B.J. Claiborne, Division of Life Sciences, The University of Texas at San Antonio, San Antonio, TX 78285, U.S.A.
0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
intracellular injection in vitro [4, 1 1]. A preliminary version of this work has been reported in abstract form [7]. The animals used in this study were Sprague-Dawley rats of both sexes (Harlan Sprague-Dawley, Inc.), ranging in age from 37 to 43 days, and weighing between 121 and 204 g. An animal was anesthetized with either Metofane or Nembutal (60 mg/kg b. w.) and decapitated. The brain was removed, immersed in ice-cold saline and the hippocampus was dissected out as described previously [3]. Transverse slices from the middle third of the hippocampus were cut at a thickness of 400/~m on a Mcllwain tissue chopper, fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 30-45 min at 4°C, and then washed in buffer. Slices not used on the same day were stored in buffer at 4°C for up to 7 days. Any slices stored for 3 days or more were re-fixed for 30 min and washed in buffer before labeling. Using the microelectrode injection techniques described by others for filling cortical neurons in fixed tissue [10], granule neurons in the suprapyramidal blade of the dentate gyrus were injected with Lucifer Yellow (2.5% in 0.5 M LiC1). After neuronal labeling, slices were again fixed in the 4% paraformaldehyde for 12 h at 4°C and washed in buffer. They were then either dehydrated in ethanol and cleared in methyl salicylate, a procedure that produced slices with a low background fluorescence suitable for photography, or cleared in a glycerol-based, anti-bleach solution (see below) which reduced fading of the Lucifer yellow and allowed camera lucida drawings to be made of the filled neurons.
250 The dendrites o f granule neurons filled with Lucifer yellow were easily visible in slice whole-mounts cleared in either methyl salicylate or the anti-bleach solution. Between one and four primary dendrites emerged from the soma and were seen to branch within the molecular layer. All branches were covered with spines. In most cases, an axon could be seen to emerge from the cell b o d y and, occasionally, could be followed to the pyramidal cell layer in field CA3. Axonal collaterals were often visible in the hilus. F o r the quantitative analyses, the same selection criteria used in previous studies by Claiborne and colleagues [4, 11] for dentate granule cells filled with horseradish peroxidase ( H R P ) in vitro were applied to the granule neurons labeled here. First, neurons were judged to be adequately labeled if the Lucifer yellow was intensely fluorescent t h r o u g h o u t the entire dendritic tree. If it appeared to fade, the cell was rejected. Secondly, neurons were rejected if any dendrite in the proximal third o f the molecular layer was cut, or if two or m o r e branches were cut in the distal portions o f the layer. O f the approximately 30 labeled granule neurons, 19 were judged to fulfill the above criteria. Qualitative observations o f these well-labeled neurons showed that almost all dendrites could be followed to the hippocampal fissure at the top o f the molecular layer (Figs. 1 and 2A), as seen previously [4, 11] for granule cells labeled in vitro with H R P (Fig. 2B). This result indicated that dendrites were filled to their most distal tips in the fixed tissue. To analyze the n u m b e r o f dendritic branches that were filled, b r a n c h counts were done either by observation t h r o u g h the microscope (n = 16) or f r o m camera lucida drawings (n = 3). The average n u m b e r o f branches per neuron was 29.8 ___0.8 (mean + S.E.M.).
Fig. 1. Micrograph of the tip of a granule cell dendrite labeled with Lucifer yellow in a fixed hippocampal slice. The dye-filled dendrite could be followed to the hippocampal fissure (dashed line) at the top of the molecular layer. Bar = 25/tm.
Slices were cleared in the anti-bleach solution by first immersing them in 1% Triton X-100 to facilitate penetration o f the glycerol. Next they were washed in buffer for 1 h, put t h r o u g h a graded series o f glycerol in distilled H20, immersed in 100% glycerol for 1 h and then placed for 12 h in the anti-bleach solution at 4°C. The antibleach solution was the same as that described by Shum a n et al. [13], except that we adjusted the p H to approximately 7.8 with N a O H pellets.
A
B
/
i, GL
~
GL
Fig. 2. Camera lucida drawings of dentate granule neurons. The original drawings were done at a magnification of x 800. A: a neuron filled with Lucifer yellow in a fixed slice. Note that all dendrites except one (*) extended to the hippocampal fissure (dashed line). B: a neuron injected with HRP in vitro and analyzed for a previous study [10]. GL, granule cell layer; ML, molecular layer. Final magnifications were the same for both drawings. Bar = 50/zm.
251 This value was similar to the average branch n u m b e r s found in two previous studies o f rat dentate granule cells labeled in vitro [4, 11]. F o r these previous studies, hippoc a m p a l slices were maintained in a recording c h a m b e r and granule neurons injected intracellularly with H R P [3, 4, 11]. T o visualize the HRP-filled neurons, slices were processed with diaminobenzidine, cleared in gycerol and complete dendritic trees were analyzed quantitatively from w h o l e - m o u n t s o f the slices. T o m a k e a valid comparison with the cells labeled in the present work, subgroups o f granule cells filled in these previous studies were chosen on the basis o f cell location (suprapyramidal blade) and age o f the animals (37-43 days). T h e subgroup o f granule neurons f r o m one study [11] had an average of 31.7_+1.8 branches per neuron (mean+ S . E . M . ; n = 10) and those f r o m the second study [4] had an average o f 30.8-+ 1 branches ( n = 2 0 ) . Neither average was significantly different (P values of 0.29 and 0.48, respectively; Student's t-test) f r o m the average o f 29.8 obtained in the present study, supporting the suggestion that all o f the granule cell dendritic branches appear to fill when labeled in fixed slices. Thus, Lucifer yellow, when injected intracellularly into granule cells in fixed tissue, filled dendritic branches to their most distal tips and labeled all branches in the dendritic tree. This study also showed that neurons filled with Lucifer yellow in fixed slices can be analyzed directly from thick w h o l e - m o u n t s o f the slices, as done previously for cells labeled in vitro [3, 4, 11]. The use o f w h o l e - m o u n t s eliminates the time-consuming steps o f sectioning the slices and then reconstructing dendritic trees f r o m the serial sections. In s u m m a r y , intracellular labeling o f granule neurons in fixed h i p p o c a m p a l slices permits quantitative analysis o f their dendritic trees. We are presently using this procedure to quantify the dendritic structures o f developing granule neurons that are difficult to fill in vitro, and to increase the n u m b e r o f neurons labeled per animal in our aging studies. Use o f this technique should also m a k e it
possible to extend our quantitative studies o f neuronal m o r p h o l o g y to h u m a n cortical tissue. We t h a n k Dr. J a i - H y o n R h o for helpful suggestions on the technique. This w o r k was s u p p o r t e d by N S F G r a n t BNS-8709366, N I A G r a n t AG07141 and N I H G r a n t RR08194-09. 1 Buhl, E.H. and L~ibke, J., Intracellular Lucifer yellow injection in fixed brain slices combined with retrograde tracing, light and electron microscopy, Neuroscience, 28 (1989) 3-16. 2 Buhl, E.H. and Schlote, W., Intracellular Lucifer yellow staining and electron microscopy of neurons in slices of fixed epitumourous human cortical tissue, Acta Neuropathol., 75 (1987) 140-146. 3 Claibome, B.J., Amaral, D.G. and Cowan, W.M., A light and electron microscopic analysis of the mossy fibers of the rat dentate gyrus, J. Comp. Neurol., 246 (1986) 435-458. 4 Claiborne, B.J., Amaral, D.G. and Cowan, W.M., A quantitative, three-dimensional analysis of HRP-labeled granule cell dendrites in the rat dentate gyrus, submitted. 5 Desmond, N.L. and Levy, W.B., A quantitative anatomical study of the granule cell dendritic fields of the rat dentate gyrus using a novel probabilistic method, J. Comp. Neurol., 2 ! 2 (1982) 131-145. 6 Einstein, G., Intracellular injection of Lucifer yellow into cortical neurons in lightly fixed sections and its application to human autopsy material, J. Neurosci. Methods, 26 (1988) 95-103. 7 Felthauser, A.M. and Claiborne, B.J., Quantitative morphological comparison of rat dentate granule cells injected in fixed slices and those filled in vitro, Soc. Neurosci. Abstr., 15 (1989) 310. 8 Germroth, P., Schwerdtfeger, W.K. and Buhl, E.H., Morphology of identified entorhinal neurons projecting to the hippocampus. A light microscopical study combining retrograde tracing and intracellular injection, Neuroscience, 30 (1989) 683-691. 9 Liibke, J. and Albus, K., The postnatal development of layer VI pyramidal neurons in the cat's striate cortex, as visualized by intracellular Lucifer yellow injections in aldehyde-fixed tissue, Dev. Brain Res., 45 (1989) 29-38. 10 Rho, J.-H. and Sidman, R.L., Intracellular injection of Lucifer yellow into lightly fixed cerebellar neurons, Neurosci. Lett., 72 (1986) 21-24. 11 Rihn, L.A. and Claiborne, B.J., Dendritic growth and regression in rat dentate granule cells during late postnatal development, Dev. Brain Res., 54 (1990) 115-124. 12 Schwerdtfeger, W.K. and Buhl, E.H., Various types of nonpyramidal hippocampal neurons project to the septum and contralateral hippocampus, Brain Res., 386 (1986) 146-154. 13 Shuman, H., Murray, J.M. and DiLullo, C., Confocal microscopy: An overview, BioTechniques, 7 (1989) 154-163.
249
Elsevier Scientific Publishers Ireland Ltd. NSL 07229
Intracellular labeling of dentate granule cells in fixed tissue permits quantitative analysis of dendritic morphology Alicia M . F e l t h a u s e r a n d B r e n d a J. C l a i b o r n e Division of Life Sciences, The University of Texas at San Antonio, San Antonio, TX 78285 (U.S.A.)
(Received 18 October 1989; Revised version received22 June 1990; Accepted 27 June 1990) Key words: Dendrite; Dentate gyrus; Fixed tissue; Fluorescent dye; Hippocampus; Rat
Although cortical neurons in fixed tissue can be labeled by intracellular dye injection, it is not yet known whether the dye diffuses to the most distal tips of the dendrites, or whether all dendritic branches of a neuron are labeled. In this study we addressed these questions by injecting granule neurons from the rat dentate gyrus in fixed, 400-gm-thick hippocampal slices with the fluorescent dye Lucifer yellow and comparing the resultant dendritic morphologies with those of neurons labeled in slices maintained in vitro. Results indicated that each dendritic branch was filled to its most distal tip and that the average number of branches per neuron in the fixed tissue was the same as that seen after in vitro labeling. Thus, intracellular labeling of neurons in fixed tissue permits quantitative studies of dendritic tree structure.
Recently, neurons in fixed slices of the m a m m a l i a n brain have been labeled intracellularly with the fluorescent dye Lucifer yellow [1, 2, 6, 8, 9, 10, 12]. This technique has a number of advantages over injections in vivo or in vitro, including the large number of neurons that can be labeled in a single experiment, the ability to inject neurons that have small cell bodies and the use of human autopsy material for cellular studies [1, 2, 6, 10]. Although the dendritic trees of neurons injected in fixed tissue appear qualitatively similar to those stained by other methods [1, 9, 10], it is not yet known if the entire tree is filled with the dye. Specific questions include whether each branch is filled to its most distal tip and whether all dendritic branches of a neuron are labeled. These issues must be addressed before neurons labeled intracellularly in fixed tissue can be used for quantitative studies. The present work was undertaken, therefore, to examine the dendritic morphologies of granule neurons from the rat dentate gyrus labeled by intracellular injection in fixed hippocampal slices. The granule cells are particularly advantageous for such a study because virtually all of their dendrites reach the top of the molecular layer [4, 5, l 1], thereby providing a reference point to assess whether dendrites fill completely in fixed tissue. In addition, the number of branches per neuron is known from previous quantitative studies of granule cells labeled by Correspondence: B.J. Claiborne, Division of Life Sciences, The University of Texas at San Antonio, San Antonio, TX 78285, U.S.A.
0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
intracellular injection in vitro [4, 1 1]. A preliminary version of this work has been reported in abstract form [7]. The animals used in this study were Sprague-Dawley rats of both sexes (Harlan Sprague-Dawley, Inc.), ranging in age from 37 to 43 days, and weighing between 121 and 204 g. An animal was anesthetized with either Metofane or Nembutal (60 mg/kg b. w.) and decapitated. The brain was removed, immersed in ice-cold saline and the hippocampus was dissected out as described previously [3]. Transverse slices from the middle third of the hippocampus were cut at a thickness of 400/~m on a Mcllwain tissue chopper, fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 30-45 min at 4°C, and then washed in buffer. Slices not used on the same day were stored in buffer at 4°C for up to 7 days. Any slices stored for 3 days or more were re-fixed for 30 min and washed in buffer before labeling. Using the microelectrode injection techniques described by others for filling cortical neurons in fixed tissue [10], granule neurons in the suprapyramidal blade of the dentate gyrus were injected with Lucifer Yellow (2.5% in 0.5 M LiC1). After neuronal labeling, slices were again fixed in the 4% paraformaldehyde for 12 h at 4°C and washed in buffer. They were then either dehydrated in ethanol and cleared in methyl salicylate, a procedure that produced slices with a low background fluorescence suitable for photography, or cleared in a glycerol-based, anti-bleach solution (see below) which reduced fading of the Lucifer yellow and allowed camera lucida drawings to be made of the filled neurons.
250 The dendrites o f granule neurons filled with Lucifer yellow were easily visible in slice whole-mounts cleared in either methyl salicylate or the anti-bleach solution. Between one and four primary dendrites emerged from the soma and were seen to branch within the molecular layer. All branches were covered with spines. In most cases, an axon could be seen to emerge from the cell b o d y and, occasionally, could be followed to the pyramidal cell layer in field CA3. Axonal collaterals were often visible in the hilus. F o r the quantitative analyses, the same selection criteria used in previous studies by Claiborne and colleagues [4, 11] for dentate granule cells filled with horseradish peroxidase ( H R P ) in vitro were applied to the granule neurons labeled here. First, neurons were judged to be adequately labeled if the Lucifer yellow was intensely fluorescent t h r o u g h o u t the entire dendritic tree. If it appeared to fade, the cell was rejected. Secondly, neurons were rejected if any dendrite in the proximal third o f the molecular layer was cut, or if two or m o r e branches were cut in the distal portions o f the layer. O f the approximately 30 labeled granule neurons, 19 were judged to fulfill the above criteria. Qualitative observations o f these well-labeled neurons showed that almost all dendrites could be followed to the hippocampal fissure at the top o f the molecular layer (Figs. 1 and 2A), as seen previously [4, 11] for granule cells labeled in vitro with H R P (Fig. 2B). This result indicated that dendrites were filled to their most distal tips in the fixed tissue. To analyze the n u m b e r o f dendritic branches that were filled, b r a n c h counts were done either by observation t h r o u g h the microscope (n = 16) or f r o m camera lucida drawings (n = 3). The average n u m b e r o f branches per neuron was 29.8 ___0.8 (mean + S.E.M.).
Fig. 1. Micrograph of the tip of a granule cell dendrite labeled with Lucifer yellow in a fixed hippocampal slice. The dye-filled dendrite could be followed to the hippocampal fissure (dashed line) at the top of the molecular layer. Bar = 25/tm.
Slices were cleared in the anti-bleach solution by first immersing them in 1% Triton X-100 to facilitate penetration o f the glycerol. Next they were washed in buffer for 1 h, put t h r o u g h a graded series o f glycerol in distilled H20, immersed in 100% glycerol for 1 h and then placed for 12 h in the anti-bleach solution at 4°C. The antibleach solution was the same as that described by Shum a n et al. [13], except that we adjusted the p H to approximately 7.8 with N a O H pellets.
A
B
/
i, GL
~
GL
Fig. 2. Camera lucida drawings of dentate granule neurons. The original drawings were done at a magnification of x 800. A: a neuron filled with Lucifer yellow in a fixed slice. Note that all dendrites except one (*) extended to the hippocampal fissure (dashed line). B: a neuron injected with HRP in vitro and analyzed for a previous study [10]. GL, granule cell layer; ML, molecular layer. Final magnifications were the same for both drawings. Bar = 50/zm.
251 This value was similar to the average branch n u m b e r s found in two previous studies o f rat dentate granule cells labeled in vitro [4, 11]. F o r these previous studies, hippoc a m p a l slices were maintained in a recording c h a m b e r and granule neurons injected intracellularly with H R P [3, 4, 11]. T o visualize the HRP-filled neurons, slices were processed with diaminobenzidine, cleared in gycerol and complete dendritic trees were analyzed quantitatively from w h o l e - m o u n t s o f the slices. T o m a k e a valid comparison with the cells labeled in the present work, subgroups o f granule cells filled in these previous studies were chosen on the basis o f cell location (suprapyramidal blade) and age o f the animals (37-43 days). T h e subgroup o f granule neurons f r o m one study [11] had an average of 31.7_+1.8 branches per neuron (mean+ S . E . M . ; n = 10) and those f r o m the second study [4] had an average o f 30.8-+ 1 branches ( n = 2 0 ) . Neither average was significantly different (P values of 0.29 and 0.48, respectively; Student's t-test) f r o m the average o f 29.8 obtained in the present study, supporting the suggestion that all o f the granule cell dendritic branches appear to fill when labeled in fixed slices. Thus, Lucifer yellow, when injected intracellularly into granule cells in fixed tissue, filled dendritic branches to their most distal tips and labeled all branches in the dendritic tree. This study also showed that neurons filled with Lucifer yellow in fixed slices can be analyzed directly from thick w h o l e - m o u n t s o f the slices, as done previously for cells labeled in vitro [3, 4, 11]. The use o f w h o l e - m o u n t s eliminates the time-consuming steps o f sectioning the slices and then reconstructing dendritic trees f r o m the serial sections. In s u m m a r y , intracellular labeling o f granule neurons in fixed h i p p o c a m p a l slices permits quantitative analysis o f their dendritic trees. We are presently using this procedure to quantify the dendritic structures o f developing granule neurons that are difficult to fill in vitro, and to increase the n u m b e r o f neurons labeled per animal in our aging studies. Use o f this technique should also m a k e it
possible to extend our quantitative studies o f neuronal m o r p h o l o g y to h u m a n cortical tissue. We t h a n k Dr. J a i - H y o n R h o for helpful suggestions on the technique. This w o r k was s u p p o r t e d by N S F G r a n t BNS-8709366, N I A G r a n t AG07141 and N I H G r a n t RR08194-09. 1 Buhl, E.H. and L~ibke, J., Intracellular Lucifer yellow injection in fixed brain slices combined with retrograde tracing, light and electron microscopy, Neuroscience, 28 (1989) 3-16. 2 Buhl, E.H. and Schlote, W., Intracellular Lucifer yellow staining and electron microscopy of neurons in slices of fixed epitumourous human cortical tissue, Acta Neuropathol., 75 (1987) 140-146. 3 Claibome, B.J., Amaral, D.G. and Cowan, W.M., A light and electron microscopic analysis of the mossy fibers of the rat dentate gyrus, J. Comp. Neurol., 246 (1986) 435-458. 4 Claiborne, B.J., Amaral, D.G. and Cowan, W.M., A quantitative, three-dimensional analysis of HRP-labeled granule cell dendrites in the rat dentate gyrus, submitted. 5 Desmond, N.L. and Levy, W.B., A quantitative anatomical study of the granule cell dendritic fields of the rat dentate gyrus using a novel probabilistic method, J. Comp. Neurol., 2 ! 2 (1982) 131-145. 6 Einstein, G., Intracellular injection of Lucifer yellow into cortical neurons in lightly fixed sections and its application to human autopsy material, J. Neurosci. Methods, 26 (1988) 95-103. 7 Felthauser, A.M. and Claiborne, B.J., Quantitative morphological comparison of rat dentate granule cells injected in fixed slices and those filled in vitro, Soc. Neurosci. Abstr., 15 (1989) 310. 8 Germroth, P., Schwerdtfeger, W.K. and Buhl, E.H., Morphology of identified entorhinal neurons projecting to the hippocampus. A light microscopical study combining retrograde tracing and intracellular injection, Neuroscience, 30 (1989) 683-691. 9 Liibke, J. and Albus, K., The postnatal development of layer VI pyramidal neurons in the cat's striate cortex, as visualized by intracellular Lucifer yellow injections in aldehyde-fixed tissue, Dev. Brain Res., 45 (1989) 29-38. 10 Rho, J.-H. and Sidman, R.L., Intracellular injection of Lucifer yellow into lightly fixed cerebellar neurons, Neurosci. Lett., 72 (1986) 21-24. 11 Rihn, L.A. and Claiborne, B.J., Dendritic growth and regression in rat dentate granule cells during late postnatal development, Dev. Brain Res., 54 (1990) 115-124. 12 Schwerdtfeger, W.K. and Buhl, E.H., Various types of nonpyramidal hippocampal neurons project to the septum and contralateral hippocampus, Brain Res., 386 (1986) 146-154. 13 Shuman, H., Murray, J.M. and DiLullo, C., Confocal microscopy: An overview, BioTechniques, 7 (1989) 154-163.