- Email: [email protected]
Extracellularly applied horseradish peroxidase increases the number of dense core vesicles in leech sensory neurons
Brain Research 967 (2003) 301–305 www.elsevier.com / locate / brainres
Short communication
Extracellularly applied horseradish peroxidase increases the number of dense core vesicles in leech sensory neurons Mei-Hui Tai a , Birgit Zipser b , * a
Department of Pediatrics and Human Development, Michigan State University, East Lansing, MI 48824, USA b Department of Physiology, Michigan State University, East Lansing, MI 48824, USA Accepted 6 January 2003
Abstract The uptake of horseradish peroxidase (HRP), applied as an extracellular tracer, is a classical method for studying endo / exocytosis of synaptic vesicles at the ultrastructural level. It is generally not considered that HRP may affect neuronal function. Reported here is the finding that extracellularly applied HRP (0.1%) perturbs dense core vesicles in the synaptic processes of leech neurons. The strength of the effect varies with neuronal class. In sensory afferents, the number of dense core vesicles increases 5-fold, while there is only a 2-fold increase in central neurons. 2003 Elsevier Science B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Staining, tracing and imaging techniques Keywords: HRP uptake; Sensory neuron; Transmitter release; Exocytosis
The uptake of HRP in membranous structures was pioneered as a method to monitor synaptic activity at the ultrastructural level [9,22]. Since then, this method has been extensively used to measure the turnover of transmitter and synaptic vesicles in different types of neurons and organisms [2,4,5,8]. Generally, HRP is thought to be an innocuous tracer without consideration that it may perturb neuronal function. However, a recent comparison of the two different horseradish peroxidase isoenzymes showed that basic HRP is taken up more efficiently than acidic HRP. This suggests that there may be interactions between HRP and neuronal membranes involving molecular charge beyond the nonspecific fluid-phase endocytosis [12]. Experimental paradigms using HRP uptake involve stimulating transmitter release using a convulsant, strychnine [22], electrical stimulation, elevated potassium [5] or sensory stimulation [17] while the preparation is exposed to HRP at concentrations ranging from 0.1 to 3%. Durations of HRP treatment vary between 20 min and 8 h. *Corresponding author. Tel.: 11-517-355-6475x1105; fax: 11-517432-1967. E-mail address: [email protected] (B. Zipser).
Following these treatments, HRP, detected by its diaminobenzidine (DAB) reaction product, is found in various membranous structures, cisternae, small synaptic vesicles, and coated synaptic vesicles. Recent observation established significant endo / exocytotic activity in the absence of experimental stimulation in cultured neurons [13,14,23]. As a first initiative to study spontaneous transmitter release in the intact central nervous system, we demonstrated HRP uptake into synaptic vesicles after treating the germinal plates of the embryonic leech with HRP. A surprising finding was that neurons cultured in the presence of HRP showed an increase of dense core vesicles compared to the control group. Our embryo culture conditions, using Leibovitz-15 medium (Gibco, Grand Island, NY, USA) supplemented with epidermal growth factor (Collaborative Research, Bedford, MA, USA), permit the normal patterning of sensory afferent projections in the central and peripheral nervous system to proceed at 92% of its normal rate [18]. The control group was cultured in defined medium while 0.1% HRP (Sigma, St. Louis, MO, USA), was added to the experimental group for the duration of 1.5 h. After a brief rinse, sensory afferents were live-stained on their surface
0006-8993 / 03 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02242-X
302
M.H. Tai, B. Zipser / Brain Research 967 (2003) 301–305
membrane by exposing both the experimental and control embryos to HRP-conjugated Lan3-2 or Laz2-369 for another 1.5 h. Monoclonal antibodies were directly conjugated with HRP according to the methods of Avrameas and Ternynck [1]. Therefore, during antibody staining, embryos were exposed to 0.01% HRP conjugated to IgG. This low concentration of HRP does not lead to DAB reaction product in synaptic vesicles [19–21]. The germinal plates were then fixed with 2% glutaraldehyde and processed for electron microscopy followed our previously
published procedure. Grids were examined with a Philips CM10 electron microscope at 100 kV [10,19–21]. Fig. 1 illustrates the ultrastructure of control afferents that were identified by staining their cell surface with mAbs Lan3-2 (Fig. 1A) and Laz2-369 (Fig. 1B). In Fig. 1A, the sectioned profiles of four large sensory afferent processes (numbered 1–4) and neuritic projections are labeled on their cell surface with DAB reaction product (triangles outline profile 1). Large ovoid axons are replete with synaptic vesicle clusters (arrow). Virtually every
Fig. 1. Electron micrographs showing sensory afferent profiles in control embryos. (A) Sensory afferents were briefly stained on their cell surface with HRP-conjugated Lan3-2. Treating live germinal plates with low concentrations of HRP-conjugated monoclonal antibodies stains the surface membrane of sensory afferents (profile 1 is outlined by triangles) but not their synaptic vesicles [19–21]. The unstained profiles belong to central neurons. Profiles of four young sensory afferents (1–4) forming a fascicle are surrounded with neuritic projections. All four large profiles possess multiple clusters of small synaptic vesicles (arrow) and an occasional dense core vesicle (arrowhead). (B) Sensory afferents were briefly stained on their cell surface with HRP-conjugated Laz2-369. Two sensory afferents (1,2) are apposed to the unstained profile of a central neuron. Profile 1 is replete with synaptic vesicles (arrowhead) and appears to be making an en passant synapse with the unstained central neuron. Bar, 0.5 mm.
M.H. Tai, B. Zipser / Brain Research 967 (2003) 301–305
axonal section of such young sensory afferents contained at least one synaptic vesicle cluster underneath the surface membrane. In addition to the clusters of clear vesicles, there are also dense core vesicles (arrowhead) and numerous mitochondria. Most of these synaptic vesicle clusters are not in contact with central neurons as shown in previous three-dimensional reconstruction of young sensory afferents [20]. In Fig. 1B, more mature sensory afferents (numbered 1, 2), stained with mAb Laz2-369, are apposed to a central neuron. The developing nerve terminal is replete with clear vesicles (arrow) and contains also some dense core vesicles (arrowhead). A mitochondrion is also present. Treating live germinal plates with low concentrations of HRP-conjugated monoclonal antibodies stains the surface membrane of sensory afferents but not their synaptic vesicles [19–21]. Fig. 2 illustrates the ultrastructure of sensory afferents in germinal plates that were cultured in the presence of 0.1% HRP. Sensory afferents are again identified by DAB reaction product on their surface membrane through stain-
303
ing with low concentrations of HRP-conjugated monoclonal antibodies. This time however, DAB reaction product is also found in small synaptic vesicles (Fig. 2A, arrows). This uptake of extracellularly applied HRP into synaptic vesicles has been the classical method for studying endo / exocytosis of synaptic vesicles at the ultrastructural level. As the embryos were not stimulated, the HRP uptake reflects basal endo / exocytosis. Spontaneous endo- and exocytosis has been observed in regenerating spinal cord neurons in culture [13,14,23] and in motor neurons in vivo [16]. A surprising observation was that sensory afferents, exposed to extracellular HRP, exhibited a dramatic increase in the number of their dense core vesicles (Fig. 2A and B, arrowheads). This increase in dense core vesicles has not been observed as a result of live-staining sensory afferents with low concentrations of HRP-conjugated monoclonal antibodies [19–21]. Therefore, it is a result of the application of 0.1% HRP as an extracellular tracer. The histogram in Fig. 3 compares the number of dense
Fig. 2. Electronmicrographs showing sensory afferents treated with 0.1% HRP. (A) A large sensory afferent profile stained with HRP-conjugated Lan3-2 shows HRP uptake into small synaptic vesicles (arrows). Multiple dense core vesicles are present (arrowhead). (B) A large afferent profile stained with HRP-conjugated Laz2-369 shows an increased number of dense core vesicles.
304
M.H. Tai, B. Zipser / Brain Research 967 (2003) 301–305
Fig. 3. HRP uptake leads to an increase in the number of dense core vesicles in sensory afferents and central neurons. Dense core vesicles were counted in electron micrographs taken through the sensory afferent target region in CNS ganglia. To correct for the difference in the magnification of the micrographs, the number of dense core vesicles was normalized to the area of the micrograph. Experimental values are listed as relative to the control values. Exposed to 0.1% HRP for 3 h, sensory afferents underwent an almost 5-fold increase (P,0.0001) in dense core vesicles while central neurons experienced a less than 2-fold increase. Significantly different than the control (*), (P,0.0001).
core vesicles in serial sections from neurons in control CNS ganglia (n54; sixty-six sections; two embryos) and CNS ganglia (n54; ninety-two sections; two embryos) that were treated with 0.1% HRP. Dense core vesicles, recognized by their dark center and their morphology as doublemembrane rounded granules, were found in both sensory afferents and other neurons. Sensory afferents were identified by DAB reaction product on their surface membrane through staining with low concentration of HRP-conjugated monoclonal antibodies Lan3-2 and Laz2-369. The unstained profiles in the electron micrographs mostly belong to central neurons. To determine the effect of HRP treatment on dense core vesicle number, we counted the absolute number of dense core vesicles / synaptic profiles divided by the area of the target region. To avoid doublesampling the same dense core vesicle, we used every other section of the serially sectioned target regions for quantification. The ratio of sensory afferents profiles / central profiles in the target regions as well as the average profile area did not change as a result of applying 0.1% HRP. Sensory afferents exposed to 0.1% HRP underwent about a 5-fold increase in dense core vesicle number. Central neurons, not stained by monoclonal antibodies, experienced about a 2-fold increase. Thus, exposure to HRP results in an increase in dense core vesicles, the magnitude of which varies with neuronal class. What distinguishes primary sensory afferents from other neurons is the abundant expression of cell type-specific neutral glycans on their surface membranes. Throughout phylogeny, different sensory modalities are labeled with
different neutral surface glycans of the complex and oligomannosidic type [7,11,15]. Using chemical and immunological methods, it has been observed that neural tissue in general expresses an unusually large amount of oligomannosidic glycans [6]. Likewise, HRP also expresses neutral glycans of the high mannose type [3]. It remains to be determined whether glycosylation is one of the features that play a role in the dramatic increase in the number of dense core vesicles in sensory neurons and the lesser increase in other neural cell types.
Acknowledgements This research was supported by National Institutes of Health Grant NS25117. We thank Brent Kauffman and Harper VanSteenhouse for discussions.
References [1] S. Avrameas, T. Ternynck, Peroxidase labeled antibody and Fab conjugates with enhanced intracellular penetration, Immunocytochemistry 8 (1971) 1175–1179. [2] L.H. Bannister, H.C. Dodson, Endocytic pathways in the olfactory and vomeronasal epithelia of the mouse: ultrastructure and uptake of tracers, Microsc. Res. Tech. 23 (1992) 128–141. [3] M.L. Bajt, B. Schmitz, M. Schachner, B. Zipser, Carbohydrate epitopes involved in neural cell recognition are conserved between vertebrates and leech, J. Neurosci. Res. 27 (1990) 276–285.
M.H. Tai, B. Zipser / Brain Research 967 (2003) 301–305 [4] M.B. Bunge, Initial endocytosis of peroxidase or ferritin by growth cones of cultured nerve cells, J. Neurocytol. 6 (1977) 407–439. [5] B. Ceccareli, W.P. Hurlbut, A. Mauro, Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction, J. Cell Biol. 57 (1973) 499–524. [6] Y.-J. Chen, D.R. Wing, G.R. Guile, R.A. Dwek, D.J. Harvey, S. Zamze, Neutral N-glycans in adult rat brain tissue, Eur. J. Biochem. 251 (1998) 691–703. [7] J. Dodd, T.M. Jessell, Lactoseries carbohydrates specify subsets of dorsal root ganglion neurons projecting to the superficial dorsal horn of rat spinal cord, J. Neurosci. 5 (1985) 3278–3294. [8] J.E. Heuser, T.S. Reese, Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction, J. Cell Biol. 57 (1973) 315–344. [9] E. Holtzman, A.R. Freeman, Stimulation-dependent alterations in peroxidase uptake at lobster neuromuscular junctions, Science 173 (1971) 733–736. [10] K. Ito, A. Konishi, S. Nomura, Y. Nakamura, T. Sugimoto, Application of coupled oxidation reaction to electron microscopic demonstration of horseradish peroxidase: cobalt–glucose oxidase method, Brain Res. 175 (1979) 341–346. [11] B. Key, R.A. Akeson, Delineation of olfactory pathways in the frog nervous system by unique glycoconjugates and N-CAM glycoforms, Neuron 6 (1991) 381–396. [12] B. Key, P.P. Giorgi, Uptake and axonal transport of horseradish peroxidase isoenzymes by different neuronal types, Neuroscience 22 (1987) 1135–1144. [13] K. Kraszewski, O. Mundig, L. Daniell, C. Verderio, M. Matteoli, P. De Camilli, Synaptic vesicle dynamics in living cultured hippocampal neurons visualized with Cy3-conjugated antibodies directed against the lumenal domain of synaptotagmin, J. Neurosci. 15 (1995) 4328–4342. [14] M. Matteoli, K. Takei, M.S. Perin, T.C. Sudhof, P. De Camilli,
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
305
Exo–endocytotic recycling of synaptic vesicles in developing processes of cultured hippocampal neurons, J. Cell Biol. 117 (1992) 849–861. L. Pays, G. Schwarting, Gal-NCAM is a differentially expressed marker for mature sensory neurons in the rat olfactory system, J. Neurobiol. 43 (2000) 173–185. L. Polo-Parada, C.M. Bose, L.T. Landmesser, Alterations in transmission, vesicle dynamics, and transmitter release machinery at NCAM-deficient neuromuscular junctions, Neuron 32 (2001) 815– 828. S. Schacher, E. Holtzman, D.C. Hood, Synaptic activity of frog retinal photoreceptors, a peroxidase uptake study, J. Cell Biol. 70 (1976) 178–192. J. Song, B. Zipser, Kinetics of the inhibition of axonal defasciculation mediated by carbohydrate markers in the embryonic leech, Dev. Biol. 168 (1995) 319–331. M.-H. Tai, M. Rheuben, D. Autio, B. Zipser, Leech photoreceptors project their galectin-containing processes into optic neuropils where they contact AP cells, J. Comp. Neurol. (1996) 235–248. M.-H. Tai, B. Zipser, Mannose-specific recognition mediates two aspects of synaptic growth in leech sensory afferents: collateral branching and formation of synaptic vesicle clusters, Dev. Biol. 201 (1998) 154–166. M.-H. Tai, B. Zipser, Sequential steps in synaptic targeting of leech sensory afferents are mediated by constitutive and developmentally regulated glycosylations of CAMs, Devel. Biol. 214 (1999) 258– 276. S. Teichberg, E. Holtzman, S.M. Crain, E.R. Peterson, Circulation and turnover of synaptic vesicle membrane in cultured fetal mammalian spinal cord neurons, J. Cell Biol. 67 (1975) 215–230. S. Zakharenko, S. Chang, M. O’Donoghue, S.V. Popov, Neurotransmitter secretion along growing nerve processes: comparison with synaptic vesicle exocytosis, J. Cell Biol. 144 (1999) 507–517.
Short communication
Extracellularly applied horseradish peroxidase increases the number of dense core vesicles in leech sensory neurons Mei-Hui Tai a , Birgit Zipser b , * a
Department of Pediatrics and Human Development, Michigan State University, East Lansing, MI 48824, USA b Department of Physiology, Michigan State University, East Lansing, MI 48824, USA Accepted 6 January 2003
Abstract The uptake of horseradish peroxidase (HRP), applied as an extracellular tracer, is a classical method for studying endo / exocytosis of synaptic vesicles at the ultrastructural level. It is generally not considered that HRP may affect neuronal function. Reported here is the finding that extracellularly applied HRP (0.1%) perturbs dense core vesicles in the synaptic processes of leech neurons. The strength of the effect varies with neuronal class. In sensory afferents, the number of dense core vesicles increases 5-fold, while there is only a 2-fold increase in central neurons. 2003 Elsevier Science B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Staining, tracing and imaging techniques Keywords: HRP uptake; Sensory neuron; Transmitter release; Exocytosis
The uptake of HRP in membranous structures was pioneered as a method to monitor synaptic activity at the ultrastructural level [9,22]. Since then, this method has been extensively used to measure the turnover of transmitter and synaptic vesicles in different types of neurons and organisms [2,4,5,8]. Generally, HRP is thought to be an innocuous tracer without consideration that it may perturb neuronal function. However, a recent comparison of the two different horseradish peroxidase isoenzymes showed that basic HRP is taken up more efficiently than acidic HRP. This suggests that there may be interactions between HRP and neuronal membranes involving molecular charge beyond the nonspecific fluid-phase endocytosis [12]. Experimental paradigms using HRP uptake involve stimulating transmitter release using a convulsant, strychnine [22], electrical stimulation, elevated potassium [5] or sensory stimulation [17] while the preparation is exposed to HRP at concentrations ranging from 0.1 to 3%. Durations of HRP treatment vary between 20 min and 8 h. *Corresponding author. Tel.: 11-517-355-6475x1105; fax: 11-517432-1967. E-mail address: [email protected] (B. Zipser).
Following these treatments, HRP, detected by its diaminobenzidine (DAB) reaction product, is found in various membranous structures, cisternae, small synaptic vesicles, and coated synaptic vesicles. Recent observation established significant endo / exocytotic activity in the absence of experimental stimulation in cultured neurons [13,14,23]. As a first initiative to study spontaneous transmitter release in the intact central nervous system, we demonstrated HRP uptake into synaptic vesicles after treating the germinal plates of the embryonic leech with HRP. A surprising finding was that neurons cultured in the presence of HRP showed an increase of dense core vesicles compared to the control group. Our embryo culture conditions, using Leibovitz-15 medium (Gibco, Grand Island, NY, USA) supplemented with epidermal growth factor (Collaborative Research, Bedford, MA, USA), permit the normal patterning of sensory afferent projections in the central and peripheral nervous system to proceed at 92% of its normal rate [18]. The control group was cultured in defined medium while 0.1% HRP (Sigma, St. Louis, MO, USA), was added to the experimental group for the duration of 1.5 h. After a brief rinse, sensory afferents were live-stained on their surface
0006-8993 / 03 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02242-X
302
M.H. Tai, B. Zipser / Brain Research 967 (2003) 301–305
membrane by exposing both the experimental and control embryos to HRP-conjugated Lan3-2 or Laz2-369 for another 1.5 h. Monoclonal antibodies were directly conjugated with HRP according to the methods of Avrameas and Ternynck [1]. Therefore, during antibody staining, embryos were exposed to 0.01% HRP conjugated to IgG. This low concentration of HRP does not lead to DAB reaction product in synaptic vesicles [19–21]. The germinal plates were then fixed with 2% glutaraldehyde and processed for electron microscopy followed our previously
published procedure. Grids were examined with a Philips CM10 electron microscope at 100 kV [10,19–21]. Fig. 1 illustrates the ultrastructure of control afferents that were identified by staining their cell surface with mAbs Lan3-2 (Fig. 1A) and Laz2-369 (Fig. 1B). In Fig. 1A, the sectioned profiles of four large sensory afferent processes (numbered 1–4) and neuritic projections are labeled on their cell surface with DAB reaction product (triangles outline profile 1). Large ovoid axons are replete with synaptic vesicle clusters (arrow). Virtually every
Fig. 1. Electron micrographs showing sensory afferent profiles in control embryos. (A) Sensory afferents were briefly stained on their cell surface with HRP-conjugated Lan3-2. Treating live germinal plates with low concentrations of HRP-conjugated monoclonal antibodies stains the surface membrane of sensory afferents (profile 1 is outlined by triangles) but not their synaptic vesicles [19–21]. The unstained profiles belong to central neurons. Profiles of four young sensory afferents (1–4) forming a fascicle are surrounded with neuritic projections. All four large profiles possess multiple clusters of small synaptic vesicles (arrow) and an occasional dense core vesicle (arrowhead). (B) Sensory afferents were briefly stained on their cell surface with HRP-conjugated Laz2-369. Two sensory afferents (1,2) are apposed to the unstained profile of a central neuron. Profile 1 is replete with synaptic vesicles (arrowhead) and appears to be making an en passant synapse with the unstained central neuron. Bar, 0.5 mm.
M.H. Tai, B. Zipser / Brain Research 967 (2003) 301–305
axonal section of such young sensory afferents contained at least one synaptic vesicle cluster underneath the surface membrane. In addition to the clusters of clear vesicles, there are also dense core vesicles (arrowhead) and numerous mitochondria. Most of these synaptic vesicle clusters are not in contact with central neurons as shown in previous three-dimensional reconstruction of young sensory afferents [20]. In Fig. 1B, more mature sensory afferents (numbered 1, 2), stained with mAb Laz2-369, are apposed to a central neuron. The developing nerve terminal is replete with clear vesicles (arrow) and contains also some dense core vesicles (arrowhead). A mitochondrion is also present. Treating live germinal plates with low concentrations of HRP-conjugated monoclonal antibodies stains the surface membrane of sensory afferents but not their synaptic vesicles [19–21]. Fig. 2 illustrates the ultrastructure of sensory afferents in germinal plates that were cultured in the presence of 0.1% HRP. Sensory afferents are again identified by DAB reaction product on their surface membrane through stain-
303
ing with low concentrations of HRP-conjugated monoclonal antibodies. This time however, DAB reaction product is also found in small synaptic vesicles (Fig. 2A, arrows). This uptake of extracellularly applied HRP into synaptic vesicles has been the classical method for studying endo / exocytosis of synaptic vesicles at the ultrastructural level. As the embryos were not stimulated, the HRP uptake reflects basal endo / exocytosis. Spontaneous endo- and exocytosis has been observed in regenerating spinal cord neurons in culture [13,14,23] and in motor neurons in vivo [16]. A surprising observation was that sensory afferents, exposed to extracellular HRP, exhibited a dramatic increase in the number of their dense core vesicles (Fig. 2A and B, arrowheads). This increase in dense core vesicles has not been observed as a result of live-staining sensory afferents with low concentrations of HRP-conjugated monoclonal antibodies [19–21]. Therefore, it is a result of the application of 0.1% HRP as an extracellular tracer. The histogram in Fig. 3 compares the number of dense
Fig. 2. Electronmicrographs showing sensory afferents treated with 0.1% HRP. (A) A large sensory afferent profile stained with HRP-conjugated Lan3-2 shows HRP uptake into small synaptic vesicles (arrows). Multiple dense core vesicles are present (arrowhead). (B) A large afferent profile stained with HRP-conjugated Laz2-369 shows an increased number of dense core vesicles.
304
M.H. Tai, B. Zipser / Brain Research 967 (2003) 301–305
Fig. 3. HRP uptake leads to an increase in the number of dense core vesicles in sensory afferents and central neurons. Dense core vesicles were counted in electron micrographs taken through the sensory afferent target region in CNS ganglia. To correct for the difference in the magnification of the micrographs, the number of dense core vesicles was normalized to the area of the micrograph. Experimental values are listed as relative to the control values. Exposed to 0.1% HRP for 3 h, sensory afferents underwent an almost 5-fold increase (P,0.0001) in dense core vesicles while central neurons experienced a less than 2-fold increase. Significantly different than the control (*), (P,0.0001).
core vesicles in serial sections from neurons in control CNS ganglia (n54; sixty-six sections; two embryos) and CNS ganglia (n54; ninety-two sections; two embryos) that were treated with 0.1% HRP. Dense core vesicles, recognized by their dark center and their morphology as doublemembrane rounded granules, were found in both sensory afferents and other neurons. Sensory afferents were identified by DAB reaction product on their surface membrane through staining with low concentration of HRP-conjugated monoclonal antibodies Lan3-2 and Laz2-369. The unstained profiles in the electron micrographs mostly belong to central neurons. To determine the effect of HRP treatment on dense core vesicle number, we counted the absolute number of dense core vesicles / synaptic profiles divided by the area of the target region. To avoid doublesampling the same dense core vesicle, we used every other section of the serially sectioned target regions for quantification. The ratio of sensory afferents profiles / central profiles in the target regions as well as the average profile area did not change as a result of applying 0.1% HRP. Sensory afferents exposed to 0.1% HRP underwent about a 5-fold increase in dense core vesicle number. Central neurons, not stained by monoclonal antibodies, experienced about a 2-fold increase. Thus, exposure to HRP results in an increase in dense core vesicles, the magnitude of which varies with neuronal class. What distinguishes primary sensory afferents from other neurons is the abundant expression of cell type-specific neutral glycans on their surface membranes. Throughout phylogeny, different sensory modalities are labeled with
different neutral surface glycans of the complex and oligomannosidic type [7,11,15]. Using chemical and immunological methods, it has been observed that neural tissue in general expresses an unusually large amount of oligomannosidic glycans [6]. Likewise, HRP also expresses neutral glycans of the high mannose type [3]. It remains to be determined whether glycosylation is one of the features that play a role in the dramatic increase in the number of dense core vesicles in sensory neurons and the lesser increase in other neural cell types.
Acknowledgements This research was supported by National Institutes of Health Grant NS25117. We thank Brent Kauffman and Harper VanSteenhouse for discussions.
References [1] S. Avrameas, T. Ternynck, Peroxidase labeled antibody and Fab conjugates with enhanced intracellular penetration, Immunocytochemistry 8 (1971) 1175–1179. [2] L.H. Bannister, H.C. Dodson, Endocytic pathways in the olfactory and vomeronasal epithelia of the mouse: ultrastructure and uptake of tracers, Microsc. Res. Tech. 23 (1992) 128–141. [3] M.L. Bajt, B. Schmitz, M. Schachner, B. Zipser, Carbohydrate epitopes involved in neural cell recognition are conserved between vertebrates and leech, J. Neurosci. Res. 27 (1990) 276–285.
M.H. Tai, B. Zipser / Brain Research 967 (2003) 301–305 [4] M.B. Bunge, Initial endocytosis of peroxidase or ferritin by growth cones of cultured nerve cells, J. Neurocytol. 6 (1977) 407–439. [5] B. Ceccareli, W.P. Hurlbut, A. Mauro, Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction, J. Cell Biol. 57 (1973) 499–524. [6] Y.-J. Chen, D.R. Wing, G.R. Guile, R.A. Dwek, D.J. Harvey, S. Zamze, Neutral N-glycans in adult rat brain tissue, Eur. J. Biochem. 251 (1998) 691–703. [7] J. Dodd, T.M. Jessell, Lactoseries carbohydrates specify subsets of dorsal root ganglion neurons projecting to the superficial dorsal horn of rat spinal cord, J. Neurosci. 5 (1985) 3278–3294. [8] J.E. Heuser, T.S. Reese, Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction, J. Cell Biol. 57 (1973) 315–344. [9] E. Holtzman, A.R. Freeman, Stimulation-dependent alterations in peroxidase uptake at lobster neuromuscular junctions, Science 173 (1971) 733–736. [10] K. Ito, A. Konishi, S. Nomura, Y. Nakamura, T. Sugimoto, Application of coupled oxidation reaction to electron microscopic demonstration of horseradish peroxidase: cobalt–glucose oxidase method, Brain Res. 175 (1979) 341–346. [11] B. Key, R.A. Akeson, Delineation of olfactory pathways in the frog nervous system by unique glycoconjugates and N-CAM glycoforms, Neuron 6 (1991) 381–396. [12] B. Key, P.P. Giorgi, Uptake and axonal transport of horseradish peroxidase isoenzymes by different neuronal types, Neuroscience 22 (1987) 1135–1144. [13] K. Kraszewski, O. Mundig, L. Daniell, C. Verderio, M. Matteoli, P. De Camilli, Synaptic vesicle dynamics in living cultured hippocampal neurons visualized with Cy3-conjugated antibodies directed against the lumenal domain of synaptotagmin, J. Neurosci. 15 (1995) 4328–4342. [14] M. Matteoli, K. Takei, M.S. Perin, T.C. Sudhof, P. De Camilli,
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
305
Exo–endocytotic recycling of synaptic vesicles in developing processes of cultured hippocampal neurons, J. Cell Biol. 117 (1992) 849–861. L. Pays, G. Schwarting, Gal-NCAM is a differentially expressed marker for mature sensory neurons in the rat olfactory system, J. Neurobiol. 43 (2000) 173–185. L. Polo-Parada, C.M. Bose, L.T. Landmesser, Alterations in transmission, vesicle dynamics, and transmitter release machinery at NCAM-deficient neuromuscular junctions, Neuron 32 (2001) 815– 828. S. Schacher, E. Holtzman, D.C. Hood, Synaptic activity of frog retinal photoreceptors, a peroxidase uptake study, J. Cell Biol. 70 (1976) 178–192. J. Song, B. Zipser, Kinetics of the inhibition of axonal defasciculation mediated by carbohydrate markers in the embryonic leech, Dev. Biol. 168 (1995) 319–331. M.-H. Tai, M. Rheuben, D. Autio, B. Zipser, Leech photoreceptors project their galectin-containing processes into optic neuropils where they contact AP cells, J. Comp. Neurol. (1996) 235–248. M.-H. Tai, B. Zipser, Mannose-specific recognition mediates two aspects of synaptic growth in leech sensory afferents: collateral branching and formation of synaptic vesicle clusters, Dev. Biol. 201 (1998) 154–166. M.-H. Tai, B. Zipser, Sequential steps in synaptic targeting of leech sensory afferents are mediated by constitutive and developmentally regulated glycosylations of CAMs, Devel. Biol. 214 (1999) 258– 276. S. Teichberg, E. Holtzman, S.M. Crain, E.R. Peterson, Circulation and turnover of synaptic vesicle membrane in cultured fetal mammalian spinal cord neurons, J. Cell Biol. 67 (1975) 215–230. S. Zakharenko, S. Chang, M. O’Donoghue, S.V. Popov, Neurotransmitter secretion along growing nerve processes: comparison with synaptic vesicle exocytosis, J. Cell Biol. 144 (1999) 507–517.