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tufted cell dendrites
Brain Research 860 Ž2000. 170–173 www.elsevier.comrlocaterbres
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Ultrastructural identification of synapses between mitralrtufted cell dendrites Dianne M. Allen ) , Kathryn A. Hamilton Department of Cellular Biology and Anatomy, Louisiana State UniÕersity Health Sciences Center, 1501 Kings Highway, ShreÕeport, LA 71130-3932, USA Accepted 28 December 1999
Abstract Asymmetrical, type 1 synapses between mitral andror tufted ŽMrT. cell dendrites were observed in the glomerular layer ŽGL. and juxtaglomerular external plexiform layer ŽEPL. of salamander olfactory bulb sections. The dendrites had electron-lucent cytoplasm containing regularly-arrayed microtubules and spherical translucent vesicles. The vesicles were clustered against a thin pre-synaptic density that was aligned with a 17–20 nm-wide synaptic cleft and a thicker post-synaptic density. These dendrodendritic synapses could be a source of the delayed, prolonged excitation that originates from the GLrEPL. During spatiotemporal encoding of odor stimuli, they could amplify or synchronize MrT cell responses. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Olfactory bulb; Mitral cell; Dendrodendritic synapse; Ultrastructure; Salamander
In response to olfactory stimulation, the mitral ŽM. and tufted ŽT. output neurons of the olfactory bulb are excited by olfactory nerve ŽON. axon terminals that form type 1 synapses with the MrT cell dendritic tufts within the olfactory bulb glomeruli w26,34x. Other potential cellular and synaptic sources of MrT cell excitation also occur in the glomerular layer ŽGL. Žsee Refs. w17,31x., however, which could assist in encoding stimulus characteristics into spatiotemporal activity patterns that are transmitted to the olfactory cortex by the MrT cell axons Žreviewed in Refs. w19,20x.. Intracellular recordings of delayed excitation from rabbit MrT cells initially prompted the suggestion that MrT cell axon collaterals might mediate recurrent excitation w22x. Intracellular recordings of delayed, prolonged depolarization and excitation from rat w2,21x and turtle w23,24x MrT cells have prompted an alternative suggestion, however, which is that the MrT cell neurotransmitter Žglutamate, see Ref. w30x. might re-excite the releasing MrT cell or nearby MrT cells w3,23x. Current source density analysis, field potential recordings, and voltagesensitive dye recordings from rat w2x, skate w7x, salamander w8x, and rabbit w18x olfactory bulbs have shown that the delayed, prolonged excitation originates from the GL and
) Corresponding author. Fax: q1-318-675-5889; e-mail: [email protected]
juxtaglomerular portion of the external plexiform layer ŽEPL.. In the course of ultrastructural studies of the salamander olfactory bulb w1x, another potential source of MrT cell excitation has now been identified. Adult male Ambystoma tigrinum and A. maculatum were obtained from a commercial supplier, housed on a 12-h lightr12-h dark cycle at 10–148C in an accredited facility, and used in accordance with NIH guidelines. Prior to surgery, the skin of the head and chest was swabbed with 4% Licocaine HCl, the animal was anesthetized with sodium pentobarbital Ž150–275 mgrkg body weight., and it was transcardially perfused with amphibian Ringer’s solution w8x followed by 0.6% paraformaldehyde q2–4% glutaraldehyde in 0.035–0.1 M Naq cacodylate buffer at pH 7.35– 7.40 w4,35x or by 4% paraformaldehyde q0.25% glutaraldehyde in 0.05 M Naq phosphate buffer at pH 7.40 w32x. The olfactory bulbs were removed, immersed for 2–16 h in the fixative, washed in the fixative buffer, post-fixed for 1 h with 2% OsO4 in the same buffer, washed in buffer, stained overnight with 1% aqueous uranyl acetate, and washed in distilled H 2 O, all at 48C. The bulbs were dehydrated and cleared in ethanol-propylene oxide series and embedded in Polybed 812 ŽPolysciences, Warrington, PA.. Semi-thin sections Ž; 1 mm thick. were cut using a Reichert-Jung Ultracut E and glass knives, stained with 0.25–1% toluidine blue, and examined with a light microscope to locate the GL and juxta-
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 0 0 . 0 2 0 1 2 - 6
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obtained at 1450–18,000 = magnification using a Philips CM-10 transmission electron microscope. Areas of interest were enlarged and the contrast adjusted to yield nearly identical prints of the synapses and organelles.
glomerular EPL. Silver and silver–gray thin sections were then cut using a diamond knife, mounted on copper mesh grids, and stained with 1% uranyl acetate and with 0.2% lead citrate. Photographic negatives Ž3.325 = 3.75 in. were
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Fig. 1. Asymmetrical, type 1 synapses between MrT cell dendrites. Ža. Synapse Ž . between two dendritic profiles in the GL of an A. tigrinum olfactory bulb. Two glomeruli ŽG1, G2. are shown, containing electron-dense ON axon terminals ŽU . and electron-lucent MrT cell dendritic profiles ŽMTd., which also demarcate the PG zone. Žb. Higher magnification of the same synapse Ž . shown in Ža.. Both the pre-synaptic ŽD1. and post-synaptic ŽD2. profiles contain the regular microtubule Žmt. arrays and spherical translucent vesicles Žsv. that are characteristic of MrT cell dendrites. Note the clustering of vesicles against the thin pre-synaptic density and the thicker post-synaptic density. m, mitochondrion; dv, dense core vesicle. Žc and d. Oblique sections through synapses Ž . between MrT cell dendritic profiles in the juxtaglomerular external plexiform layer of an A. maculatum olfactory bulb. The preŽD1. and post- ŽD2. synaptic profiles exhibit the characteristic microtubule Žmt. arrays and spherical vesicles Žsv. of MrT cell dendrites. m, mitochondria. Že. Synapse Ž . from a MrT dendritic profile ŽD1. onto a PG cell dendritic profile ŽPGd.. The PG cell profile contains predominantly pleomorphic and flattened Ž-. vesicles, and it lacks a microtubule array. m, mitochondria. Scales are 2 mm Ža. and 0.2 mm Žb–e..
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D.M. Allen, K.A. Hamiltonr Brain Research 860 (2000) 170–173
Profiles of pre- and post-synaptic cells were identified using previously established ultrastructural criteria. As shown in Fig. 1a, within the glomerular neuropil, ON axons were identified by their electron-dense cytoplasm and ON axon terminals were identified by their densely packed spherical translucent vesicles w26,29x. Within the glomeruli and in the surrounding periglomerular ŽPG. zones, MrT cell dendrites were identified by their electron-lucent cytoplasm containing numerous, regularly arrayed microtubules and predominantly spherical translucent vesicles w13,25,26,28,34x. As shown in Fig. 1a and at higher magnification in Fig. 1b, in the PG zone, some of the MrT cell dendrites formed asymmetrical synapses. The synapse shown in Fig. 1b exhibited typical type 1 characteristics w9,11x, including clustering of predominantly spherical, 30–50 nm-diameter translucent vesicles against the pre-synaptic density. The synaptic cleft ranged from 17 to 20 nm wide along the length of the post-synaptic density, which was thicker than the pre-synaptic density. Fig. 1c and d show two other type 1 synapses between MrT cell dendrites in the juxtaglomerular EPL of a section from another animal. Synapses between MrT cell dendrites were observed in five of 26 thin sections through 1–3 glomeruli from four animals. Because so few glomeruli were examined, it is not yet known if this frequency of occurrence Ž19%. is representative. In the rat EPL, type 1 synapses have been observed from MrT cell dendrites onto processes of superficial short-axon cells, Van Gehuchten cells, and multipolar cells that are immunoreactive for parvalbumin, w33x. Type 1 synapses have more commonly been observed in the rodent GL, from MrT cell dendrites onto PG cell dendrites w26,27,34x. Type 1 synapses between homologous cell types have not commonly been observed, however. Hinds Žw13x, Fig. 3. showed a type 1 synapse from a presumptive T cell dendrite onto an unidentified dendrite exhibiting regularly arrayed microtubules. Goheen et al. w10x described symmetrical, type 2 synapses from tyrosine hydroxylase-containing external T cell dendrites onto presumptive M or internal T cell dendrites. White w34x observed type 2 synapses between PG cell dendrites. In the present study, profiles of PG cell dendrites were also observed. As shown in Fig. 1e, the PG cell dendritic profiles received type 1 synapses from typical MrT cell dendritic profiles. Like rat PG cell dendrites, the salamander PG cell dendrites contained translucent vesicles that were predominantly pleomorphic and flattened, and they contained few if any microtubules w13,25,26,34x. Many of the salamander PG and MrT cell dendritic profiles also contained a few dense core vesicles Že.g., Fig. 1b.. Dense core vesicles have previously been observed in the GL dendrites of Xenopus olfactory bulb neurons w4x. In a recent model of spatiotemporal odor encoding mechanisms, Hoshino et al. w14x pointed out that better odor recognition would be possible if the M cells were connected by both excitatory and inhibitory synapses,
within an associative network. There is considerable evidence that MrT cells receive inhibition from PG cells within the GL and from granule cells within the EPL Žreviewed in Refs. w19,20x.. In the present study, type 1 synapses between MrT cell dendrites were observed near the junction of the two cellular layers. These synapses could provide the previously unidentified excitatory connections, which may serve to amplify weak signals or to synchronize MrT cell activity between parallel odor processing modules Žsee Refs. w2,14,16,20x.. In salamander MrT output cells w6,12,15x and in moth olfactory projection neurons w5x, many odor stimuli evoke complex temporal activity patterns in which excitation follows initial hyperpolarization. Type 1 synapses between MrT cells could contribute to the delayed excitation observed in the salamander. Because different MrT cell types exhibit similar ultrastructural features w25,26x, and different T cell types might release different neurotransmitters or modulators Žsee Ref. w17x., the net effect of the type 1 synapses on the MrT output cell population might not be excitatory, however. To understand how the type 1 dendrodendritic and other synapses could assist in generating specific temporal activity patterns in salamander MrT cells, quantitative ultrastructural studies are being conducted of the salamander GL and juxtaglomerular EPL.
Acknowledgements We thank Wanda Green for the technical assistance, the Department of Cellular Biology and Anatomy for the use of electron microscopy facilities, and the Biomedical Research Foundation of Northwest Louisiana for the grant support. This research was conducted in partial fulfilment of the PhD requirements of the School of Graduate Studies, LSU Health Sciences Center.
References w1x D.M. Allen, Quantitative Ultrastructure of Olfactory Bulb Synapses that may contribute to the Early Inhibition of MitralrTufted Neurons, Cellular Biology and Anatomy, LSU Health Sciences Center in Shreveport, PhD dissertation, in preparation. w2x V. Aroniadou-Anderjaska, M. Ennis, M.T. Shipley, Glomerular synaptic responses to olfactory nerve input in rat olfactory bulb slices, Neuroscience 79 Ž1997. 425–434. w3x V. Aroniadou-Anderjaska, M. Ennis, M.T. Shipley, Current-source density ŽCSD. analysis in the rat main olfactory bulb ŽMOB.: laminar distribution of AMPA and NMDA receptor-mediated currents, Neurosci. Abstr. 24 Ž1998. 1884. w4x C.A. Byrd, G.D. Burd, Development of the olfactory bulb in the clawed frog, Xenopus laeÕis: a morphological and quantitative analysis, J. Comp. Neurol. 314 Ž1991. 79–90. w5x T.A. Christensen, T. Heinbockel, J.G. Hildebrand, Olfactory information processing in the brain: encoding chemical and temporal features of odors, J. Neurobiol. 30 Ž1996. 82–91. w6x A.R. Cinelli, K.A. Hamilton, J.S. Kauer, Salamander olfactory bulb neuronal activity observed by video rate, voltage-sensitive dye imag-
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ing: III. Spacial and temporal properties of responses evoked by odorant stimulation, J. Neurophysiol. 73 Ž1995. 2053–2071. A.R. Cinelli, B.M. Salzberg, Multiple site optical recording of transmembrane voltage ŽMSORTV., single-unit recordings, and evoked field potentials from the olfactory bulb of skate Ž Raja erinacea., J. Neurophysiol. 64 Ž1990. 1767–1790. A.R. Cinelli, B.M. Salzberg, Dendritic origin of late events in optical recordings from salamander olfactory bulb, J. Neurophysiol. 68 Ž1992. 786–806. M. Colonnier, Synaptic patterns on different cell types in the different laminae of the cat visual cortex. An electron microscope study, Brain Res. 9 Ž1968. 268–287. B.L. Goheen, J.N. Kott, L.E. Westrum, Tyrosine hydroxylase expression in rat olfactory bulb transplants: an electron microscope study, Synapse 23 Ž1996. 132–141. E.G. Gray, Axo-somatic and axo-dendritic synapses of the cerebral cortex: an electron microscopic study, J. Anat., 1959, pp. 420–433. K.A. Hamilton, J.S. Kauer, Patterns of intracellular potentials in salamander mitralrtufted cells in response to odor stimulation, J. Neurophysiol. 62 Ž1989. 609–625. J.W. Hinds, Reciprocal and serial dendrodendritic synapses in the glomerular layer of the rat olfactory bulb, Brain Res. 17 Ž1970. 530–534. O. Hoshino, Y. Kashimori, T. Kambara, An olfactory recognition model based on spatio-temporal encoding of odor quality in the olfactory bulb, Biol. Cybern. 79 Ž1998. 109–120. J.S. Kauer, Response patterns of amphibian olfactory bulb neurones to odour stimulation, J. Physiol. 243 Ž1974. 695–715. J.S. Kauer, A.R. Cinelli, Are there structural and functional modules in the vertebrate olfactory bulb?, Microsc. Res. Tech. 24 Ž1993. 157–167. K. Kosaka, K. Toida, Y. Aika, T. Kosaka, How simple is the organization of the olfactory glomerulus?: the heterogeneity of so-called periglomerular cells, Neurosci. Res. 30 Ž1998. 101–110. D.P. Martinez, W.J. Freeman, Periglomerular cell action on mitral cells in olfactory bulb shown by current source density analysis, Brain Res. 308 Ž1984. 223–233. K. Mori, Membrane and synaptic properties of identified neurons in the olfactory bulb, Prog. Neurobiol. 29 Ž1987. 275–320. K. Mori, H. Nagao, Y.F. Sasaki, Computation of molecular information in mammalian olfactory systems, Network Comput. Neural Syst. 9 Ž1998. R79–R102.
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w21x W.T. Nickell, M.T. Shipley, M.M. Behbehani, Orthodromic synaptic activation of rat olfactory bulb mitral cells in isolated slices, Brain Res. Bull. 39 Ž1996. 57–62. w22x R.A. Nicoll, Recurrent excitation of secondary olfactory neurons: a possible mechanism for signal amplification, Science 171 Ž1971. 824–826. w23x R.A. Nicoll, C.E. Jahr, Self-excitation of olfactory bulb neurones, Nature 296 Ž1982. 441–444. w24x M.C. Nowycky, K. Mori, G.M. Shepherd, Blockade of synaptic inhibition reveals long-lasting synaptic excitation in isolated turtle olfactory bulb, J. Neurophysiol. 46 Ž1981. 649–658. w25x A.J. Pinching, T.P.S. Powell, The neuron types of the glomerular layer of the olfactory bulb, J. Cell Sci. 9 Ž1971. 305–345. w26x A.J. Pinching, T.P.S. Powell, The neuropil of the glomeruli of the olfactory bulb, J. Cell Sci. 9 Ž1971. 347–377. w27x A.J. Pinching, T.P.S. Powell, The neuropil of the periglomerular region of the olfactory bulb, J. Cell Sci. 9 Ž1971. 379–409. w28x J.L. Price, T.P.S. Powell, The mitral and short axon cells of the olfactory bulb, J. Cell Sci. 7 Ž1970. 631–651. w29x T.S. Reese, M.W. Brightman, Olfactory surface and central olfactory connexions in some vertebrates, in: G.E.W. Wostenholme, J. Knight ŽEds.., Taste and Smell in Vertebrates, A C.I.B.A. Fdn. Symp., Churchill, London, 1970, pp. 115–149. w30x N.E. Schoppa, J.M. Kinzie, Y. Sahara, T.P. Segerson, G.L. Westbrook, Dendrodendritic inhibition in the olfactory bulb is driven by NMDA receptors, J. Neurosci. 18 Ž1998. 6790–6802. w31x M.T. Shipley, M. Ennis, Functional organization of olfactory system, J. Neurobiol. 30 Ž1996. 123–176. w32x M.T. Shipley, L. Fu, M. Ennis, W.-L. Liu, G. Aston-Jones, Dendrites of locus coeruleus neurons extend preferentially into two pericoerulear zones, J. Comp. Neurol. 365 Ž1996. 56–68. w33x K. Toida, K. Kosaka, C.W. Heizmann, T. Kosaka, Electron microscopic serial-sectioningrreconstruction study of parvalbumin-containing neurons in the external plexiform layer of the rat olfactory bulb, Neuroscience 72 Ž1996. 449–466. w34x E.L. White, Synaptic organization of the mammalian olfactory glomerulus: new findings including an intraspecific variation, Brain Res. 60 Ž1973. 299–313. w35x B.S. Zielinski, M.L. Getchell, R.L. Wenokur, T.V. Getchell, Ultrastructural localization and identification of adrenergic and cholinergic nerve terminals in the olfactory mucosa, Anat. Rec. 225 Ž1989. 232–245.
Short communication
Ultrastructural identification of synapses between mitralrtufted cell dendrites Dianne M. Allen ) , Kathryn A. Hamilton Department of Cellular Biology and Anatomy, Louisiana State UniÕersity Health Sciences Center, 1501 Kings Highway, ShreÕeport, LA 71130-3932, USA Accepted 28 December 1999
Abstract Asymmetrical, type 1 synapses between mitral andror tufted ŽMrT. cell dendrites were observed in the glomerular layer ŽGL. and juxtaglomerular external plexiform layer ŽEPL. of salamander olfactory bulb sections. The dendrites had electron-lucent cytoplasm containing regularly-arrayed microtubules and spherical translucent vesicles. The vesicles were clustered against a thin pre-synaptic density that was aligned with a 17–20 nm-wide synaptic cleft and a thicker post-synaptic density. These dendrodendritic synapses could be a source of the delayed, prolonged excitation that originates from the GLrEPL. During spatiotemporal encoding of odor stimuli, they could amplify or synchronize MrT cell responses. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Olfactory bulb; Mitral cell; Dendrodendritic synapse; Ultrastructure; Salamander
In response to olfactory stimulation, the mitral ŽM. and tufted ŽT. output neurons of the olfactory bulb are excited by olfactory nerve ŽON. axon terminals that form type 1 synapses with the MrT cell dendritic tufts within the olfactory bulb glomeruli w26,34x. Other potential cellular and synaptic sources of MrT cell excitation also occur in the glomerular layer ŽGL. Žsee Refs. w17,31x., however, which could assist in encoding stimulus characteristics into spatiotemporal activity patterns that are transmitted to the olfactory cortex by the MrT cell axons Žreviewed in Refs. w19,20x.. Intracellular recordings of delayed excitation from rabbit MrT cells initially prompted the suggestion that MrT cell axon collaterals might mediate recurrent excitation w22x. Intracellular recordings of delayed, prolonged depolarization and excitation from rat w2,21x and turtle w23,24x MrT cells have prompted an alternative suggestion, however, which is that the MrT cell neurotransmitter Žglutamate, see Ref. w30x. might re-excite the releasing MrT cell or nearby MrT cells w3,23x. Current source density analysis, field potential recordings, and voltagesensitive dye recordings from rat w2x, skate w7x, salamander w8x, and rabbit w18x olfactory bulbs have shown that the delayed, prolonged excitation originates from the GL and
) Corresponding author. Fax: q1-318-675-5889; e-mail: [email protected]
juxtaglomerular portion of the external plexiform layer ŽEPL.. In the course of ultrastructural studies of the salamander olfactory bulb w1x, another potential source of MrT cell excitation has now been identified. Adult male Ambystoma tigrinum and A. maculatum were obtained from a commercial supplier, housed on a 12-h lightr12-h dark cycle at 10–148C in an accredited facility, and used in accordance with NIH guidelines. Prior to surgery, the skin of the head and chest was swabbed with 4% Licocaine HCl, the animal was anesthetized with sodium pentobarbital Ž150–275 mgrkg body weight., and it was transcardially perfused with amphibian Ringer’s solution w8x followed by 0.6% paraformaldehyde q2–4% glutaraldehyde in 0.035–0.1 M Naq cacodylate buffer at pH 7.35– 7.40 w4,35x or by 4% paraformaldehyde q0.25% glutaraldehyde in 0.05 M Naq phosphate buffer at pH 7.40 w32x. The olfactory bulbs were removed, immersed for 2–16 h in the fixative, washed in the fixative buffer, post-fixed for 1 h with 2% OsO4 in the same buffer, washed in buffer, stained overnight with 1% aqueous uranyl acetate, and washed in distilled H 2 O, all at 48C. The bulbs were dehydrated and cleared in ethanol-propylene oxide series and embedded in Polybed 812 ŽPolysciences, Warrington, PA.. Semi-thin sections Ž; 1 mm thick. were cut using a Reichert-Jung Ultracut E and glass knives, stained with 0.25–1% toluidine blue, and examined with a light microscope to locate the GL and juxta-
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 0 0 . 0 2 0 1 2 - 6
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obtained at 1450–18,000 = magnification using a Philips CM-10 transmission electron microscope. Areas of interest were enlarged and the contrast adjusted to yield nearly identical prints of the synapses and organelles.
glomerular EPL. Silver and silver–gray thin sections were then cut using a diamond knife, mounted on copper mesh grids, and stained with 1% uranyl acetate and with 0.2% lead citrate. Photographic negatives Ž3.325 = 3.75 in. were
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Fig. 1. Asymmetrical, type 1 synapses between MrT cell dendrites. Ža. Synapse Ž . between two dendritic profiles in the GL of an A. tigrinum olfactory bulb. Two glomeruli ŽG1, G2. are shown, containing electron-dense ON axon terminals ŽU . and electron-lucent MrT cell dendritic profiles ŽMTd., which also demarcate the PG zone. Žb. Higher magnification of the same synapse Ž . shown in Ža.. Both the pre-synaptic ŽD1. and post-synaptic ŽD2. profiles contain the regular microtubule Žmt. arrays and spherical translucent vesicles Žsv. that are characteristic of MrT cell dendrites. Note the clustering of vesicles against the thin pre-synaptic density and the thicker post-synaptic density. m, mitochondrion; dv, dense core vesicle. Žc and d. Oblique sections through synapses Ž . between MrT cell dendritic profiles in the juxtaglomerular external plexiform layer of an A. maculatum olfactory bulb. The preŽD1. and post- ŽD2. synaptic profiles exhibit the characteristic microtubule Žmt. arrays and spherical vesicles Žsv. of MrT cell dendrites. m, mitochondria. Že. Synapse Ž . from a MrT dendritic profile ŽD1. onto a PG cell dendritic profile ŽPGd.. The PG cell profile contains predominantly pleomorphic and flattened Ž-. vesicles, and it lacks a microtubule array. m, mitochondria. Scales are 2 mm Ža. and 0.2 mm Žb–e..
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D.M. Allen, K.A. Hamiltonr Brain Research 860 (2000) 170–173
Profiles of pre- and post-synaptic cells were identified using previously established ultrastructural criteria. As shown in Fig. 1a, within the glomerular neuropil, ON axons were identified by their electron-dense cytoplasm and ON axon terminals were identified by their densely packed spherical translucent vesicles w26,29x. Within the glomeruli and in the surrounding periglomerular ŽPG. zones, MrT cell dendrites were identified by their electron-lucent cytoplasm containing numerous, regularly arrayed microtubules and predominantly spherical translucent vesicles w13,25,26,28,34x. As shown in Fig. 1a and at higher magnification in Fig. 1b, in the PG zone, some of the MrT cell dendrites formed asymmetrical synapses. The synapse shown in Fig. 1b exhibited typical type 1 characteristics w9,11x, including clustering of predominantly spherical, 30–50 nm-diameter translucent vesicles against the pre-synaptic density. The synaptic cleft ranged from 17 to 20 nm wide along the length of the post-synaptic density, which was thicker than the pre-synaptic density. Fig. 1c and d show two other type 1 synapses between MrT cell dendrites in the juxtaglomerular EPL of a section from another animal. Synapses between MrT cell dendrites were observed in five of 26 thin sections through 1–3 glomeruli from four animals. Because so few glomeruli were examined, it is not yet known if this frequency of occurrence Ž19%. is representative. In the rat EPL, type 1 synapses have been observed from MrT cell dendrites onto processes of superficial short-axon cells, Van Gehuchten cells, and multipolar cells that are immunoreactive for parvalbumin, w33x. Type 1 synapses have more commonly been observed in the rodent GL, from MrT cell dendrites onto PG cell dendrites w26,27,34x. Type 1 synapses between homologous cell types have not commonly been observed, however. Hinds Žw13x, Fig. 3. showed a type 1 synapse from a presumptive T cell dendrite onto an unidentified dendrite exhibiting regularly arrayed microtubules. Goheen et al. w10x described symmetrical, type 2 synapses from tyrosine hydroxylase-containing external T cell dendrites onto presumptive M or internal T cell dendrites. White w34x observed type 2 synapses between PG cell dendrites. In the present study, profiles of PG cell dendrites were also observed. As shown in Fig. 1e, the PG cell dendritic profiles received type 1 synapses from typical MrT cell dendritic profiles. Like rat PG cell dendrites, the salamander PG cell dendrites contained translucent vesicles that were predominantly pleomorphic and flattened, and they contained few if any microtubules w13,25,26,34x. Many of the salamander PG and MrT cell dendritic profiles also contained a few dense core vesicles Že.g., Fig. 1b.. Dense core vesicles have previously been observed in the GL dendrites of Xenopus olfactory bulb neurons w4x. In a recent model of spatiotemporal odor encoding mechanisms, Hoshino et al. w14x pointed out that better odor recognition would be possible if the M cells were connected by both excitatory and inhibitory synapses,
within an associative network. There is considerable evidence that MrT cells receive inhibition from PG cells within the GL and from granule cells within the EPL Žreviewed in Refs. w19,20x.. In the present study, type 1 synapses between MrT cell dendrites were observed near the junction of the two cellular layers. These synapses could provide the previously unidentified excitatory connections, which may serve to amplify weak signals or to synchronize MrT cell activity between parallel odor processing modules Žsee Refs. w2,14,16,20x.. In salamander MrT output cells w6,12,15x and in moth olfactory projection neurons w5x, many odor stimuli evoke complex temporal activity patterns in which excitation follows initial hyperpolarization. Type 1 synapses between MrT cells could contribute to the delayed excitation observed in the salamander. Because different MrT cell types exhibit similar ultrastructural features w25,26x, and different T cell types might release different neurotransmitters or modulators Žsee Ref. w17x., the net effect of the type 1 synapses on the MrT output cell population might not be excitatory, however. To understand how the type 1 dendrodendritic and other synapses could assist in generating specific temporal activity patterns in salamander MrT cells, quantitative ultrastructural studies are being conducted of the salamander GL and juxtaglomerular EPL.
Acknowledgements We thank Wanda Green for the technical assistance, the Department of Cellular Biology and Anatomy for the use of electron microscopy facilities, and the Biomedical Research Foundation of Northwest Louisiana for the grant support. This research was conducted in partial fulfilment of the PhD requirements of the School of Graduate Studies, LSU Health Sciences Center.
References w1x D.M. Allen, Quantitative Ultrastructure of Olfactory Bulb Synapses that may contribute to the Early Inhibition of MitralrTufted Neurons, Cellular Biology and Anatomy, LSU Health Sciences Center in Shreveport, PhD dissertation, in preparation. w2x V. Aroniadou-Anderjaska, M. Ennis, M.T. Shipley, Glomerular synaptic responses to olfactory nerve input in rat olfactory bulb slices, Neuroscience 79 Ž1997. 425–434. w3x V. Aroniadou-Anderjaska, M. Ennis, M.T. Shipley, Current-source density ŽCSD. analysis in the rat main olfactory bulb ŽMOB.: laminar distribution of AMPA and NMDA receptor-mediated currents, Neurosci. Abstr. 24 Ž1998. 1884. w4x C.A. Byrd, G.D. Burd, Development of the olfactory bulb in the clawed frog, Xenopus laeÕis: a morphological and quantitative analysis, J. Comp. Neurol. 314 Ž1991. 79–90. w5x T.A. Christensen, T. Heinbockel, J.G. Hildebrand, Olfactory information processing in the brain: encoding chemical and temporal features of odors, J. Neurobiol. 30 Ž1996. 82–91. w6x A.R. Cinelli, K.A. Hamilton, J.S. Kauer, Salamander olfactory bulb neuronal activity observed by video rate, voltage-sensitive dye imag-
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