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Neuronal gap junctions in the polymorph layer of the rat dentate gyrus
Brain Research, 277 (1983)347-351 Elsevier
347
Short Communications
Neuronal gap junctions in the polymorph layer of the rat dentate gyrus TOSHIO KOSAKA National Institute for Physiological Sciences, Myodaiji, Okazaki 444 (Japan)
(Accepted June 14th, 1983) Key words: gap junction - - interneuron - - dentate gyrus- - hippocampus- - rat - - mammal
In thin sections of the rat dentate gyrus, neuronal gap junctions were observed in the polymorphlayer. Gap junctions were seen on dendritic stems, on smooth and/or varicose dendrites, on spine-like appendages, and in one case on a soma. Somata of gap junctionbearing neurons showed indented nuclei with intranuclear inclusions, and received many asymmetrical and a few symmetrical synapses. Gap junctions are ubiquitously encountered between various kinds of cells, and are regarded as intercellular communicating junctions 5. In the central nervous system (CNS), they are considered to be the morphological correlates of electrotonic coupling, and have been reported in various parts of mammalian and non-mammalian CNSX2. They are assumed to play important roles in neuronal functions, e.g. in synchronization of neuronal activity2,12. The present paper is a continuation of a prior description3 of gap junctions between non-pyramidal cell dendrites in CA1 and CA3 regions of the rat hippocampus, and is concerned with neuronal gap junctions in the rat dentate gyrus. The animals and histological procedures used here have been described in detail in a previous paper 3. Briefly, thin sections were cut, mounted in ribbons on either mesh grids or Formvar-coated single slot grids (1 x 2 mm), double-stained with uranyl acetate and lead citrate and examined with a JEM 100CX electron microscope using a tilting stage. The infrapyramidal blade of the dentate gyrus was examined in the same materials used in the previous investigations of hippocampal CA1 and CA3 regions. Despite examination of all 3 layers of the dentate gyrus (the molecular layer, the granule cell layer and the polymorph layer)n.6, 7 during the initial phase of this study, 0006-8993/83/$03.00© 1983Elsevier Science Publishers B.V.
neuronal gap junctions were encountered only in the polymorph layer. Thus the attention of the later phase of this study concentrated on the polymorph layer, where neuronal gap junctions were encountered frequently, apparently more frequently than in CA1 or CA3 regions. Gap junction-bearing dendrites in the polymorph layer of the dentate gyrus closely resembled those in the hippocampal CA1 and CA3 regions in their structural features. Most of them were smooth or varicose, about 0,5-3/~m in diameter with few, if any, spine-like appendages, filled with a pale cytoplasmic matrix, and impinged upon by many synaptic terminals. Most of the latter contained many spherical vesicles along with a few dense-cored vesicles and made asymmetrical synapses (Figs. 1, 3 and 4). In two cases (Figs. 2 and 5) gap junctions were observed on spine-like appendages arising from dendritic shafts. Fig. 1 shows a neuron located in the polymorph layer immediately deep to the granule cell layer, which had an indented nucleus and an intranuclear rod composed of filaments. This neuron had an abundant perikaryal cytoplasm containing well developcd Golgi apparatus and lamellae of rough endoplasmic reticulum. It gave rise to at least 4 smooth or somewhat varicose dendrites about 1-2.5/~m in diameter which extended almost parallel to the granule cell
348
349 layer. Two of these dendrites were in close apposi-
dentate gyrus (unpublished observations) and by sev-
tion to two dendrites of u n k n o w n origin near their origins from the soma, respectively (C and E in Fig. 1). At these apposed areas, gap junctions and
eral large to medium-sized presumable i n t e r n e u r o n s
some specialized junctions accompanying them such as puncta adherentia and intermediary junctionsl3 were observed. Many synaptic terminals impinged upon the soma and dendrites of this n e u r o n , most of which contained spherical vesicles and made asymmetrical synapses but a few contained pleomorphic
in the cat hippocampus TM. Previous Golgi studies 1,6,9 have identified several types of neurons 19cated in the polymorph layer and/or extending their dendrites there, Without the intimate cooperation of light and electron microscopic investigations, it may be impossible to identify and characterize these neuron types at the ultrastructural level.
vesicles and made symmetrical synapses.
The structural and synaptic features of gap junction-bearing dendrites and somata in the dentate gy-
Fig. 2 shows another n e u r o n located in the polymorph layer about 50 ~ m deep to the granule cell lay-
rus as well as those in hippocampal CA1 and CA3 regions 3, appear to resemble those in motor and senso-
er. The nucleus of this cell, as in the case of the neuron in Fig. 1, was indented and contained an intranu-
ry neocortex H~.I1 and cerebellar cortex 13. Therefore, it is tempting to speculate that most of these gap junc-
clear sheet, while the a b u n d a n t perikaryal cytoplasm was filled with organelles such as lamellae of rough endoplasmic reticulum, mitochondria and Golgi apparatus. Many synaptic terminals impinged upon this cell, most of which contained spherical vesicles and
tion-bearing dendrites in various brain areas originate from similar kinds of neurons, i.e. presumably inhibitory interneurons. In their review on neuronal gap junctions in vertebrate CNS, Sotelo and Korn 12
made asymmetrical synapses. A spine-like appendage arising from a dendritic shaft was found in direct contact with a part of the soma of this n e u r o n , where gap junctions and puncta adherentia were seen. The structural features of the n e u r o n s shown in Figs. 1 and 2 closely resemble those of pyramidal basket cells reported previouslys. However, this ultrastructural resemblance may not necessarily indicate that the gap j u n c t i o n - b e a r i n g n e u r o n s are pyramidal basket cells, for their ultrastructural characteristics appear to be also shared by some presumable i n t e r n e u r o n s in the molecular layer of the rat
ventured that, '... several of them (inhibitory interneurons) can be activated as a closely knit group. It is possible that the synchronous activation of inhibitory interneurons thereby provided by gap junctions is of considerable importance in the organization of neuronal nets in the cerebellum'. It seems very plausible that this holds true in other brain areas. The author wishes to thank Drs. K. H a m a and A. T. Ishida for their valuable discussions and critical reading of the manuscript and Mr. Y. Hataguchi for his technical assistance.
Fig. 1. Electron micrographs of a gap junction-bearingneuron located in the polymorph layer beneath the granule cell layer (GL). A: low magnification electron micrograph of the neuron, illustrating indentations in the nucleus, an intranuclear rod of filaments (arrows) and an abundant cytoplasm rich in cell organelles. The intranuclear rod was somewhat wavy, thus appears to be fragmentary in thin sections. A somewhat varicose dendrite extends from the soma parallel to the granule cell layer. Arrowhead indicates an area in close apposition to an obliquely sectioned dendrite where a gap junction is seen as shown in C at higher magnification. Scale bar - 10 ~m. B: detail of the intranuclear rod indicated by arrow in A, which is composed of filaments. Scale bar = 11tm. C: detail of the closely apposed area indicated by arrowhead in A in an adjacent section, showing a gap junction (arrow) and an intermediary junction beside it (arrowhead). Cytoplasmic semidense material appears to undercoat the plasma membrane at the gap junction, while somewhat denser material is seen at the adjacent intermediary junction. A synaptic terminal (asterisk) making an asymmetrical synapse on the longitudinally sectioned dendrite (a) was found in subsequent sections to also make an asymmetrical synapse on the obliquely sectioned dendrite (b). Scale bar = 1/~m. D: two synaptic terminals impinging upon the soma, each containing spherical vesicles and making an asymmetrical synapse. Scale bar = 1ktm. E: another dendrite arising from the soma of the neuron shown in A, but about 40 sections away from that in A. This dendrite (a) is in close apposition with a cross-sectioned dendrite (b), and in addition several synaptic terminals impinge upon it. The closely apposed area between these two dendrites exhibits a specialized junctional zone (between arrowheads) which is shown in F and G at higher magnificatiom Scale bar = 1~m. F and G: detail of the specialized juntional zone shown in E in two adjacent sections. In F small gap junctions (arrows) and intermediary junctions (arrowheads) are placed in an alternating fashion. In G a rather large gap junction is seen between two puncta adherentia (PA). Scale bars = 0.1 Bm.
350
Fig. 2, A: part of a neuronal soma located about 5 0 p m deep to the granule cell layer, showing an indented nucleus with a commashaped intranuclear sheet (arrow) and several synaptic terminals impinging upon it. A dendrite with pale cytoplasmic matrix (asterisk) gives rise to a spine-like appendage which makes a specialized junctional zone with the soma (arrowhead). Scale bar = 1 pro. B: detail of a part of the specialized junctional zone between the soma and the spine-like appendage (asterisk) shown in A. Arrows indicate gap
351 1 Amaral, D. G., A Golgi study of cell types in the hilar region of the hippocampus in the rat, J. comp. Neurol., 182 (1978) 851-914. 2 Korn, H. and Farber, D. S., Electrical interactions between vertebrate neurons: field effects and electronic coupling. In F. O. Schmitt and F. G. Worden (Eds.), The Neurosciences Fourth Study Program, MIT Press, Cambridge, MA 1979, pp. 333-358. 3 Kosaka, T., Gap junctions between nonpyramidal cell dendrites in the rat hippocampus (CA1 and CA3 regions), Brain Research, 271 (1983) 157-161. 4 Laatsch, R. H. and Cowan, W. M., Electron microscopic studies of the dentate gyrus of the rat. I. Normal structure with special reference to synaptic organization, J. comp. Neurol., 128 (1966) 359-396. 5 Loewenstein, W. R., Junctional intercellular communication: the cell-to-cell membrane channel, Physiol. Rev., 61 (1981) 829-913. 6 Lorente de N6, R., Studies of the cerebral cortex. II. Continuation of the study of the ammonic system, J. Psychol. Neurol., 46 (1934) 113-177. 7 Ram6n y Cajal, S., The structure of Ammon's Horn, Trans-
8
9
10 11
12
13
14
lated by L. M. Kraft, Charles Thomas, Springfield, IL, 1968, 78 pp. Ribak, C. E. and Anderson, L., Ultrastructure of the pyramidal basket cell in the dentate gyrus in the rat, J. comp. Neurol., 192 (1980) 903-916. Seress, L. and Pokorny, J., Structure of the granular layer of the rat dentate gyrus. A light microscopic and Golgi study, J. Anat. (Lond.), 133 (1981) 181-195. Sloper, J. J., Gap junctions between dendrites in the primate cortex, Brain Research, 44 (1972) 641-646. Sloper, J. J., An electron microscopic study of the neurons of the primate motor and somatic sensory cortices, J. Neurocytol., 2 (1973) 351-359. Sotelo, C. and Korn, H., Morphological correlates of electrical and other interactions through low-resistance pathways between neurons of the vertebrate central nervous system, Int. Rev. Cytol., 55 (1978) 67-107. Sotelo, C. and Llin~is, R., Specialized membrane junctions between neurons in the vertebrate cerebellar cortex, J. Cell Biol., 53 (1972) 271-289. T6mb61, T., Babosa, M., Hajdt~, F. and Somogyi, Gy., Interneurons: an electron microscopic study of the cat's hippocampal formation, II, Acta morph. Acad. Sci. Hung., 27 (1979) 297-313.
junction. Scale bar = 0.1 pm. C: detail of the intranuclear sheet indicated by arrow in A, but about 10 sections beyond. Scale bar = 0.1 pm. Fig. 3. A: varicose dendrite in the polymorph layer beneath the granule cell layer. Many asymmetrical synapses are seen on it. Arrow indicates a gap junction with a punctum adherens beside it. Scale bar = 1/~m. B: detail of gap junction and punctum adherens (PA) indicated by arrow in A. Cytoplasmic semidense material (arrowheads) undercoats the junctional membrane. Scale bar = 0.1 pm. Fig. 4. A: two varicose dendrites in close apposition which were traced serially in 40 consecutive sections. A bundle of several thin nerve fibers (arrowhead) appears to pierce between these two dendrites. Arrow indicates a gap junction between the thick portion of dendrite a and the transition portion between thick and thin portions of dendrite b. Asterisk indicates the thin portion of dendrite a. Scale bar = 1/~m. B: detail of the gap junction indicated by arrow in A. Semidense cytoplasmic material undercoats the junctional membrane (arrowhead). PA, punctum adherens. Scale bar = 0.1/~m. Fig. 5. Spine-like appendage arising from a varicose dendrite bears a gap junction on its tip (arrow). An asymmetrical synapse is made upon this appendage. Scale bar = 1/~m.
347
Short Communications
Neuronal gap junctions in the polymorph layer of the rat dentate gyrus TOSHIO KOSAKA National Institute for Physiological Sciences, Myodaiji, Okazaki 444 (Japan)
(Accepted June 14th, 1983) Key words: gap junction - - interneuron - - dentate gyrus- - hippocampus- - rat - - mammal
In thin sections of the rat dentate gyrus, neuronal gap junctions were observed in the polymorphlayer. Gap junctions were seen on dendritic stems, on smooth and/or varicose dendrites, on spine-like appendages, and in one case on a soma. Somata of gap junctionbearing neurons showed indented nuclei with intranuclear inclusions, and received many asymmetrical and a few symmetrical synapses. Gap junctions are ubiquitously encountered between various kinds of cells, and are regarded as intercellular communicating junctions 5. In the central nervous system (CNS), they are considered to be the morphological correlates of electrotonic coupling, and have been reported in various parts of mammalian and non-mammalian CNSX2. They are assumed to play important roles in neuronal functions, e.g. in synchronization of neuronal activity2,12. The present paper is a continuation of a prior description3 of gap junctions between non-pyramidal cell dendrites in CA1 and CA3 regions of the rat hippocampus, and is concerned with neuronal gap junctions in the rat dentate gyrus. The animals and histological procedures used here have been described in detail in a previous paper 3. Briefly, thin sections were cut, mounted in ribbons on either mesh grids or Formvar-coated single slot grids (1 x 2 mm), double-stained with uranyl acetate and lead citrate and examined with a JEM 100CX electron microscope using a tilting stage. The infrapyramidal blade of the dentate gyrus was examined in the same materials used in the previous investigations of hippocampal CA1 and CA3 regions. Despite examination of all 3 layers of the dentate gyrus (the molecular layer, the granule cell layer and the polymorph layer)n.6, 7 during the initial phase of this study, 0006-8993/83/$03.00© 1983Elsevier Science Publishers B.V.
neuronal gap junctions were encountered only in the polymorph layer. Thus the attention of the later phase of this study concentrated on the polymorph layer, where neuronal gap junctions were encountered frequently, apparently more frequently than in CA1 or CA3 regions. Gap junction-bearing dendrites in the polymorph layer of the dentate gyrus closely resembled those in the hippocampal CA1 and CA3 regions in their structural features. Most of them were smooth or varicose, about 0,5-3/~m in diameter with few, if any, spine-like appendages, filled with a pale cytoplasmic matrix, and impinged upon by many synaptic terminals. Most of the latter contained many spherical vesicles along with a few dense-cored vesicles and made asymmetrical synapses (Figs. 1, 3 and 4). In two cases (Figs. 2 and 5) gap junctions were observed on spine-like appendages arising from dendritic shafts. Fig. 1 shows a neuron located in the polymorph layer immediately deep to the granule cell layer, which had an indented nucleus and an intranuclear rod composed of filaments. This neuron had an abundant perikaryal cytoplasm containing well developcd Golgi apparatus and lamellae of rough endoplasmic reticulum. It gave rise to at least 4 smooth or somewhat varicose dendrites about 1-2.5/~m in diameter which extended almost parallel to the granule cell
348
349 layer. Two of these dendrites were in close apposi-
dentate gyrus (unpublished observations) and by sev-
tion to two dendrites of u n k n o w n origin near their origins from the soma, respectively (C and E in Fig. 1). At these apposed areas, gap junctions and
eral large to medium-sized presumable i n t e r n e u r o n s
some specialized junctions accompanying them such as puncta adherentia and intermediary junctionsl3 were observed. Many synaptic terminals impinged upon the soma and dendrites of this n e u r o n , most of which contained spherical vesicles and made asymmetrical synapses but a few contained pleomorphic
in the cat hippocampus TM. Previous Golgi studies 1,6,9 have identified several types of neurons 19cated in the polymorph layer and/or extending their dendrites there, Without the intimate cooperation of light and electron microscopic investigations, it may be impossible to identify and characterize these neuron types at the ultrastructural level.
vesicles and made symmetrical synapses.
The structural and synaptic features of gap junction-bearing dendrites and somata in the dentate gy-
Fig. 2 shows another n e u r o n located in the polymorph layer about 50 ~ m deep to the granule cell lay-
rus as well as those in hippocampal CA1 and CA3 regions 3, appear to resemble those in motor and senso-
er. The nucleus of this cell, as in the case of the neuron in Fig. 1, was indented and contained an intranu-
ry neocortex H~.I1 and cerebellar cortex 13. Therefore, it is tempting to speculate that most of these gap junc-
clear sheet, while the a b u n d a n t perikaryal cytoplasm was filled with organelles such as lamellae of rough endoplasmic reticulum, mitochondria and Golgi apparatus. Many synaptic terminals impinged upon this cell, most of which contained spherical vesicles and
tion-bearing dendrites in various brain areas originate from similar kinds of neurons, i.e. presumably inhibitory interneurons. In their review on neuronal gap junctions in vertebrate CNS, Sotelo and Korn 12
made asymmetrical synapses. A spine-like appendage arising from a dendritic shaft was found in direct contact with a part of the soma of this n e u r o n , where gap junctions and puncta adherentia were seen. The structural features of the n e u r o n s shown in Figs. 1 and 2 closely resemble those of pyramidal basket cells reported previouslys. However, this ultrastructural resemblance may not necessarily indicate that the gap j u n c t i o n - b e a r i n g n e u r o n s are pyramidal basket cells, for their ultrastructural characteristics appear to be also shared by some presumable i n t e r n e u r o n s in the molecular layer of the rat
ventured that, '... several of them (inhibitory interneurons) can be activated as a closely knit group. It is possible that the synchronous activation of inhibitory interneurons thereby provided by gap junctions is of considerable importance in the organization of neuronal nets in the cerebellum'. It seems very plausible that this holds true in other brain areas. The author wishes to thank Drs. K. H a m a and A. T. Ishida for their valuable discussions and critical reading of the manuscript and Mr. Y. Hataguchi for his technical assistance.
Fig. 1. Electron micrographs of a gap junction-bearingneuron located in the polymorph layer beneath the granule cell layer (GL). A: low magnification electron micrograph of the neuron, illustrating indentations in the nucleus, an intranuclear rod of filaments (arrows) and an abundant cytoplasm rich in cell organelles. The intranuclear rod was somewhat wavy, thus appears to be fragmentary in thin sections. A somewhat varicose dendrite extends from the soma parallel to the granule cell layer. Arrowhead indicates an area in close apposition to an obliquely sectioned dendrite where a gap junction is seen as shown in C at higher magnification. Scale bar - 10 ~m. B: detail of the intranuclear rod indicated by arrow in A, which is composed of filaments. Scale bar = 11tm. C: detail of the closely apposed area indicated by arrowhead in A in an adjacent section, showing a gap junction (arrow) and an intermediary junction beside it (arrowhead). Cytoplasmic semidense material appears to undercoat the plasma membrane at the gap junction, while somewhat denser material is seen at the adjacent intermediary junction. A synaptic terminal (asterisk) making an asymmetrical synapse on the longitudinally sectioned dendrite (a) was found in subsequent sections to also make an asymmetrical synapse on the obliquely sectioned dendrite (b). Scale bar = 1/~m. D: two synaptic terminals impinging upon the soma, each containing spherical vesicles and making an asymmetrical synapse. Scale bar = 1ktm. E: another dendrite arising from the soma of the neuron shown in A, but about 40 sections away from that in A. This dendrite (a) is in close apposition with a cross-sectioned dendrite (b), and in addition several synaptic terminals impinge upon it. The closely apposed area between these two dendrites exhibits a specialized junctional zone (between arrowheads) which is shown in F and G at higher magnificatiom Scale bar = 1~m. F and G: detail of the specialized juntional zone shown in E in two adjacent sections. In F small gap junctions (arrows) and intermediary junctions (arrowheads) are placed in an alternating fashion. In G a rather large gap junction is seen between two puncta adherentia (PA). Scale bars = 0.1 Bm.
350
Fig. 2, A: part of a neuronal soma located about 5 0 p m deep to the granule cell layer, showing an indented nucleus with a commashaped intranuclear sheet (arrow) and several synaptic terminals impinging upon it. A dendrite with pale cytoplasmic matrix (asterisk) gives rise to a spine-like appendage which makes a specialized junctional zone with the soma (arrowhead). Scale bar = 1 pro. B: detail of a part of the specialized junctional zone between the soma and the spine-like appendage (asterisk) shown in A. Arrows indicate gap
351 1 Amaral, D. G., A Golgi study of cell types in the hilar region of the hippocampus in the rat, J. comp. Neurol., 182 (1978) 851-914. 2 Korn, H. and Farber, D. S., Electrical interactions between vertebrate neurons: field effects and electronic coupling. In F. O. Schmitt and F. G. Worden (Eds.), The Neurosciences Fourth Study Program, MIT Press, Cambridge, MA 1979, pp. 333-358. 3 Kosaka, T., Gap junctions between nonpyramidal cell dendrites in the rat hippocampus (CA1 and CA3 regions), Brain Research, 271 (1983) 157-161. 4 Laatsch, R. H. and Cowan, W. M., Electron microscopic studies of the dentate gyrus of the rat. I. Normal structure with special reference to synaptic organization, J. comp. Neurol., 128 (1966) 359-396. 5 Loewenstein, W. R., Junctional intercellular communication: the cell-to-cell membrane channel, Physiol. Rev., 61 (1981) 829-913. 6 Lorente de N6, R., Studies of the cerebral cortex. II. Continuation of the study of the ammonic system, J. Psychol. Neurol., 46 (1934) 113-177. 7 Ram6n y Cajal, S., The structure of Ammon's Horn, Trans-
8
9
10 11
12
13
14
lated by L. M. Kraft, Charles Thomas, Springfield, IL, 1968, 78 pp. Ribak, C. E. and Anderson, L., Ultrastructure of the pyramidal basket cell in the dentate gyrus in the rat, J. comp. Neurol., 192 (1980) 903-916. Seress, L. and Pokorny, J., Structure of the granular layer of the rat dentate gyrus. A light microscopic and Golgi study, J. Anat. (Lond.), 133 (1981) 181-195. Sloper, J. J., Gap junctions between dendrites in the primate cortex, Brain Research, 44 (1972) 641-646. Sloper, J. J., An electron microscopic study of the neurons of the primate motor and somatic sensory cortices, J. Neurocytol., 2 (1973) 351-359. Sotelo, C. and Korn, H., Morphological correlates of electrical and other interactions through low-resistance pathways between neurons of the vertebrate central nervous system, Int. Rev. Cytol., 55 (1978) 67-107. Sotelo, C. and Llin~is, R., Specialized membrane junctions between neurons in the vertebrate cerebellar cortex, J. Cell Biol., 53 (1972) 271-289. T6mb61, T., Babosa, M., Hajdt~, F. and Somogyi, Gy., Interneurons: an electron microscopic study of the cat's hippocampal formation, II, Acta morph. Acad. Sci. Hung., 27 (1979) 297-313.
junction. Scale bar = 0.1 pm. C: detail of the intranuclear sheet indicated by arrow in A, but about 10 sections beyond. Scale bar = 0.1 pm. Fig. 3. A: varicose dendrite in the polymorph layer beneath the granule cell layer. Many asymmetrical synapses are seen on it. Arrow indicates a gap junction with a punctum adherens beside it. Scale bar = 1/~m. B: detail of gap junction and punctum adherens (PA) indicated by arrow in A. Cytoplasmic semidense material (arrowheads) undercoats the junctional membrane. Scale bar = 0.1 pm. Fig. 4. A: two varicose dendrites in close apposition which were traced serially in 40 consecutive sections. A bundle of several thin nerve fibers (arrowhead) appears to pierce between these two dendrites. Arrow indicates a gap junction between the thick portion of dendrite a and the transition portion between thick and thin portions of dendrite b. Asterisk indicates the thin portion of dendrite a. Scale bar = 1/~m. B: detail of the gap junction indicated by arrow in A. Semidense cytoplasmic material undercoats the junctional membrane (arrowhead). PA, punctum adherens. Scale bar = 0.1/~m. Fig. 5. Spine-like appendage arising from a varicose dendrite bears a gap junction on its tip (arrow). An asymmetrical synapse is made upon this appendage. Scale bar = 1/~m.