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Gap junctions on CA3 pyramidal cells of guinea pig hippocampus shown by freeze-fracture
Brain Research, 217 (1981) 175-178 O Elsevier/North-Holland Biomedical Press
175
Gap junctions on CA3 pyramidal cells of guinea pig hippocampus shown by freeze-fracture
H E N N I N G S C H M A L B R U C H and H E N R I K JAHNSEN
Institute of Neurophysiology, University of Copenhagen, The Panum Institute, Blegdamsvej 3 C, DK2200 Copenhagen N (Denmark) (Accepted March 19th, 1981)
Key words: hippocampus - - gap junction - - freeze fracture - - guinea pig - - mammal In freeze-fracture replicas of the hippocampus of guinea pig, gap junctions were found on membranes identified as belonging to the apical dendrites or somata of the CA3 pyramidal cells. The junctions were not part of mixed synapses. It is concluded that adjacent pyramidal cells, which in places lacked an interposed glial sheath are electrically coupled.
The normal synchronous activity of nerve cells in the hippocampus may, in part, be due to electrotonic coupling. In rats, intracellularly injected current and Lucifer yellow spread into neighbouring CA3 neurones 3.4. The intercellular diffusion of dye might indicate that neurones in the CA3 area are coupled by gap junctions. We here describe gap junctions on the proximal part of the apical dendrite of the CA3 pyramidal cells. The brains of adult guinea pigs were fixed by vascular perfusion with glutaraldehyde and paraformaldehyde in Ringer's solution. On a Vibratome (Oxford), the hippocampus was cut into slices 500/~m thick, the CA3 area was isolated, and either embedded for thin sections, or after glycerination frozen in nitrogen slush and fractured and replicated in a Balzers BAE 120 unit with double fracture device as described earlier 1°. Freeze-fractures showed gap junctions on the surface of tubes 2-7 /~m in diameter (Fig. 1), or on membrane surfaces which were plane and measured more than 5 / t m in each direction. Axons and synaptic boutons of mossy fibres were seen adjacent to membranes with gap junctions; dendritic spines were not associated with gap junctions. This suggested that the gap junctions were localized on the thick stem of the apical dendrite and perhaps on the soma of the pyramidal cells as well. Gap junctions were not found on synaptic boutons or on unmyelinated axons. Gap junctions were scarce and tended to be grouped. On fortunate fractures exposing 50-100 sq. # m membrane area, at most 3 junctions were found.Their localization was not related to that of aggregations of large particles attached to the E-face of the postsynaptic membrane characterizing chemical synapses. The mean area of the
Fig. 1. Freeze-fracture. Pyramidal cell of the hippocampus of guinea pig. The P-face of an apical dendrite is exposed; the diameter of the dendrite increased from top to bottom indicating that the soma was below. Two gap junctions (straight arrows), and shallow impressions caused by crossing projections of other cells are visible. The intramembrane particles form strands bordering the shallow impressions. A thin branch of the dendrite carries a synaptic bouton (curved arrow). Two myelinated axons (M) are seen (25,000 × ). Inset: higher magnification of one of the gap junctions showing the irregular array of particles and corresponding pits of the attached E-face (100,000 ×).
177 gap junctions was 0.12 sq. /~m (S.D. = 0.07 sq. #m; n = 16). The gap junction particles seen on P-faces were irregularly arranged and did not show a crystalline array. Corresponding pits on the E-faces were distinct (Fig. 1 ; inset). The disorderly array of particles of gap junctions is reported to indicate low-resistance coupling, whereas high-resistance couplings are characterized by an hexagonal packing of the particlesS-V,L For lens fibres, however, this has not been confirmed 2. The neuronal membranes carrying gap junctions showed shallow impressions caused by transversely or obliquely running axons or dendritic branches. Intramembranous particles were scarce within these grooves. In thin sections, no gap junctions on neurones could be identified unequivocally. This might be due to the fact that gap junctions were rare, and possibly also because the particles within the gap junctions were loosely packed. Also in retina cells, gap junctions were numerous in freeze-facture replicas but could not be identified in thin sections s. The somata and dendrites of the pyramidal cells were usually separated by at least one layer of glia cell projections, but occasionally these glial sheaths were incomplete and the plasma membranes of adjacent cells were closely apposed (Fig. 2). Between glia cell projections several gap junctions were found which were well preserved and showed a distinct central 'gap'. Hence, the failure to find gap junctions between neurons in sections was probably not due to improper fixation. In freeze-fractures, only gap junctions localized on large membrane faces could be identified as neuronal; junctions found on small membrane areas of irregular shapes belonged to glia cells or neurones. It is most likely that the gap junctions on neuronal membranes connect adjacent pyramidal cells which are in direct contact in places where the glial sheaths are incompletel; no gap junctions being part of mixed synapses have been seen. It is unlikely that the gap junctions connect glia and pyramidal cells because physiological or morphological coupling of different cell types have not been observed. Yamamoto 14 discussed the possibility that mossy fibres and CA3 pyramidal cells are electrically coupled. If electrically mediated synaptic transmission does take place between mossy fibres and pyramidal neurones, it must be by 'ephatic' interaction.
Fig. 2. Thin section. Pyramidal cells of the hippocampus of guinea pig. The plasma membranes of two somata are partly separated by a thin projection of a glia cell (right), but partly in direct contact (left)
0oo,ooo x).
178 The fast prepotentials of pyramidal cells described by Spencer a n d Kande111 are t h o u g h t to be spikes generated by patches of dendritic m e m b r a n e c o n t a i n i n g sodium channels12,1~. However, they may also be explained as action potentials of adjacent neurones conducted t h r o u g h gap junctions. I f this is so, gap j u n c t i o n s should be expected between CA1 n e u r o n e s as well, because these cells also have fast prepotentials. Electrotonic coupling of the p y r a m i d a l cells m a y be related to their tendency to epileptic discharges a n d it might be worthwhile to study gap j u n c t i o n s in species more prone to seizures t h a n guinea pigs.
1 Hamlyn, H., An electron microscope study of pyramidal neurons in the Ammon's horn of the rabbit, J. Anat. (Lond.), 97 (1963) 189-201. 2 Goodenough, D. A., Intercellular communication in cell and tissue systems, Europ. J. Cell Biol., 22 (1980) 246. 3 MacVicar, B. A. and Dudek, F. E., Evidence for electrotonic coupling between CA3 pyramidal cells of rat hippocampus: dye-coupling and simultaneous intracellular recordings, Neurosci. Abstr., 6 (1980) abstract 137A.5, 407. 4 MacVicar, B. A. and Dudek, F. E., Dye-coupling between CA3 pyramidal cells in slices of rat hippocampus, Brain Research, 196 (1980) 494-497. 5 Peracchia, C., Calcium effects on gap junction structure and cell coupling, Nature (Lond.), 271 (1978) 669-671. 6 Peracchia, C. and Peracchia, L. L., Gap junction dynamics: reversible effects of divalent cations, J. Cell Biol., 87 (1980) 708-718. 7 Peracchia, C. and Peracchia, L. L., Gap junction dynamics: reversible effects of hydrogen ions, J. Cell Biol., 87 (1980) 719-727. 8 Raviola, E. and Gilula, N. B., Intramembrane organization of specialized contacts in the outer plexiform layer of the retina. A freeze-fracture study in monkeys and rabbits, J. Cell BioL, 65 (1975) 192-222. 9 Raviola, E., Goodenough, D. A. and Raviola, G., The native structure of gap junctions rapidly frozen at 4 °K, J. Cell Biol., 79 (1978) 229. 10 Schmalbruch, H., Delayed fixation alters the pattern of intramembrane particles in mammalian muscle fibers, J. Ulstrastruct. Res., 70 (1980) 15-20. 11 Spencer, W. A. and Kandel, E. R., Electrophysiology of hippocampal neurons. IV. Fast prepotentials, J. Neurophysiol., 24 (1961) 272 285. 12 Traub, R. D. and Llimis, R., Hippocampal pyramidal cells: Significance of dendritic ionic conductances for neuronal function and epileptogenesis, J. Neurophysiol., 42 (1979) 476-496. 13 Wong, R. K. S., Prince, D. A. and Basbaum, A. I., lntradendritic recordings from hippocampal neurons, Proc. nat. Acad. Sci. (Wash.), 76 (1979)986--990. 14 Yamamoto, C., Activation of hippocampal neurons by mossy fiber stimulation in thin brain sections in vitro, Exp. Brain Res., 14 (1972) 423 435.
175
Gap junctions on CA3 pyramidal cells of guinea pig hippocampus shown by freeze-fracture
H E N N I N G S C H M A L B R U C H and H E N R I K JAHNSEN
Institute of Neurophysiology, University of Copenhagen, The Panum Institute, Blegdamsvej 3 C, DK2200 Copenhagen N (Denmark) (Accepted March 19th, 1981)
Key words: hippocampus - - gap junction - - freeze fracture - - guinea pig - - mammal In freeze-fracture replicas of the hippocampus of guinea pig, gap junctions were found on membranes identified as belonging to the apical dendrites or somata of the CA3 pyramidal cells. The junctions were not part of mixed synapses. It is concluded that adjacent pyramidal cells, which in places lacked an interposed glial sheath are electrically coupled.
The normal synchronous activity of nerve cells in the hippocampus may, in part, be due to electrotonic coupling. In rats, intracellularly injected current and Lucifer yellow spread into neighbouring CA3 neurones 3.4. The intercellular diffusion of dye might indicate that neurones in the CA3 area are coupled by gap junctions. We here describe gap junctions on the proximal part of the apical dendrite of the CA3 pyramidal cells. The brains of adult guinea pigs were fixed by vascular perfusion with glutaraldehyde and paraformaldehyde in Ringer's solution. On a Vibratome (Oxford), the hippocampus was cut into slices 500/~m thick, the CA3 area was isolated, and either embedded for thin sections, or after glycerination frozen in nitrogen slush and fractured and replicated in a Balzers BAE 120 unit with double fracture device as described earlier 1°. Freeze-fractures showed gap junctions on the surface of tubes 2-7 /~m in diameter (Fig. 1), or on membrane surfaces which were plane and measured more than 5 / t m in each direction. Axons and synaptic boutons of mossy fibres were seen adjacent to membranes with gap junctions; dendritic spines were not associated with gap junctions. This suggested that the gap junctions were localized on the thick stem of the apical dendrite and perhaps on the soma of the pyramidal cells as well. Gap junctions were not found on synaptic boutons or on unmyelinated axons. Gap junctions were scarce and tended to be grouped. On fortunate fractures exposing 50-100 sq. # m membrane area, at most 3 junctions were found.Their localization was not related to that of aggregations of large particles attached to the E-face of the postsynaptic membrane characterizing chemical synapses. The mean area of the
Fig. 1. Freeze-fracture. Pyramidal cell of the hippocampus of guinea pig. The P-face of an apical dendrite is exposed; the diameter of the dendrite increased from top to bottom indicating that the soma was below. Two gap junctions (straight arrows), and shallow impressions caused by crossing projections of other cells are visible. The intramembrane particles form strands bordering the shallow impressions. A thin branch of the dendrite carries a synaptic bouton (curved arrow). Two myelinated axons (M) are seen (25,000 × ). Inset: higher magnification of one of the gap junctions showing the irregular array of particles and corresponding pits of the attached E-face (100,000 ×).
177 gap junctions was 0.12 sq. /~m (S.D. = 0.07 sq. #m; n = 16). The gap junction particles seen on P-faces were irregularly arranged and did not show a crystalline array. Corresponding pits on the E-faces were distinct (Fig. 1 ; inset). The disorderly array of particles of gap junctions is reported to indicate low-resistance coupling, whereas high-resistance couplings are characterized by an hexagonal packing of the particlesS-V,L For lens fibres, however, this has not been confirmed 2. The neuronal membranes carrying gap junctions showed shallow impressions caused by transversely or obliquely running axons or dendritic branches. Intramembranous particles were scarce within these grooves. In thin sections, no gap junctions on neurones could be identified unequivocally. This might be due to the fact that gap junctions were rare, and possibly also because the particles within the gap junctions were loosely packed. Also in retina cells, gap junctions were numerous in freeze-facture replicas but could not be identified in thin sections s. The somata and dendrites of the pyramidal cells were usually separated by at least one layer of glia cell projections, but occasionally these glial sheaths were incomplete and the plasma membranes of adjacent cells were closely apposed (Fig. 2). Between glia cell projections several gap junctions were found which were well preserved and showed a distinct central 'gap'. Hence, the failure to find gap junctions between neurons in sections was probably not due to improper fixation. In freeze-fractures, only gap junctions localized on large membrane faces could be identified as neuronal; junctions found on small membrane areas of irregular shapes belonged to glia cells or neurones. It is most likely that the gap junctions on neuronal membranes connect adjacent pyramidal cells which are in direct contact in places where the glial sheaths are incompletel; no gap junctions being part of mixed synapses have been seen. It is unlikely that the gap junctions connect glia and pyramidal cells because physiological or morphological coupling of different cell types have not been observed. Yamamoto 14 discussed the possibility that mossy fibres and CA3 pyramidal cells are electrically coupled. If electrically mediated synaptic transmission does take place between mossy fibres and pyramidal neurones, it must be by 'ephatic' interaction.
Fig. 2. Thin section. Pyramidal cells of the hippocampus of guinea pig. The plasma membranes of two somata are partly separated by a thin projection of a glia cell (right), but partly in direct contact (left)
0oo,ooo x).
178 The fast prepotentials of pyramidal cells described by Spencer a n d Kande111 are t h o u g h t to be spikes generated by patches of dendritic m e m b r a n e c o n t a i n i n g sodium channels12,1~. However, they may also be explained as action potentials of adjacent neurones conducted t h r o u g h gap junctions. I f this is so, gap j u n c t i o n s should be expected between CA1 n e u r o n e s as well, because these cells also have fast prepotentials. Electrotonic coupling of the p y r a m i d a l cells m a y be related to their tendency to epileptic discharges a n d it might be worthwhile to study gap j u n c t i o n s in species more prone to seizures t h a n guinea pigs.
1 Hamlyn, H., An electron microscope study of pyramidal neurons in the Ammon's horn of the rabbit, J. Anat. (Lond.), 97 (1963) 189-201. 2 Goodenough, D. A., Intercellular communication in cell and tissue systems, Europ. J. Cell Biol., 22 (1980) 246. 3 MacVicar, B. A. and Dudek, F. E., Evidence for electrotonic coupling between CA3 pyramidal cells of rat hippocampus: dye-coupling and simultaneous intracellular recordings, Neurosci. Abstr., 6 (1980) abstract 137A.5, 407. 4 MacVicar, B. A. and Dudek, F. E., Dye-coupling between CA3 pyramidal cells in slices of rat hippocampus, Brain Research, 196 (1980) 494-497. 5 Peracchia, C., Calcium effects on gap junction structure and cell coupling, Nature (Lond.), 271 (1978) 669-671. 6 Peracchia, C. and Peracchia, L. L., Gap junction dynamics: reversible effects of divalent cations, J. Cell Biol., 87 (1980) 708-718. 7 Peracchia, C. and Peracchia, L. L., Gap junction dynamics: reversible effects of hydrogen ions, J. Cell Biol., 87 (1980) 719-727. 8 Raviola, E. and Gilula, N. B., Intramembrane organization of specialized contacts in the outer plexiform layer of the retina. A freeze-fracture study in monkeys and rabbits, J. Cell BioL, 65 (1975) 192-222. 9 Raviola, E., Goodenough, D. A. and Raviola, G., The native structure of gap junctions rapidly frozen at 4 °K, J. Cell Biol., 79 (1978) 229. 10 Schmalbruch, H., Delayed fixation alters the pattern of intramembrane particles in mammalian muscle fibers, J. Ulstrastruct. Res., 70 (1980) 15-20. 11 Spencer, W. A. and Kandel, E. R., Electrophysiology of hippocampal neurons. IV. Fast prepotentials, J. Neurophysiol., 24 (1961) 272 285. 12 Traub, R. D. and Llimis, R., Hippocampal pyramidal cells: Significance of dendritic ionic conductances for neuronal function and epileptogenesis, J. Neurophysiol., 42 (1979) 476-496. 13 Wong, R. K. S., Prince, D. A. and Basbaum, A. I., lntradendritic recordings from hippocampal neurons, Proc. nat. Acad. Sci. (Wash.), 76 (1979)986--990. 14 Yamamoto, C., Activation of hippocampal neurons by mossy fiber stimulation in thin brain sections in vitro, Exp. Brain Res., 14 (1972) 423 435.