BR I E F COM MUNICAT10 N
Astrocytic Gap Junctions in the Rat Lateral Hypothalamic Area JACK C. SIPE AND ROBERT Y. MOORE Department of Neurosciences, University of California, S a n Diego and the V e t e r a n s Administration Hospital, L a JoIZa, California 92037 U.S.A.
ABSTRACT An extensive system of gap or nexus junctions has been found between astrocytic processes in the neuropil of the lateral hypothalamic area in the albino rat. These specialized interastrocytic junctions occur in regions of high synaptic density where neural processes are separated by the interconnected glial system. In this study, 909h of the gap junctions observed in the lateral hypothalamic neuropil are in the immediate proximity of synaptic terminals. The close morphological relationship of these glial gap junctions with synaptic contacts suggests that they may play a significant role in the process of synaptic transmission.
Localized modifications of the plasma membrane occur in the vertebrate nervous system at sites of intercellular junction. Among these the gap or nexus junction has generated considerable recent interest in the correlation of cellular structure and function. Gap junctions appear to serve at least two cellular functions; intercellular adhesion, a property shared by adherens and occludens junctions, and intercellular communication through channels in the apposed cell membranes (Brightman and Reese, '69; McNutt and Weinstein, '73; Weinstein and McNutt, '72). One well-documented site of gap junctions is between neurons where free passage of ions and some molecules across the junction appears to participate i n the intercellular propagation of an action potential (Pappas et al., '71; Payton et al., '69; Weinstein and McNutt, '72). In the central nervous system, gap junctions have been observed not only between electrically excitable cells but between ependymal cells and glial cells (Brightman and Reese, '67, '69). Interastrocytic gap junctions are found with the greatest frequency between perivascular endfoot processes and between interdigitating subpial processes (Brightman and Reese, '69; Reese and Karnovsky, '6'7). And, although the presence of gap junctions between astrocytes has been well documented in these regions of brain, their distribution within the new ropil of most parenchymal areas has not
been identified. In this study we describe the organization of an extensive population of interastrocytic gap junctions observed in the neuropil of the rat lateral hypothalamic area (LHA). METHODS
Male albino rats, 250-300 gm, of the Spr ague-Dawley strain were anesthetized with pentobarbital (40 mg/kg) and perfused by intracardiac administration of a glut araldehyde-paraf ormaldehyde mixture in cacodylate buffer at 37°C as previously described (Sipe et al., '73). The perfusion pressure varied from 150-200 m m Hg and the animals were perfused first with 500 ml of a week glutaraldehyde-paraformaldehyde mixture followed by 500 rnl of a strong mixture (Karnovsky, '65). Tissue blocks, approximately 1 m m in each dimension, were dissected from the lateral hypothalamic area between the optic chiasm and the mamillary bodies. The blocks were further fixed in cacodylatebuffered oxmium tetroxide and maleatebuffered uranyl acetate prior to dehydration in graded alcohols (Karnovsky, '67). Blocks embedded in Epon were sectioned at 1.5 for light microscopy and 400-800 for electron microscopy. Sections from the sagittal, coronal and horizontal planes were examined. Electron microscopy was performed using a Siemens 101 electron microscope operating at 80 KV. QuantitaReceived July 9,'75. Accepted Jan. 13, '76.
247 ANAT. REC., 1 8 5 : 247-252.
248
JACK C. SIPE AND ROBERT Y. MOORE
tive data were obtained by counting 100 gap junctions and contiguous structures in a series of unselected electron micrographs from the LHA. OBSERVATIONS
The LHA contains the axons of the medial forebrain bundle, scattered neurons and a population of neuroglia. The neuronal architecture of the LHA will be analyzed in detail in a separate report (Sipe and Moore, ’76, in preparation). In the LHA, the neuroglia are represented by satellite and interfascicular oligodendrocytes together with protoplasmic astrocytes. Cell bodies of the latter are distributed uniformly through the LHA and numerous astrocytic processes are arranged throughout the neuropil in such a manner as to separate or isolate the active surfaces of neuronal perikarya, dendrites and axon terminals. These slender glial processes contain ribosomes, endoplasmic reticulum and sparsely distributed 90 A glial filaments. Many of the glial processes are interconnected by gap, or nexus, junctions espcially in regions densely populated by synaptic terminals. In low power electron micrographs of the rat LHA one or more gap junctions are routinely observed (fig. 1 j . The distribution of the junction appears to be in relation to synaptic terminals and this can be corroborated by quantitative analysis. One hundred consecutive gap junctions encountered in unselected electron micrographs from the rat LHA were analyzed for their relationship to synaptic terminals. Using the criterion that a gap junction is in direct apposition to a synapse if one or both glial processes joined by the junction are immediately contiguous to a synaptic terminal, separated only by the 150-200 A extracellular space, 90 of the 100 gap junctions counted fulfilled this criterion. These data indicate that the gap junctions i n the LHA are distributed in close proximity to synaptic terminals. The junctions are formed by the convergence of the outer leaflets of adjacent glial plasma membranes to form a minute cleft or gap 20-30 A wide (fig. 2 ) . With “en bloc” uranyl acetate staining, a ladder-like junctional structure with periodic dense lines every 80-90 A is evident (fig.
2 , inset). The overall width of the septalaminar junction measures 140-160 A and the overall length ranges 0.2-1.5 p. Characteristic gap junctions sectioned tangential to the plasma membranes appear to have a polygonal unit substructure similar to that described by Brightman and Reese (’69). Not only do abundant gap junctions exist between astrocytic processes in the neuropil of the LHA, including areas adjacent to blood vessels, but occasional astrocytic-oligodendrocytic gap junctions are evident in the vicinity of neuronal perikaria. These interglial junctions do not differ in morphology from those between astrocytes. DISCUSSION
A number of studies have reported gap junctions between neuronal elements but there have been few accounts of interastrocytic gap junctions (Coggeshall, ’74) and the precise distribution of these intercellular junctions in the mammalian nervous system is unknown. Gap junctions between glial cells have been found with the greatest prevalence at the apical ends of ependyma1 cells (Brightman and Reese, ’69) and between astrocytes of the glia limitans and perivascular regions (Brightman and Reese, ’69; Reese and Karnovsky, ’67). Gap junctions have also been described between oligodendrocyte processes in the goldfish brain (Brightman and Reese, ’69). In the present study we have identified an extensive system of gap junctions between astrocytic processes in the neuropil of the rat LHA. The distribution of these junctions is such that they are most prevalent in areas of neuropil containing large numbers of synaptic terminals. In such areas axon terminals appear to be separated from surrounding neural elements, axons, axon terminals and dendrites by large numbers of thin, astrocytic processes (Peters and Palay, ’65). In some instances, several contiguous glial processes are joined by a gap junction network of discontinuous plaques. The ultrastructure of the gap junctions does not differ from that described previously by others in a number of regions in the vertebrate central nervous system (Brighton and Reese, ’69; McNutt and Weinstein, ’73; Sotelo and Palay, ’70;
HYPOTHALAMIC GAP JUNCTIONS
249
Fig. 1 Rat lateral hypothalamic area. In this area of neuropil at moderate magnification, five typical gap junctions are evident (arrows). Four of these junctions are in immediate proximity to synaptic terminals. x 34,000.
250
JACK C. SIPE AND ROBERT Y. MOORE
Fig. 2 Rat LHA. Three slender glial processes immediately contiguous with synaptic terminals (center) are joined by gap junctions. x 51,500. The inset is a higher magnification view showing this astrocytic gap junction with a narrow 25 A gap and ladder-like arrangement. x 189,500.
H rYUlHALAl\’IIC
Sotelo and Taxi, ’70; Sotelo and Llinas, ’72). Using the “en bloc” uranyl acetate staining technique, the junctions exhibit a typical seven-layered appearance. With a favorable plane of section through a junction, it is possible to identify compactly organized polygonal subunits similar to those described in material prepared with colloidal lanthanum (Brightman and Reese, ’67) and in material stained “en bloc” with uranyl acetate (Sotelo and Llinas, ’72). This study demonstrates the presence of gap junctions i n the rat LHA with a remarkable predilection for direct contiguity with synaptic contacts in the LHA neuropil. The functional implication of this distinctive arrangement of gap junctions is of considerable interest. Such junctions are known to provide pathways of low electrical resistance between cells (Kuffler and Nicholls, ’66; Payton et al., ’69; Weinstein and McNutt, ’72) and, also, to participate in the intercellular exchange of ions via transnexus channels (Payton et al., ’69). Their presence in the immediate vicinity of synaptic terminals in the LHA neuropil suggests that they may play a significant role in the function of astrocytes during synaptic transmission in this region of the rat brain. ACKNOWLEDGMENTS
This work was supported by a grant from the Veterans Administration and by NIH Grant NS-12080. LITERATURE CITED Brightman, M. W., and T.S. Reese 1967 Astrocytic and ependymal junctions i n the mouse brain. J. Cell Biol., 35; 16A (abstract). 1969 Junctions between intimately apposed cell membranes in the vertebrate brain. J. Cell Biol., 40: 648-677.
Coggeshall, R. E. 1974 Gap junctions between identified glial cells i n the leech. J. Neurocytol., 5: 463-467. Karnovsky, M. J. 1965 A formaldehyde-glutaraldehyde fixative of high osmolality for use i n electron microscopy. J. Cell Biol., 27: 137A ( abstract). 1967 The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J. Cell Biol., 35; 213-236. Kuffler, S. W., and J. G. Nicholls 1966 The physiology of neuroglial cells. Ergebn. Physiol., 57: 1-90. McNutt, N. S., and R. S. Weinstein 1973 Membrane ultrastructure at mammalian intercellular junctions. Prog. Biophys. Molec. Biol., 26: 45-101. Pappas, G . D., Y. Asada and M. V. L. Bennett 1971 Morphological correlates of increased coupling resistance at a n electrotonic synapse. J. Cell Biol., 49: 173-188. Payton, B. W., M. V. L. Bennett and G. D. Pappas 1969 Permeability and structure of junctional membranes at an electrotonic synapse. Science, 166: 1641-1643. Peters, A,, and S. L. Palay 1965 A n electron microscopic study of the distribution and pattern of astroglial processes in the central nervous system. J. Anat. (London), 99: 419. Reese, T. S., and M. J. Karnovsky 1967 Fine structural localization of a blood-brain barrier to exogenous peroxidase. J. Cell Biol., 34: 207217. Sipe, J. C., and R. Y. Moore (1976, in preparation) The fine structure of the rat lateral hypothalamic area. Sipe, J. C., N. A. Vick, S. Schulman and C . Fernandez 1973 Plasmocid encephalopathy in the Rhesus monkey: A study of selective vulnerability. J. Neuropath. Exp. Neurol., 32: 446457. Sotelo, C., and R. Llinas 1972 Specialized membrane junctions between neurons in the vertebrate cerebellar cortex. J. Cell Biol., 53: 271-289. Sotelo, C . , and S . L. Palay 1970 The fine structure of the lateral vestibular nucleus in the rat. 11. Synaptic organization. Brain Res., 18: 93-115. Sotelo, C., and J. Taxi 1970 Ultrastructural aspects of electrotonic junctions i n spinal cord of the frog. Brain Res., 17: 137-141. Weinstein, R. S . , and N. S . McNutt 1972 Cell Junctions. New Eng. J. Med., 286: 521-524.
Astrocytic Gap Junctions in the Rat Lateral Hypothalamic Area JACK C. SIPE AND ROBERT Y. MOORE Department of Neurosciences, University of California, S a n Diego and the V e t e r a n s Administration Hospital, L a JoIZa, California 92037 U.S.A.
ABSTRACT An extensive system of gap or nexus junctions has been found between astrocytic processes in the neuropil of the lateral hypothalamic area in the albino rat. These specialized interastrocytic junctions occur in regions of high synaptic density where neural processes are separated by the interconnected glial system. In this study, 909h of the gap junctions observed in the lateral hypothalamic neuropil are in the immediate proximity of synaptic terminals. The close morphological relationship of these glial gap junctions with synaptic contacts suggests that they may play a significant role in the process of synaptic transmission.
Localized modifications of the plasma membrane occur in the vertebrate nervous system at sites of intercellular junction. Among these the gap or nexus junction has generated considerable recent interest in the correlation of cellular structure and function. Gap junctions appear to serve at least two cellular functions; intercellular adhesion, a property shared by adherens and occludens junctions, and intercellular communication through channels in the apposed cell membranes (Brightman and Reese, '69; McNutt and Weinstein, '73; Weinstein and McNutt, '72). One well-documented site of gap junctions is between neurons where free passage of ions and some molecules across the junction appears to participate i n the intercellular propagation of an action potential (Pappas et al., '71; Payton et al., '69; Weinstein and McNutt, '72). In the central nervous system, gap junctions have been observed not only between electrically excitable cells but between ependymal cells and glial cells (Brightman and Reese, '67, '69). Interastrocytic gap junctions are found with the greatest frequency between perivascular endfoot processes and between interdigitating subpial processes (Brightman and Reese, '69; Reese and Karnovsky, '6'7). And, although the presence of gap junctions between astrocytes has been well documented in these regions of brain, their distribution within the new ropil of most parenchymal areas has not
been identified. In this study we describe the organization of an extensive population of interastrocytic gap junctions observed in the neuropil of the rat lateral hypothalamic area (LHA). METHODS
Male albino rats, 250-300 gm, of the Spr ague-Dawley strain were anesthetized with pentobarbital (40 mg/kg) and perfused by intracardiac administration of a glut araldehyde-paraf ormaldehyde mixture in cacodylate buffer at 37°C as previously described (Sipe et al., '73). The perfusion pressure varied from 150-200 m m Hg and the animals were perfused first with 500 ml of a week glutaraldehyde-paraformaldehyde mixture followed by 500 rnl of a strong mixture (Karnovsky, '65). Tissue blocks, approximately 1 m m in each dimension, were dissected from the lateral hypothalamic area between the optic chiasm and the mamillary bodies. The blocks were further fixed in cacodylatebuffered oxmium tetroxide and maleatebuffered uranyl acetate prior to dehydration in graded alcohols (Karnovsky, '67). Blocks embedded in Epon were sectioned at 1.5 for light microscopy and 400-800 for electron microscopy. Sections from the sagittal, coronal and horizontal planes were examined. Electron microscopy was performed using a Siemens 101 electron microscope operating at 80 KV. QuantitaReceived July 9,'75. Accepted Jan. 13, '76.
247 ANAT. REC., 1 8 5 : 247-252.
248
JACK C. SIPE AND ROBERT Y. MOORE
tive data were obtained by counting 100 gap junctions and contiguous structures in a series of unselected electron micrographs from the LHA. OBSERVATIONS
The LHA contains the axons of the medial forebrain bundle, scattered neurons and a population of neuroglia. The neuronal architecture of the LHA will be analyzed in detail in a separate report (Sipe and Moore, ’76, in preparation). In the LHA, the neuroglia are represented by satellite and interfascicular oligodendrocytes together with protoplasmic astrocytes. Cell bodies of the latter are distributed uniformly through the LHA and numerous astrocytic processes are arranged throughout the neuropil in such a manner as to separate or isolate the active surfaces of neuronal perikarya, dendrites and axon terminals. These slender glial processes contain ribosomes, endoplasmic reticulum and sparsely distributed 90 A glial filaments. Many of the glial processes are interconnected by gap, or nexus, junctions espcially in regions densely populated by synaptic terminals. In low power electron micrographs of the rat LHA one or more gap junctions are routinely observed (fig. 1 j . The distribution of the junction appears to be in relation to synaptic terminals and this can be corroborated by quantitative analysis. One hundred consecutive gap junctions encountered in unselected electron micrographs from the rat LHA were analyzed for their relationship to synaptic terminals. Using the criterion that a gap junction is in direct apposition to a synapse if one or both glial processes joined by the junction are immediately contiguous to a synaptic terminal, separated only by the 150-200 A extracellular space, 90 of the 100 gap junctions counted fulfilled this criterion. These data indicate that the gap junctions i n the LHA are distributed in close proximity to synaptic terminals. The junctions are formed by the convergence of the outer leaflets of adjacent glial plasma membranes to form a minute cleft or gap 20-30 A wide (fig. 2 ) . With “en bloc” uranyl acetate staining, a ladder-like junctional structure with periodic dense lines every 80-90 A is evident (fig.
2 , inset). The overall width of the septalaminar junction measures 140-160 A and the overall length ranges 0.2-1.5 p. Characteristic gap junctions sectioned tangential to the plasma membranes appear to have a polygonal unit substructure similar to that described by Brightman and Reese (’69). Not only do abundant gap junctions exist between astrocytic processes in the neuropil of the LHA, including areas adjacent to blood vessels, but occasional astrocytic-oligodendrocytic gap junctions are evident in the vicinity of neuronal perikaria. These interglial junctions do not differ in morphology from those between astrocytes. DISCUSSION
A number of studies have reported gap junctions between neuronal elements but there have been few accounts of interastrocytic gap junctions (Coggeshall, ’74) and the precise distribution of these intercellular junctions in the mammalian nervous system is unknown. Gap junctions between glial cells have been found with the greatest prevalence at the apical ends of ependyma1 cells (Brightman and Reese, ’69) and between astrocytes of the glia limitans and perivascular regions (Brightman and Reese, ’69; Reese and Karnovsky, ’67). Gap junctions have also been described between oligodendrocyte processes in the goldfish brain (Brightman and Reese, ’69). In the present study we have identified an extensive system of gap junctions between astrocytic processes in the neuropil of the rat LHA. The distribution of these junctions is such that they are most prevalent in areas of neuropil containing large numbers of synaptic terminals. In such areas axon terminals appear to be separated from surrounding neural elements, axons, axon terminals and dendrites by large numbers of thin, astrocytic processes (Peters and Palay, ’65). In some instances, several contiguous glial processes are joined by a gap junction network of discontinuous plaques. The ultrastructure of the gap junctions does not differ from that described previously by others in a number of regions in the vertebrate central nervous system (Brighton and Reese, ’69; McNutt and Weinstein, ’73; Sotelo and Palay, ’70;
HYPOTHALAMIC GAP JUNCTIONS
249
Fig. 1 Rat lateral hypothalamic area. In this area of neuropil at moderate magnification, five typical gap junctions are evident (arrows). Four of these junctions are in immediate proximity to synaptic terminals. x 34,000.
250
JACK C. SIPE AND ROBERT Y. MOORE
Fig. 2 Rat LHA. Three slender glial processes immediately contiguous with synaptic terminals (center) are joined by gap junctions. x 51,500. The inset is a higher magnification view showing this astrocytic gap junction with a narrow 25 A gap and ladder-like arrangement. x 189,500.
H rYUlHALAl\’IIC
Sotelo and Taxi, ’70; Sotelo and Llinas, ’72). Using the “en bloc” uranyl acetate staining technique, the junctions exhibit a typical seven-layered appearance. With a favorable plane of section through a junction, it is possible to identify compactly organized polygonal subunits similar to those described in material prepared with colloidal lanthanum (Brightman and Reese, ’67) and in material stained “en bloc” with uranyl acetate (Sotelo and Llinas, ’72). This study demonstrates the presence of gap junctions i n the rat LHA with a remarkable predilection for direct contiguity with synaptic contacts in the LHA neuropil. The functional implication of this distinctive arrangement of gap junctions is of considerable interest. Such junctions are known to provide pathways of low electrical resistance between cells (Kuffler and Nicholls, ’66; Payton et al., ’69; Weinstein and McNutt, ’72) and, also, to participate in the intercellular exchange of ions via transnexus channels (Payton et al., ’69). Their presence in the immediate vicinity of synaptic terminals in the LHA neuropil suggests that they may play a significant role in the function of astrocytes during synaptic transmission in this region of the rat brain. ACKNOWLEDGMENTS
This work was supported by a grant from the Veterans Administration and by NIH Grant NS-12080. LITERATURE CITED Brightman, M. W., and T.S. Reese 1967 Astrocytic and ependymal junctions i n the mouse brain. J. Cell Biol., 35; 16A (abstract). 1969 Junctions between intimately apposed cell membranes in the vertebrate brain. J. Cell Biol., 40: 648-677.
Coggeshall, R. E. 1974 Gap junctions between identified glial cells i n the leech. J. Neurocytol., 5: 463-467. Karnovsky, M. J. 1965 A formaldehyde-glutaraldehyde fixative of high osmolality for use i n electron microscopy. J. Cell Biol., 27: 137A ( abstract). 1967 The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J. Cell Biol., 35; 213-236. Kuffler, S. W., and J. G. Nicholls 1966 The physiology of neuroglial cells. Ergebn. Physiol., 57: 1-90. McNutt, N. S., and R. S. Weinstein 1973 Membrane ultrastructure at mammalian intercellular junctions. Prog. Biophys. Molec. Biol., 26: 45-101. Pappas, G . D., Y. Asada and M. V. L. Bennett 1971 Morphological correlates of increased coupling resistance at a n electrotonic synapse. J. Cell Biol., 49: 173-188. Payton, B. W., M. V. L. Bennett and G. D. Pappas 1969 Permeability and structure of junctional membranes at an electrotonic synapse. Science, 166: 1641-1643. Peters, A,, and S. L. Palay 1965 A n electron microscopic study of the distribution and pattern of astroglial processes in the central nervous system. J. Anat. (London), 99: 419. Reese, T. S., and M. J. Karnovsky 1967 Fine structural localization of a blood-brain barrier to exogenous peroxidase. J. Cell Biol., 34: 207217. Sipe, J. C., and R. Y. Moore (1976, in preparation) The fine structure of the rat lateral hypothalamic area. Sipe, J. C., N. A. Vick, S. Schulman and C . Fernandez 1973 Plasmocid encephalopathy in the Rhesus monkey: A study of selective vulnerability. J. Neuropath. Exp. Neurol., 32: 446457. Sotelo, C., and R. Llinas 1972 Specialized membrane junctions between neurons in the vertebrate cerebellar cortex. J. Cell Biol., 53: 271-289. Sotelo, C . , and S . L. Palay 1970 The fine structure of the lateral vestibular nucleus in the rat. 11. Synaptic organization. Brain Res., 18: 93-115. Sotelo, C., and J. Taxi 1970 Ultrastructural aspects of electrotonic junctions i n spinal cord of the frog. Brain Res., 17: 137-141. Weinstein, R. S . , and N. S . McNutt 1972 Cell Junctions. New Eng. J. Med., 286: 521-524.
