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Synapse formation and disappearance in adult rat supraoptic nucleus during different hydration states
Brain Research, 309 (1984) 373-376 Elsevier
373
BRE 20356
Synapse formation and disappearance in adult rat supraoptic nucleus during different hydration states CHARLES D. TWEEDLE 1. and GLENN I. HATFON2 1Department of Anatomy, and 2Neuroscience Program, Michigan State University, East Lansing, M14882 4-1117 (U.S.A.)
(Accepted May 8th, 1984) Key words: neuroplasticity - - supraoptic nucleus - - dehydration - - neurosecretion - - astrocytic movement
Activation of the adult rat supraoptic nucleus by a chronic stimulus (10 days of drinking 2% NaCI instead of tap water) brought about the appearance of newly formed specialized synapses onto the magnocellular neurosecretory cells. The increase in these synapses was reversed when the animals were allowed to rehydrate by drinking tap water. The apparent retraction and reinsertion of the thin glial processes from between the neurosecretory cell somata, which results in significant changes in soma-somatic direct membrane appositions, is most likely involved in the formation and elimination of these synapses.
The magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) in the hypothalamus produce either oxytocin or vasopressin. These nonapeptide hormones are transported by axoplasmic flow to the neurohypophysis where, reflecting hormone demand and MNC cell firing, they are released into the perivascular spaces of fenestrated capillaries 3. Chronic activation of the rat SON by lactation led to the ultrastructural appearance of synapses on the MNC somata of a type extremely rare in adult virgin females1,8, 9. These novel synapses involve one presynaptic terminal making synaptic contacts simultaneously with two adjacent neurosecretory cell somata, hence called 'double' synapses (see Fig. 1). Such synapses did not appear in appreciable numbers with either short-term (1 day) water deprivation10,11, pregnancy or parturition t. Double synapses onto MNC dendrites in the SON dendritic zone have also been shown to vary with the chronic stimuli of gestation and lactation s but not with acute (1 day) water deprivation6. We provide here evidence for the formation of double synapses during the stimulus of chronic dehydration and the disappearance of those synapses when the stimulation is terminated. Recent evidence indicates that weaning following lactation is also accompanied by a loss of SON double synapses 8.
Twenty-five 100-day-old rats (Holtzman Sprague-Dawley), were divided into the following treatment groups: (1) controls, given ad libitum tap water (4 males and 4 females); (2) dehydrated, given 2% NaCI to drink for 10 days (4 males and 4 females); (3) 5-day rehydrated, dehydrated as above, then given water to drink again for 5 days (4 females); and (4) 14-day rehydrated, dehydrated as above, then given water to drink for 14 days (5 females). Animals were perfused with a buffered mixed aldehyde fixative10,11; the brains were removed and left overnight in fixative. The hypothalamus was dissected out and cut into 500/~m thick slices with a tissue chopper. The SON was removed with an electrolytically etched 18-gauge hypodermic needle. These 'cores' of SON tissue were osmicated for 1 h in 1% OsO4 in 0.1 M cacodylate buffer at p H 7.4, stained en bloc in aqueous uranyl acetate, and embedded in Spurr's resin in an orientation such that coronal sections could be obtained. Ultrathin sections of SON magnocellular neurons were collected, further stained with lead citrate, and examined with a Philips 201C electron microscope. From several levels of the SON 12-14 electron micrographs were taken per animal at x3000 (final print magnification was ×7500). Data from these prints were summed to give a single value
Correspondence: C. D. Tweedle, Department of Anatomy, Michigan State University, East Lansing, M148824-1117, U.S.A.
374 for that animal for each p a r a m e t e r measured. A n image analyzer was used to measure the total length of SON somatic m e m b r a n e and of S O N somatic m e m b r a n e in direct apposition. The percentage of m e m b r a n e in direct soma-somatic apposition was assessed by dividing the length of somatic m e m b r a n e in apposition by the total length of somatic m e m b r a n e . Profiles of axonal terminals with synaptic vesicles and thickenings simultaneously contacting two postsynaptic SON somata were quantified. Confirmation of possibly ambiguous double synapses was obtained by examination under high p o w e r ( x 10,000-20,000). The percentage of cell bodies with double synapses was c o m p u t e d as the ratio of cells contacted by double synapses to the total n u m b e r of cells observed. The heterogeneity of variance due to the presence of numerous 'zeros' associated with certain t r e a t m e n t groups dictated the use of medians and interquartile ranges 4. However, similar results were obtained when the parametric measures, means and standard errors were used in analyses. Since analyses of the p a r a m e t e r s to be considered here revealed no differences between the data from male and female rats,
Fig. 1. Synapses seen in the supraoptic nucleus following chronic dehydration. A: a double synapse between two adjacent MNC somata. It has both smaller, clear vesicles and larger dense core vesicles. B: a vesicle-filled profile apposed to two adjacent cell bodies. There is an area of somatic membrane thickening of unknown function adjacent to this profile (arrow). C: a double synapse showing the large size that they can display in the appropriate plane of section. Bar = 0.5 #m.
2O 16 12.tz
84-
O
0
._~
10S
5R
14R
10S
5R
14R
6-
X ._.q
4 3
0
Days of Saline Drinking or Rehydration
Fig. 2. The percent of cells with double synapses and the percent of MNC somatic membrane in direct apposition in the different treatment groups. Presented as medians and interquartile ranges. 10S, 10 days saline treatment; 5R, 5 days rehydration; and 14R, 14 days rehydration.
these data were c o m b i n e d for statistical purposes. The K r u s k a l - W a l l i s H-test and M a n n - W h i t n e y U-test 7 were used for comparisons of the d a t a among the various treatment groups. Ten days of drinking 2% saline instead of water brought about striking morphological changes in the SON, although the rats a p p e a r e d to be in g o o d general health. There were statistically significant changes in the percentages of cells contacted by double synapses (overall test, H = 20.1, P < 0.001). These data are presented in Figs. 1 and 2. Individual p l a n n e d comparisons showed that the percentage of cells contacted by such synapses (over 20% in saline-treated vs 0% observed on the MNCs of control rats) was significantly elevated by 10 days of saline drinking (U = 0, P = 0.001). Five days of rehydration produced no reliable decrease in the presence of double synapses, whereas the decrease in the 14-day group was significant (U = 3, P = 0.005). These synapses were similar in a p p e a r a n c e and n u m b e r to those found in lactating rats~. Measurements of profiles of M N C s o m a t a from sa-
375 line-treated rats indicated increases in cell area of at least 50%, with an increase in percentage of direct neuronal soma-somatic membrane apposition from less than 0.05% of somal membrane in contact to 6.25% (U = 0, P = 0.001). The percentage of MNC somatic profiles in contact increased from 1.5% to 28.9% (U = 0, P = 0.001). This increased contact is due to withdrawal of glial processes from between the neurons. Rehydration for 5 days brought the amount of cell-cell membrane apposition back to normal, but the number of double synapses remained relatively unchanged. Rehydration for 14 days, however, as noted above was accompanied by a return of double synapse occurrence to normal values (Fig. 2). In a preliminary experiment using older (10-monthold) female rats, 10 days of saline treatment likewise increased the incidence of double synapses, although to a lesser degree, only 7.1% of the cells being contacted by these specialized synapses. Thus, double synapses increase in the SON of adult rats during chronic activation of the nucleus whether this activation be elicited by suckling (causing predominant oxytocin release) or by a dehydration stimulus in either male or female rats (releasing both oxytocin and vasopressin) 2. This indicates that (a) lactation-associated or sex-specific circulating hormones are not necessary for the new synaptic connection to form and (b) these double synapses are not necessarily related to patterns of activity seen during milk ejection. The new synapses as well as the increased cell-cell contact have been thought heretofore to be an adaptation specifically allowing more synchronization of oxytocinergic neuronsS,9. Notable here is the observation of decreased cell-cell apposition prior to a statistically significant decline in the percentage of MNCs contacted by double synapses. This indicates that a considerable amount ofglial reinsertion can occur upon reversal of the dehydrating conditions without altering the synaptic morphology. However, whether the glia ultimately participate in the removal of one or both of the synaptic specializations of the double synapses is not presently known. The axonal origin or the nature of the neurotransmitter(s) in the double synapses are not yet known.
Double synapses on SON somata in our material are 'symmetrical' and contain predominantly 30-40 nm clear, round vesicles with a few 90-100 nm dense core vesicles (Fig. 1). They do not show reaction product in rats treated with 5-hydroxydopamine, indicating that they do not contain catecholamines (unpublished observations). It is also not known whether the number of other types of synapses in SON, less conspicuous than the double synapses, also change in frequency of occurrence. In need of investigation is the identity of the hormone contained in the postsynaptic cells. The new synaptic contacts form concomitantly with a large increase in direct soma-somatic appositions and with the retraction of intervening glial processes. This glial retraction could put existing terminals, heretofore on one MNC, in a position to form a second synaptic thickening on a newly juxtaposed second MNC and, thus, become a double synapse. Axonal sprouting, therefore, might not be required for their formation. No degenerating synaptic terminals or obvious retraction bulbs have been seen during rehydration when synapse elimination occurs. Possible stimuli for the formation of these novel synapses could be from a number of sources including: (a) altered extracellular levels of chemical factors or ions released by rapidly firing presynaptic or postsynaptic neural elements; (b) glial withdrawal from between adjacent MNC somata exposing new, potentially postsynaptic membrane to existing synapses; (c) increased areas of receptive postsynaptic membrane brought about by MNC volume increases, or (d) some combination of these factors. Similar glial and synaptic plasticity as described here may occur elsewhere in the adult mammalian CNS under physiological conditions. The particular synaptic and neuronal cell arrangements of the SON have facilitated detecting such changes in the present study.
1 Hatton, G. I. and Tweedle, C. D., Magnocellularpeptidergic neurons in hypothalamus: increases in membrane apposition and number of specialized synapses from pregnancy to lactation, Brain Res. Bull., 8 (1982) 197-204.
2 Jones, C. W. and Pickering, B. T., Comparison of the effects of water deprivation and sodium chloride imbibition on the hormone content of the neurohypophysisof the rat, J. Physiol. (Lond. ), 203 (1969) 449-458.
Supported by N.I.H. Grant NS 09140. We thank P. Cobbett, W. M. Falls and L. Perlmutter for suggestions on an earlier version of the manuscript and B. Schmidt and J. Harper for typing assistance.
376 3 Mason, C. A. and Bern, H. A., Cellular biology of the neurosecretory neuron. In E. R. Kandel (Ed.), Handbook of Physiology, Vol. 1, Amer. Physiol. Soc., Bethesda, 1977, pp. 651-690. 4 Mosteller, F. and Tukey, J. W., Data Analysis and Regression, Addison-Wesley, Reading, MA, 1977. 5 Perlmutter, L. S., Tweedle, C. D. and Hatton, G. I., Neuronai/glial plasticity in the supraoptic dendritic zone: dendritic bundling and double synapse formation at parturition, Neuroscience, in press. 6 Perlmutter, L. S., Tweedle, C. D. and Hatton, G. I., Plasticity in supraoptic neurons: dendritic bundle formation in response to short-term dehydration and rehydration, Anat. Rec., 208 (1984) 136-137A. 7 Siegel, S., Non-parametric Statistics, McGraw-Hill, New York, NY, 1956.
8 Theodosis, D. T. and Poulain, D. A., Evidence for structural plasticity in the supraoptic nucleus of the rat hypothalamus in relation to gestation and lactation, Neuroscience, 11 (1983) 183-192. 9 Theodosis, D. T., Poulain, D. A. and Vincent, J.-D., Possible morphological bases for synchronization of neuronal firing in the rat supraoptic nucleus during lactation, Neuroscience, 6 (1981) 919-929. 10 Tweedle, C. D. and Hatton, G. I., Ultrastructurai comparisons of neurons of supraoptic and circularis nuclei in normal and dehydrated rats, Brain Res. Bull., 1 (1976) 103-118. 11 Tweedle, C. D. and Hatton, G. I., Ultrastructural changes in rat hypothalamus neurosecretory cells and their associated glia during minimal dehydration and rehydration, Cell Tiss. Res., 181 (1977) 59-72.
373
BRE 20356
Synapse formation and disappearance in adult rat supraoptic nucleus during different hydration states CHARLES D. TWEEDLE 1. and GLENN I. HATFON2 1Department of Anatomy, and 2Neuroscience Program, Michigan State University, East Lansing, M14882 4-1117 (U.S.A.)
(Accepted May 8th, 1984) Key words: neuroplasticity - - supraoptic nucleus - - dehydration - - neurosecretion - - astrocytic movement
Activation of the adult rat supraoptic nucleus by a chronic stimulus (10 days of drinking 2% NaCI instead of tap water) brought about the appearance of newly formed specialized synapses onto the magnocellular neurosecretory cells. The increase in these synapses was reversed when the animals were allowed to rehydrate by drinking tap water. The apparent retraction and reinsertion of the thin glial processes from between the neurosecretory cell somata, which results in significant changes in soma-somatic direct membrane appositions, is most likely involved in the formation and elimination of these synapses.
The magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) in the hypothalamus produce either oxytocin or vasopressin. These nonapeptide hormones are transported by axoplasmic flow to the neurohypophysis where, reflecting hormone demand and MNC cell firing, they are released into the perivascular spaces of fenestrated capillaries 3. Chronic activation of the rat SON by lactation led to the ultrastructural appearance of synapses on the MNC somata of a type extremely rare in adult virgin females1,8, 9. These novel synapses involve one presynaptic terminal making synaptic contacts simultaneously with two adjacent neurosecretory cell somata, hence called 'double' synapses (see Fig. 1). Such synapses did not appear in appreciable numbers with either short-term (1 day) water deprivation10,11, pregnancy or parturition t. Double synapses onto MNC dendrites in the SON dendritic zone have also been shown to vary with the chronic stimuli of gestation and lactation s but not with acute (1 day) water deprivation6. We provide here evidence for the formation of double synapses during the stimulus of chronic dehydration and the disappearance of those synapses when the stimulation is terminated. Recent evidence indicates that weaning following lactation is also accompanied by a loss of SON double synapses 8.
Twenty-five 100-day-old rats (Holtzman Sprague-Dawley), were divided into the following treatment groups: (1) controls, given ad libitum tap water (4 males and 4 females); (2) dehydrated, given 2% NaCI to drink for 10 days (4 males and 4 females); (3) 5-day rehydrated, dehydrated as above, then given water to drink again for 5 days (4 females); and (4) 14-day rehydrated, dehydrated as above, then given water to drink for 14 days (5 females). Animals were perfused with a buffered mixed aldehyde fixative10,11; the brains were removed and left overnight in fixative. The hypothalamus was dissected out and cut into 500/~m thick slices with a tissue chopper. The SON was removed with an electrolytically etched 18-gauge hypodermic needle. These 'cores' of SON tissue were osmicated for 1 h in 1% OsO4 in 0.1 M cacodylate buffer at p H 7.4, stained en bloc in aqueous uranyl acetate, and embedded in Spurr's resin in an orientation such that coronal sections could be obtained. Ultrathin sections of SON magnocellular neurons were collected, further stained with lead citrate, and examined with a Philips 201C electron microscope. From several levels of the SON 12-14 electron micrographs were taken per animal at x3000 (final print magnification was ×7500). Data from these prints were summed to give a single value
Correspondence: C. D. Tweedle, Department of Anatomy, Michigan State University, East Lansing, M148824-1117, U.S.A.
374 for that animal for each p a r a m e t e r measured. A n image analyzer was used to measure the total length of SON somatic m e m b r a n e and of S O N somatic m e m b r a n e in direct apposition. The percentage of m e m b r a n e in direct soma-somatic apposition was assessed by dividing the length of somatic m e m b r a n e in apposition by the total length of somatic m e m b r a n e . Profiles of axonal terminals with synaptic vesicles and thickenings simultaneously contacting two postsynaptic SON somata were quantified. Confirmation of possibly ambiguous double synapses was obtained by examination under high p o w e r ( x 10,000-20,000). The percentage of cell bodies with double synapses was c o m p u t e d as the ratio of cells contacted by double synapses to the total n u m b e r of cells observed. The heterogeneity of variance due to the presence of numerous 'zeros' associated with certain t r e a t m e n t groups dictated the use of medians and interquartile ranges 4. However, similar results were obtained when the parametric measures, means and standard errors were used in analyses. Since analyses of the p a r a m e t e r s to be considered here revealed no differences between the data from male and female rats,
Fig. 1. Synapses seen in the supraoptic nucleus following chronic dehydration. A: a double synapse between two adjacent MNC somata. It has both smaller, clear vesicles and larger dense core vesicles. B: a vesicle-filled profile apposed to two adjacent cell bodies. There is an area of somatic membrane thickening of unknown function adjacent to this profile (arrow). C: a double synapse showing the large size that they can display in the appropriate plane of section. Bar = 0.5 #m.
2O 16 12.tz
84-
O
0
._~
10S
5R
14R
10S
5R
14R
6-
X ._.q
4 3
0
Days of Saline Drinking or Rehydration
Fig. 2. The percent of cells with double synapses and the percent of MNC somatic membrane in direct apposition in the different treatment groups. Presented as medians and interquartile ranges. 10S, 10 days saline treatment; 5R, 5 days rehydration; and 14R, 14 days rehydration.
these data were c o m b i n e d for statistical purposes. The K r u s k a l - W a l l i s H-test and M a n n - W h i t n e y U-test 7 were used for comparisons of the d a t a among the various treatment groups. Ten days of drinking 2% saline instead of water brought about striking morphological changes in the SON, although the rats a p p e a r e d to be in g o o d general health. There were statistically significant changes in the percentages of cells contacted by double synapses (overall test, H = 20.1, P < 0.001). These data are presented in Figs. 1 and 2. Individual p l a n n e d comparisons showed that the percentage of cells contacted by such synapses (over 20% in saline-treated vs 0% observed on the MNCs of control rats) was significantly elevated by 10 days of saline drinking (U = 0, P = 0.001). Five days of rehydration produced no reliable decrease in the presence of double synapses, whereas the decrease in the 14-day group was significant (U = 3, P = 0.005). These synapses were similar in a p p e a r a n c e and n u m b e r to those found in lactating rats~. Measurements of profiles of M N C s o m a t a from sa-
375 line-treated rats indicated increases in cell area of at least 50%, with an increase in percentage of direct neuronal soma-somatic membrane apposition from less than 0.05% of somal membrane in contact to 6.25% (U = 0, P = 0.001). The percentage of MNC somatic profiles in contact increased from 1.5% to 28.9% (U = 0, P = 0.001). This increased contact is due to withdrawal of glial processes from between the neurons. Rehydration for 5 days brought the amount of cell-cell membrane apposition back to normal, but the number of double synapses remained relatively unchanged. Rehydration for 14 days, however, as noted above was accompanied by a return of double synapse occurrence to normal values (Fig. 2). In a preliminary experiment using older (10-monthold) female rats, 10 days of saline treatment likewise increased the incidence of double synapses, although to a lesser degree, only 7.1% of the cells being contacted by these specialized synapses. Thus, double synapses increase in the SON of adult rats during chronic activation of the nucleus whether this activation be elicited by suckling (causing predominant oxytocin release) or by a dehydration stimulus in either male or female rats (releasing both oxytocin and vasopressin) 2. This indicates that (a) lactation-associated or sex-specific circulating hormones are not necessary for the new synaptic connection to form and (b) these double synapses are not necessarily related to patterns of activity seen during milk ejection. The new synapses as well as the increased cell-cell contact have been thought heretofore to be an adaptation specifically allowing more synchronization of oxytocinergic neuronsS,9. Notable here is the observation of decreased cell-cell apposition prior to a statistically significant decline in the percentage of MNCs contacted by double synapses. This indicates that a considerable amount ofglial reinsertion can occur upon reversal of the dehydrating conditions without altering the synaptic morphology. However, whether the glia ultimately participate in the removal of one or both of the synaptic specializations of the double synapses is not presently known. The axonal origin or the nature of the neurotransmitter(s) in the double synapses are not yet known.
Double synapses on SON somata in our material are 'symmetrical' and contain predominantly 30-40 nm clear, round vesicles with a few 90-100 nm dense core vesicles (Fig. 1). They do not show reaction product in rats treated with 5-hydroxydopamine, indicating that they do not contain catecholamines (unpublished observations). It is also not known whether the number of other types of synapses in SON, less conspicuous than the double synapses, also change in frequency of occurrence. In need of investigation is the identity of the hormone contained in the postsynaptic cells. The new synaptic contacts form concomitantly with a large increase in direct soma-somatic appositions and with the retraction of intervening glial processes. This glial retraction could put existing terminals, heretofore on one MNC, in a position to form a second synaptic thickening on a newly juxtaposed second MNC and, thus, become a double synapse. Axonal sprouting, therefore, might not be required for their formation. No degenerating synaptic terminals or obvious retraction bulbs have been seen during rehydration when synapse elimination occurs. Possible stimuli for the formation of these novel synapses could be from a number of sources including: (a) altered extracellular levels of chemical factors or ions released by rapidly firing presynaptic or postsynaptic neural elements; (b) glial withdrawal from between adjacent MNC somata exposing new, potentially postsynaptic membrane to existing synapses; (c) increased areas of receptive postsynaptic membrane brought about by MNC volume increases, or (d) some combination of these factors. Similar glial and synaptic plasticity as described here may occur elsewhere in the adult mammalian CNS under physiological conditions. The particular synaptic and neuronal cell arrangements of the SON have facilitated detecting such changes in the present study.
1 Hatton, G. I. and Tweedle, C. D., Magnocellularpeptidergic neurons in hypothalamus: increases in membrane apposition and number of specialized synapses from pregnancy to lactation, Brain Res. Bull., 8 (1982) 197-204.
2 Jones, C. W. and Pickering, B. T., Comparison of the effects of water deprivation and sodium chloride imbibition on the hormone content of the neurohypophysisof the rat, J. Physiol. (Lond. ), 203 (1969) 449-458.
Supported by N.I.H. Grant NS 09140. We thank P. Cobbett, W. M. Falls and L. Perlmutter for suggestions on an earlier version of the manuscript and B. Schmidt and J. Harper for typing assistance.
376 3 Mason, C. A. and Bern, H. A., Cellular biology of the neurosecretory neuron. In E. R. Kandel (Ed.), Handbook of Physiology, Vol. 1, Amer. Physiol. Soc., Bethesda, 1977, pp. 651-690. 4 Mosteller, F. and Tukey, J. W., Data Analysis and Regression, Addison-Wesley, Reading, MA, 1977. 5 Perlmutter, L. S., Tweedle, C. D. and Hatton, G. I., Neuronai/glial plasticity in the supraoptic dendritic zone: dendritic bundling and double synapse formation at parturition, Neuroscience, in press. 6 Perlmutter, L. S., Tweedle, C. D. and Hatton, G. I., Plasticity in supraoptic neurons: dendritic bundle formation in response to short-term dehydration and rehydration, Anat. Rec., 208 (1984) 136-137A. 7 Siegel, S., Non-parametric Statistics, McGraw-Hill, New York, NY, 1956.
8 Theodosis, D. T. and Poulain, D. A., Evidence for structural plasticity in the supraoptic nucleus of the rat hypothalamus in relation to gestation and lactation, Neuroscience, 11 (1983) 183-192. 9 Theodosis, D. T., Poulain, D. A. and Vincent, J.-D., Possible morphological bases for synchronization of neuronal firing in the rat supraoptic nucleus during lactation, Neuroscience, 6 (1981) 919-929. 10 Tweedle, C. D. and Hatton, G. I., Ultrastructurai comparisons of neurons of supraoptic and circularis nuclei in normal and dehydrated rats, Brain Res. Bull., 1 (1976) 103-118. 11 Tweedle, C. D. and Hatton, G. I., Ultrastructural changes in rat hypothalamus neurosecretory cells and their associated glia during minimal dehydration and rehydration, Cell Tiss. Res., 181 (1977) 59-72.