Charles D. Tweedle

Ultrastructural changes in rat hypothalamic neurosecretory cells and their associated glia during minimal dehydration and rehydration

SummaryA quantitative ultrastructural study was performed to determine the changes in the neurosecretory neurons of the supraoptic (SON) and circularis (NC) nuclei following 4–24 h of water deprivation (WD) and subsequent rehydration (12 and 24 h). In both nuclei, the amount of direct soma-somatic contact increased throughout WD, apparently by retraction of fine glial processes from between the cells. Rehydration reversed these changes. The number of smaller (<1600 Å) neurosecretory granules (NSGs) decreased in both nuclei at 4 h of WD but returned to control levels by 24 h of WD and remained so during rehydration. Larger (<1600 Å) NSGs decreased in number at 4 h of WD in SON and then returned to control levels by 24 h of WD and remained the same throughout rehydration. In NC, these NSGs did not change in number with WD, but significantly increased between 12 and 24 h of rehydration. No cells with dilated rough endoplasmic reticulum were seen in NC during this study. In SON, however, the percentage of such cells increased at 4 and 12 h of dehydration only to decrease to control levels at 24 h of dehydration and throughout rehydration. Lysosomes decreased at 4 h of dehydration in SON and returned to control levels thereafter. In NC, lysosomes tended to decrease with dehydration and increase with rehydration. These findings indicate that detectable morphological changes take place in the course of alterations in hydration state that are well within the physiological range.

Ultrastructural comparisons of neurons of supraoptic and circularis nuclei in normal and dehydrated rats

A quantitative ultrastructural investigation was undertaken to compare the nucleus circularis (NC) and supraoptic nucleus (SON) of the rat both under normal and water-deprived conditions. NC was found to have dramatically more of its cells and membrane surface involved in direct soma-somatic contact than the SON. Water deprivation, even for one day, brought about a significant increase in both percentage of cells and membrane surface in contact in both nuclei, apparently by the retraction of fine glial processes from between the somata. The normal NC was made up of only one ultrastructurally identifiable cell type. The normal NC had no cells showing expanded endoplasmic reticulum, although these were seen following 5 days (but not 1 day) of water deprivation. The normal SON did have 4.4% of its cells showing expanded endoplasmic reticulum. This percentage significantly increased following water deprivation. The vesicle population per area of cytoplasm was very similar between the two normal nuclei. One day of water deprivation brought about a significant increase in less than 800 A vesicles in NC but not the SON. Five days of water deprivation resulted in a significant decrease in the lysosomal population per unit area in both nuclei. Vesicle changes have been discussed in relation to the volume changes in the cells.

Magnocellular neuropeptidergic neurons in hypothalamus: Increases in membrane apposition and number of specialized synapses from pregnancy to lactation

Morphological changes which have been hypothesized to accompany functional alterations in magnocellular neuropeptidergic cells (MNCs) were studied in female rats. Direct soma-somatic appositions between the MNC profiles of two nuclear groups, the supraoptic nucleus (SON) and nucleus circularis (NC) were investigated at the ultrastructural level in 4 groups of animals: virgin females, immediately pre-partum pregnant rats, post-partum and 14-day lactating animals. The percentage of SON MNC profiles in soma-somatic apposition and the amount of membrane in direct contact significantly increased over control levels by the last day of pregnancy. Further significant increases in these measures were observed in lactating rats. MNCs in NC showed steady gradual increases on these measures with significant differences from controls occurring in the post-partum group. The percentage of SON cell profiles with double synapses (i.e., presynaptic terminals making synaptic contact with two postsynaptic neurons) was significantly elevated in lactating rats (approximately 10%) over the next highest group (approximately 1% for post-partum rats). In NC, approximately 10% of the cell profiles sampled had such synapses but no differences among treatments occurred. The changes during late pregnancy suggest that close appositions may serve to enhance the metabolic activity of MNCs at a time when there is a build-up of stored oxytocin. Further increases in cell-cell contact and the addition of double synapses on possibly electrotonically coupled MNCs during lactation may serve a synchronizing function, particularly in the oxytocin cells participating in the milk ejection reflex.

Synapse formation and disappearance in adult rat supraoptic nucleus during different hydration states

Activation of the adult rat supraoptic nucleus by a chronic stimulus (10 days of drinking 2% NaCl 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.

Brain slice preparation: hypothalamus.

Methods, materials and procedures for producing viable hypothalamic slices are described in detail. Also described are the results of methodological experiments dealing with combatting the problem of evaporative water loss which produces subsequent increases in concentration of the bathing medium. A formula is given by which the amounts of evaporative loss may be calculated and compensated for without direct measurement of the medium osmotic pressure. Finally, ultrastructural data are presented which indicate that paraventricular nucleus neurosecretory cells in the slices undergo a loss of dense core vesicles during the initial 3 hr in vitro, then recover by 5 hr and maintain a relatively constant state for up to 9 hr, the longest time sampled.

Magnocellular neuropeptidergic terminals in neurohypophysis: Rapid glial release of enclosed axons during parturition

The ultrastructure of pituicytes as well as their relationship with neurosecretory axons were measured from the following groups of adult rats: virgin females; immediately pre-partum; post-partum; and following 14 days of lactation. Both parturition and lactation brought about a significant decrease in the number of pituicytes with neurosecretory axons completely surrounded by their cytoplasm. Since these two physiological states are each characterized by increased neurohypophysial hormone release, the data suggest that pituicyte enclosure of neurosecretory axons occurs mainly during conditions of low hormone release. Examination of pituicyte size and number of lipid inclusions, and the number of synaptoid contacts from neurosecretory axons onto pituicytes showed no differences among groups.

Neuronal/glial plasticity in the supraoptic dendritic zone in response to acute and chronic dehydration

The magnocellular neurosecretory cells of the supraoptic nucleus increase production and secretion of oxytocin and/or vasopressin in response to dehydration, gestation and lactation. Dynamic neuronal/glial interactions have also been shown to occur in response to these stimuli, resulting in a reversible increase in soma-somatic direct membrane apposition at these times. Chronic (lactation, 10 days of saline drinking) but not acute stimuli (4-24 h water deprivation) are further accompanied by the reversible formation of axo-somatic double synapses (one presynaptic terminal contacting two postsynaptic elements), which are virtually absent in control animals. The dendrites of these cells course ventrolaterally toward the ventral glial lamina, and have also been shown to be involved in this plasticity: dendro-dendritic direct membrane apposition and axo-somatic double synapses significantly vary with gestation and parturition. The present study investigated the dendritic zone response to both chronic and acute dehydration and rehydration. Increased dendro-dendritic membrane contacts resulted from both stimuli. Rehydration following acute dehydration resulted in a dose-dependent return to control levels, while rehydrated chronic dehydrates did not show such a return until 35 days of rehydration. The percentage of dendrites contacted by double synapses did not vary with treatment, and there were no sex differences. The recalcitrance on the part of the dendrites to return to normal following chronic dehydration may reflect a readiness to respond to renewed hormone demand.

Ultrastructure of neurons in the paraventricular nucleus of normal, dehydrated and rehydrated rats.

Previous reports have shown that the percentage of neuronal somatic membrane in soma-somatic apposition (without intervening glia) increased with brief periods of dehydration (4--24 hr) and decreased with rehydration in the rat supraoptic and circularis nuclei. In the present study, the percentage of somal membrane in soma-somatic appositions was found to increase in the primarily vasopressin-containing lateral portion of the rat paraventricular nucleus with twelve hours of dehydration. Further evidence for altered cellular function in this nucleus was a decrease in the number of smaller dense core vesicles (< 2600A) per unit cytoplasmic area during initial dehydration (4--12 hr). No changes were detected, however, in the number of larger dense core vesicles (> 4000 A) or lysosomes (> 4000 A) per unit cytoplasm. Intranuclear membrane-bound vacuoles were found primarily in hydrated and rehydrated animals. No reliable changes were seen in the dilation of granular endoplasmic reticulum. Cilia were found in the neuropil and were occasionally traced to magnocellular somata. Differences in the patterns of morphological responses among the magnocellular hypothalamic nuclei suggest specializations in their roles, and further support a functional significance of neuronal membrane appositions.

Reevaluation of the plasticity in the rat supraoptic nucleus after chronic dehydration using immunogold for oxytocin and vasopressin at the ultrastructural level

It has been shown that during physiological stimuli, such as dehydration, supraoptic nucleus (SON) neurons undergo profound morphological changes. However, little is known about how much each type of cell, oxytocin (OT) or vasopressin (VP), contributes to this plasticity during dehydration. Using postembedding immunogold cytochemistry for both OT and VP hormones at the electron microscopic level, we address this question. Rats were chronically dehydrated (given 2% saline to drink for 10 days) and their SON neurons were studied morphologically. The results were compared to control animals with free access to water. Both VP and OT somata showed an enlargement in size in dehydrated animals. Percentage of somasomatic/dendritic membrane contact increased significantly in both VP and OT neurons, with no significant changes in percentage of coverage of the cells by astrocytic membrane. Only the VP cells had a lesser amount of axosomatic membrane contact after dehydration, possibly due to an increase in cell size rather than a decrease in synaptic contact. Multiple synapses (MSs) (i.e., terminals that form more than one synapse with adjacent somata and or dendrites) occurred only between positively labeled cells and between negatively labeled cells, but not between positively and negatively labeled cells. The number of MSs per 100 microns OT somatic membrane or per 100 OT cells was significantly higher in dehydrated rats but was unchanged with regard to VP neurons. These findings indicate that both VP and OT neurons undergo morphological changes during chronic dehydration and, thus, that plasticity is not limited to OT cells as some earlier reports have suggested.

Horseradish peroxidase evidence for a spinal projection from the preoptic area of the goldfish, a light and electron microscopic study

Horseradish peroxidase (HRP) was applied to the transected spinal cord of goldfish labeled neurons in the preoptic area. Since leakage of HRP into the blood could produce the labeling of neurosecretory cells, intraperitoneal (i.p.) injections of HRP were made with a wide range of dosages in order to intentionally label preoptic neurosecretory cells. The distribution of preoptic neurons labeled after spinal HRP application was far more restricted than the labeling via uptake of HRP from the blood, even when cells in the spinal cord-transected fish were intensely labeled. Furthermore, in HRP electron microscopic material, morphological differences were observed between neurons labeled by the two procedures. Large numbers of dense core vesicles and well-developed stacks of rough endoplasmic reticulum, features typical of cells projecting to the pituitary, were not observed in cells labeled via the spinal cord. These findings indicate that in goldfish a direct projection exists from the preoptic area to the spinal cord which could be homologous to one arising from the paraventricular nucleus of mammals. Both i.p. injection and spinal transection also produced labeling of more caudal periventricular diencephalic cells which resemble preoptic cells in efferent projections as well as ultrastructural features.