Catherine S. Woolley

Gonadal steroids regulate dendritic spine density in hippocampal pyramidal cells in adulthood

Gonadal steroids are known to influence hippocampal physiology in adulthood. It is presently unknown whether gonadal steroids influence the morphology of hippocampal neurons in the adult intact rat brain. In order to determine whether female sex hormones influence hippocampal morphology in the intact adult, we performed Golgi impregnation on brains from ovariectomized rats and ovariectomized rats which received estradiol or estradiol and progesterone replacement. Removal of circulating gonadal steroids by ovariectomy of adult female rats resulted in a profound decrease in dendritic spine density in CA1 pyramidal cells of the hippocampus. Estradiol replacement prevented the observed decrease in dendritic spine density; progesterone augmented the effect of estradiol within a short time period (5 hr). Ovariectomy or gonadal steroid replacement did not affect spine density of CA3 pyramidal cells or granule cells of the dentate gyrus. These results demonstrate that gonadal steroids are necessary for the maintenance of normal adult CA1 hippocampal pyramidal cell structure. The short time course required to observe these effects (3 d for the estradiol effect and 5 hr for the progesterone effect) implies that CA1 pyramidal cell dendritic spine density may fluctuate during the normal (4–5 d) rat estrous cycle.

Differentiation of newly born neurons and glia in the dentate gyrus of the adult rat

In order to determine whether newly born cells in the dentate gyrus of the adult rat express the neuronal marker, neuron-specific enolase, or the glial marker, glial fibrillary acidic protein, we performed combined immunohistochemistry and autoradiography on brains from adult rats perfused at various times ranging from 1 h to four weeks following [3H]thymidine administration. Light-microscopic examination revealed a negligible number of [3H]thymidine-labeled cells showing neuron-specific enolase immunoreactivity during mitosis. However, by two weeks after [3H]thymidine administration, a significant increase in the density of [3H]thymidine-labeled neuron-specific enolase-immunoreactive cells was detected. Three weeks following [3H]thymidine injection the majority of [3H]thymidine-labeled cells (> 70%) were immunoreactive for the neuronal marker. At the four-week time-point, [3H]thymidine-labeled neuron-specific enolase-immunoreactive cells were indistinguishable from neighboring granule cells. In contrast, glial fibrillary acidic protein immunoreactivity was observed in a small but significant number of [3H]thymidine cells at the 1-h time-point and the proportion of labeled cells that were immunoreactive for this cell marker did not increase with time. [3H]Thymidine-labeled cells that were immunoreactive for glial fibrillary acidic protein typically showed morphologic characteristics of radial glia at all time-points. At the 1-h time-point, the majority of [3H]thymidine-labeled cells were observed in the hilus (> 60%) with the remainder being located in the granule cell layer. However, with a four-week survival-time most [3H]thymidine-labeled cells (> 85%) were located in the granule cell layer. The majority of newly born cells in the adult dentate gyrus differentiate into neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Exposure to excess glucocorticoids alters dendritic morphology of adult hippocampal pyramidal neurons

We have used Golgi-impregnated tissue to demonstrate that exposure to excess glucocorticoids alters dendritic morphology in a specific population of neurons in the adult rat hippocampus. Daily injection of 10 mg of corticosterone for 21 days resulted in decreased numbers of apical dendritic branch points and decreased total apical dendritic length measured in a 100-microns-thick section in CA3 pyramidal cells compared to sham-injected and non-injected controls. In contrast, no changes were observed in CA3 pyramidal cell basal dendritic morphology. Furthermore, no changes were observed in the dendritic morphology of CA1 pyramidal cells or granule cells of the dentate gyrus. Cross-sectional cell body area of any of the 3 cell types examined in this study was unaffected by corticosterone treatment. Finally, qualitative analysis of Nissl-stained tissue from the same brains revealed increased numbers of darkly staining, apparently shrunken CA3 pyramidal cells in corticosterone treated compared to control brains. The changes in dendritic morphology we have observed may be indicative of neurons in the early stages of degeneration, as prolonged exposure to high levels of corticosterone has been shown by others to result in a loss of CA3 pyramidal cells. Additionally, these results suggest possible structural alterations which may occur under physiological conditions in which corticosterone levels are chronically elevated such as in aged animals.

Short-term glucocorticoid manipulations affect neuronal morphology and survival in the adult dentate gyrus.

In order to determine whether short-term glucocorticoid manipulations influence the morphology and survival of neurons in the adult mammalian hippocampal formation, we performed quantitative analyses of Golgi-impregnated and Nissl-stained tissue from the brains of sham operated male rats, adrenalectomized male rats and adrenalectomized male rats which received corticosterone replacement. Three days after adrenalectomy, massive cell death, as detected by a dramatic increase in number of pyknotic cells, was observed in the granule cell layer of the dentate gyrus. By seven days following adrenalectomy, the numbers of pyknotic cells were even greater. Moreover, significant decreases in cross-sectional cell body area and numbers of dendritic branch points of Golgi-impregnated dentate gyrus granule cells were detected at seven days after adrenalectomy. Replacement of corticosterone to adrenalectomized rats prevented the appearance of large numbers of pyknotic cells as well as the decrease in granule cell cross-sectional cell body area and the numbers of dendritic branch points. In contrast, no obvious signs of degeneration were detected in the pyramidal cell layers of the CA1 and CA3 regions of the hippocampus at either three or seven days following adrenalectomy. In addition, no significant changes in morphological characteristics were observed in CA1 or CA3 pyramidal cells with adrenalectomy. These results show that dentate gyrus granule cells require glucocorticoids for their survival and for the maintenance of normal morphology and suggest that granule cell morphology and/or survival may undergo constant fluctuation in response to diurnal rhythms or stress-induced changes in glucocorticoid levels.

ESTRADIOL AND PROGESTERONE REGULATE NEURONAL STRUCTURE AND SYNAPTIC CONNECTIVITY IN ADULT AS WELL AS DEVELOPING BRAIN

Until recently, it has been widely believed that the adult brain does not undergo changes in its structure, particularly in relation to the actions of circulating hormones. It has now become clear that estradiol and progesterone have important effects on adult brain structure and function. Single section Golgi silver staining and electron microscopy have been used to analyze numbers of spines on dendrites and to count synapses on dendritic spines. In the adult female rat brain, we find that dendrites of neurons in the ventromedial hypothalamus and CA1 region of the hippocampus sprout increased numbers of spines on dendrites and then lose them during the 4- or 5-day estrous cycle. Increased spine numbers are accompanied by increased numbers of synapses on spines. In the hippocampus, the loss of spines and spine synapses occurs during a 24-h period between the time of maximum sexual receptivity on the day of proestrus and the next day, the day of estrus. This loss is not due solely to the decline in estradiol; however, giving progesterone speeds up the decline, and administering the antiprogestin, Ru486, on proestrus blocks the natural decline of synapse density. The changes of synaptic density in the hypothalamus are responsible, at least in part, for the cyclicity of sexual behavior, whereas the cyclicity of synapses in the hippocampus may subserve functions related to spatial learning and memory. In human subjects, cyclic fluctuations in gonadal hormones are associated with cyclic changes in performance on a variety of cognitive and motor tasks.

Estrogen increases synaptic connectivity between single presynaptic inputs and multiple postsynaptic CA1 pyramidal cells: A serial electron-microscopic study

Dendritic spines are sites of the vast majority of excitatory synaptic input to hippocampal CA1 pyramidal cells. Estrogen has been shown to increase the density of dendritic spines on CA1 pyramidal cell dendrites in adult female rats. In parallel with increased spine density, estrogen has been shown also to increase the number of spine synapses formed with multiple synapse boutons (MSBs). These findings suggest that estrogen-induced dendritic spines form synaptic contacts with preexisting presynaptic boutons, transforming some previously single synapse boutons (SSBs) into MSBs. The goal of the current study was to determine whether estrogen-induced MSBs form multiple synapses with the same or different postsynaptic cells. To quantify same-cell vs. different-cell MSBs, we filled individual CA1 pyramidal cells with biocytin and serially reconstructed dendrites and dendritic spines of the labeled cells, as well as presynaptic boutons in synaptic contact with labeled and unlabeled (i.e., different-cell) spines. We found that the overwhelming majority of MSBs in estrogen-treated animals form synapses with more than one postsynaptic cell. Thus, in addition to increasing the density of excitatory synaptic input to individual CA1 pyramidal cells, estrogen also increases the divergence of input from individual presynaptic boutons to multiple postsynaptic CA1 pyramidal cells. These findings suggest the formation of new synaptic connections between previously unconnected hippocampal neurons.

Gonadal Steroids Modify Dendritic Spine Density in Ventromedial Hypothalamic Neurons: A Golgi Study in the Adult Rat

Neurons in the adult rat ventromedial hypothalamic nucleus (VMN, 4-6 neurons per brain; 3-7 brains per group) were studied under various hormonal conditions using the single-section Golgi impregnation technique. Intact rats of both sexes and ovariectomized females treated with oil, estrogen or estrogen and progesterone were used. Golgi-impregnated neurons in the VMN were analyzed to determine possible differences in cell body size, number of primary dendrites, number of dendritic branch-points and spine density. The only significant differences found were in spine density. In the VMN of ovariectomized rats given oil, there were significantly fewer spines on primary dendrites than in either estrogen-treated, estrogen plus progesterone-treated or intact female rats. There were no differences between intact male and female rats observed in any parameter. In addition, when spine density of VMN neurons was assessed throughout the estrous cycle, it was determined that spine density was significantly lower at diestrus than proestrus.

Estradiol increases the frequency of multiple synapse boutons in the hippocampal CA1 region of the adult female rat

The effect of estradiol to increase the density of dendritic spines and axospinous synapses on hippocampal CA1 pyramidal cells in the adult female rat has been well‐documented. However, presynaptic involvement in this process of synapse elimination and formation in the adult is unknown. To address this issue, we have reconstructed 410 complete presynaptic boutons through coded serial electron micrographs of CA1 stratum radiatum to determine the: (1) frequency of multiple (MSB) vs. single (SSB) synapse boutons; (2) number of synaptic contacts per MSB; (3) bouton volume and surface area; and (4) types of spines in synaptic contact with MSBs and SSBs in ovariectomized, estradiol‐treated animals (OVX + E) versus ovariectomized oil‐treated controls (OVX + O). Quantitative analysis of this tissue revealed that, in OVX + E animals, 45.0% of presynaptic boutons form multiple synaptic contacts with dendritic spines compared to 27.3% in controls (P < 0.01); the average number of synapses per MSB was 2.7 in OVX + E animals compared to 2.3 in controls (P < 0.05). This represents a 25.5% increase in the number of synapses formed by a given number of presynaptic boutons in estradiol‐treated animals (P < 0.01) which largely accounts for the previously observed estradiol‐induced increase in axospinous synapse density. There was no treatment effect on bouton size; however, because MSBs are larger than SSBs, the increased frequency of MSBs in estradiol‐treated tissue results in a trend toward an estradiol‐induced increase in average bouton size. Additionally, MSBs were found to be more irregular in shape, i.e., significantly less spherical, than SSBs. Our results indicate that estradiol‐induced dendritic spines form synapses primarily with preexisting boutons in stratum radiatum and that these boutons enlarge and change shape as they accommodate new synapses. Such findings suggest a relatively active role for dendrites in the process of adult synapse formation.

Effects of aldosterone or RU28362 treatment on adrenalectomy-induced cell death in the dentate gyrus of the adult rat.

Previous studies have shown that granule cells of the adult dentate gyrus require adrenal steroids for their survival. In order to investigate whether activation of type I or type II adrenal steroid receptors can mediate granule cell survival, we have analyzed the density of pyknotic cells in the granule cell, CA1 and CA3 pyramidal cell layers in Nissl stained hippocampal sections from adult male rats which were either sham operated, adrenalectomized, or adrenalectomized and treated with aldosterone as a specific type I receptor agonist or RU28362 as a specific type II receptor agonist. Aldosterone treatment completely protected the dentate gyrus from adrenalectomy-induced cell death, while treatment with RU28362 resulted in only a partial protection against cell death in this region. These results indicate that type I adrenal steroid receptor activation is sufficient to protect against adrenalectomy-induced cell death.

Effects of estrogen in the CNS

Awareness of estrogens effects on neural function is broadening rapidly. Areas of recent progress include increased understanding of estrogen signaling through both genomic and nongenomic pathways, as well as the mechanisms by which estrogen can induce or maintain synapses and protect neurons from a variety of insults. Findings in these areas demonstrate a role for estrogen that goes beyond direct control of reproductive function.