Involvement of protein kinase-C in the mitogenic effect of insulin-like growth factor-I on rat astrocytes.

Insulin-like growth factor-I (IGF-I) stimulates the proliferation of many cell types, including astrocytes. Astrocytes are a population of brain cells highly enriched in IGF-I receptors, which unlike neurons, retain the ability to proliferate in the adult brain. Although astrocyte proliferation in response to IGF-I is well documented, the intracellular mechanisms that mediate this phenomenon are poorly defined. Interestingly, activation of protein kinase-C (PKC) by IGF-I has been observed in several cell types. In this report we first characterized the mitogenic effects of IGF-I on highly purified type I rat astrocyte cultures. Next, we determined whether IGF-I activates PKC in our cultures. Finally, since astrocyte proliferation is stimulated by both IGF-I and the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA), we decided to test the possible involvement of PKC in the mitogenic activity of IGF-I on astrocytes. IGF-I stimulated the DNA synthesis rate in rat astrocytes. Analysis of the time course revealed that IGF-I (10 nM) induces maximal stimulation of [3H]thymidine incorporation (a 4-fold increase) 16-18 h after exposure. TPA also stimulated mitogenesis in our cultures. The dose-response of [3H]thymidine incorporation induced by IGF-I and TPA indicated that 10 nM was the lowest concentration producing a maximal effect for both agents. Analysis of proteins by Western blot revealed that 10 nM IGF-I translocates PKC(alpha), the predominant PKC isoform in astrocyte cultures, from the cytosol to the membrane fraction within 20 min. A similar activation of PKC was achieved with 100 nM TPA. When astrocytes were exposed to IGF-I (10 nM) and TPA (10 nM) in combination, [3H]thymidine uptake was significantly higher than the uptake induced by either IGF-I (10 nM) or TPA (10 nM) alone. However, the effect of IGF-I plus TPA was not fully additive. In a second experiment, the mitogenic effect of IGF-I was partially abolished in cells depleted of PKC by preincubation with high concentrations of TPA (300 nM). Finally, incubation of astrocytes with the PKC inhibitor H-7 at 20 microM, a concentration that completely blocked the mitogenic action of TPA, only reduced the ability of IGF-I to stimulate DNA synthesis by 50%. In summary, our results demonstrate that IGF-I can rapidly activate PKC in astrocytes, and that PKC activation is involved in the mitogenic effect of IGF-I on these cells. However, we conclude that IGF-I also stimulates astrocyte proliferation through PKC-independent pathways.

Gap junctions in the hypothalamic arcuate neurons of ovariectomized and estradiol-treated rats.

Freeze-fracture methodology was used to study the organization of the neuronal plasma membrane in the rat arcuate nucleus, an estrogen sensitive area of the hypothalamus. Freeze-fracture replicas were prepared from 6 adult ovariectomized rats injected with a single dose of 17 beta-estradiol and from 6 ovariectomized littermates injected with vehicle. Rats were sacrificed 2 days after the injection. Occasional gap junctions were observed in freeze-fractured neuronal membranes from both groups of animals and their incidence was increased (P less than 0.01) in estradiol treated rats. This study demonstrates gap junctions in arcuate neurons and suggests that these structures may be affected by gonadal hormones.

Sex differences in plasma membrane concanavalin A binding in the rat arcuate neurons.

Previous studies have shown that synaptic connections and organization of neuronal membranes are sexually dimorphic in the arcuate nucleus of developing and adult rats. These sex differences can be abolished by the perinatal androgenization of females. In this study the label-fracture method of Pinto da Silva and Kan was used in order to determine whether membrane sex differences are related to the glycoconjugates in neuronal plasma membranes. Six Sprague-Dawley female rats treated with testosterone on the day of birth, six control females injected with vehicle and six intact males were studied when they were 100 days old. The arcuate nucleus was dissected and incubated for 2 hours in a solution of 0.25 mg/ml concanavalin A, washed in buffer and incubated for 3 hours in a suspension of horseradish peroxidase-coated colloidal gold. Then, freeze-fracture replicas of the arcuate nucleus were prepared. Colloidal gold labeling was observed to be codistributed with intramembrane particles in the outer membrane face of the neuronal perikaryal plasma membrane. The numerical density of small (less than 10 nm) intramembrane particles and colloidal gold particles was significantly greater in control female membranes when compared to males or to androgenized females. The labeling was significantly reduced when the arcuate nucleus was incubated with concanavalin A in presence of 0.5 M methyl-alpha-manopyranoside. These findings indicate a sex difference in the density and distribution of glycoconjugates and intramembranous particles in the neuronal plasma membrane that is dependent on the perinatal levels of sex steroids and is concordant with, and could be the cause of, sex differences in the pattern of synaptic contacts.

Synaptic remodeling in the rat arcuate nucleus during the estrous cycle.

Adult female rats showing regular vaginal cycles were studied in order to determine the number of axosomatic synapses in thin sections of the arcuate nucleus. The number of synapses per length of perikaryal membrane was significantly decreased in estrus, compared to other days of the estrous cycle (P less than 0.05). The reduction in the number of synapses in estrus was accompanied by a decrease in the percentage of the average length of perikaryal membrane covered by presynaptic terminals and by an increase in the percentage of membrane in close apposition of glial processes. Since the average perikaryal perimeter was not significantly changed during the estrous cycle, these results indicate a net decrease in the number of arcuate nucleus axosomatic synapses between proestrus and estrus, with a reinnervation of arcuate neurons between estrus and metestrus. These results suggest that there is a physiological synaptic turnover in the arcuate nucleus of the rat during the estrous cycle.

Sexual differentiation of the neuronal plasma membrane: neonatal levels of sex steroids modulate the number of exo-endocytotic images in the developing rat arcuate neurons.

Exo-endocytotic images and intramembrane protein particles (IMP) were quantitatively assessed in freeze-fracture replicas from the plasma membrane of arcuate neurons of rats aged 0 (newborns), 10, 20 and 100 days postpartum. Membranes contained significantly (P less than 0.02) more IMPs in females than in males. Exo-endocytotic images were increased in newborn and 10-day-old males when compared to adult males or to developing females (48 +/- 6 vs 6 +/- 1 images/100 micron 2 in 10-day-old male and female rats, respectively). Androgenization of females with a single injection of testosterone propionate on the day of birth resulted in an increased number of exo-endocytotic images in developing animals (75 +/- 9 images/100 micron 2, 10-day-old rats) and in the abolishment of the sex differences in the number of IMPs.

The distribution of glial fibrillary acidic protein in the adult rat brain is influenced by the neonatal levels of sex steroids.

Sex steroids during the perinatal period are able to modify the postnatal development of neurons within steroid-sensitive areas in the rat brain. This study was designed to test the possible influence of the early postnatal levels of sex steroids on the morphology of the astrocytes. The experimental manipulation of the neonatal levels of sex steroids was performed by the androgenization of females with a single injection of testosterone propionate and by the orchidectomy of males on the day of birth. Control females received a single injection of vehicle and control males were sham operated. All the animals were sacrificed at 3 months of age postnatally. The immunohistochemical distribution of the glial fibrillary acidic protein (GFAP), a marker of astrocytic filaments, was studied on coronal sections of the dorsal hippocampus, the globus pallidus and the hypothalamic arcuate nucleus. The number of GFAP immunoreactive cells, the number of GFAP immunoreactive primary processes per cell and the surface density of the GFAP immunoreactive material were evaluated. This morphometric evaluation revealed a decreased surface density of GFAP immunoreactive material in the hippocampus, globus pallidus and the ventral part of the arcuate nucleus of orchidectomized males when compared to control males. Sex differences in the distribution of GFAP immunoreactivity were detected in the hippocampus and globus pallidus. These differences were abolished by the androgenization of females. The number of GFAP immunoreactive cells was similar in all the experimental groups, indicating that the differences in surface density represent an effect of sex steroids on the growth of astrocytic processes rather than on the proliferation of astrocytes.

Neuronal membrane remodelling during the oestrus cycle: a freeze-fracture study in the arcuate nucleus of the rat hypothalamus.

Freeze-fracture methodology was used to study the organization of the neuronal plasma membrane in the rat arcuate nucleus, an oestrogen sensitive area of the hypothalamus. The quantitative evaluation of freeze-fracture replicas of the perikarya, dendritic shafts and dendritic spines revealed that the numerical density of intramembranous particles varied during the ovarian cycle. The number of small (less than 10 nm) particles was decreased, and the number of large (greater than 10 nm) particles was increased, in the P-face of the perikaryal plasma membranes in prooestrus and oestrus when compared to dioestrus. This change was associated with a significant increase in the number of exo-endocytotic images in the perikaryal plasma membrane in prooestrus. Changes in intramembranous particles during the ovarian cycle were not detected in arcuate nucleus dendritic shafts and dendritic spines.

Estradiol--induced redistribution of glial fibrillary acidic protein immunoreactivity in the rat brain.

The immunohistochemical distribution of the glial fibrillary acidic protein (GFAP), a marker of glial filaments, was studied on coronal sections of the globus pallidus, the area CA4 of the hippocampus and the arcuate nucleus of the hypothalamus, 3 estrogen-sensitive areas of the rat brain. The number and the surface density of the GFAP-immunoreactive cells were evaluated in 6 adult ovariectomized rats injected with a single dose (20 mg/kg) of estradiol valerate (OVX + E2 rats) and in 6 ovariectomized littermates injected with vehicle (OVX rats). Two days after the injection, a similar distribution of the GFAP was observed in the arcuate nucleus of OVX + E2 rats when compared to OVX rats, whereas a significantly (P less than 0.001) increased surface density of GFAP immunoreactive material was observed in the globus pallidus and hippocampus of estradiol-treated rats. Since the number of GFAP-positive cells was unchanged by the estradiol injection, the enhanced surface density of GFAP immunoreactive material in the hippocampus and globus pallidus suggest a possible influence of estradiol on GFAP-immunoreactive glial processes.