Rochelle S. Cohen

MAP5: A novel brain microtubule-associated protein under strong developmental regulation

SummaryA novel microtubule-associated protein, MAP5, is described, whose chemical properties and cytological distribution distinguish it from other known microtubule-associated proteins (MAPs). Its status as a MAP is indicated by the observations that (i) it co-assembles efficiently with microtubulesin vitro, (ii) it is localized on microtubules in brain sections by immunogold staining with monoclonal antibody against MAP5 and (iii) immunoaffinity purified MAP5 stimulates tubulin polymerization. Immunoperoxidase staining of brain sections showed that MAP5 is present in neurons throughout the brain and that in them it is evenly distributed throughout axons, dendrites and cell bodies. In this respect it differs from previously described MAP5 (1, 2, 3 and tau) which are differentially compartmentalized in brain neurons. MAP5 is not present in axon terminals, dendritic spines or other synaptic elements. It is present at substantially higher levels in neonatal brain than adult and it is more abundant than either MAP1 or MAP2a up to postnatal day 10. The fall in amount of MAP5, from juvenile to adult levels, is completed between postnatal days 10 and 20. This suggests that MAP5 is particularly important in modulating microtubule function during the formation of neuronal processes.

Immunocytochemical localization of actin in dendritic spines of the cerebral cortex using colloidal gold as a probe.

Summary1.Immunocytochemical localization of actin in rat cerebral cortex embedded in the resin LR White was performed using 5 nm colloidal gold as a probe. Antigenicity is maintained throughout the embedding procedure and the low electron opacity of LR White permits fine filamentous structures to be visualized. Control experiments included incubating the sections with normal goat serum or mouse IgG instead of the primary antibody, preadsorbing the antibody with actin from bovine muscle or liver acetone powder, and heat treating the primary antibody.2.Immunoreactive actin was identified primarily in dendritic spines, particularly in the postsynaptic density (PSD), the subsynaptic web, and the spine apparatus and endothelial and smooth muscle cells of blood vessels.3.Within dendritic spines, actin which is labeled in the PSD is in continuity with the filaments of the subsynaptic web. These filaments, in turn, are in continuity with the spine apparatus and/or the spine membranes adjacent to the PSD. The PSD may therefore function like other submembranous filamentous arrays which communicate events occurring at the membrane, in this case, the postsynaptic membrane, to the underlying cytoskeletal network, i.e., the subsynaptic web of the spine. It is also suggested that the actin present in the spine may play a role in changes in spine shape and synaptic curvature. Some actin was also seen in the presynaptic process in association with synaptic vesicles, the filamentous network that is contiguous with the synaptic vesicle membrane, and the presynaptic dense projections. Actin may be involved in dynamic processes in the presynaptic ending which include vesicle translocation.

Ultrastructure of neurons in the ventromedial nucleus or the hypothalamus in ovariectomized rats with or without estrogen treatment

SummaryThe fine structure of the ventrolateral and dorsomedial subdivisions of the ventromedial nucleus (VMN) of the hypothalamus was examined in ovariectomized/control and ovariectomized/estrogen-treated rats to compare neurons of these areas to other neurons (specifically the ventrolateral thalamus), and to determine the effects of estrogen on these cells. The neurons of the VMN contain a large nucleus with a prominent nucleolus, rough endoplasmic reticulum (RER), polysomes, a Golgi complex, coated, uncoated and dense-cored vesicles, lysosome-like bodies, inclusion bodies, multivesicular bodies, whorl bodies and myelin figures. Similar organelles were present in the neurons of the ventrolateral thalamus, although polysomes were more prominent, and the cells lacked dense-cored vesicles in the perikarya. Differences in the cells of the VMN between ovariectomized/control and ovariectomized/estrogen-treated rats included a more conspicuous stacking of the RER and greater number of dense-cored vesicles in the estrogen-treated group in both the ventrolateral and dorsomedial subdivisions. In both areas the differences were statistically significant, although more marked in the ventrolateral subdivision. In both VMN subdivisions, the increased stacking of the RER could be correlated with the greater number of dense-cored vesicles and may reflect increased biosynthesis of a secretory product.

Effects of estrogen treatment on expression of brain-derived neurotrophic factor and cAMP response element-binding protein expression and phosphorylation in rat amygdaloid and hippocampal structures.

Clinical studies indicate an effect of estrogen (E2) on affect and cognition, which may be mediated by the cAMP response element-binding protein (CREB) pathway and CREB-related gene target brain-derived neurotrophic factor (BDNF). We investigated the effect of E2 on CREB expression and phosphorylation and BDNF expression in the amygdala and hippocampus, areas involved in emotional processing. Ovariectomized rats were given 10 µg 17β-estradiol or vehicle for 14 days and expression of components of the CREB signaling pathway, i.e., CREB, phosphorylated CREB (pCREB), and BDNF in amygdala and hippocampus were investigated using immunogold labeling. Levels of BDNF mRNA were determined by in situ reverse-transcriptase polymerase chain reaction. We also examined the effect of E2 on calcium/calmodulin kinase (CaMK IV) immunolabeling in the hippocampus. E2 increased immunolabeling and mRNA levels of BDNF in the medial and basomedial amygdala and CA1 and CA3 regions of the hippocampus, but not in any other amygdaloid or hippocampal regions examined. E2 increased immunolabeling of CREB and pCREB in the medial and basomedial, but not central or basolateral amygdala. E2 also increased CaMK IV and pCREB immunolabeling in the CA1 and CA3 regions, but not CA2 region or dentate gyrus, of the hippocampus. There was no change in immunolabeling of CREB in any hippocampal region. These data identify a signaling pathway through which E2 increases BDNF expression that may underlie some actions of E2 on affective behavior and indicate neuroanatomical heterogeneity in the E2 effect within the amygdala and hippocampus.

Estrogen modulation of the cyclic AMP response element-binding protein pathway. Effects of long-term and acute treatments.

Actions of estrogen include mechanisms leading to alterations in gene transcription that may be independent of nuclear estrogen receptors, as well as those involving direct action of the estrogen receptor on the genome. Also, the influence of estrogen in the brain appears to extend well beyond areas associated with reproduction and may include forebrain areas linked to affective and cognitive behaviors. We investigated the effects of acute and long-term estradiol benzoate (E2) treatment on total and phosphorylated cyclic AMP responsive element-binding (CREB) protein levels and on cyclic AMP response element (CRE)-DNA binding in forebrain areas of ovariectomized (OVX) rats. Long-term E2 treatment increased CRE-DNA binding in the amygdala but not in hippocampus, frontal cortex, or cerebellum. The increase in CRE-DNA binding in the amygdala was associated with increased levels of total and phosphorylated CREB (pCREB) protein during protracted E2 exposure. To localize the estrogenic effect in the amygdala and determine if an effect in one hippocampal region was masked by a lack of effect in another subregion, we performed immunolabeling of pCREB in brain structures of chronically treated OVX animals with or without E2. This treatment resulted in a significant increase in relative total immunolabeled nuclei in the anteroventral subdivision of the medial amygdala. In the hippocampus, a significant increase in relative total immunolabeled nuclei was seen in the CA1 and CA3 regions, but not in the dentate gyrus or hilus of the dentate gyrus. Acute E2 treatment resulted in increased CRE-DNA binding in the frontal cortex but not in amygdala, hippocampus, or cerebellum. However, no changes in levels of total CREB or pCREB protein were observed in the frontal cortex under E2 treatment. No changes were observed either in basal or cAMP-stimulated protein kinase A (PKA) activity or in PKA-α catalytic subunit immunoreactivity in the amygdala or the frontal cortex. Our study indicates that both long-term and acute treatments with estrogens influence the function of CREB in specific brain structures.

Alteration by estrogen of the nucleoli in nerve cells of the rat hypothalamus

SummaryEstrogen is accumulated from the blood by nerve cells in the ventromedial nucleus of the hypothalamus and can facilitate female reproductive behavior by acting on this region of the brain. This cell group was examined in ovariectomized female rats, given estrogen or control treatment, by use of light and electron microscopy. A significantly greater portion of the nerve cells in the estrogen-treated animals had protuberances on their nucleolar surfaces, apparent under the light microscope. The fine structure of such protuberances included dense, aggregated material, which is shown to contain DNA by the sodium tungstate staining technique. Because increased numbers of such protuberances were found in nuclei of cells of the experimental group where previous studies demonstrated a significant increase in ultrastructural signs of biosynthetic activity, they may be associated with increased RNA synthesis. Thus, they could indicate, ultrastructurally, increased synthetic rates for RNA in nerve cells through which estrogen promotes reproductive behavior.

Ventromedial hypothalamic neurons in the mediation of long-lasting effects of estrogen on lordosis behavior

Abbreviations 423

Regulation of neuronal nitric oxide synthase mRNA in lordosis-relevant neurons of the ventromedial hypothalamus following short-term estrogen treatment

Short-term estrogenic regulation of neuronal nitric oxide synthase (nNOS) mRNA in the ventrolateral subdivision of the ventromedial nucleus (VLVMN), an area central to lordosis, was demonstrated using in situ hybridization. Estrogen-treated animals showed a significantly greater signal in the VLVMN, but not the arcuate or supraoptic nuclei, compared to ovariectomized controls. Neuronal NOS may be involved in early actions of estrogen in the VLVMN.

Glyceraldehyde-3-Phosphate Dehydrogenase Activity and F-Actin Associations in Synaptosomes and Postsynaptic Densities of Porcine Cerebral Cortex

Abstract1. Glyceraldehyde-3-phosphate dehydrogenase (G3PD) is a glycolytic enzyme that has also been implicated in a wide variety of functions within neurons. Because of the well-documented role of G3PD as an actin-binding protein, we sought evidence for a G3PD–actin complex in synaptosomes and postsynaptic densities (PSDs).2. We have shown G3PD association with 0.5-μm synaptosomal particles by immunofluorescence as similarly demonstrated for actin (Toh et al., Nature264:648–650, 1976). An immunoblot analysis also showed G3PD and actin to be enriched in synaptosomes. Further analysis of subcellular fractions from synaptosomes showed the PSD but not the synaptosomal plasma membranes to be enriched in G3PD and actin.3. Highest levels of G3PD catalytic activity were found in synaptosomes and PSDs. Although synaptosomes showed significant activity for phosphoglyceratekinase (PGK), an enzyme in sequence with G3PD for ATP production in the glycolytic pathway, no such activity was detected in the PSD fraction.4. Our studies indicate that a G3PD–actin complex may exist at the synapse. A physical association of G3PD with endogenous F-actin in synaptosomes and PSDs was demonstrated by combined phalloidin shift velocity sedimentation/immunoblot studies. By this approach, synaptosomal G3PD–actin complexes were also found to be significantly less dense than the PSD G3PD–actin complexes.5. G3PD and PGK catalytic activity in synaptosomes suggests a role in glycolysis, as well as actin binding, in the presynaptic terminals. On the other hand, the high levels of G3PD activity in PSDs but lack of PGK activity suggests that G3PD is involved in nonglycolytic functions, such as actin binding and actin filament network organization.

NADPH diaphorase activity and nitric oxide synthase immunoreactivity in lordosis-relevant neurons of the ventromedial hypothalamus

The distribution of the enzymes NADPH diaphorase and nitric oxide synthase in the ventromedial nucleus of the hypothalamus of cycling and ovariectomized/estrogen-treated and control female rats was demonstrated using histochemical and immunocytochemical methods. Serial section analysis of vibratome sections through the entire ventromedial nucleus showed that NADPH diaphorase cellular staining was localized primarily in the ventrolateral subdivision. NADPH diaphorase staining was visible in both neuronal perikarya and processes. Light microscopic immunocytochemistry using affinity-purified polyclonal antibodies to brain nitric oxide synthase revealed a similar pattern of labelling within the ventromedial nucleus and within neurons of the ventrolateral subdivision of the ventromedial nucleus. Control experiments involved omitting the primary antibodies; no labelling was visible under these conditions. Some, but not all, neurons in the ventrolateral subdivision of the ventromedial nucleus contained both NADPH diaphorase and brain nitric oxide synthase as demonstrated by co-localization of these two enzymes in individual cells of this area. That NADPH diaphorase and brain nitric oxide synthase were found in estrogen-binding cells was shown by co-localization of NADPH diaphorase and estrogen receptor and brain nitric oxide synthase and estrogen receptor at the light and ultrastructural levels, respectively. Our studies suggest that brain nitric oxide synthase is present and may be subject to estrogenic influences in lordosis-relevant neurons in the ventrolateral subdivision of the ventromedial nucleus. The hypothalamus is a primary subcortical regulatory center controlling sympathetic function. Therefore, not only is nitric oxide likely to be important for reproductive behavior, but also for the regulation of responses to emotional stress and other autonomic functions.