Sex difference among nonneuronal cells precedes sexually dimorphic neuron growth and survival in an avian song control nucleus

In zebra finches only males sing, and several song control nuclei contain more neurons in adult males than in females. In the robust nucleus of the archistriatum (RA), this sex difference in neuron number arises because neuron survival is greater in young males than in females. The events initiating this sex difference in neuron survival are not known, but in earlier studies we observed that during sexual differentiation the proliferation and/or survival of RA cells exhibiting glial morphology is greater in males than in females. Because glia and glia-derived molecules are known to exert trophic effects on developing neurons, we wanted to determine when the sex difference in RA glia develops relative to the sexually dimorphic growth and survival of RA neurons. Male and female zebra finches were injected twice daily with ³[H]thymidine for 2 days beginning either on day 15 or 27. Two days later (day 18 or 30) sections through the RA were processed for autoradiography. Virtually all of the ³[H]thymidine labeled cells within the RA exhibited morphological features characteristic of glia and were not immunoreactive for the neuron-specific antigen, Hu. The number of these ³[H]thymidine labeled cells was measured, as were the number and soma size of RA neurons. Sex differences in RA neuron number and soma size were not evident at day 18, but emerged by day 30. However, at both ages the density of ³[H]thymidine labeled RA cells and their total number/RA neuron were significantly greater in males than in females. No such sexual dimorphism in the density of ³[H]thymidine labeled cells was evident in the archistriatum lateral to the RA, or within the RA of adult birds. These data indicate that sexually dimorphic gliogenesis is an early event in the sexual differentiation of the RA, preceding sex differences in RA neuron growth and survival. The possibility that glia (or glia-derived substances) may contribute to the neurotrophic effects of masculinization within the RA is discussed. © 1996 John Wiley & Sons, Inc.     

Developmental regulation of NMDA receptor 2B subunit mRNA and ifenprodil binding in the zebra finch anterior forebrain

In passerine songbirds, song learning often is restricted to an early sensitive period and requires the participation of several discrete regions within the anterior forebrain. Activation of N-methyl-D-aspartate (NMDA) receptors is implicated in song learning and in one forebrain song region, the lateral magnocellular nucleus of the anterior neostriatum (IMAN), NMDA receptors decrease in density, their affinity for the antagonist MK-801 increases, and their currents decay more quickly as young male zebra finches lose the ability to imitate new song elements. These developmental changes in NMDA receptor pharmacology and physiology suggest that the subunit composition of NMDA receptors changes developmentally. Here, we have used in situ hybridization and [3H]ifenprodil receptor autoradiography to study the developmental regulation of the NMDA receptor 2B subunit (NR2B) within the anterior forebrain of male zebra finches. NR2B mRNA expression within the IMAN was twice as great in 30-day-old males (early in the sensitive period for song learning) as in adult males, and this developmental decrease in NR2B mRNA expression was mirrored by a decrease in high-affinity (NR2B-associated) [3H]ifenprodil binding within this song region. In another anterior forebrain song region, Area X, NR2B mRNA also declined significantly after 30 days posthatch, but this decline was not accompanied by a significant decrease in [3H]ifenprodil binding. The results are consistent with the hypothesis that developmental changes in NMDA receptor function mediated by regulation of subunit composition contribute to the sensitive period for vocal learning in birds.     

Overlapping but distinct gene regulation profiles by glucocorticoids and progestins in human breast cancer cells

Glucocorticoids and progestins bind to receptors that share many structural and functional similarities, including virtually identical DNA recognition specificity. Nonetheless, the two hormones mediate very distinct biological functions. For example, progestins are associated with the incidence and progression of breast cancer, whereas glucocorticoids are growth suppressive in mammary cancer cells. To understand the mechanisms that engender biological specificity, it is necessary to identify genes that are differentially regulated by the two receptors. Here we employ Affymetrix oligonucleotide arrays to compare glucocorticoid- and progestin-regulated gene expression in a human breast cancer cell line. This global analysis reveals that the two hormones regulate overlapping but distinct sets of genes, including 31 genes that are differentially regulated. Surprisingly, the set of differentially regulated genes was almost as large as the set of genes regulated by both hormones. Examination of the set of differentially regulated genes suggests mechanisms behind the distinct growth effects of the two hormones in breast cancer. The differential regulation of four genes representing different regulatory patterns was confirmed by RT-PCR and Northern blot analyses. Treatment with cycloheximide or RU486 indicates that the regulation is a primary, receptor-mediated event. Detailed analyses of genes identified in these studies will furnish a mechanistic understanding of differential regulation by glucocorticoids and progestins.     

Estrogen accumulation in zebra finch song control nuclei: implications for sexual differentiation and adult activation of song behavior

In zebra finches the gonadal steroid estradiol (E2) directs the sexual differentiation of neural regions controlling song and synergizes with androgens to stimulate song in adulthood. To identify regions where E2 may act to exert these effects, steroid autoradiographic techniques were used to assess cellular accumulation of 3[H]-E2 or its metabolites within various nuclei of the zebra finch brain. In Experiment 1 we examined brains from juvenile females, still within the critical period for E2's effect on sexual differentiation. In Experiment 2 the pattern and extent of labeling in adult male brains was determined following injection of 3[H]-E2, 3[H]-testosterone, or 3[H]-dihydrotestosterone. The results suggest that, both during development and in adulthood, most song-control nuclei contain few E2-accumulating cells. In contrast, many cells densely labeled by 3[H]-E2 or its metabolites are present in the hypothalamus and in close proximity to one song-control region, the hyperstriatum ventralis pars caudalis (HVc). The distribution of these latter cells overlaps with cells that project to another song-related nucleus, Area X. Thus, in Experiment 3 fluorescent retrograde tracing and steroid autoradiographic techniques were combined to determine if E2-accumulating cells project to Area X in adult males. Although a few retrogradely labeled cells were lightly labeled by 3[H]-E2 or its metabolites, for the most part these appear to be two distinct populations of cells. The sparse accumulation of E2 in the zebra finch song system contrasts with that described in other song birds and has important implications as to the mechanism of E2 action on the developing and mature song system.     

Anatomical and synaptic substrates for avian song learning

In songbirds, vocal learning occurs during periods of major cellular and synaptic change. This neural reorganization includes massive synaptogenesis associated with the addition of new neurons into the vocal motor pathway, as well as pruning of connections between song regions. These observations, coupled with behavioral evidence that song development requires NMDA receptor activation in specific song nuclei, suggest that experiences associated with vocal learning are encoded by activity driven, Hebbianlike processes of synaptic change akin to those implicated in many other forms of developmental plasticity and learning. In this review we discuss the hypothesis that develpmental and/or seasonal changes in NMDA receptor function and the availability of new synapses may modulate thresholds for plasticity and thereby define sensitive periods for vocal learning. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 532–548, 1997     

Cytoplasmic filaments and cellular wound healing in amoeba proteus

The flexibility and self-healing properties of animal cell surface membranes are well known. These properties have been best exploited in various micrurgical studies on living cells (2, 3), especially in amoebae (7, 20). During nuclear transplantation in amoebae, the hole in the membrane through which a nucleus passes can have a diameter of 20-30 μm, and yet such holes are quickly sealed, although some cytoplasm usually escapes during the transfer. While enucleating amoebae in previous studies, we found that if a very small portion of a nucleus was pushed through the membrane and exposed to the external medium, the amoeba expelled such a nucleus on its own accord. When this happened, a new membrane appeared to form around the embedded portion of the nucleus and no visible loss of cytoplasm occurred during nuclear extrusion. In the present study, we examined amoebae that were at different stages of expelling partially exposed nuclei, to follow the sequence of events during the apparent new membrane formation. Unexpectedly, we found that a new membrane is not formed around the nucleus from inside but a hole is sealed primarily by a constriction of the existing membrane, and that cytoplasmic filaments are responsible for the prevention of the loss of cytoplasm.