Effects of neonatal treatment with valproic acid on vasopressin immunoreactivity and olfactory behaviour in mice.

Recent findings demonstrate that epigenetic modifications are required for the sexual differentiation of the brain. For example, neonatal administration of the histone deacetylase inhibitor, valproic acid, blocks masculinisation of cell number in the principal nucleus of the bed nucleus of the stria terminalis (BNST). In the present study, we examined the effects of valproic acid on neurochemistry and behaviour, focusing on traits that are sexually dimorphic and linked to the BNST. Newborn mice were treated with saline or valproic acid and the effect on vasopressin immunoreactivity and olfactory preference behaviour was examined in adulthood. As expected, males had more vasopressin immunoreactive fibres than females in the lateral septum and medial dorsal thalamus, which are two projection sites of BNST vasopressin neurones. Neonatal valproic acid increased vasopressin fibre density specifically in females in the lateral septum, thereby reducing the sex difference, and increased vasopressin fibres in both sexes in the medial dorsal thalamus. The effects were not specific to BNST vasopressin projections, however, because valproic acid also significantly increased vasopressin immunoreactivity in the anterior hypothalamic area in both sexes. Subtle sex-specific effects of neonatal valproic acid treatment were observed on olfactory behaviour. As predicted, males showed a preference for investigating female-soiled bedding, whereas females showed a preference for male-soiled bedding. Valproic acid did not significantly alter olfactory preference, per se, although it increased the number of visits females made to female-soiled bedding and the overall time females spent investigating soiled versus clean bedding. Taken together, these results suggest that a transient disruption of histone deacetylation at birth does not have generalised effects on sexual differentiation, although it does produce lasting effects on brain neurochemistry and behaviour.

Absence of progestin receptors alters distribution of vasopressin fibers but not sexual differentiation of vasopressin system in mice.

Perinatal estrogens increase the number of vasopressin-expressing cells and the density of vasopressin-immunoreactive fibers observed in adult male rodents. The mechanism of action of estrogens on sexual differentiation of the extra-hypothalamic vasopressin system is unknown. We hypothesized that the sexually dimorphic expression of progestin receptors (PRs) during development would masculinize vasopressin expression in mice. We compared the number of vasopressin-expressing cells in the bed nucleus of the stria terminalis (BNST) and medial amygdala and the density of vasopressin-immunoreactive fibers in several brain regions of male and female wild type and PRKO mice using in situ hybridization and immunohistochemistry. As expected, sex differences in vasopressin cell number were observed in the BNST and medial amygdaloid nucleus. Vasopressin-immunoreactive fiber density was sexually dimorphic in the lateral septum, lateral habenular nucleus, medial amygdaloid nucleus, and mediodorsal thalamus. Sex differences were also observed in the principal nucleus of the BNST and medial preoptic area but not in the dorsomedial hypothalamus, which are thought to receive vasopressin innervation from the suprachiasmatic nucleus. Deletion of PRs did not alter the sex difference in vasopressin mRNA expression and vasopressin fiber immunoreactivity in any area examined. However, deletion of PRs increased the density of vasopressin fiber immunoreactivity in the lateral habenular nucleus. Our data suggest that PRs modulate vasopressin levels, but not sexual differentiation of vasopressin innervation in mice.

Conformational changes in the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum detected using phosphorescence polarization.

The technique of time-averaged phosphorescence has been used to study the interaction of calcium ions and ATP with the (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum vesicles. The presence of excess calcium ions was found to cause a 20% decrease in the phosphorescence emission anisotropy. This is interpreted as being due to a conformational change in the protein and is supported by data from time-resolved phosphorescence measurements which also show a lowering of the anisotropy. This change in the decay of the emission anisotropy is associated with only minor changes in the rotational relaxation time of the protein and is again suggestive of a conformational change in the protein. In some cases ATP was also observed to lower the time-averaged phosphorescence anisotropy possibly via an interaction with the low-affinity regulatory site of the protein.

Rotational diffusion of calcium-dependent adenosine-5'-triphosphatase in sarcoplasmic reticulum: a detailed study.

The Ca2+-Mg2+ adenosine-5'-triphosphatase (ATPase) in sarcoplasmic reticulum has been covalently labeled with the phosphorescent triplet probe erythrosinyl 5-isothiocyanate. The rotational diffusion of the protein in the membrane at 25 degrees C was examined by measuring the time dependence of the phosphorescence emission anisotropy. Detailed analysis of both the total emission S(t) = Iv(t) + 2IH(t) and anisotropy R(t) = [Iv(t) - IH(t)]/[Iv(t) + 2IH(t)] curves shows the presence of multiple components. The latter is incompatible with a simple model of protein movement. The experimental data are consistent with a model in which the sum of four exponential components defines the phosphorescence decay. The anisotropy decay corresponds to a model in which the phosphor itself or a small phosphor-bearing segment reorients on a sub-microsecond time scale about an axis attached to a larger segment, which in turn reorients on a time scale of a few microseconds about an axis fixed in the frame of the ATPase. A fraction of the protein molecules rotate on a time scale of 100-200 microseconds about the normal to the bilayer, while the rest are rotationally stationary, at least on a sub-millisecond time scale.

Monitoring membrane protein rotational diffusion using time-averaged phosphorescence.

Rotational motions of membrane proteins have previously been measured using time-dependent phosphorescence techniques. This paper discusses a method of examining membrane protein mobility at temperatures relevant to biological systems, using a technique similar to steady-state fluorescence. The method is demonstrated using sarcoplasmic reticulum ATPase labelled with erythrosin isothiocyanate, both in its natural condition and crosslinked by incubation with glutaraldehyde. The experimentally-observed dependence of phosphorescence anisotropy on temperature is compared to a calculated anisotropy-temperature curve. Comparison is made between the anisotropy decay curves obtained by time-averaged phosphorescence and steady-state fluorescence.