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Notes: Tunable Fabry–Perot modulators, consisting of strained InGaAs-GaAs multiple quantum well (MQW) layers, have been shown to yield a contrast over 1200:1. Tuning is achieved by varying the index of refraction of the MQW layers forming the cavity using a quantum confined Stark effect. The mirrors are realized by AlAs-GaAs quarter wave λ/4 dielectric stacks having 12 and 15.5 periods, respectively. The device has the potential of achieving even higher tunable contrast ratios when the number of periods of the λ/4 mirrors are increased. A contrast ratio of 6000:1 has been achieved for a nontunable structure. Measured data on optical transmission and contrast ratio are presented for various wavelengths as a function of applied bias. Results of simulation of transmitted output are also discussed.

Notes: Melatonin was recently identified as playing a role in fine-tuning the effects of gonadal steroids in the regulation of seasonal neuroplasticity within the telencephalic song control system of European starlings. The present study investigated possible seasonal regulation of melatonin receptors (MelR) within the starling song control system, in the presence or absence of gonadal steroids. Brains were sampled from photosensitive starlings exposed to short days, photostimulated starlings exposed to long days and photorefractory starlings also exposed to long days. Each condition contained a group of gonad-intact birds and a group of castrated birds. Melatonin receptor distribution was assessed in vitro by 125–iodomelatonin (IMEL) receptor autoradiography. In general, MelR distribution was similar to that described in other songbird species. However, there was a striking downregulation of MelR in the song control nucleus Area X of intact and castrated photostimulated birds on long days compared to their photorefractory counterparts on the same long days and to the short-day groups. Downregulation of MelR occurred independently of gonadal steroids. Nevertheless, superimposed on this general pattern of MelR downregulation during photostimulation, IMEL binding was observed in a medial subdivision of Area X when gonadal steroids were present. Downregulation of MelR in Area X during the short breeding season has implications for seasonal regulation of the song control system. Subsequent upregulation of MelR as birds become photorefractory, in the absence of any change in photoperiod, gonadal steroids or melatonin signal is the first description of photoperiod-independent regulation of MelR in adults of any vertebrate class.

Notes: In seasonally breeding songbirds, seasonal fluctuations occur in serum testosterone (T) concentrations and reproductive behaviours. Many T-dependent behaviours are regulated by the activity of androgenic and oestrogenic metabolites within specific brain regions. Male European starlings breed in spring when circulating T concentrations peak. T and its metabolites act within portions of the diencephalon to regulate the pituitary-gonadal axis and to activate courtship and copulation. Song in male starlings is critical for mate attraction during the breeding season and is regulated by steroid-sensitive nuclei in the telencephalon and diencephalon. Outside the breeding season, T is undetectable, however, males continue to sing at high levels. This suggests that singing outside of the breeding season might not be T-dependent as it appears to be in the spring. Alternatively, singing when T is low might continue to be regulated by T due to increased sensitivity of the brain to the action of the steroid. This increased sensitivity could be mediated by changes in intracellular T metabolism leading to increased production of active or decreased production of inactive metabolites. To explore the relationship between T-metabolism and reproductive behaviour, we analysed seasonal changes in the activity of four brain T-metabolizing enzymes: aromatase, 17β-hydroxysteroid dehydrogenase (17β-HSDH), 5α-reductase (all three convert T into active metabolites) and 5β-reductase (converts T into an inactive metabolite) in the diencephalon and telencephalon. In the anterior and posterior diencephalon, the highest aromatase was observed in spring when this region is critical for courtship and copulation. In the telencephalon, aromatase was highest and 5β-reductase was lowest throughout the winter months well prior to the reproductive season and these enzymes presumably maximize T-activity within this region. Although these data do not indicate whether the metabolic changes occur specifically within song nuclei, these findings are compatible with the idea that singing in male starlings outside the breeding season may be regulated by steroids despite the presence of low serum T concentrations. Overall, seasonal changes in T-metabolizing enzymes appear to play a significant role in seasonal changes in behaviour and reproductive physiology.

Notes: In songbirds, the initiation of song behaviour and the neural substrate of this system are highly influenced by gonadal steroids. Receptors for gonadal steroid hormones, such as androgens and oestrogens, have been localized within select nuclei of the song system. An important step in steroid receptor action is the recruitment of nuclear receptor coactivators. The coactivator, cAMP response element binding protein (CREB)-binding protein (CBP), has been implicated in both androgen and oestrogen receptor transactivation. Although the role of CBP in transcriptional mechanisms has been widely studied, little is known about CBP expression in the brain. The association between the distribution of CBP and oestrogen receptors in the hippocampus has been related to long-term memory. However, the distribution of brain CBP has not been related to the expression of gonadal steroid receptors in a system as relevant to reproductive behaviour as the avian song system. Western immunoblotting of European starling (Sturnus vulgaris) brain tissue reveals a band at 265 kDa. Immunohistochemical localization of CBP in starling brain indicates wide, but heterogeneous expression. CBP-immunoreactive (CBP-ir) cells define the boundaries of song control nuclei. In HVc (sometimes called the High Vocal Center) and the robust nucleus of the archistriatum (RA), there is a higher density of CBP-ir cells within the boundaries of these nuclei than in adjacent neostriatum or archistriatum, for HVc and RA, respectively. We also report that the distribution of CBP-ir cells varies among different nuclei within the song control system. CBP-ir cells within area X (also a part of the song system) and HVc are densely packed into clusters, whereas cells can be easily discriminated in RA. CBP is also highly expressed in hypothalamic areas, indicating that areas rich in steroid receptors also contain CBP. These data suggest that CBP is important for modulating transcriptional activities in the song system and other sites in the songbird brain that express gonadal steroid receptors.

Notes: Oestrogens derived from the neural aromatisation of testosterone play a key role in the activation of male sexual behaviour in many vertebrates. Besides their slow action on gene transcription mediated by the binding to nuclear receptors, oestrogens have now been recognised to have more rapid membrane-based effects on brain function. Rapid changes in aromatase activity, and hence in local oestrogen concentrations, could thus rapidly modulate behavioural responses. We previously demonstrated that calcium-dependent kinases are able to down-regulate aromatase activity after incubations of 10–15 min in phosphorylating conditions. In the present study, in quail hypothalamic homogenates, we show that Ca2+ or calmodulin alone can very rapidly change aromatase activity. Preincubation with 1 mM EGTA or with a monoclonal antibody raised against calmodulin immediately increased aromatase activity. The presence of calmodulin on aromatase purified by immunoprecipitation and electrophoresis was previously identified by western blot and two consensus binding sites for Ca2+-calmodulin are identified here on the deduced amino acid sequence of the quail brain aromatase. The rapid control of brain aromatase activity thus appears to include two mechanisms: (i) an immediate regulatory process that involves the Ca2+-calmodulin binding site and (ii) a somewhat slower phosphorylation by several protein kinases (PKC, PKA but also possibly Ca2+-calmodulin kinases) of the aromatase molecule.

Notes: Prairie voles (Microtus ochrogaster) typically stop breeding during winter. Male prairie voles respond to winter day lengths with gonadal regression, whereas female voles are relatively unresponsive to photoperiod. Unlike commonly studied laboratory rodents, female prairie voles do not exhibit spontaneous oestrous cycles. Instead, females are induced into oestrus by chemosensory cues from conspecific male urine. The present study investigated the interaction among day length, chemosensory cues and the initial brain responses during oestrus induction in female voles. A single drop of male conspecific urine, saline or skimmed milk was applied to the nares of female prairie voles housed for 9 weeks in either long (LD 16 : 8 h) or short (LD 8 : 16 h) days. Animals were killed 0.5, 1, 2 or 24 h after chemosensory treatment and their brains were processed for Fos immunocytochemistry. Body mass and ovarian fat pad mass were higher, but uterine and ovarian mass were lower, in short-day compared to long-day females. Regardless of photoperiod, Fos- immunoreactivity increased in the granule layer of the accessory olfactory bulb (AOB), the supraoptic nucleus and bed nucleus of the stria terminalis (BNST) (anterior medial) in females treated with male urine compared to the two control groups. Fos staining intensified in the AOB, medial and posterocortical medial amygdala and BNST (posterior ventral), 1 h and 2 h after urine treatment. In the medial preoptic area, anterior and lateral hypothalamus, and ventromedial nucleus of the hypothalamus, Fos-immunoreactivity was elevated in females 2 h after receiving urine. Overall, long-day females displayed higher Fos expression in response to urine than females maintained in short days. These results identify a putative neural circuitry of oestrus induction in this species, and provide an approximate time line of activation in the brain circuit responsible for oestrus induction in prairie voles.

Notes: Aromatization of testosterone into oestradiol plays a key role in the activation of male sexual behaviour in many vertebrate species. Rapid changes in brain aromatase activity have recently been identified and the resulting changes in local oestrogen bioavailability could modulate fast behavioural responses to oestrogens. In quail hypothalamic homogenates, aromatase activity is down-regulated within minutes by calcium-dependent phosphorylations in the presence of ATP, MgCl2 and CaCl2 (ATP/Mg/Ca). Three kinases (protein kinases A and C and calmodulin kinase; PKA, PKC and CAMK) are potentially implicated in this process. If kinases decrease aromatase activity in a reversible manner, then it would be expected that the enzymatic activity would increase and/or return to baseline levels in the presence of phosphatases. We showed previously that 0.1 mM vanadate (a general inhibitor of protein phosphatases) significantly decreases aromatase activity but specific protein phosphatases that could up-regulate aromatase activity have not been identified to date. The reversibility of aromatase activity inhibition by phosphorylations was investigated in the present study using alkaline and acid phosphatase (Alk and Ac PPase). Unexpectedly, Alk PPase inhibited aromatase activity in a dose-dependent manner in the presence, as well as in the absence, of ATP/Mg/Ca. By contrast, Ac PPase completely blocked the inhibitory effects of ATP/Mg/Ca on aromatase activity, even if it moderately inhibited aromatase activity in the absence of ATP/Mg/Ca. However, the addition of Ac PPase was unable to restore aromatase activity after it had been inhibited by exposure to ATP/Mg/Ca. Taken together, these data suggest that, amongst the 15 potential consensus phosphorylation sites identified on the quail aromatase sequence, some must be constitutively phosphorylated for the enzyme to be active whereas phosphorylation of the others is involved in the rapid inhibition of aromatase activity by the competitive effects of protein kinases and phosphatases. Two out of these 15 putative phosphorylation sites occur in an environment corresponding to the consensus sites for PKC, PKA (and possibly a CAMK) and, in all probability, represent the sites whose phosphorylation rapidly blocks enzyme activity.

Notes: Many actions of androgens require their conversion via the enzyme aromatase into oestrogens. Changes in brain aromatase activity are thought to take place via changes in enzyme concentration mediated by effects of sex steroids on aromatase transcription. These changes are relatively slow which fits in well with the fact that oestrogens are generally viewed as slow-acting messengers that act via changes in gene transcription. More recently, fast actions of oestrogens, presumably at the level of the cell membrane, have been described both in the female brain and in the male brain after the conversion of testosterone to oestradiol. It is difficult to reconcile the slow regulation of oestrogen synthesis (that occurs via changes in aromatase concentration) with a rapid action at the membrane level. Even if fast transduction mechanisms are available, this will not result in rapid changes in brain function if the availability of the ligand does not also change rapidly. Here, we report that aromatase activity in neural tissue of male Japanese quail (Coturnix japonica) is rapidly downregulated in the presence of Mg2+, Ca2+ and ATP in hypothalamic homogenates and in brain explants exposed to high Ca2+ levels following a K+-induced depolarization or the stimulation of glutamate receptors. The K+-induced inhibition of aromatase activity is observed within minutes and reversible. Given that aromatase is present in presynaptic boutons, it is possible that rapidly changing levels of locally produced oestrogen are available for nongenomic regulation of neuronal physiology in a manner more akin to the action of a neuropeptide than previously hypothesized.