Quail Brain Research Articles

Distribution of a novel avian gonadotropin-inhibitory hormone in the quail brain.

We recently identified a novel hypothalamic neuropeptide inhibiting gonadotropin release in the quail brain and termed it gonadotropin inhibitory hormone (GnIH). In this study, we investigated the localization and distribution of GnIH in both sexes of adult quails by immunohistochemistry with a specific antiserum against GnIH and in situ hybridization. Quantitative analysis demonstrated that the concentration of GnIH in the diencephalon was greater than that in the mesencephalon without sex difference. GnIH concentrations in the cerebrum and cerebellum were below the level of detectability. Clusters of GnIH-like immunoreactive (GnlH-ir) cell bodies were localized in the paraventricular nucleus (PVN) of the hypothalamus. There was no significant difference in the number of GnlH-ir cells in the PVN between males and females. By double immunostaining with antisera reacting with GnIH or avian posterior pituitary hormones (vasotocin and mesotocin), GnIH-ir cells were found to be parvocellular neurons in the ventral portion of PVN, which showed no immunoreaction with the antisera against vasotocin and mesotocin. In situ hybridization revealed the cellular localization of GnIH mRNA in the PVN. GnIH-ir nerve fibers were however widely distributed in the diencephalic and mesencephalic regions. Dense networks of immunoreactive fibers were found in the ventral paleostriatum, septal area, preoptic area, hypothalamus, and optic tectum. The most prominent fibers were seen in the median eminence of the hypothalamus and the dorsal motor nucleus of the vagus in the medulla oblongata. Thus, GnIH may participate not only in neuroendocrine functions, but also in behavioral and autonomic mechanisms.

Anatomical relationships between aromatase-immunoreactive neurons and nitric oxide synthase as evidenced by NOS immunohistochemistry or NADPH diaphorase histochemistry in the quail forebrain

In Japanese quail ( Coturnix japonica), previous studies indicated that the distribution of reduced nicotinamide dinucleotide phosphate (NADPH) diaphorase overlaps with steroid-sensitive areas that contain dense populations of aromatase-immunoreactive (ARO-ir) cells. We investigated here the anatomical relationships between aromatase (ARO) and nitric oxide synthase (NOS)-containing cells that were visualized both by NOS-immunohistochemistry and NADPH-histochemistry. The distribution of ARO-ir and of NADPH-positive cells in the forebrain observed here matched exactly the distribution previously reported. The distribution of NOS-immunoreactive material in the vicinity of ARO-ir cell groups appeared similar to the distribution of NADPH-positive structures previously identified by histochemistry. The number of NOS-immunoreactive cells was similar to the number of NADPH-positive cells and they were found in the same brain regions. In contrast, few NOS-immunoreactive fibers were observed whereas numerous NADPH-positive fibers and punctuate structures were present in many areas. Major groups of NOS-immunoreactive/NADPH-positive neurons were adjacent to the main ARO-ir cell groups, such as the medial preoptic nucleus, the bed nucleus of the stria terminalis and the nucleus ventromedialis hypothalamic. However, examination of adjacent sections indicated that there is very little overlap between the NOS-immunoreactive and ARO-ir cell populations. This notion got further support by double-labeled sections where no double-labeled cells could be identified. In sections stained simultaneously by histochemistry for NADPH and immunohistochemistry for ARO, many NADPH-positive fibers and punctate structures were closely associated with ARO-ir perikarya. Taken together, the present data indicate that NOS is not or very rarely colocalized with ARO but that NOS inputs are closely associated with ARO-ir cells. Based on previous work in a variety of model systems, it can be hypothesized that these inputs modulate the expression or activity of ARO in the quail brain.

Androgen biosynthesis in the quail brain

We have demonstrated that the quail brain possesses the cholesterol side-chain cleavage enzyme (cytochrome P450scc) and 3β-hydroxysteroid dehydrogenase/Δ 5-Δ 4-isomerase (3β-HSD) and produces pregnenolone, pregnenolone sulfate and progesterone from cholesterol. We have also demonstrated the expression of cytochrome P450 17α-hydroxylase/c17,20-lyase ( P450 17α,lyase) and the conversion of progesterone to 17α-hydroxyprogesterone in the same avian species. Therefore, the present study was conducted to investigate androgen biosynthesis from progesterone in the avian brain. Employing biochemical techniques combined with HPLC and TLC analyses, the conversion of progesterone to androstenedione, an androgen precursor, was found in quail brain. The present biochemical analysis further revealed the conversion of androstenedione to testosterone, indicating the presence of 17β-hydroxysteroid dehydrogenase (17β-HSD) in the quail brain. The formation of testosterone from progesterone was also detected in the brain. Testosterone formation was more intense in the diencephalon, whereas the concentration of endogenous testosterone in the diencephalon was lower than those in other brain regions in castrated quails. However, the concentration of endogenous estradiol, a metabolite of testosterone by cytochrome P450arom, was highest in the diencephalon of castrated quails. These results suggest that testosterone biosynthesis occurs in the quail brain, in particular the diencephalon. Testosterone may subsequently be converted to estradiol.

Antioxidant systems of the developing quail embryo are compromised by mycotoxin aurofusarin

The effects of aurofusarin in the quail diet on the antioxidant systems of the developing embryo are investigated. Thirty eight 45-day-old Japanese quails ( Coturnix japonica) were divided into two groups and were fed on a corn-soya diet or the same diet supplemented with aurofusarin at the level of 26.4 mg/kg feed in the form of Fusarium graminearum culture enriched with aurofusarin. Eggs obtained after 7 weeks of feeding were incubated. Samples of quail tissues were collected at day 17 of embryonic development and from day old hatchlings. Antioxidants and malondialdehyde were analysed by HPLC-based methods. Inclusion of aurofusarin in the maternal diet was associated with decreased concentrations of α- and γ-tocopherols, α- and γ-tocotrienols, retinol, lutein and zeaxanthin in egg yolk. The vitamin E (tocopherols and tocotrienols) concentration in the liver and yolk sac membrane (YSM) of the day 17 embryos and the hatchlings from aurofusarin-fed group was significantly decreased. Alpha-tocopherol concentration was also reduced in kidney, lung, heart, muscle and brain of day-old quails. In the liver of day-old quails, concentrations of lutein, zeaxanthin, retinol, retinyl linoleate, retinyl oleate, retinyl palmitate and retinyl stearate were also reduced. As a result of these diminished antioxidant concentrations, tissue susceptibility to lipid peroxidation was significantly increased. It is suggested that a compromised antioxidant system of the egg yolk and embryonic tissues could predispose quails to increased mortality at late stages of their embryonic development.

Purification of soluble acetylcholinesterase from Japanese quail brain by affinity chromatography

The purification of a soluble acetylcholinesterase from Japanese quail brain using affinity chromatography on concanavalin A–Sepharose and edrophonium–Sepharose is described. The affinity matrix was synthesized by coupling an inhibitor edrophonium to epoxy-activated Sepharose. Acetylcholinesterase was purified 10 416-fold with a specific activity of 2500 U/mg protein. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol gave only one band with a molecular weight of 62.5 kDa. The molecular weight of the purified acetylcholinesterase was estimated to be 245.5 kDa by gel chromatography on Sephacryl S-200 under nondenaturing conditions. Based on the molecular weight obtained by both SDS-PAGE and gel filtration the purified acetylcholinesterase was assumed to be a tetrameric form.

Neuroectodermal origin of brain pericytes and vascular smooth muscle cells.

The origin of vascular pericytes (PCs) and smooth muscle cells (vSMCs) in the brain has hitherto remained an open question. In the present study, we used the quail-chick chimerization technique to elucidate the lineage of cranial PCs/vSMCs. We transplanted complete halves of brain anlagen, or dorsal (presumptive neural crest [NC]) or ventral cranial neural tube. Additional experiments included transplantations of neuroectoderm into limb mesenchyme, and of head mesoderm or limb mesenchyme into paraxial head mesoderm. After interspecific transplantation of quail brain rudiment, graft-derived vSMCs were found in the vessel walls of the grafted brain. Notably, transplanted ventral neural tube also gave rise to vSMCs. After grafting of quail head mesoderm, quail endothelial cells were found in the host brain, but no vSMCs of donor origin. Grafting of quail whole or ventral neural tube into the limb bud led to endowment of graft and host vessels with graft-derived vSMCs. Quail limb bud mesenchyme contributed to vSMCs in the ectopic neural graft, but, when transplanted into paraxial head mesenchyme, it did not form intraneural vSMCs. After orthotopic transplantation of cranial NC, graft-derived vSMCs were not only found in meninges and brain of the operated side, but also on the contralateral side. Our results show that 1) avian cranial neuroectoderm is able to differentiate into vSMCs of the brain; 2) this potential is not restricted to the prospective NC; and 3) neither cranial mesoderm nor cranially transplanted limb bud mesoderm can give rise to brain vSMC.

Phosphorylation processes mediate rapid changes of brain aromatase activity

The enzyme aromatase (also called estrogen synthase) that catalyzes the transformation of testosterone (T) into estradiol plays a key limiting role in the action of T on many aspects of reproduction. The distribution and regulation of aromatase in the quail brain has been studied by radioenzyme assays on microdissected brain areas, immunocytochemistry, RT-PCR and in situ hybridization. High levels of aromatase activity (AA) characterize the sexually dimorphic, steroid-sensitive medial preoptic nucleus (POM), a critical site of T action and aromatization for the activation of male sexual behavior. The boundaries of the POM are clearly outlined by a dense population of aromatase-containing cells as visualized by both immunocytochemistry and in situ hybridization histochemistry. Aromatase synthesis in the POM is controlled by T and its metabolite estradiol, but estradiol receptors alpha (ERα) are not normally co-localized with aromatase in this brain area. Estradiol receptor beta (ERβ) has been recently cloned in quail and localized in POM but we do not yet know whether ERβ occurs in aromatase cells. It is therefore not known whether estrogens regulate aromatase synthesis directly or by affecting different inputs to aromatase cells as is the case with the gonadotropin releasing hormone neurons. The presence of aromatase in presynaptic boutons suggests that locally formed estrogens may exert part of their effects by non-genomic mechanisms at the membrane level. Rapid effects of estrogens in the brain that presumably take place at the neuronal membrane level have been described in other species. If fast transduction mechanisms for estrogen are available at the membrane level, this will not necessarily result in rapid changes in brain function if the availability of the ligand does not also change rapidly. We demonstrate here that AA in hypothalamic homogenates is rapidly down-regulated by exposure to conditions that enhance protein phosphorylation (addition of Ca 2+, Mg 2+, ATP). This inhibition is blocked by kinase inhibitors which supports the notion that phosphorylation processes are involved. A rapid (within minutes) and reversible regulation of AA is also observed in hypothalamic explants incubated in vitro and exposed to high Ca 2+ levels (K +-induced depolarization, treatment by thapsigargin, by kainate, AMPA or NMDA). The local production and availability of estrogens in the brain can therefore be rapidly changed by Ca 2+ based on variation in neurotransmitter activity. Locally-produced estrogens are as a consequence available for non-genomic regulation of neuronal physiology in a manner more akin to the action of a neuropeptide/neurotransmitter than previously thought.

Expression and localization of cytochrome P450 17α-hydroxylase/c17,20-lyase in the avian brain

Steroids synthesized de novo from cholesterol in the brain are generally called neurosteroids. We have recently demonstrated, using biochemical and molecular biological methods, that certain structures in the quail brain possess cytochrome P450 side-chain cleavage enzyme ( P450scc) and 3β-hydroxysteroid dehydrogenase/Δ 5-Δ 4-isomerase (3β-HSD) and produce pregnenolone, pregnenolone sulfate and progesterone. To clarify the biosynthetic pathway of neurosteroids in the avian brain, therefore, we examined the expression of messenger RNA (mRNA) encoding for the enzyme cytochrome P450 17α-hydroxylase/c17,20-lyase ( P450 17α,lyase), which converts pregnenolone to dehydroepiandrosterone via 17α-hydroxypregnenolone or progesterone to androstenedione via 17α-hydroxyprogesterone. RT-PCR analysis followed by Southern hybridization indicated the expression of P450 17α,lyase mRNA in the brain of sexually mature birds without a clear-cut sex difference. Employing biochemical techniques combined with HPLC analysis, the conversion of progesterone to 17α-hydroxyprogesterone was also found in brain slices of the mature male. P450 17α,lyase mRNA was detected in various brain regions, but there was a clear regional difference in the expression. The expressions of P450 17α,lyase mRNA in the diencephalon and mesencephalon were significantly higher than those in the cerebrum and cerebellum, unlike 3β-HSD mRNA, which showed no regional difference in the expression. In situ hybridization revealed the cellular localization of P450 17α,lyase mRNA. The cells expressing P450 17α,lyase mRNA were detected several diencephalic and mesencephalic regions, such as the preoptic area, the anterior hypothalamus, the dorsolateral thalamus, the optic tectum and the ventral midbrain. The expression was also localized in the septum, the hyperstriatum accessorium, and the ventral portions of the archistriatum in the telencephalon. Cerebellar Purkinje cells also expressed P450 17α,lyase mRNA. These results suggest that the avian brain possesses P450 17α,lyase as well as P450scc and 3β-HSD in both sexes. The expression of P450 17α,lyase in the avian brain may be region-dependent.

Developmental changes in progesterone biosynthesis and metabolism in the quail brain

We have recently demonstrated that the quail brain possesses the cholesterol side-chain cleavage enzyme (cytochrome P450scc) and 3β-hydroxysteroid dehydrogenase/Δ 5-Δ 4-isomerase (3β-HSD) and produces pregnenolone, pregnenolone sulfate and progesterone from cholesterol. To elucidate the developmental changes in progesterone biosynthesis and its metabolism in the quail brain, we examined the expression and activity of 3β-HSD and progesterone metabolite(s) during embryonic and post-hatched ages. Both the progesterone concentration and 3β-HSD mRNA expression in the brain were almost constant during embryonic and post-hatched ages. The conversion of pregnenolone to progesterone (net 3β-HSD enzymatic activity) was also constant during development and at maturity. However, without radioinert progesterone, the production of progesterone was drastically reduced in the embryonic brain, indicating active progesterone metabolism at the embryonic stage. Biochemical analysis together with HPLC and TLC revealed that only the embryonic brain actively produced 5β-dihydroprogesterone from progesterone. Thus, progesterone production may be constant during embryonic and post-hatched development and in adulthood, whereas 5β-dihydroprogesterone may be produced actively only in embryonic life due to 5β-reductase.

Characterization of a cDNA encoding a novel avian hypothalamic neuropeptide exerting an inhibitory effect on gonadotropin release.

We previously isolated a novel dodecapeptide containing a C-terminal -Arg-Phe-NH(2) sequence, SIKPSAYLPLRF-NH(2) (RFamide peptide), from the quail brain. This quail RFamide peptide was shown to decrease gonadotropin release from the cultured anterior pituitary and to be located at least in the quail hypothalamo-hypophysial system. We therefore designated this RFamide peptide gonadotropin inhibitory hormone (GnIH). In the present study we characterized the GnIH cDNA from the quail brain by a combination of 3' and 5' rapid amplification of cDNA ends ('RACE'). The deduced GnIH precursor consisted of 173 amino acid residues, encoding one GnIH and two putative gene-related peptide (GnIH-RP-1 and GnIH-RP-2) sequences that included -LPXRF (X=L or Q) at their C-termini. All these peptide sequences were flanked by a glycine C-terminal amidation signal and a single basic amino acid on each end as an endoproteolytic site. Southern blotting analysis of reverse-transcriptase-mediated PCR products demonstrated a specific expression of the gene encoding GnIH in the diencephalon including the hypothalamus. Furthermore, mass spectrometric analyses detected the mass numbers for matured GnIH and GnIH-RP-2, revealing that both peptides are produced from the precursor in the diencephalon as an endogenous ligand. Taken together, these results lead to the conclusion that GnIH is a hypothalamic factor responsible for the negative regulation of gonadotropin secretion. Furthermore, the presence of a novel RFamide peptide family containing a C-terminal -LPXRF-NH(2) sequence has been revealed.

Characterization of a cDNA encoding a novel avian hypothalamic neuropeptide exerting an inhibitory effect on gonadotropin release

We previously isolated a novel dodecapeptide containing a C-terminal -Arg-Phe-NH2 sequence, SIKPSAYLPLRF-NH2 (RFamide peptide), from the quail brain. This quail RFamide peptide was shown to decrease gonadotropin release from the cultured anterior pituitary and to be located at least in the quail hypothalamo-hypophysial system. We therefore designated this RFamide peptide gonadotropin inhibitory hormone (GnIH). In the present study we characterized the GnIH cDNA from the quail brain by a combination of 3′ and 5′ rapid amplification of cDNA ends (‘RACE’). The deduced GnIH precursor consisted of 173 amino acid residues, encoding one GnIH and two putative gene-related peptide (GnIH-RP-1 and GnIH-RP-2) sequences that included -LPXRF (X = L or Q) at their C-termini. All these peptide sequences were flanked by a glycine C-terminal amidation signal and a single basic amino acid on each end as an endoproteolytic site. Southern blotting analysis of reverse-transcriptase-mediated PCR products demonstrated a specific expression of the gene encoding GnIH in the diencephalon including the hypothalamus. Furthermore, mass spectrometric analyses detected the mass numbers for matured GnIH and GnIH-RP-2, revealing that both peptides are produced from the precursor in the diencephalon as an endogenous ligand. Taken together, these results lead to the conclusion that GnIH is a hypothalamic factor responsible for the negative regulation of gonadotropin secretion. Furthermore, the presence of a novel RFamide peptide family containing a C-terminal -LPXRF-NH2 sequence has been revealed.

Distribution of DARPP-32 immunoreactive structures in the quail brain: anatomical relationship with dopamine and aromatase

We recently demonstrated that dopamine (DA) as well as different DA receptor agonists and antagonists are able to decrease within a few minutes the aromatase activity (AA) measured in vitro in homogenates or in explants of the quail preoptic area — hypothalamus. In addition, DA also appears to regulate AA, in vivo presumably by modifying enzyme synthesis. The cellular mechanisms and the anatomical substrate that mediate these controls of AA by DA are poorly understood. Tyrosine hydroxylase-immunoreactive (TH-ir) fibers and punctate structures have been previously observed in close vicinity of aromatase-immunoreactive (ARO-ir) cells in the quail medial preoptic nucleus (POM) and bed nucleus striae terminalis (BST) but these fibers could reflect a noradrenergic innervation. We also do not know whether aromatase cells are dopaminoceptive. The main goal of the present study was therefore to bring more information on the anatomical relationships between aromatase expressing neurons and the dopaminergic system in the quail brain. The visualization by immunocytochemistry of DA and of the D1 receptor associated protein DARPP-32 was used to address these questions. DA-ir fibers were observed in the quail forebrain and overlapped extensively with nuclei that contain high densities of ARO-ir cells such as the POM and BST. This confirms that the previously reported TH-ir innervation of ARO-ir cells is, at least in part, of dopaminergic nature. DARPP-32-immunoreactive cells were found in periventricular position throughout the hypothalamus. DARPP-32-ir cells were also observed in telencephalic and mesencephalic areas (hyperstriatum accessorium, paleostriatum, nucleus intercollicularis, optic tectum). DARPP-32-ir fibers were widespread in tel-, di-, and mes-encephalic areas. The highest densities of immunoreactive fibers were detected in the lobus parolfactorius, paleostriatum augmentatum and substantia nigra/area ventralis of Tsai. In double-labeled sections, appositions between DARPP-32 fibers and ARO-ir cells were present in the dorsolateral POM and BST but DARPP-32 immunoreactivity was not detected in the ARO-ir perikarya (no colocalization). These data confirm the presence of a dopaminoceptive structures within the main cell clusters of ARO-ir cells in the quail brain but provide no evidence that these ARO-ir cells are themselves dopaminoceptive. Because DARPP-32 is not present in all types of cells expressing DA receptors, the presence of DA receptors that would not be associated with DARPP-32 in ARO-ir cells still remains to be investigated

Dopamine D 2-like receptor binding in the brain of male Japanese quail ( Coturnix japonica)

Japanese quail ( Coturnix japonica) have been used extensively to study appetitive behaviors. However, little is known about the appetitive-relevant neurochemical systems in this species. The present investigation examined the distribution of D 2-like dopamine receptors in the quail brain. [ 3H]Spiperone was incubated in brain tissue homogenates and non-specific binding was defined using (−)-sulpiride. Scatchard analysis of whole brain without cerebellum and forebrain alone indicated approximate K d's of 0.08 and 0.04 nM, respectively. In addition, the preferential D 3 agonist (±)-2-dipropylamino-7-hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (7-OH-DPAT) did not displace [ 3H]spiperone binding in quail forebrain. Finally, regional analysis showed that the highest densities of D 2-like receptors were located in the forebrain. Overall, these results indicate that there is some conservation of dopaminergic mechanisms between aves and mammals. Thus, Japanese quail may be useful for examining the neuropharmacological mechanisms of dopaminergic stimulant drugs that work via D 2-like receptor activation.

A Novel Avian Hypothalamic Peptide Inhibiting Gonadotropin Release

The neuropeptide control of gonadotropin secretion at the level of the anterior pituitary gland is primarily through the stimulatory action of the hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH), which was originally isolated from mammals and subsequently from nonmammals. To date, however, an inhibitory peptide of gonadotropin release is unknown in vertebrates. Here we show, in a bird, that the hypothalamus also contains a novel peptide which inhibits gonadotropin release. Acetic acid extracts of quail brains were passed through C-18 reversed-phase cartridges, and then the retained material was subjected to the reversed-phase and cation-exchange high-performance liquid chromatography (HPLC). The peptide was isolated from avian brain and shown to have the sequence Ser-Ile-Lys-Pro-Ser-Ala-Tyr-Leu-Pro-Leu-Arg-Phe-NH2. Cell bodies and terminals containing this peptide were localized immunohistochemically in the paraventricular nucleus and median eminence, respectively. This peptide inhibited, in a dose-related way, gonadotropin release from cultured quail anterior pituitaries. This is the first hypothalamic peptide inhibiting gonadotropin release reported in a vertebrate. We therefore term it gonadotropin-inhibitory hormone (GnIH).

Fos induction in the Japanese quail brain after expression of appetitive and consummatory aspects of male sexual behavior

We investigated the expression of Fos, the protein product of the immediate early gene c- fos in the brain of male Japanese quail after they engaged in either appetitive or consummatory sexual behavior (i.e., copulation). For 1 h, castrated males treated with testosterone were either allowed to copulate with a female or to exhibit a learned social proximity response indicative of appetitive sexual behavior. Control birds were either left in their home cage or placed in the experimental chamber but did not exhibit the appetitive sexual behavior because they had never learned it. Fos expression was studied with an immunocytochemical procedure in two sets of adjacent sections through the entire forebrain. These sections were immunolabelled with 2 different antibodies raised against a synthetic fragment corresponding to the 21 carboxy-terminal residues of the chicken Fos sequence. Contrary to the results of a previous study in which gonadally intact birds were used, Fos induction was observed neither in the medial preoptic nucleus nor in the nucleus intercollicularis in birds that had interacted for 1 h with a female. This may be related to a lower frequency of copulation in the testosterone-implanted birds than in intact birds, or to differences in the time the brains were collected after the birds engaged in sexual behavior between the two studies (60 min in this study, 120 min in the previous study). The performance of copulation and/or appetitive sexual behavior increased the number of Fos-immunoreactive cells in the ventral hyperstriatum, medial archistriatum, and nucleus striae terminalis. These increases were observed using both antibodies, although each antibody produced minor differences in the number of Fos-immunoreactive cells observed. Using one of the antibodies, but not the other, increases in Fos immunoreactivity were also observed in the nucleus accumbens and hyperstriatum after either copulation or appetitive sexual behavior. These differences illustrate how minor technical variations in the Fos immunocytochemical procedure influence the results obtained. These differences also show that Fos induction in a number of brain regions is observed after performance of consummatory (copulation) as well as appetitive (looking at the female) sexual behavior. This induction is, therefore, not related solely to the control of copulatory acts but, presumably, also to the processing in a variety of telencephalic association areas of stimuli originating from the female. The observation that increased Fos immunoreactivity is present in birds that had learned the response indicative of appetitive sexual behavior, and not in those that had not learned the behavior, further indicates that it is not simply the sight of the female that results in this Fos induction, but the analysis of the relevant stimuli in a sexually explicit context. Conditioned neural activity resulting from a learned association between the stimulus female and the performance of copulatory behavior may also explain some aspects of the brain activation observed in birds viewing, but not allowed to interact with, the female.

Protein kinase and phosphatase modulation of quail brain GABA A and non-NMDA receptors co-expressed in Xenopus oocytes

The GABA A receptor and the non-NMDA subtype of the ionotropic glutamate receptor were co-expressed in Xenopus oocytes by injection of quail brain mRNA. The oocytes were treated with various protein kinase (PK) and protein phosphatase (PP) activators and inhibitors and the effects on receptor functioning were monitored. Two phorbol esters, 4-β-phorbol 12-myristate-13-acetate (PMA) and 4-β-phorbol 12,13-dibutyrate (PDBu); the cGMP-dependent PK activators sodium nitroprusside (SNP) and S-nitrosoglutathione (SNOG); and the PP inhibitor okadaic acid (OA) reduced the amplitude of the GABA-induced currents, whilst the PK inhibitor staurosporine potentiated it. In addition, PMA, PDBu, SNP, and OA reduced the desensitization of the GABA-induced response. Identical treatments generally had similar but less pronounced effects on responses generated by kainate (KA) but the desensitization characteristic of the non-NMDA receptor was not affected. None of the treatments had any effect on the reversal potentials of the induced currents. Immunoblots revealed that the oocytes express endogenous PKG and guanylate cyclase. The results are discussed in terms of the molecular structures of GABA A and non-NMDA receptors and the potential functional consequences of phosphorylation/dephosphorylation.

From Behaviour To Genes: Sex in the Japanese Quail

In Japanese quail (Coturnix japonica) like in most vertebrates, testosterone (T) activates male copulatory behaviour through its action in the preoptic area (POA). The transformation of T into estradiol by the enzyme aromatase localized in the POA is critical for this behavioral activation. We studied the distribution of aromatase in the quail brain and its regulation by steroids by radioenzyme assays on microdissected brain areas (quantification of enzymatic activity), by immunocytochemistry (visualization of the protein) and by RT PCR/in situ hybridization (quantification and visualization of the corresponding mRNA). Partial cloning of the aromatase gene in quail also allowed us to produce a specific antibody to this enzyme raised against the quail sequence expressed in Escherichia coli. Aromatase synthesis in the POA is controlled at the pre-translational level by T and its metabolite estradiol, but estradiol receptors alpha (ERα) are not normally co-localized with aromatase. A new form of ER (ERβ) has been identified and we have recently cloned this receptor in quail. It is located in all brain regions that contain aromatase but whether ERβ is specifically present in aromatase-immunoreactive cells still needs to be investigated.

Avian galanin: Cloning of complementary DNAs and characterization of transcripts in different tissues

Avian galanin was first isolated from the chicken intestine, and subsequently from the quail oviduct. Avian galanin is a 29-residue peptide including an amidated threonine at the C-terminus and differs at several positions from the C-terminal part of mammalian galanins. In contrast to extensive mammalian studies, no information is available on the structure of precursor molecule of avian galanin. In this study, therefore, we conducted complementary DNA (cDNA) cloning of the avian galanin gene from quail brain RNA using degenerated 3'RACE and 5'RACE techniques. A cloned cDNA for avian galanin contained an open reading frame consisting of 117 amino acids which had overall amino acid homology of 63%, 60%, 54%, 58%, and 62% with bovine, human, mouse, porcine, and rat galanins, respectively. Another cDNA containing a 69-nucleotide insertion, which gave an additional 23 amino acids to mature avian galanin, was also cloned, suggesting the presence of two transcripts by alternative splicing. Northern blot analysis revealed that avian galanin mRNA was expressed, as a shorter transcript, in the quail brain, ovary, and intestine, unlike the liver and oviduct. A larger mRNA of avian galanin may be further expressed only in the ovary.

Effects of testosterone on sexually dimorphic parvocellular neurons expressing vasotocin mRNA in the male quail brain

In situ hybridization with a P 33-labelled cDNA probe was used to analyze the effects of castration and replacement therapy by testosterone on the number of neurons expressing vasotocin mRNA in the male quail brain. Castration completely eliminated neurons expressing vasotocin mRNA in the previously described parvocellular vasotocin cell groups, located in the medial preoptic nucleus and in the anterior and posterior part of the medial subdivision of the bed nucleus of the stria terminalis. These effects were completely reversed by a 3-week treatment with exogenous testosterone. No marked change in vasotocin expression could be detected in the magnocellular cell groups located in the paraventricular and supraoptic nuclei. These data indicate that the testosterone-induced changes in the vasotocinergic innervation of the quail medial preoptic region and bed nucleus of the stria terminalis result from controlling mechanisms at the pretranslational, presumably transcriptional level. These control mechanisms are therefore very similar to those described for the rat brain despite the existence of major differences in the neuroanatomical organization of this peptidergic system in the two species.

Performance of appetitive or consummatory components of male sexual behavior is mediated by different brain areas: a 2-deoxyglucose autoradiographic study

The in vivo autoradiographic deoxyglucose method was used to identify the functional brain circuits that are involved in the performance of appetitive and consummatory components of male sexual behavior in Japanese quail ( Coturnix japonica). Two groups of castrated, testosterone-treated male quail were trained during 12 sessions to associate the view of a female behind a window with the opportunity to interact freely and to copulate with her. They developed, as a consequence, a social proximity response (staying close and looking through the window providing a view of the female) that has been used in previous experiments to measure appetitive sexual behavior. A third control group (also castrated and treated with testosterone) was allowed to view the female but not to copulate with her and therefore did not develop this proximity response. 2- 14C-deoxyglucose was then injected i.p. to these birds and they were allowed to either copulate freely with a female (consummatory sexual behavior group) or express the social proximity response (appetitive sexual behavior group). The control group was provided a view of the female but these birds, although they were exposed to the same stimuli as birds in the appetitive group, did not express the social proximity response because they had never learned the association with the opportunity to copulate. Birds were killed 45 min after the deoxyglucose injection and their brains were processed for autoradiography. Densitometric analyses of the autoradiograms revealed that the expression of appetitive or consummatory aspects of male sexual behavior was associated with significant increases by comparison with the control group in the deoxyglucose incorporation in the nucleus mesencephalicus lateralis, pars dorsalis and in the nucleus leminsci lateralis. In addition, an increase in the deoxyglucose incorporation was specifically observed in the paleostriatum primitivum, rostral preoptic area, nucleus intercollicularis, nucleus interpeduncularis and third nerve but a decrease was observed in the dorsomedial part of the hippocampus and in the nucleus nervi oculomotori in birds of the consummatory sexual behavior group by comparison with controls. By contrast, in the appetitive sexual behavior group, significant increases in deoxyglucose incorporation were observed in two telencephalic areas, the intermediate hyperstriatum ventrale and neostriatum caudolaterale by comparison with the controls, but decreases were detected in the stratum griseum et fibrosum superficiale of optic tectum by comparison with the consummatory behavior group. These studies demonstrate that the performance of appetitive or consummatory components of male sexual behavior affects in a specific manner the deoxyglucose uptake and accumulation in specific regions of the quail brain. Changes in metabolic activity were observed in steroid-sensitive areas, in auditory, visual and vocal brain regions, and in brain nuclei related to motor behavior but also in association telencephalic and limbic structures. These changes in oxidative metabolism overlap to some extent with metabolic changes as revealed by immunocytochemistry for the immediate early gene products Fos and Zenk, but many specific reactions are also detected indicating that these techniques are not necessarily redundant and, together, they can provide a more complete picture of the brain circuits that are implicated in the control and performance of complex behaviors.