Retinotopic map plasticity in adult cat visual cortex is accompanied by changes in Ca2+/calmodulin-dependent protein kinase II alpha autophosphorylation.

In adult cats, the induction of homonymous binocular central retinal lesions causes a dramatic reorganization of the topographic map in the sensory-deprived region of the primary visual cortex. To investigate the possible involvement of the alpha-subunit of the calcium/calmodulin dependent protein kinase type II (alphaCaMKII) in this form of brain plasticity, we performed in situ hybridization and Western blotting experiments to analyze mRNA, protein and autophosphorylation levels of this multifunctional kinase. No differences in the mRNA or protein levels were observed between the central, sensory-deprived and the peripheral, non-deprived regions of area 17 of retinal lesion animals or between corresponding cortical regions of normal control animals. Western blotting with an alphaCaMKII threonine-286 phosphorylation-state specific antiserum consistently showed a small, albeit not significant, increase of alphaCaMKII autophosphorylation in the central versus the peripheral region of cortical area 17, and this both in normal subjects as well as in retinal lesion animals with a 3-day post-lesion survival time. In contrast, a post-lesion survival time of 14 days resulted in a alphaCaMKII autophosphorylation level that was four times higher in visually-deprived area 17 than in the non-deprived cortical region. This increased phosphorylation state is not a direct consequence of the decrease in visual activity in these neurons, because we would have expected to see a similar change at shorter or longer post-lesion survival times or in the visually deprived visual cortex of animals in which the left optic tract and the corpus callosum were surgically cut. No such changes were observed, leading to the conclusion that the phosphorylation changes observed at 14 days are related to a delayed reorganization of the retinotopic map of the striate cortex.

Immunohistochemical localization of chromogranin A in gonadotrophs and somatotrophs of the turkey and chicken pituitary.

In the course of producing monoclonal antibodies to turkey prolactin, three monoclonal antibodies to turkey chromogranin A (CgA) were also produced, apparently arising from minor contamination of the turkey prolactin immunogen with peptide fragments of CgA. The identity of the antigen recognized by these antibodies was established by tandem mass spectrometry de novo sequencing of seven tryptic peptides from a turkey pituitary protein purified by immunoaffinity chromatography. These peptides showed high homology with distinctly separate regions of mammalian and ostrich CgA, and in silico cloned chicken CgA sequences. Chromogranin A immunostaining patterns on Western blots and pituitary tissue sections differed from those of prolactin, growth hormone, or luteinizing hormone (LH). Dual-label fluorescent immunohistochemistry revealed that CgA was co-localized with LH in most avian gonadotrophs in young chickens and turkeys, but not in adult, laying birds. Conversely, CgA was found in a majority of somatotrophs in laying birds but was absent from somatotrophs in young, growing chickens and turkeys. Lactotrophs contained no detectable CgA immunoreactivity in the tissues studied. These results suggest that CgA may modulate hormone secretion by gonadotrophs and somatotrophs in a manner that differs between cell type with age or reproductive state.

FSH- and LH-cells originate as separate cell populations and at different embryonic stages in the chicken embryo.

The histological distribution of gonadotrophs containing either LH or FSH, but not both gonadotropins, has been demonstrated before in the juvenile and adult chicken throughout the caudal and cephalic anterior pituitary lobes. In the present investigation, the distribution of FSH- and/or LH-containing gonadotrophs was further investigated in the chicken embryo by use of the same homologous antibodies as used in our earlier study. Fluorescent dual-labeling immunohistochemistry revealed that during embryogenesis LH and FSH reside exclusively in separate gonadotrophs, as has been described before in the post hatch bird. LH-immunoreactive cells were observed for the first time at day 9 of embryogenesis. This is as much as 4 days earlier than the FSH-immunoreactive cells, which appeared at day 13 of embryogenesis. Our results confirm that FSH- and LH-containing gonadotrophs are distributed throughout both lobes of the anterior pituitary. No conspicuous differences were observed between the sexes in any of the aspects investigated. The described situation is unique in that it seems to imply the existence of separate cell lineages for FSH- and LH-producing cells, as opposed to the single gonadotrope lineage described in all other species studied so far, with the exception of bovine. Our data indeed raise the question as to which signaling and/or transcription factors may cause the unique dichotomy observed in the chicken gonadotrophs.

Neurofilament protein: a selective marker for the architectonic parcellation of the visual cortex in adult cat brain.

In this immunocytochemical study, we examined the expression profile of neurofilament protein in the cat visual system. We have used SMI-32, a monoclonal antibody that recognizes a nonphosphorylated epitope on the medium- and high-molecular-weight subunits of neurofilament proteins. This antibody labels primarily the cell body and dendrites of pyramidal neurons in cortical layers III, V, and VI. Neurofilament protein-immunoreactive neurons were prominent in 20 visual cortical areas (areas 17, 18, 19, 20a, 20b, 21a, 21b, and 7; posteromedial lateral, posterolateral lateral, anteromedial lateral, anterolateral lateral, dorsal lateral, ventral lateral, and posterior suprasylvian areas; anterior ectosylvian, the splenial, the cingulate, and insular visual areas; and the anterolateral gyrus area). In addition, we have also found strong immunopositive cells in the A laminae of the dorsal part of the lateral geniculate nucleus (dLGN) and in the medial interlaminar nucleus, but no immunoreactive cells were present in the parvocellular C (1-3) laminae of the dLGN, in the ventral part of the LGN and in the perigeniculate nucleus. This SMI-32 antibody against neurofilament protein revealed a characteristic pattern of immunostaining in each visual area. The size, shape, intensity, and density of neurofilament protein-immunoreactive neurons and their dendritic arborization differed substantially across all visual areas. Moreover, it was also obvious that several visual areas showed differences in laminar distribution and that such profiles may be used to delineate various cortical areas. Therefore, the expression of neurofilament protein can be used as a specific marker to define areal patterns and topographic boundaries in the cat visual system.

In vivo microdialysis in the visual cortex of awake cat. I: surgery, animal training and sampling.

Sampling and monitoring release of excitatory and inhibitory amino acids in the striate cortex of mammals will provide important information for visual system research. A method allowing repeated microdialysis in the cortical layers of area 17 of the awake cat is described. Under visual control through a surgical microscope and using a stereotactic instrument, four probe guides are permanently implanted in area 17 of one hemisphere of the anesthetized animal and two fixation bars are mounted on the skull to allow fixation of the cat in a stereotactic frame. The implantation of four probe guides in the same hemisphere allows simultaneous sampling from different cortical regions subserving different parts of the visual field. A removable transparent cover protects the probe guides. After recovery from surgery the awake cats are trained to adapt to a fixation in a stereotaxic apparatus. Once adapted to that situation, the cats are used for 5 h in vivo microdialysis experiments without anesthesia.

In vivo microdialysis in the visual cortex of awake cat. II: sample analysis by microbore HPLC-electrochemical detection and capillary electrophoresis-laser-induced fluorescence detection.

Sampling and monitoring release of excitatory and inhibitory amino acids in the striate cortex of mammals will provide important information for visual system research. Two microbore high performance liquid chromatography-electrochemical detection methods and a capillary electrophoresis-laser induced fluorescence detection were developed to determine the inhibitory amino acid, gamma-aminobutyric acid and the excitatory amino acids, glutamate and aspartate in microdialysates of cat striate cortex. In the liquid chromatography method, samples were derivatized using OPA-TBT. Ten microliters of derivatized product was injected onto the microbore column (100 x 1 mm i.d., C8) for quantitative analysis. Electrochemical detection was employed. In the capillary electrophoresis method, samples were derivatized using fluorescein isothiocyanate and separated in borate buffer within 15 min, then detected by a laser-induced fluorescence detector.

In vivo microdialysis in the visual cortex of awake cat. III: histological verification.

In vivo microdialysis sampling extracellular excitatory and inhibitory amino acids from the striate cortex of mammals will provide important information for visual system research. To facilitate the interpretation of microdialysis results, this protocol critically examines: (1) the location of probe implantation in the visual cortex using Nissl staining; (2) the morphological changes after probe implantation by visualization of neurons containing glutamate; (3) the morphological changes after probe implantation by visualization of gliosis using glial fibrillary acidic protein (GFAP) immunocytochemistry; (4) the implantation of the probe in sensory-deprived versus non-deprived cortical regions by visualization of neurons containing c-Fos protein after limited retinal lesion. The histochemical and immunocytochemical methods of Glu, GFAP and c-Fos used are described.

Analysis of extracellular gamma-aminobutyric acid, glutamate and aspartate in cat visual cortex by in vivo microdialysis and capillary electrophoresis-laser induced fluorescence detection.

To investigate the influence of a partial sensory deprivation on the extracellular concentration of amino acid neurotransmitters in cat visual cortex, a capillary electrophoresis method was developed for the quantification of gamma-aminobutyric acid (GABA), glutamate (Glu) and aspartate (Asp) in in vivo microdialysis samples of cat brain. Microdialysis samples from different regions of area 17 were obtained every 15-min using CMA 12 2-mm probes perfused with synthetic cerebrospinal fluid and derivatized using fluorescein isothiocyanate (FITC). Laser-induced fluorescence (LIF) detection was employed. Good selectivity was obtained with a borate buffer (20 mM, pH 10.25). The whole procedure, including the washing step takes only 15 min. The conditions for derivatization and separation were optimized. The parameters for validation such as linearity, precision and detection limit are also reported. The results are consistent with those of HPLC but, as the sample volumes needed are only 1--5 nl, a much better time resolution can be obtained.

Expression of the high-affinity glutamate transporter EAAT4 in mammalian cerebral cortex.

RT-PCR, immunocytochemistry and Western blotting were used to study the expression of the glutamate transporter EAAT4 in the cerebral cortex of cat and mouse. By means of RT-PCR we were able to detect EAAT4 mRNA in the cerebral cortex of both species. Sequencing ensured the specificity of the amplified fragment. Immunocytochemistry and Western blotting enabled us to localize EAAT4 protein in cat and mouse cerebral cortex. Intense EAAT4 immunoreactivity was found in the soma and dendrites of neurons mainly of layers II, III and V. For both species, the signal in the cerebellum was very intense and confined to the molecular and Purkinje cell layer.

Effect of partial sensory deprivation on monoaminergic neuromodulators in striate cortex of adult cat.

The role of monoaminergic neuromodulators in the reorganization of cortical topography following limited sensory deprivation in the adult cat was investigated. The total concentrations of dopamine, noradrenaline, serotonin and their major metabolites were measured in the visual cortex of both normal control and experimental animals using microbore high-performance liquid chromatography coupled with electrochemical detection. The experimental animals were subjected to a binocular retinal lesion corresponding to the central 10 degrees of vision and killed two weeks post-lesion. The sensory deprivation was confirmed in area 17 by measuring immediate-early gene zif-268 messenger RNA expression. Following the retinal lesion, the total concentrations of noradrenaline and dopamine were significantly higher in the non-deprived cortex of retinal lesion cats than in the deprived cortex of retinal lesion cats and the cortex of normal animals. This pattern follows the release of the excitatory neurotransmitter glutamate under the same conditions. Serotonin levels were significantly lower in the deprived cortex, and its metabolite 5-hydroxyindole-3-acetic acid was significantly higher in the non-deprived cortex than in deprived cortex and normal cortex. From these results, we suggest that the modulation of noradrenaline, dopamine and serotonin is regulated by visual afferent activity.

Cooperative changes in GABA, glutamate and activity levels: the missing link in cortical plasticity.

Different intracortical mechanisms have been reported to contribute to the substantial topographic reorganization of the mammalian primary visual cortex in response to matching lesions in the two retinas: an immediate expansion of receptive fields followed by a gradual shift of excitability into the deprived area and finally axonal sprouting of laterally projecting neurons months after the lesion. To gain insight into the molecular mechanisms of this adult plasticity, we used immunocytochemical and bioanalytical methods to measure the glutamate and GABA neurotransmitter levels in the visual cortex of adult cats with binocular central retinal lesions. Two to four weeks after the lesions, glutamate immunoreactivity was decreased in sensory-deprived cortex as confirmed by HPLC analysis of the glutamate concentration. Within three months normal glutamate immunoreactivity was restored. In addition, the edge of the unresponsive cortex was characterized by markedly increased glutamate immunoreactivity 2-12 weeks postlesion. This glutamate immunoreactivity peak moved into the deprived area over time. These glutamate changes corresponded to decreased spontaneous and visually driven activity in unresponsive cortex and to strikingly increased neuronal activity at the border of this cortical zone. Furthermore, the previously reported decrease in glutamic acid decarboxylase immunoreactivity was found to reflect decreased GABA levels in sensory-deprived cortex. Increased glutamate concentrations and neuronal activity, and decreased GABA concentrations, may be related to changes in synaptic efficiency and could represent a mechanism underlying the retinotopic reorganization that occurs well after the immediate receptive field expansion but long before the late axonal sprouting.

Investigation of cortical reorganization in area 17 and nine extrastriate visual areas through the detection of changes in immediate early gene expression as induced by retinal lesions.

The effect of binocular central retinal lesions on the expression of the immediate early genes c-fos and zif268 in the dorsal lateral geniculate nucleus (dLGN) and the visual cortex of adult cats was investigated by in situ hybridization and immunocytochemistry. In the deafferented region of the dLGN, the c-fos mRNA level was decreased within 3 days. The dimensions of the geniculate region showing decreased amounts of c-fos mRNA matched the predictions based on the lesion size and the retinotopic maps of Sanderson ([1971] J. Comp. Neurol. 143:101-118). We did not detect zif268 mRNA in the dLGN. At the cortical level, both c-fos and zif268 mRNA expression decreased in the sensory-deprived region of area 17. In addition, the portions of areas 18, 19, 21a, 21b, and 7, as well as the posterior medial lateral suprasylvian area, the posterior lateral lateral suprasylvian area, the ventral lateral suprasylvian area, and the dorsal lateral suprasylvian area corresponding to the retinal lesions also displayed decreased c-fos and zif268 mRNA levels. Immunocytochemistry revealed similar changes for Zif268 and Fos protein. Three days post lesion, the dimensions of the lesion-affected cortical loci exceeded the predictions in relation to the size of the retinal lesions and the available retinotopic maps. Longer postlesion survival times clearly resulted in a time-dependent restoration of immediate early gene expression from the border to the center of the lesion-affected cortical portions. Our findings represent a new approach for investigating the capacity of adult sensory systems to undergo plastic changes following sensory deprivation and for defining the topographic nature of sensory subcortical and cortical structures.

Distribution of pituitary adenylate cyclase-activating polypeptide and pituitary adenylate cyclase-activating polypeptide type I receptor mRNA in the chicken brain.

To map in detail the brain areas in which pituitary adenylate cyclase-activating polypeptide (PACAP) may play a significant role in birds, the distribution of PACAP and PACAP type I receptor (PAC(1)-R) mRNA was examined throughout the entire chicken brain by using in situ hybridization histochemistry. Widespread distribution of both PACAP and its receptor mRNA was found. The telencephalic areas where the most intense signals for PACAP mRNA were found included the hyperstriatum accessorium, the hippocampus, and the archistriatum. In the diencephalon, a group of neurons that highly expressed PACAP mRNA was observed from the anterior medial hypothalamic nucleus to the inferior hypothalamic nucleus. Moderate expression was found in the paraventricular nucleus and the preoptic region. A second large group of neurons containing PACAP message was found within the nucleus dorsolateralis anterior thalami and extended caudally to the area around the nucleus ovoidalis and the nucleus paramedianus internus thalami. Furthermore, expression of PACAP message was observed within the bed nucleus of the pallial commissure, nucleus spiriformis medialis, optic tectum, cerebellar cortex, olfactory bulbs, and several nuclei within the brainstem (dorsal vagal and parabrachial complex, reticular formation). The highest expression of PAC(1)-R mRNA was found in the dorsal telencephalon, olfactory bulbs, lateral septum, optic tectum, cerebellum, and throughout the hypothalamus and thalamus. The presence of PACAP and PAC(1)-R mRNA in a variety of brain areas in birds suggests that PACAP mediates several physiologically important processes in addition to regulating the activity of the pituitary gland.

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.

Sampling extracellular aspartate, glutamate and gamma-aminobutyric acid in striate cortex of awake cat by in vivo microdialysis: surgical and methodological aspects.

A method which permits repeated microdialysis in the cortical layers of area 17 of the awake cat is described. Under visual control through a surgical microscope and using a stereotactic instrument, four probe guides are permanently implanted in area 17 of one hemisphere of the anesthetized animal and two fixation bars are mounted on the skull to allow fixation of the cat in a stereotactic frame. The implantation of four probe guides in the same hemisphere allows simultaneous sampling from different cortical regions serving different parts of the visual field. A removable transparent cover protects the probe guides. After recovery from surgery the awake cats are trained to adapt to a fixation of 5 h in a stereotaxic apparatus. Once adapted to that situation, the cats are ready for microdialysis experiments without anesthesia. The day of the experiment, the awake animal was fixed in the stereotactic frame and the probes inserted into the guides. To test the validity of the method, the basal efflux and the depolarization efflux, triggered by the addition of 65 mM K(+) to the artificial cerebrospinal fluid, of the amino acids aspartate, glutamate and gamma-aminobutyric acid are measured by two HPLC-electrochemical detection methods. The exact localization of the probes and the reaction of the surrounding tissue is studied using immunocytochemistry for glutamate and glial fibrilary acidic protein. Our neurochemical and morphological results suggest the feasibility of multiple and repeated probe insertions for microdialysis experiments in the cerebral cortex of awake and behaving cat. This method provides a new tool to investigate the cortical plasticity.

A new cat Fos antibody to localize the immediate early gene c-fos in mammalian visual cortex after sensory stimulation.

We developed a novel antibody against cat Fos by immunizing rabbits with a 26-amino-acid peptide. Immunocytochemistry on visual cortex of cats undergoing different visual manipulations was applied to test the reliability and the efficacy of this antiserum. One hour of light stimulation after an overnight dark adaptation resulted in strongly induced Fos expression in supra- and infragranular layers of cat primary visual cortex. Short-term monocular deprivation changed the Fos expression profile into a columnar immunostaining related to ocular dominance columns. Fos expression has also been analyzed in cats in which visual input was confined to the right hemisphere by sectioning the left optic tract and the corpus callosum. In the right hemisphere, visual stimulation elicited Fos induction, whereas in the contralateral hemisphere a very low Fos signal was observed. The specificity of this newly synthesized antibody was confirmed by Western blotting. To further establish the applicability of this Fos antiserum, we performed immunostaining on monkey and rat visual cortex. This new cat Fos antibody appears to be excellent for study of Fos expression as a marker for mapping neuronal activity in mammalian brain.

Colocalization of arginine-vasotocin and chicken luteinizing hormone-releasing hormone-I (cLHRH-I) in the preoptic-hypothalamic region of the chicken.

To characterize a possible relationship between chicken luteinizing hormone-releasing hormone-I (cLHRH-I) and arginine-vasotocin (AVT) we performed immunocytochemical double-stainings throughout the preoptic-hypothalamic region of the chicken brain. This study clearly reveals a partial colocalization between both neuropeptides. Single-labeled neurons, containing either cLHRH-I or AVT are found intermingled with double stained cells, immunoreactive (ir) for both peptides. A significant number of double-labeled perikarya is found in the preoptic area, more specifically in the ventral and external portion of the supraoptic nucleus (SOv and SOe) and in the medial preoptic nucleus (MPOv). At the level of the anterior hypothalamus, double-labeled cells are predominantly observed near the third ventricle in the nucleus paraventricularis magnocellularis (PVN) and the nucleus periventricularis hypothalami (PHN). Next to this colocalization, a number of cLHRH-I-ir cell bodies are found in close apposition to AVT-ir fiber profiles in the very same areas. Taken together, these data are the first to provide morphological evidence indicating that the AVT system might be involved in the regulation of cLHRH-I release and thus of reproductive functions in birds.

Sequence and distribution of pro-opiomelanocortin in the pituitary and the brain of the chicken (Gallus gallus).

Although pro-opiomelanocortin (POMC) is a well-known hormone precursor in many species, molecular information about avian POMCs is still relatively scarce. In a former study (Berghman et al., [1998] Mol Cell Endocrinol. 142:119-130) the nucleotide and amino acid sequence of N-terminal POMC in the chicken were reported. To complete the nucleotide sequence of the precursor, rapid amplification of 3' and 5' cDNA end reactions were performed and the polymerase chain reaction (PCR) products were cloned and sequenced. The chicken POMC coding region appears to consist of 678 base pairs in the pituitary and also in the hypothalamus, as assessed by reverse transcriptase PCR. Overall nucleotide sequence homology with other species ranges from 41% (in bovine) to 57% (in rat). The distribution of the POMC mRNA in pituitary and brain was analyzed by in situ hybridization by using 33P-labelled oligonucleotides. Expression of POMC mRNA in the pituitary was restricted to the cephalic lobe, whereas in the brain, the signal was limited to the hypothalamic region. As assessed by Northern blot analysis, the length of the POMC mRNA in both the pituitary and the hypothalamus was approximately 1,200 nucleotides. By using antisera to N-terminal POMC, alpha-melanotropin and beta-endorphin, POMC-containing cells were observed in the cephalic lobe of the pituitary and immunopositive perikarya were localized in the infundibular nucleus and median eminence of the hypothalamus. Immunoreactive fibers were found in the preoptic area and in the medial basal hypothalamus surrounding the third ventricle and more dorsally in the thalamus. Double-staining experiments in the pituitary clearly indicated a complete overlap of the signals generated by these antisera.

Validation of a new antiserum directed towards the synthetic c-terminus of the FOS protein in avian species: immunological, physiological and behavioral evidence.

In the past 10 years, the study of the expression of immediate early genes, such as c-fos, in the brain has become a common method for the identification of brain areas involved in the regulation of specific physiological and behavioral functions. The use of this method in avian species has been limited by the paucity of suitable antibodies that cross-react with the FOS protein in birds. We describe in this paper the preparation of an antibody directed against a synthetic fragment of the protein product of the c-fos gene in chickens (Gallus domesticus). We demonstrate that this new antibody can be used in several avian species to study FOS expression induced by a variety of pharmacological, physiological and behavioral stimuli. Western blot studies indicated that this antibody recognizes a protein of the expected size (47 kDa) but also cross reacts to some extent with proteins of lower molecular weight that share sequence homology with FOS (Fos-related antigens). FOS immunocytochemistry was performed with this antibody in four species of birds in three different laboratories utilizing diverse variants of the immunocytochemical procedure. In all cases the antibody provided a reliable identification of the FOS antigen. The new antibody described here appears to be suitable for the study of FOS expression in several different avian species and situations. It is available in substantial amounts and will therefore make it possible to use FOS expression as a tool to map brain activity in birds as has now been done for several years in mammalian species.

Molecular cloning and expression of a chicken pituitary adenylate cyclase-activating polypeptide receptor.

Although, since the isolation of pituitary adenylate cyclase-activating polypeptide (PACAP), a wealth of literature has been published describing its localization, binding sites, and biological activities in a variety of mammalian tissues, only very little is known about PACAP in avian species. Therefore, in order to find out the sites of actions of PACAP and to elucidate its physiological significance in birds, we identified a chicken PACAP receptor homologue of the mammalian type I receptors (PAC(1)-Rs). The chicken PACAP type I cDNA sequence was obtained using reverse transcriptase-polymerase chain reaction (RT-PCR) in combination with 3'- and 5'-RACE PCR. This cDNA encodes a 471 amino acid precursor protein, sharing 81-83% sequence identity with mammalian analogs and 76% amino acid identity with the goldfish type I PACAP receptor. Northern blot analysis of chicken brain poly(A)(+)-rich RNA revealed the presence of a 5.5 kb and 7.5 kb PAC(1) receptor transcript. RT-PCR revealed that the chicken PACAP receptor is mainly expressed in the brain and gonads. A smaller amount of the receptor mRNA was found in pituitary, adrenal gland, kidney, intestine, pancreas, lung, and heart tissue. In situ hybridization with specific antisense oligodeoxynucleotide probes showed a widespread distribution of PAC(1) receptor mRNA in the chicken brain, with the highest expression being found in the dorsal telencephalon, olfactory bulb, hypothalamus, optic tectum, and cerebellar cortex. These findings suggest that PACAP affect a variety of functions both in the brain and peripheral tissues of the chicken.