Brain Behav Evol 2014;83:82–92 DOI: 10.1159/000357834

Received: December 8, 2013 Accepted: December 9, 2013 Published online: April 24, 2014

Revival of Calcium-Binding Proteins for Neuromorphology: Secretagogin Typifies Distinct Cell Populations in the Avian Brain Georgina Gáti a Dávid Lendvai a Tomas Hökfelt b, c Tibor Harkany d, e Alán Alpár a, d, f a

Department of Anatomy, Histology and Embryology, Semmelweis University, Budapest, Hungary; b Department of Neuroscience, c Science for Life Laboratory, and d Division of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden; e Department of Molecular Neuroscience, Medical University of Vienna, Vienna, Austria; f Research Group of Experimental Neuroanatomy and Developmental Biology, Hungarian Academy of Sciences, Budapest, Hungary

Key Words Comparative neuroanatomy · Cortex · Evolution · Pallium

Abstract In the vertebrate nervous system, the Ca2+-binding proteins parvalbumin, calbindin and calretinin have been extensively used to elaborate the molecular diversity of neuronal subtypes. Secretagogin is a phylogenetically conserved Ca2+binding protein, which marks neuronal populations largely distinct from other Ca2+-binding proteins in mammals. Whether secretagogin is expressed in nonmammalian vertebrates, particularly in birds, and, if so, with a brain cytoarchitectonic design different from that of mammals is unknown. Here, we show that secretagogin is already present in the hatchlings’ brain with continued presence into adulthood. Secretagogin-immunoreactive neurons primarily accumulate in the olfactory bulb, septum, subpallial amygdala, hippocampus, hypothalamus, habenular nuclei and deep layers of the optic tectum of adult domestic chicks (Gallus domesticus). In the olfactory bulb, secretagogin labels periglomerular neurons as well as a cell continuum ascending dorsomedially, reaching the ventricular wall. Between the hippocampus and septal nuclei, the interconnecting thin septal tissue

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harbors secretagogin-immunoreactive neurons that contact the ventricular wall with their ramifying dendritic processes. Secretagogin is also present in the neuroendocrine hypothalamus, with particularly rich neuronal clusters seen in its suprachiasmatic and infundibular nuclei. Secretagogin expression identified a hitherto undescribed cell contingent along intratelencephalic cell-free laminae separating brain regions or marking the palliosubpallial boundary, as well as a dense neuronal population in the area corticoidea lateralis. In both the telencephalon and midbrain, secretagogin complemented the distribution of the canonical ‘neuronal’ Ca2+binding proteins. Our findings identify novel neuronal subtypes, connectivity patterns in brain areas functionally relevant to olfaction, orientation, behavior as well as endocrine functions, which will help refine existing concepts on the neuronal diversity and organizational principles of the avian brain. © 2014 S. Karger AG, Basel

Georgina Gáti and Dávid Lendvai contributed equally to the present study.

Alán Alpár Department of Anatomy, Histology and Embryology Semmelweis University, Tuzolto utca 58 HU–1094 Budapest (Hungary) E-Mail Alpar.Alan @ med.semmelweis-univ.hu

Introduction

The excitability of and second messenger signaling in neurons are critically dependent upon tightly controlled intracellular calcium (Ca2+) levels. This is achieved, in part, by Ca2+-binding proteins, which can either rapidly sequester excess cytosolic Ca2+ (‘Ca2+ buffers’) or initiate signal transduction through protein-protein interactions (‘Ca2+ sensors’) [Skelton et al., 1994]. Secretagogin is a recently described, 276-amino-acid-long protein primarily localized to the cytosol and to a lesser extent to the nucleus [Wagner et al., 1998, 2000]. Given its relatively low Ca2+-binding affinity and conformational modifications upon Ca2+ binding, secretagogin qualifies as a Ca2+ sensor [Rogstam et al., 2007]. The predicted interactome of secretagogin includes members of SNARE (soluble NSF attachment protein receptor) superfamily of proteins, suggesting its involvement in regulating secretory and synaptic vesicle trafficking and activity-dependent release [Rogstam et al., 2007; Bauer et al., 2011; Hasegawa et al., 2013]. Secretagogin is expressed in essentially all organ systems [Wagner et al., 2000; Alpár et al., 2012]. Initial studies demonstrated secretagogin-containing(+) cells in the pancreas, adrenal gland, thyroid, pituitary and luminous organs of the gastrointestinal system, with particularly high secretagogin levels in insulin-producing β cells of the pancreas and enteroendocrine cells of the gut [Wagner et al., 2000]. Secretagogin generated considerable interest in neurobiology, when it was exploited to characterize spatially segregated neuronal subsets, likely conferring novel network modalities in the mammalian brain [Mulder et al., 2009, 2010], including humans [Attems et al., 2007, 2008, 2012]. Secretagogin expression temporally precedes many of the ‘canonical’ neuronal Ca2+-binding proteins used as selective typifying markers, including a contingent of adult-born neurons in lower mammals [Mulder et al., 2009]. Cytoarchitectural appearance of the avian brain, particularly its telencephalon, is strikingly different from the mammalian cerebrum: telencephalic areas are not laminated. Instead, nuclear structures exist, propelling misconceptions about the homology of the bird’s ‘striatum’ and the purported lack of pallial (cortical) structures in the avian brain [Reiner et al., 1984]. Nevertheless, based on hodological, developmental and neurochemical evidence, several domains of the avian brain were identified as pallial or subpallial structures, and being homologous to cortical or subpallial domains of the mammalian brain [Reiner et al., 2004; Jarvis et al., 2005]. Secretagogin in the Avian Brain

The identification of cell contingents and homogenous neuronal subsets in species with different phylogenetic backgrounds is a powerful tool to understand basic architectonic principles of the nervous system. We hypothesized that secretagogin is expressed in the avian cerebrum, where it might reveal undescribed neuronal arrangements in cytoarchitectonics and/or connectivity. Here, we describe the distribution pattern of secretagogin-positive(+) neurons in the chicken brain (Gallus domesticus) with notable cell densities in the olfactory bulb, septum, bed nucleus of stria terminalis, hypothalamus and deeper layers of the optic tectum. Secretagogin+ cells preferentially occur at the telencephalic surface (e.g. ventricular wall) and along the dorsolateral convexity of the forebrain. If homologous between the avian and mammalian brain, neuronal territories harbor secretagogin+ cells in similar patterns and densities. Yet divergent secretagogin+ cell populations also exist in the chicken foreand midbrain. These results will facilitate the understanding of the cellular functions of this Ca2+-binding protein, as well as expanding on the anatomical and functional organization of the birds’ brain.

Materials and Methods Animals and Tissue Processing Chickens (G. domesticus, Hunnia broilers) were used at the age of 1, 13 and 90 days (n = 12 animals in total). For immunohistochemistry, n = 2 chickens/age were processed according to published protocols [Gáti et al., 2010]. Briefly, animals were overdosed with CO2 and perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer (PB). Whole brains were removed and postfixed using the same fixative at 4 ° C overnight. After cryoprotection in 30% sucrose in PB overnight at 4 ° C, 50-μm-thick coronal sections were cut on a cryostat microtome and processed. For biochemistry, fore- and midbrains were homogenized (n = 2 chickens/age) and processed for quantitative protein analysis. Experimental procedures on birds were approved by the Semmelweis University and conformed to the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (protocols: ETS No. 170 and 123).  

 

 

 

Immunohistochemistry and Microscopy Free-floating sections were rinsed in PB (pH 7.4) and pretreated with 0.3% Triton X-100 (in PB) for 1 h at 22–24 ° C to enhance the penetration of primary antibodies. Nonspecific immunoreactivity was suppressed by incubating our specimens in a cocktail of 5% normal donkey serum (NDS; Jackson), 1% BSA (Sigma, St. Louis, Mo., USA) and 0.3% Triton X-100 (Sigma) in PB for 1 h at 22– 24 ° C. Sections were exposed (72 h at 4 ° C) to select combinations of primary antibodies (table 1) diluted in PB to which 0.1% NDS and 0.3% Triton X-100 had been added. After extensive rinsing in PB, sections were processed for chromogenic or immunofluorescence detection as described [Mulder et al., 2009; Gáti et al., 2010].  

 

 

Brain Behav Evol 2014;83:82–92 DOI: 10.1159/000357834

 

 

 

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Table 1. List of markers used for immunohistochemistry (IH) and Western blotting (WB) Marker

Source

Host

IH dilution

WB dilution

Reference

β-Actin Calbindin Calretinin ChAT GFAP MAP2 Parvalbumin Secretagogin Secretagogin Secretagogin

Sigma SWant SWant Millipore Synaptic Systems Sigma SWant Atlas Antibodies R&D Systems Atlas Antibodies

mouse, mc mouse, mc goat, pc goat, pc guinea pig, pc mouse, mc rabbit, pc mouse, mc goat, pc rabbit, pc

n.a. 1:2,000 1:2,000 1:200 1:500 1:200 1:5,000 1:5,000 1:1,000 1:2,000

1:10,000 n.a. n.a. n.a. n.a. n.a. n.a. 1:5,000 1:2,000 1:2,000

Mulder et al., 2009 Celio, 1990 Schwaller et al., 1993 Li and Furness, 1998 this study Mulder et al., 2009 Celio, 1990 Mulder et al., 2009 Attems et al., 2012 Mulder et al., 2009

ChAT = Choline acetyltransferase; GFAP = glial fibrillary acidic protein; MAP2 = microtubule-associated protein 2; mc = monoclonal antibody; pc = polyclonal antibody.

In single-labeling experiments, sections were exposed to biotinylated anti-mouse, rabbit or goat IgG raised in donkey [1:1,000 (Jackson), 2 h at 22–24 ° C] followed by the addition of preformed avidin-biotin complexes for 1 h at 22–24 ° C (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, Calif., USA; 1:1,000). Secretagogin immunosignals were visualized by applying 3,3′-diaminobenzidine (Sigma, 0.025%) as chromogen intensified with Ni-ammonium sulfate (0.05%, Merck) in the presence of 0.001% H2O2 as substrate (dissolved in 0.05 M Tris buffer, pH 8.0). In multiple immunofluorescence labeling experiments, a cocktail of primary antibodies (table 1) was applied and immunoreactivity was detected by species-specific carbocyanine 2, 3 or 5-tagged secondary antibodies raised in donkey [1:200 (Jackson), 2 h at 22– 24 ° C]. Glass-mounted sections were air-dried and coverslipped with Entellan (VWR, Dublin, Ireland). Survey images were captured on a Nikon Eclipse microscope using 4×, 10×, 20× and 100× objectives (Plan-Apochromat 4×/0.2, 10×/0.45, 20×/0.8 or 100×/1.46). Sections processed for multiple immunofluorescence histochemistry were inspected and images acquired on a 700LSM confocal laser-scanning microscope (Zeiss, Oberkochen, Germany) at 10×, 20× or 63× primary magnification (Plan-Apochromat 10×/0.45, 20×/0.8 or 63×/1.40). Emission spectra for each dye were limited as follows: Cy2 (505–530 nm), Cy3 (560–610 nm) and Cy5 (650–720 nm). Multipanel figures were assembled in CorelDRAW X5 (Corel, Ottawa, Ont., Canada). Distribution patterns of secretagogin+ cells were analyzed by NeurolucidaTM (MicroBrightField, Brattleboro, Vt., USA) and mapped/projected onto schematic drawings of coronal brain sections (templates: http://www.avianbrain.org/atlases.html).  

 

 

 

 

 

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Western Blotting Tissues were homogenized in TNE buffer (50 mM Tris-HCl, 100 mM NaCl, 0.1 mM EDTA, pH 7.4) also containing 0.5% Triton X-100 (Sigma), 1% octyl-β-D-glucopyranoside (Calbiochem), 5 mM NaF, 100 μM Na3VO4 and a cocktail of protease inhibitors (cOmpleteTM; Roche, Basel, Switzerland) by sonication. The particulate fraction was pelleted by centrifugation (800 g, 10 min at 4 ° C). Protein concentrations were determined by Bradford’s col 

orimetric method [Bradford, 1976]. Samples were diluted to a final protein concentration of 2 μg/μl, denatured in 5× Laemmli buffer and analyzed by SDS-PAGE on 8% resolving gels. After transferring onto Immobilon-FL polyvinylidene difluoride membranes (Millipore, Billerica, Mass., USA), membrane-bound protein samples were blocked in 3% BSA and 0.5% Tween-20 diluted in Trisbuffered saline for 1.5 h, and subsequently exposed to secretagogin antibodies (table 1) overnight at 4 ° C. Appropriate combinations of horseradish peroxidase-conjugated secondary antibodies were used for signal detection (Jackson; from goat, rabbit or mouse hosts; 1:10,000, 2 h). Image acquisition and analysis were aided by a Bio-Rad XRS+ imaging platform. β-Actin (1:10,000; Sigma) was used as loading control throughout.

Brain Behav Evol 2014;83:82–92 DOI: 10.1159/000357834

Results

Secretagogin Expression in Neurons of the Chicken Fore- and Midbrain Three antibodies (table 1) were used to reveal secretagogin expression in the chick brain. We validated our antibodies by Western blotting using the rat olfactory bulb with known secretagogin expression as positive control [Mulder et al., 2009]. In samples from 1-, 13- and 90-dayold chick brain homogenates, we detected distinct and major bands with all three antibodies at 32 kDa, the predicted molecular weight of secretagogin and identical in size to that seen in rat olfactory bulb homogenates (fig. 1a). The rabbit anti-secretagogin antibody likely can resolve secretagogin isoforms in the chicken brain, as suggested by the protein tandem appearing at around the anticipated molecular weight of secretagogin (fig. 1a). All three antibodies showed similar staining patterns, as exempliGáti /Lendvai /Hökfelt /Harkany /Alpár  

 

 

 

 

a

c

e

b

b1

b2

e1

f

f1

c1

d

Fig. 1. Secretagogin (SCGN) is a neuronal marker in the chicken brain. a Secretagogin from 1- and 13-day- and 3-month-old chicken brains showing a primary protein band at 32 kDa. Rat olfactory bulb with outstanding secretagogin expression was used as positive control. Goat and rabbit secretagogin antibodies give a secondary protein band at nearby molecular weight in rat or chicken samples, respectively. b–b2 Anti-secretagogin antibodies produced in different hosts showed a largely similar expression pattern in the chicken brain. c, c1 Secretagogin colocalizes with microtubule-as-

sociated protein 2 (MAP2). Arrowheads point to colabeled processes. d Secretagogin+ cells do not show glial fibrillary acidic protein immunoreactivity. e, e1 Secretagogin labels the total somatodendritic compartment of a neuron in the hyperpallium apicale. Open arrowheads in e1 indicate spines. f, f1 Secretagogin+ neurons are more densely distributed in the 1-day-old than in the 13-dayold chicken brain. HA = Hyperpallium apicale; Hp = hippocampus; s = soma. Scale bars = 100 (b–b2), 50 (f, f1), 10 (e, c1, d1) and 3 μm (c1, d, e1).

fied in the dorsomedial domain, including the hyperpallium apicale and hippocampus, of 13-day-old chicks’ brain (fig.  1b–b2). Secretagogin+ cells co-expressed microtubule-associated protein 2 (fig.  1c, c1) but not glial fibrillary acidic protein (fig.  1d), confirming neuronal identity. In most brain regions and at all ages investigated, secretagogin immunohistochemistry revealed the whole somatodendritic dimension of the cell: in addition to strong somatic labeling, the dendritic arbor could be traced for well over 100 μms (fig. 1e), also allowing the refined visualization of dendritic spines (fig. 1e1). Secretagogin+ neuronal density decreased after hatching; secretagogin+ cells populated the 1-day-old brain more densely than that of 13-day-old chicks, as shown in the hippocampus and the neighboring periventricular region of the forebrain (fig. 1f, f1).

Distribution of Secretagogin+ Neurons in the Brain of 13-Day-Old Chicks Chickens show rapid functional and morphological brain maturation immediately after hatching [Sisken et al., 1993; Tömböl, 1995]. Neuronal morphology is fully differentiated by the end of the second week with virtually no further changes into adulthood [Tömböl, 1995], and similar results were shown regarding the stability of Ca2+-binding protein expression in the chicken midbrain with altered patterns only during aging [Santana et al., 2003]. Therefore, we used the 13-day-old chicken brain to map secretagogin expression.

Secretagogin in the Avian Brain

Olfactory Bulb and Pallium Secretagogin+ neurons appeared at peak density at the most cranial pole of the forebrain in the olfactory bulb Brain Behav Evol 2014;83:82–92 DOI: 10.1159/000357834

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Color version available online

Fig. 2. Distribution of secretagogin+ cells in

the chicken fore- and midbrain. Secretagogin+ cells (solid red circles) occur in different densities in the avian brain. Olfactory bulb (OB), septum, periventricular regions, the bed nucleus of stria terminalis (lateral part; BSTL), hypothalamic nuclei and deep layers of the optic tectum (OT) are loci with highest densities of immunoreactive cells. Cranial distance in millimeters from the interaural line is indicated in the left upper corner of each panel. AA = Arcopallium anterius; AD = arcopallium dorsale; CDL = area corticoidea dorsolateralis; DSv = nucleus decussationis supraopticae, pars ventralis; E = entopallium; GP = globus pallidus; H = habenula; HA = hyperpallium apicale; HD = hyperpallium densocellulare; Hp = hippocampus; Hy = hypothalamus; LH = lateral hypothalamus; LPS = lamina pallio-subpallialis; LSt = lateral striatum; M = mesopallium; MSt = medial striatum; N = nidopallium; NC = nidopallium caudale; NI = nidopallium intermedium; OM = occipitomesencephalic tract; PoA = posterior pallial amygdala; SL = nucleus septalis lateralis; SM = nucleus septalis medialis; SpA = subpallial amygdala; TnA = nucleus taeniae of the amygdala.

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Brain Behav Evol 2014;83:82–92 DOI: 10.1159/000357834

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a

a1

b

b2

b1

b2

b1

a1

c

d

e

f

Fig. 3. Secretagogin is expressed by distinct neuronal subsets in the chicken brain. a, a1 Secretagogin+ neurons condense in the olfactory bulb (OB) and in the continuing septal region. In the bulb, immunoreactive cells surround the circular domains of glomeruli. b–b2 Secretagogin+ cells populate the ventricular wall in extreme density (open arrowheads) with their processes contacting the ventricular wall (for higher-power images, see b1, b2) and outline the location of the septal nuclei (arrowheads). c The lateral part of the bed nucleus of stria terminalis (BSTL) is recognized via the large number of secretagogin+ neurons. d, e In the diencephalon, the supraoptic nucleus (nucleus decussationis su-

praopticae; DSv) and a cell column (open arrowheads in e) reaching the medial part of the bed nucleus of stria terminalis (BSTM) and extending along the medial aspect of the occipitomesencephalic tract (OM) showed secretagogin immunoreactivity. f The hypothalamus, especially its infundubular nucleus (IN), harbored a large number of secretagogin+ cells. Gl = Glomerulus; LH = lateral hypothalamus; N = nidopallium; v = ventricle. Scale bars = 100 (a, b, c, d, e, f), 10 (a1, b1) and 2 μm (b2). Distances from the interaural line are indicated in millimeters in the left bottom corner (for overview, please also use fig. 2).

(fig. 2, 3a). These neurons surrounded the glomeruli with 5- to 8-μm-large somata and only occasionally visible short dendrites (fig.  3a1). Many bi- or multipolar cells were identified in the hyperpallium, especially in its apical division, concentrated along the lateral margin of the ventricle or along the dorsal and dorsomedial brain surface (fig. 2). The dendrites of these neurons were either smooth or in other cases heavily decorated with spines (fig. 1e, e1). In contrast to the hippocampus, which was densely populated by secretagogin+ neurons (fig. 2), the densocellular and ventral parts of the hyperpallium were relatively spared of secretagogin immunoreactivity with residual secretagogin+ cells lining the border of the apical and densocellular hyperpallium and outlining the lamina frontalis suprema (fig. 2). The entopallium, mesopallium as well as the frontal and intermediate nidopallium were practically spared from immunoreactivity, with the medial-most/periventricular division of the nidopallium be-

ing a unique exception (fig. 3b, b1). Here, neuronal processes often ran transversally and contacted the ventricular wall (fig. 3b2). Similarly, evenly spaced neurons were seen in the caudal nidopallium (fig. 2). We also observed densely packed secretagogin+ neurons in the lateral and medial septal nuclei (fig. 3b).

Secretagogin in the Avian Brain

Brain Behav Evol 2014;83:82–92 DOI: 10.1159/000357834

Striatum and Globus Pallidus The region ventrally adjacent to the lamina pallio-subpallialis contains the medial and lateral parts of the striatum (previously referred to as the lobus parolfactorius and the paleostriatum augmentatum, respectively [Reiner et al., 2004]) and, ventromedially to these, the globus pallidus (‘paleostriatum primitivum’). Secretagogin+ neurons appeared in the caudal part of the globus pallidus, while they were infrequently seen in the striatal complex (fig. 2).

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Amygdala In mammals, subpallial and extended amygdala regions harbor many secretagogin+ cells, and this cell continuum is clearly segregated from surrounding territories [Mulder et al., 2010]. In the chick brain, we identified a distinct subset of secretagogin+ neurons in the bed nucleus of the stria terminalis (fig. 3c), the subpallial amygdala and in a more moderate number in the caudal part of the taenial amygdala (fig. 2). Coincidently, both the ventral and dorsal parts of the arcopallium as well as the posterior pallial amygdala contained secretagogin+ neurons (fig. 2). Diencephalon In the diencephalon, densely packed secretagogin+ neurons were found in the supraoptic nucleus (nucleus decussationis supraopticae; fig. 3d). Furthermore, secretagogin+ neurons were identified along the medial aspect of the occipitomesencephalic tract, reaching the medial part of the bed nucleus of stria terminalis (fig. 3e). These neurons formed a continuum from the supraoptic nucleus to the horizontal limb of the occipitomesencephalic tract (fig.  2). Moreover, the paraventricular and infundibular hypothalamic nuclei were densely populated by secretagogin+ neurons (fig. 2, 3f). Midbrain The avian midbrain has an intricately developed optic tectum, a multilayered structure that receives and processes retinal afferents and visual information, respectively [Tömböl, 1998]. Secretagogin+ neurons densely populated its deeper layers (fig. 2, 4a, a1); bipolar or pyramidal-shaped neurons with their large somata residing in layers 11/12 and their dendrites extending radially up to layer 5 occurred throughout the whole dorsoventral and craniocaudal dimensions of the optic tectum. Cells with smaller cell bodies in layers 7–10 but also in the deepest layers (layer 13) of the tectum were identified in considerable numbers (fig. 2, 4a, a1). At the dorsomedial aspect of the midbrain, the taenial nuclei harbored immunoreactive neurons in high density, similarly to mammals (fig. 2) [Mulder et al., 2009]. Secretagogin’s Segregation from Other Ca2+-Binding Proteins Neurons are typically committed to certain Ca2+-binding proteins, and even their genetic deletion remains usually uncompensated for by the expression of another EFhand family member [Schwaller, 2010]. Secretagogin distribution largely complemented that of the major 88

Brain Behav Evol 2014;83:82–92 DOI: 10.1159/000357834

‘neuronal’ Ca2+-binding proteins in the avian brain (fig. 4a–e1). Thus, neither parvalbumin nor calbindin colocalized with secretagogin in neurons of the optic tectum (fig. 4a–b1) or in any regions of the telencephalon (fig. 4d– e1). Similarly, calretinin+ cells remained immunonegative for secretagogin in the optic tectum and telencephalon (fig. 4c). However, secretagogin coexisted with parvalbumin and calbindin immunoreactivities in few cases in the nidopallium (fig. 4e1). We also assessed the relationship between cholinergic and secretagogin+ cell groups, since acetylcholine is widely expressed in the avian visual system, especially the optic tectum [Wang et al., 2006], and acetylcholine can colocalize with secretagogin in primates [Mulder et al., 2009]. Yet no colocalization was found (fig. 4f, f1). These data indicate that secretagogin+ neurons represent a distinct neuronal pool in the chicken brain.

Discussion

Secretagogin in the Avian Brain: A Tool for New Discoveries Neurons express Ca2+-binding proteins to control their Ca2+ homeostasis, which is essential to maintain their excitability and survival [Schwaller, 2010]. Ca2+binding proteins are expressed by well-defined neuronal populations [Celio, 1990] and likely contribute to shaping their action potential thresholds, frequency and forms of short-term synaptic plasticity. This is best exemplified by the fast-spiking nature of parvalbumin+ cells [Caillard et al., 2000; Collin et al., 2005]. Secretagogin was recently characterized as a neuronal Ca2+-binding protein whose distribution identifies novel neuronal subtypes and hierarchical organizing principles in the mammalian brain [Mulder et al., 2009, 2010]. Nevertheless, secretagogin expression in the avian brain remained unexplored. In birds, Ca2+-binding proteins were successfully used to identify anatomical and functional units of diverse brain areas [Montagnese et al., 1993; Heyers et al., 2008; Husband and Shimizu, 2011; Logerot et al., 2011; GarciaCalero and Scharff, 2013]. Here, we show that secretagogin labels select neuronal populations in the chick brain, including neurons of the olfactory bulb, septal nuclei, hippocampus, hypothalamus or in nuclei acknowledged as mammalian (extended) amygdala homologues. Thus, secretagogin appears as a novel neuronal marker of the avian brain, which can be used as a valuable tool to support future anatomical and functional studies.

Gáti /Lendvai /Hökfelt /Harkany /Alpár  

 

 

 

 

a

a1

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Fig. 4. Secretagogin (SCGN) shows complementary expression with other Ca2+-binding proteins and choline acetyltransferase. a, a1 Secretagogin labels neurons with large somata and vertically

oriented dendrites in layers 10/11 of the optic tectum. Smallersized neurons occur in layers 7–10. a1’–c Throughout the midbrain and within the optic tectum, secretagogin+ cells did not coexpress parvalbumin (PV), calbindin (CB) or calretinin (CR). d Secretagogin selectively labels a dense cellular locus at the ventricular wall. e, e1 Complementary distribution of secretagogin,

Secretagogin in the Avian Brain

parvalbumin and calbindin in the chicken telencephalon with eventual colocalization only between parvalbumin and calbindin (arrowhead in e1). f, f1 Secretagogin+ somata and dendrites do not co-express choline acetyltransferase (ChAT), a major neurochemical marker of the optic tectum. L3, L7, L11 = Layers of the optic tectum; Imc = nucelus isthmi, pars magnocellularis; IPS = nucleus interstitio-pretecto-subpretectalis; N = nidopallium; OT = optic tectum; SP = nucleus subpretectalis; V = ventricle. Scale bars = 100 (a2), 50 (a1, a1’, b, f), 20 (b1, c–e) and 10 μm (e1, f1).

Brain Behav Evol 2014;83:82–92 DOI: 10.1159/000357834

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Distribution of Secretagogin+ Neurons in Telencephalic Domains We have identified secretagogin in both hatchlings’ and adult chicks’ brains. System-wide neurochemical mapping revealed that fore- and midbrain regions harbored secretagogin+ neurons in broadly varying densities. The olfactory bulb, septum, bed nucleus of stria terminalis, hypothalamus and the deep layers of the optic tectum exhibited highest secretagogin+ neuron counts. This pattern shows similarities to mammals [Mulder et al., 2009, 2010] and suggests a preserved role of this Ca2+-binding protein in brain evolution. Secretagogin labeled periglomerular neurons in the olfactory bulb as well as a cell continuum dorsomedial to the olfactory bulb approaching the ventricle. In mammals, secretagogin+ neurons densely populate the rostral migratory stream as well as the periglomerular domain of olfactory glomeruli [Mulder et al., 2009]. Whilst hitherto unrevealed in chicken, the avian olfactory bulb has recently been shown to recruit a significant number of newborn cells in adult songbirds [Vellema et al., 2010]. It is tempting to speculate that secretagogin specifically might label newborn cell contingents recruited to a migratory stream of the avian olfactory system throughout adulthood. Despite the many cytological, immunohistochemical and connectivity analyses, the exact definition of the mammalian amygdala remains a continuing conundrum amongst neuroscientists [McDonald, 2003]. The discovery that secretagogin selectively labels subpallial amygdala neurons, including extended amygdala cell groups [Mulder et al., 2010], offers a new perspective: in birds, hodological, developmental, neurochemical and behavioral evidence supports the subpallial amygdaloid nature of the taenial nucleus, the (lateral and medial parts of) bed nucleus of stria terminalis and the subpallial amygdala [Aste et al., 1998; Absil et al., 2002; Kuenzel et al., 2011]. These areas were densely populated by secretagogin+ neurons. Furthermore, the medial part of the bed nucleus marked the dorsal end of a cell contingent that had aligned the medial aspect of the occipitomesencephalic tract. In turn, the ‘posterior archistriatum’, the region equivalent to the pallial amygdala homologue in birds [Zeier and Karten, 1971; Davies et al., 1997; Puelles et al., 2000], contained secretagogin+ neurons at apparently lower densities. These data support the concept that a ‘subpallial amygdala domain’ can be visualized in birds using secretagogin as a neurochemical marker. Structures at the medial surface of the chicken forebrain, including the hippocampus, as well as medial and 90

Brain Behav Evol 2014;83:82–92 DOI: 10.1159/000357834

lateral septal nuclei harbored many secretagogin+ cells. Whilst these data correspond with those described in mammals, we focused on the thinnest part of the chick septum, connecting the above regions medial to the ventricle, which was decorated by exceptionally dense secretagogin+ cell clusters. In mammals, the cells of the indusium griseum together with the anterior hippocampal continuation contain a chain of secretagogin+ cells that contact the ventricular surface with their dendrites and show similar firing properties to those of dentate granule cells [Mulder et al., 2009]. In birds, corresponding structures have remained unexplored due to the absence of dentate gyrus or fasciola cinerea homologues. Nonetheless, secretagogin+ cells in this thin brain tissue with their dendrites contacting the ventricular surface could form the avian counterparts of the above mammalian cell group. Hot Spots: Secretagogin+ Neurons at Specific Loci in the Avian Brain In addition to their above relation to anatomical brain regions or systems, secretagogin+ cells occurred at specific loci in the avian brain. First, secretagogin+ cell groups were seen at high densities at the ventricular wall. We hypothesize that these cells may contact the ventricular surface with their processes or serve as a site for adult neurogenesis. Whilst at this point either argument is speculative, they are conceivable since, firstly, secretagogin has been shown (i) to be involved in both release mechanisms [Gartner et al., 2007; Hasegawa et al., 2013] and (ii) the adult olfactory proliferation and migration system [Mulder et al., 2009], respectively. Secondly, secretagogin+ neurons line along cell-free laminae that separate brain regions with different pallial or subpallial origins. Thirdly, chains of cells decorate the telencephalic surface on its dorsolateral aspect, a field termed area corticoidea lateralis, an avian homologue of the cingulate cortex in mammals [Montagnese et al., 2003; Csillag and Montagnese, 2005]. The functional or evolutionary role of these cell groups remains elusive. Secretagogin+ Neurons in the Diencephalon and Midbrain – Complementary Distribution with Other Neuronal Ca2+-Binding Proteins The endocrine axis of mammals is decorated by secretagogin-expressing cells [Wagner et al., 2000]. In addition to peripheral expression sites like pancreatic β cells or enteroendocrine cells of the digestive system, the orchestrating center of the neuroendocrine system, the hypothalamus, contains secretagogin+ cells at remarkable denGáti /Lendvai /Hökfelt /Harkany /Alpár  

 

 

 

 

sities. Here, we show that the supraoptic nucleus (nucleus decussationis supraopticae, pars ventralis) is densely packed with secretagogin+ neurons with further cell groups present in the lateral hypothalamus, and paraventricular and infundibular nuclei. Since secretagogin was shown to impact vesicle secretion and release [Gartner et al., 2007; Bauer et al., 2011; Hasegawa et al., 2013], it is conceivable that secretagogin plays a role in the control of hypothalamic hormone secretion/release. Birds process visual stimuli in centers with unparalleled complexity to mammals. We show that secretagogin is expressed by neurons in distinct layers of the optic tectum. These cells failed to coexpress other Ca2+-binding proteins or to identify cholinergic cells, which suggests that secretagogin identifies distinct subsets of neurons in the largely heterogeneous cell population of layers 10/11 of the tectum where they could shape signal transduction and take part in synchronizing tectal channels [Wang et al., 2006]. In conclusion, we identified a novel neuronal marker that labels distinct neuronal subsets in the chicken brain.

Due to its specific expression pattern in different systems, secretagogin shall serve as a powerful tool to break functional, behavioral as well as comparative anatomical enigmas of the avian brain. Acknowledgments The authors thank Mathias Uhlén and Ludwig Wagner for anti-secretagogin antibodies. The technical assistance of Andrea Németh and Péterné Horváth is kindly acknowledged. This work was supported by the National Brain Research Program of the Hungarian Academy of Sciences (NAP; A.A.), the Doctoral School of the Semmelweis University (G.G. and A.A.), the Swedish Medical Research Council (T.Ha. and T.Hö.), the Swedish Brain Foundation (‘Hjärnfonden’; T.Ha.), Novo Nordisk Foundation (Nordic Endocrinology Research Initiative; T.Ha.) and the Petrus and Augusta Hedlunds’ Foundation (T.Ha. and T.Hö.).

Disclosure Statement The authors declare no conflict of interest.

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