The Ca2+-binding proteins (CBPs) calbindin D28k, calretinin and parvalbumin are phenotypic markers of functionally diverse subclasses of neurons in the adult brain. The developmental dynamics of CBP expression are precisely timed: calbindin and calretinin are present in prospective cortical interneurons from mid-gestation, while parvalbumin only becomes expressed during the early postnatal period in rodents. Secretagogin (scgn) is a CBP cloned from pancreatic β and neuroendocrine cells. We hypothesized that scgn may be expressed by particular neuronal contingents during prenatal development of the mammalian telencephalon. We find that scgn is expressed in neurons transiting in the subpallial differentiation zone by embryonic day (E)11 in mouse. From E12, scgn+ cells commute towards the extended amygdala and colonize the bed nucleus of stria terminalis, the interstitial nucleus of the posterior limb of the anterior commissure, the dorsal substantia innominata (SI) and the central and medial amygdaloid nuclei. Scgn+ neurons can acquire a cholinergic phenotype in the SI or differentiate into GABA cells in the central amygdala. We also uncover phylogenetic differences in scgn expression as this CBP defines not only neurons destined to the extended amygdala but also cholinergic projection cells and cortical pyramidal cells in the fetal nonhuman primate and human brains, respectively. Overall, our findings emphasize the developmentally shared origins of neurons populating the extended amygdala, and suggest that secretagogin can be relevant to the generation of functional modalities in specific neuronal circuitries.
Temporal and spatial coordination of intracellular Ca2+signalling is essential to a cell’s ability for continuous dynamic adaptation to microenvironmental stimuli. Ca2+signalling in neurons is particularly important in controlling presynaptic Ca2+dynamics that underpin neurotransmitter release, transducing the activity of synaptic inputs (Burnashev & Rozov, 2005) and orchestrating multimodal intracellular signalling tuning neuronal excitability (Wang et al., 2004; Cohen & Greenberg, 2008). Both the homeostatic maintenance of intracellular [Ca2+] and the precise temporal control of its activity-dependent transients require effective mechanisms including Ca2+extrusion by plasmalemmal Ca2+-ATPases (Strehler et al., 2007), dissipating Ca2+oscillations via Ca2+uptake by intracellular stores (Nicholls, 2009), and chelation of free cytosolic Ca2+ by Ca2+-binding proteins (CBPs) (Andressen et al., 1993).
CBPs are generally viewed as ‘buffers’ to attenuate stochastic Ca2+ peaks in neurons (Andressen et al., 1993). Members of the EF-hand family of CBPs invariably contain a 3-D motif to bind Ca2+ (Heizmann, 1986). Ancestral representatives of the CBP family, e.g. calmodulin, are ubiquitously expressed with a high degree of evolutionary conservation, and control fundamental cellular functions ranging from the cell cycle, cell motility and axon polarization to synaptic signalling (Andressen et al., 1993). In contrast, the parvalbumin (PV) and calbindin subfamilies, the latter including the vitamin D-dependent 28 kDa isoform of calbindin (CB) and calretinin (CR), exhibit phylogenetically preserved tissue-specific expression patterns in vertebrates (Freund & Buzsaki, 1996; Klausberger & Somogyi, 2008), and are restricted to morphologically distinct subpopulations of GABAergic interneurons and local projection cells in rodent, primate and human corticolimbic circuits and extended amygdala (EA), the exception being CB, which is also expressed by cortical pyramidal and dentate granule cells (Celio, 1990).
The consensus exists that, although their developmental dynamics are different, CBPs are late markers of postmitotic GABA cells in both cortical and striatal territories (Flames & Marin, 2005; Wonders & Anderson, 2006): CB+ pioneer neurons populate the cerebral cortex by embryonic day (E)14 in mouse (Sanchez et al., 1992), and are also present in human fetal brain by week 14 of pregnancy (Brun et al., 1987). CR+ neurons invade the developing cerebrum by mid-gestation in both rodents and human (Verney & Derer, 1995; Meyer et al., 1998). While PV first appears at E13 in the spinal sensory system, the onset of PV expression in forebrain GABAergic neurons is restricted to the first postnatal week (Solbach & Celio, 1991), except in human telencephalon where PV+ Cajal–Retzius cells were noted by gestational weeks 20–24 (Verney & Derer, 1995).
Secretagogin (scgn) is a recently discovered CBP harbouring six putative EF-hand motifs (Rogstam et al., 2007) that was cloned from β cells of the pancreatic islands of Langerhans and endocrine cells of the gastrointestinal tract (Wagner et al., 2000). Although the distribution and neurochemical specificity of scgn+ neurons in the adult mouse, primate (Mulder et al., 2009b) and human brain (Attems et al., 2007) have recently been explored, scgn expression in the developing nervous system remains elusive. We report that scgn is expressed in mouse telencephalon from E11, and identifies neurons inhabiting the EA in mouse, primate and human brains.
Materials and methods
Animals and tissue preparation
Mouse embryos at E10.5–18.5 (n =4–6 per time point, n ≥2 pregnancies/analysis) were obtained from time-mated C57Bl6/N, glutamate decarboxylase (GAD)67-GFP (Δneo; Tamamaki et al., 2003) or choline-acetyltransferase (ChAT)(BAC)-EGFP mice (Tallini et al., 2006). We considered the day when vaginal smear was found in females as E0.5. Neonates were killed by decapitation on postnatal day (P)0, P1 or P2. Whole brains were immersion-fixed in 4% paraformaldehyde (PFA) in Na-phosphate buffer (PB; 0.1M, pH7.4) overnight. Tissue samples were cryoprotected in an ascending gradient of sucrose in PB (up to 30%) for at least 48 h prior to cryostat sectioning (14-μm thickness). Sections were thaw-mounted on SuperFrost+ glass slides.
Adult GAD67-GFP (n =3) and ChAT(BAC)-EGFP (n =2) reporter mice were anesthetized with isoflurane (5%; 1L/min flow rate) and transcardially perfused with 4% PFA in PB (100 mL; 3–4 mL/min flow rate) that was preceded by a pre-rinse with ice-cold physiological saline. Whole brains were removed from the skull, divided into fore- and hindbrain regions and post-fixed in the same fixative overnight. Tissue blocks were cryoprotected in 30% sucrose as above and serial 40-μm-think coronal sections were cut on a cryostat microtome.
Grey mouse lemur (Microcebus murinus, Primates) embryos and fotuses were obtained from a colony bred in captivity for the past ∼35 years with stock originating from the dry forest of the South-western coast of Madagascar (Perret & Aujard, 2001). Pregnant female mouse lemurs were maintained in isolation in appropriate cages, mimicking wild breeding conditions (Perret, 1992), with constant temperature and humidity. Food and water were provided ad libitum. The mean duration of gestation in mouse lemurs is 61 ± 0.2 days (Perret, 1990). The embryos (n =2) used in this report were harvested freshly from a spontaneous abortion at day 33 of intrauterine development. Fetal brain (n =1) was collected by hysterectomy under ketamine anesthesia that was indicated because of abnormal bleeding of the female on day 50 of pregnancy. None of the offspring were viable. Whole embryos and fetal brain samples were fixed in 4% PFA in PB for 48 h, rinsed in phosphate-buffered saline (PBS; 0.01M, pH 7.4) and kept in PBS also containing 0.1% NaN3 until processing. Primate tissues were cryoprotected and sectioned (14-μm thickness) onto SuperFrost+ glass slides. Brains of adult grey mouse lemurs were processed as described (Mulder et al., 2009b).
All experiments on animals conformed to the European Communities Council Directive (86/609/EEC) and were approved by the Home Office of the United Kingdom (mice) or the Ministry of Agriculture, France (#A91.114.1; lemurs). Particular care was taken to minimize the number of animals used for these experiments.
Total RNA isolated from tissues microdissected from C57Bl6/N embryos at E12 – P2 was subjected to scgn expression analysis after confirming RNA integrity (Supporting Information, Fig. S1A). Quantitative real-time PCR (qPCR) reactions were validated by preliminary testing of amplification efficacy and by excluding the possibility of genomic DNA contamination in the presence (+) or absence (−) of reverse transcriptase in parallel and running the samples on 1.5% agarose gel (supporting Fig. S1A1). qPCR reactions were performed with custom-designed primers for scgn (supporting Fig. S1A2–A4; Mulder et al., 2009b). TATA binding protein (forward primer, 5′-ACCCTTCACCAATGACTCCTATG-3′; reverse primer, 5′-ATGACTGCAGCAAATCGCTTGG-3′) was used to normalize scgn expression.
Protein samples were analyzed under denaturing conditions. After electrophoresis, proteins were transferred onto Immobilon-FL PVDF membranes (Millipore, Billerica, MA, USA) and probed with rabbit anti-scgn (1 : 2000) and mouse anti-β-actin (1 : 4000) primary antibodies (Mulder et al., 2009b). Immunoreactivities were revealed using IRDye680 and IRDye800 secondary antibodies (Invitrogen/Molecular Probes, Paisley, UK). Blots were scanned on a Li-Cor Odyssey-IR imager (Li-Cor Biosciences, Lincoln, NA, USA).
Generation of anti-secretagogin antibodies and histochemistry
Within the framework of the Human Protein Atlas program (http://www.proteinatlas.org), a rabbit antibody against a recombinant fragment of human scgn [amino acids (AA) 135-273] was generated (Mulder et al., 2009a). The specificity of the ensuing anti-scgn antibody has been extensively evaluated (Mulder et al., 2009b) in accordance with existing guidelines on the application of primary antibodies (Fritschy, 2008). We have further validated our novel anti-scgn antibody by comparing its labelling pattern with that of a commercial polyclonal anti-scgn antibody raised in goat against scgn’s AA164-276 fragment (R & D Systems, Minneapolis, MN, USA; supporting Fig. S2A) by both Western blotting (supporting Fig. S2B) and histochemistry (supporting Fig. S2C). We find that these antibodies unequivocally recognize a major protein band corresponding to scgn’s calculated molecular weight in Western applications (supporting Fig. S2B), and reveal the same neuron populations in E15 mouse forebrain (Fig. 3 and supporting Fig. S2C). Furthermore, our anti-scgn antibody produces a staining pattern in the olfactory bulb that is indistinguishable from that of a polyclonal anti-scgn antibody generated against the complete human scgn sequence (Wagner et al., 2000) (J. Attems & L. Wagner, personal communication).
Multiple immunofluorescence histochemistry with cocktails of primary antibodies (Table 1) was performed in both species studied (Mulder et al., 2009b). Immunosignals were visualized using combinations of carbocyanine (Cy) 2-, Cy3- and Cy5-conjugated antibodies raised in donkey (1 : 200–1 : 400; Jackson ImmunoResearch, West Grove, PA, USA). Hoechst 35,528 (Sigma, St Louis, MA, USA), a nuclear dye, was applied to reveal tissue architecture. Tissue autofluorescence in sections from adult mouse and primate brains was quenched with Sudan Black B (Schnell et al., 1999).
Table 1. List of antibodies used for multiple immunofluorescence labelling.
Panel of antibodies applied to study the distribution of scgn+ neurons in the developing rodent and primate nervous systems. Staining methods (Riedel et al., 2002; Mulder et al., 2009b) and antibody specificities were described in detail elsewhere (McDonald & Pearson, 1989; Celio, 1990; Karlsen et al., 1991; Schwaller et al., 1993; Hartig et al., 1998; Kitanaka et al., 1998; Arita et al., 2002; Bernier et al., 2002; Nagatsuka et al., 2003; Bossolasco et al., 2005; Aguado et al., 2006; Berghuis et al., 2007 and Keays et al., 2007). In particular images, immunofluorescence signals were color-coded for a better identification of fine structures. Abbreviation: Cy, carbocyanine.
Sections were inspected and representative images of immunoreactivity were acquired on a Zeiss 710LSM confocal laser-scanning microscope (Zeiss, Jena, Germany) equipped to separate emission signals through spectral detection and unmixing. Emission spectra for each dye was limited as follows: Hoechst (420–485 nm), Cy2 (505–530 nm), Cy3 (560–610 nm) and Cy5 (640-720 nm). Image surveys were generated using the tile scan function with optical zoom varied from 0.6× to 1.5× at 10× primary magnification (objective: EC Plan-Neofluar 10×/0.30). Co-localization was defined as immunosignals being preset without physical signal separation in ≤ 1.0-μm optical slices at 40× (Plan-Neofluar 40×/1.30) or 63× (Plan-Apochromat 63×/1.40) primary magnification (Mulder et al., 2009b). Images were processed using the ZEN2009 software (Zeiss). Multi-panel figures were assembled in CorelDraw X3 (Corel Corp., Ottawa, ON, Canada).
Quantitative morphometry, 3-D rendering and statistics
The diameter of scgn+ neurons was measured after capturing images of scgn+ cell assemblies in pallidal and EA territories at 40× primary magnification. The somatic diameter of individual neurons was measured on the premise that scgn is a cytosolic protein (Attems et al., 2007) and is homogenously distributed throughout the neuronal perikarya. Only neurons with smooth surfaces and processes were included in our analysis to avoid bias due to partial profiles of cell fragments.
Serial coronal sections (sampling interval, 140 μm) spanning the entire forebrain from an E15 mouse were double-labelled to reveal scgn+ neurons and cell nuclei (Hoechst 35,528). Single x-y plane images were acquired (Zeiss 710LSM), and 3-D-reconstructed using the BioVis3D software (BioVis3D, Montevideo, Uruguay).
Data are expressed as means ± SEM and analyzed using Student’s t-test (spss v.16.0, SPSS Inc., Chicago, IL, USA). A P-value of < 0.05 was considered statistically significant.
In situ hybridization in human fetal brain
Human fetal specimens at mid-gestation (21–22 weeks of gestation; n =3) were obtained from saline-induced abortions (Wang et al., 2004; Hurd et al., 2005). Protocols were approved by the local institutional review board (Institutional Review Boards of Kings County Hospital Center and Downstate Medical Center, State University of New York) as part of a large-scale study to evaluate the molecular effects of prenatal drug exposure on human neurodevelopment (Hurd et al., 2005). Specimens were fixed in 1% PFA and frozen at −80°C. Coronal cryosections (20 μm) spanning corticolimbic areas including the amygdaloid complex were cut. In situ hybridization was conducted as described (Wang et al., 2004): cDNA fragments of human scgn (NM_006998) were obtained from human brain total RNA by reverse transcription PCR using the primer pairs 5′-GGTTGATTTTCAGCCCAAGA-3′ (sense) and 5′-TTTGCACAAGGTGAAACAGC-3′ (anti-sense). The RNA probe was transcribed in the presence of [35S]-uridine 5′-[α-thio]triphosphate (specific activity 1000–1500 Ci/mmol; New England Nuclear, Boston, MA, USA). In situ hybridization was carried out as described (Hurd, 2003) by applying the labelled probe to the brain sections at a concentration of 2 × 103 cpm/mm2 of the coverslip area overnight at 55°C in a humidified chamber. Two adjacent sections from each subject were studied. The slides were then apposed to Imaging Plates (FUJIFILM Corporation, Tokyo, Japan) along with 14C-standards (American Radiolabelled Chemicals, St Louis, MO, USA). Films were developed with a phosphoimaging analyzer (FLA-7000), and images were analyzed using the MultiGauge software (FUJIFILM Corporation).
We have adopted the nomenclature of Paxinos & Franklin (2001) to describe the organization of the developing mouse brain. In addition, we have relied on the nomenclature introduced by Bons et al. (1998) for the adult mouse lemur to identify brain areas in the developing grey mouse lemur brain. A comprehensive list of abbreviations of neuroanatomical structures can be found in supporting Table S1.
Expression profiling in developing brain
Recent findings demonstrate that scgn is a CBP identifying neurochemically heterogeneous subsets of neurons in adult rodent, primate (Mulder et al., 2009b) and human forebrain (Attems et al., 2007). However, it remains unknown whether scgn is expressed during neurodevelopment. We assessed scgn mRNA levels in the mouse cerebral cortex (including hippocampus; Fig. 1A) and amygdaloid complex (Fig. 1A1) by qPCR analysis (supporting Fig. S1) during mid- and late-gestation, and in neonates. We established that pallial scgn mRNA expression was robust by E14.5, whilst moderate to low between E16 and P2 in the developing mouse neocortex and hippocampus (Fig. 1A). In contrast, scgn mRNA levels in the amygdaloid complex remained largely stable until birth with a marked decline being apparent after P1 (Fig. 1A1).
Within the framework of the Human Protein Atlas program (Uhlen et al., 2005; Mulder et al., 2009a), we have generated antibodies to > 8000 proteins, including a polyclonal antibody recognizing a phylogenetically conserved scgn epitope (Mulder et al., 2009b). Here, we confirmed that this antibody recognized a single protein target in samples prepared from neonatal mouse forebrain that is identical in size to that seen in adult brain (Fig. 1B; supporting Fig. S2), and corresponds to scgn’s calculated molecular weight of 32 kDa (http://www.ensembl.org). We explored whether scgn is expressed in the developing central nervous system at the protein level by detecting scgn protein upon loading fetal and neonatal forebrain lysates (40 μg/lane) on denaturing SDS-PAGE (Fig. 1C).
The developmental dynamics of scgn mRNA expression suggest that this CBP may be transiently expressed by select cell populations in the fetal brain. Alternatively, scgn+ cells may be born by ∼E14.5 with their population size remaining stable over time. Thus, the gradual decrease in scgn mRNA expression may merely reflect a proportional reduction in the prevalence of scgn+ cells during the progressive expansion of the embryonic forebrain until birth.
Scgn expression during organogenesis
We have tested scgn’s expression sites at mid-gestation by analyzing horizontal sections spanning the whole body of mouse (E13) and grey mouse lemur (E33) embryos. We used grey mouse lemurs because detailed information is available on both the intrauterine development of this prosimian primate (Perret, 1990) and the neurochemical specificity of scgn+ neurons in the adult lemur brain (Mulder et al., 2009b). Since the distinct timelines of rodent and primate embryogenesis may be a potential confounding factor in comparative analyses, we have chosen developmental stages in either species at which the general (supporting Fig. S3) and organ systems anatomy (Fig. 2) of the embryos are similar. We found significant scgn immunolabeling in the heart, pancreas, kidney and gonads of both mouse (Fig. 2A) and lemur embryos (Fig. 2B), corroborating prior findings in human tissues (Wagner et al., 2000; Lai et al., 2006). We also showed that scgn+ putative enteroendocrine cells (Lai et al., 2006; Gartner et al., 2007) populated the developing stomach in both species (Fig. 2B1). Whilst we failed to detect scgn immunosignal in the mouse dorsal root ganglion (DRG; Fig. 2C) at E13, scgn+ neurons co-expressing doublecortin (Fig. 2C1) were present in the lemur DRG. Scgn is not expressed in the liver during adulthood (Mulder et al., 2009b). Therefore, scgn immunoreactivity in embryonic liver may either indicate transient expression of this CBP or represent a methodological artifact due to unexpected tissue immunogenicity. Overall, our results suggest that scgn is expressed in several organ systems of mid-gestation mammalian embryos.
Emergence of scgn in postmitotic neurons in the developing mouse forebrain
We find scgn+ cells at E11 in the mouse telencephalon (Fig. 3A and B). Clusters of scgn+ cells could be observed at least at two locations in the wall of the cerebral vesicle: in its anterior wall forming the olfactory bulb (OB; Fig. 3A) and in the subpial area of the ganglionic eminence (GE). At E12, scgn+ cells transit in the differentiation zone that commits neurons to the prospective globus pallidus (GP; Fig. 3C). Scgn+ cells were immunoreactive for β-III-tubulin, but not nestin (neural progenitor), RC2 (radial glia) or Brn-1 (neocortical pyramidal cell) during the period of E11-12, suggesting that scgn marks postmitotic, non-pyramidal neurons at the subpial surface of the telencephalic vesicle. The scgn+ cell pool expands by E13 with cells traversing the palliosubpallial boundary in two directions: a contingent of cells adopts scgn+/GABA+ phenotype upon entering the OB (Fig. 5C and C1). In the present study, we focused on scgn+ cells that migrate in the subpallium caudally (Fig. 3D–D4) and commit neurons to the EA (Fig. 3D1 and D2). Although we did not engage in performing detailed analysis of migratory routes, the gradual colonization of the bed nucleus of the stria terminalis (BST) and amygdaloid territories by scgn+ neurons between E13 and E15 (Fig. 3E–E6) suggests that these cells first migrate caudally in the lateral subpallium before turning, and migrating in the lateral-to-medial direction within the EA. In sum, our analysis revealed that scgn+ cells cytoarchitecturally resembling migrating neurons formed a continuous stream along the palliosubpallial boundary before reaching their final destinations in the OB or EA (Fig. 4).
Next, we analyzed the distribution of scgn+ neurons in neonatal mouse brain. We observed that the migration of scgn+ cells concluded by birth and scgn+ neurons inhabited, in an anterior-to-posterior direction, the spatially interrelated nuclei of the BST, interstitial nucleus of the posterior limb of the anterior commissure (IPAC), ventral pallidum (VP), dorsal substantia innominata (SI), and the central and medial amygdaloid nuclei (CA and MA; Fig. 5A–A7). Morphometric analysis revealed that scgn identifies divergent neuron subpopulations with different somatic diameters in the VP and EA (Fig. 5B). By using genetically tagged reporter mice we demonstrated that scgn+ neurons either adopted a GABA phenotype along the longitudinal axis of the EA (Fig. 5D and D1), similar to scgn+/GABA+ neurons in the embryonic OB (Fig. 5C and C1), or co-express ChAT when found in small-diameter cholinergic neurons of the dorsal SI (Fig. 5E and E1). Collectively, our data suggest that by E18 scgn+ neurons can acquire a distribution pattern resembling that in the adult brain, and differentiate into neurochemically distinct subtypes of EA neurons.
Scgn expression in fetal primate brain
Systematic analysis along the longitudinal axis of the fetal primate brain revealed the first contingent of scgn+ neurons in the granular and glomerular layers of the OB (Fig. 6A). However, unlike in the adult primate brain (Mulder et al., 2009b), neuroblasts migrating in the prenatal rostral migratory stream (Pencea & Luskin, 2003) did not harbour scgn expression (Fig. 6A). Pallial areas were devoid of discernible scgn immunoreactivity. In the basal forebrain, scgn+ neurons were seen in the horizontal diagonal band, nucleus accumbens, medial septum, VP, GP and SI (Fig. 6A1–A7). In contrast to scgn distribution in the neonatal rodent brain, scgn+ cells were only infrequently found in either the CA or MA. In the hypothalamus, substantial scgn+ neuron populations occupied the paraventricular and periverticular nuclei and the supraoptic nucleus (Fig. 6A5–A7). A morphological dichotomy of scgn+ neurons was evident in the basal telencephalon (Fig. 6B): small-to-medium-sized scgn+ neurons populated the horizontal diagonal band, SI and CA. In contrast, large-diameter scgn+ neurons were found in the IPAC and GP. Clusters of large-diameter neurons enveloping the dorsal and ventral tips of the GP as well as medium-sized neurons heterogeneously invading the ventral subdivision of this territory were ChAT+/CB+ cholinergic neurons (Fig. 6C–C2; Geula et al., 1993). These data demonstrate that the cholinergic identity of scgn+ cells is acquired by the third trimester of pregnancy.
Scgn in the adult amygdaloid complex
To evaluate the fate of scgn+ neurons in the EA we analyzed sections from ChAT-EGFP and GAD67-GFP reporter mice, allowing precise delineation of the anatomical boundaries of individual amygdaloid nuclei (Fig. 7A and B). Scgn expression was limited to two morphologically distinct types of neurons in the EA (Fig. 7C): scgn+ neurons with oval perikarya and short ramifying dendrites (Fig. 7Ca) predominate in the CA and IPAC. In contrast, scgn+ neurons as above are intermingled with stellate-like cells with fusiform perikarya and long, smooth primary dendrites in the MA (Fig. 7Cb–Cd). ChAT+/scgn+ neurons were exclusively identified in the VP and dorsal segment of the SI (Mulder et al., 2009b) but not amygdaloid nuclei (Fig. 7D). GAD67+/scgn+ small-diameter neurons were frequently encountered in the CA and MA (Fig. 7E) but not the IPAC (Fig. 7E1) or the intraamygdaloid segment of the BST (Fig. 7E2).
A clear phylogenetic difference in the distribution of scgn+ neurons was the complete absence of scgn immunoreactivity in small-diameter GABAergic neurons of the primate CA and MA. Instead, fusiform scgn+ neurons populated the MA (Fig. 7F).
Based on their cellular origins and connectivity maps, the lateral, basolateral, basomedial and cortical amygdaloid nuclei are classified as pallial structures. In contrast, the BST, CA, MA and SI are of subpallial origin. BST and SI neurons are thought to originate in the pallidal ridge, while neurons inhabiting the CA and MA share their birthplace with striatal neurons (Swanson & Petrovich, 1998). Therefore, the selective expression of scgn in pallidal amygdaloid territories further illustrates the above developmental dichotomy.
Scgn expression in fetal human brain
Distinct anatomical organization of scgn mRNA expression with heterogeneous signal in a number of structures was evident in the fetal human brain (Fig. 8). Scgn mRNA distribution patterns were similar in all subjects studied. Invariably strong scgn anti-sense probe hybridization signal was observed throughout the cortical plate of the cerebral cortex (Fig. 8A and B) and in the amygdaloid complex (Fig. 8B). Moderate scgn mRNA expression was observed in the hippocampus, subiculum, thalamic territories and germinal layers, whereas low signal intensity was seen in the caudate nucleus. These data demonstrate that scgn expression in the mammalian amygdaloid complex is phylogenetically conserved. In addition, our results highlight that scgn expression in pyramidal cells is developmentally regulated and can endure into adulthood in this cell type (Attems et al., 2007).
Our report identifies the developmental dynamics of scgn expression including the migratory routes and final positions of subpallial neurons expressing this CBP in rodent, primate and human fetal brain. Distinct temporal expression patterns of the three ‘classical’ CBPs, CB, CR and PV, have generated broad interest because of the importance of GABAergic neurons in refining the physiological output of neuronal networks (Freund & Buzsaki, 1996; Klausberger & Somogyi, 2008). Here, we show that scgn is expressed earlier than CB, CR or PV in pioneer neurons exiting the pallidal differentiation zone by E11 in mouse. Histochemically noticeable scgn expression is restricted to postmitotic neurons because scgn+ cells lack the expression of RC2 and nestin, radial glia and neural stem/progenitor cell markers (Carleton et al., 2003), respectively. The majority of scgn+ cells we identified migrated towards the prospective EA, and selectively inhabited its subpallial domain by forming a continuum of scgn+ neurons extending from the anterior tip of the VP towards the CA and MA. The lack of Brn-1, a POU homeodomain protein specifying neocortical pyramidal cells (Sugitani et al., 2002), supports the idea that scgn+ cell contingents are destined towards subpallial territories.
Scgn+ neurons commute in at least two major migratory streams along the palliosubpallial boundary and clearly avoid venturing into neocortical territories during forebrain development. Scgn+ neurons populating the OB travel in the anterior direction and upon reaching the olfactory granular layer frequently (> 20%) acquire GAD67+/GABA+ phenotypes. In contrast, scgn+ neurons travelling caudally to colonize the EA exhibit a substantially lower percentage (7–9%) of co-localization with GAD67 en route to their final positions. The diversity of neuronal contingents destined to the EA is first demonstrated by the bifurcation of their migratory stream at the level of the IPAC: small- to-medium-sized scgn+ neurons, many of which are GABAergic (Fig. 5), invade the CA and MA. Whilst we show that scgn can developmentally co-exist with GAD, our prior (Mulder et al., 2009b) and present analysis in adult mouse and primate forebrain reveal a limited likelihood of co-expression of scgn with the other known neuron-specific CBPs, particularly CR and CB. Alternatively, scgn+ neurons can co-express ChAT, a ubiquitous cholinergic marker (Riedel et al., 2002), upon populating the SI.
Intracellular Ca2+signalling underpins the responsiveness of developing neurons to extracellular guidance cues. We unexpectedly found that scgn is already plentiful in subsets of neurons engaged in long-distance migration with histochemically-detectable levels of this CBP maintained throughout neuronal morphogenesis. This notion may pinpoint that the scgn-mediated control of intracellular Ca2+signalling can play a role in generating adequate cellular responses to microenvironmental stimuli that are specifically present at the palliosubpallial boundary. Otherwise, scgn may be one of the early molecular determinants required for amygdala neurons to integrate into neuronal networks and to acquire specialized functions therein.
In the postnatal nervous system, CBPs are important cytosolic modulators of Ca2+signalling in neurons whose presence has been associated with the maintenance of distinct electric discharge patterns. In the basolateral amygdala, PV+ interneurons form a primary local modulatory neuronal subnetwork affecting the integration of polymodal sensory information by excitatory principal cells (Woodruff & Sah, 2007a,b). Our discovery that scgn+ neurons are only present in circumcised clusters in the EA present a number of intriguing possibilities both at the single-cell and neuronal network levels: secretagogin is an EF-hand CBP capable of simultaneously binding four Ca2+ions at physiological intracellular Ca2+levels (Rogstam et al., 2007), with an affinity similar to those of the classical neuronal CBPs. Therefore, when scgn is present in neurons otherwise lacking PV, CB or CR, this CBP may contribute to the refinement of intracellular Ca2+signalling with an as yet unknown impact on cellular excitability and integration of afferent inputs. When scgn is co-expressed with CR or CB it could account for a substantially enhanced Ca2+-buffering capacity, thus sub-diversifying the responsiveness and network contribution of a particular neuron. However, we also entertain the possibility that scgn identifies a hitherto unknown but neurochemically distinct class of GABAergic neurons in the CA. Therefore, subsequent studies aimed to elucidate scgn’s functional significance will undoubtedly advance our understanding of the neurobiological principles that govern the organization and function of amygdaloid neuronal circuitries.
Scgn expression exhibits robust phylogenetic differences across mammalian species. Scant scgn expression is found in the SI in rodent brain. However, virtually all cholinergic basal forebrain projection neurons are scgn+ and/or scgn+/CB+ in primate brain. This difference suggests that cholinergic lineage commitment associates with a selective upregulation of scgn expression in higher-order mammals. This evolutionary transitions can be significant in explaining the differential sensitivity of rodent and primate cholinergic neurons to both physiological and noxious stimuli, and might impact cholinergic neurotransmission both at the presynaptic (neurotransmitter release) and postsynaptic (second messenger signalling) levels. Such changes may be required to accommodate the increased complexity and diversity of information processed upon expansion of isocortical areas, the primary targets of cholinergic basal forebrain afferents (Mesulam et al., 1983). A critical difference between scgn expression in prosimian primate and human brain is the unique scgn expression in pyramidal neurons of the human hippocampus (Attems et al., 2007, 2008). Our in situ hybridization data in mid-gestational human embryos corroborate and extend these findings by demonstrating scgn mRNA expression in the neocortex (cortical plate), hippocampus, and prospective amygdala.
In conclusion, our present report establishes scgn as a fourth developmentally-regulated, neuron-specific CBP, whose phylogenetic preservation and selective association with neurochemically distinct subsets of neurons suggest novel dimensions of functional modalities within the extended amygdala circuitry.
The authors thank Dr B. Leete (Zeiss Microscopy UK) for help with image processing and analysis. Drs K.M. Sousa (University of Michigan) and O. Kiehn (Karolinska Institute) are acknowledged for their critical comments on this manuscript. This work was supported by the Scottish Universities Life Science Alliance (T. Harkany), Alzheimer’s Association (T. Harkany), Alzheimer’s Research Trust (ART) UK (J.M. & T. Harkany), European Molecular Biology Organization Young Investigator Programme (T. Harkany), National Institutes of Health grant DA023214 (T. Harkany, Y.L.H.), Swedish Medical Research Council (T. Hökfelt, T. Harkany), European Commission (HEALTH-F2–2007-201159; T. Harkany), Grants-in-Aid for Scientific Research from the MEXT, Japan (Y.Y.), Takeda Science Foundation (Y.Y.), and Knut and Alice Wallenberg Foundation (M.U.). J.M. is the recipient of a postdoctoral fellowship from ART UK. L.S. is a Medical Research Council-Integrated Toxicology Training Partnership (MRC-ITTP) postgraduate fellow.
bed nucleus of stria terminalis
central amygdaloid nucleus
dorsal root ganglion
glutamic acid decarboxylase
interstitial nucleus of the posterior limb of the anterior commissure