Calcium (Ca²⁺) is essential for life, as it is necessary for the formation of teeth and bones, muscular contractions, and nervous system functioning. Among the multiple functions of Ca²⁺ in the nervous system, probably the most important one is acting as a second messenger, allowing neurotransmitter release and thus synaptic transmission (Augustine et al., 1987). To accomplish its functions, Ca²⁺ levels must be very finely regulated. One of the mechanisms involved in Ca²⁺ concentration control is the so-called calcium-binding proteins, which bind Ca²⁺ with appropriate affinity and specificity to maintain its concentration at specific levels (Brini et al., 2013). These calcium-binding proteins can be classified in two different families regarding their function: Ca²⁺ sensors and Ca²⁺ buffers. The first ones translate the signal of an increase in Ca²⁺ concentration into different biochemical responses, mainly by a conformational change. The best-known example is calmodulin. The other ones, Ca²⁺ buffers, modulate Ca²⁺ signals, as they bind free ions to remove them from the cytoplasm when they reach harmful levels or to transmit the Ca²⁺ signal throughout the cell (Gifford et al., 2007). Some examples of Ca²⁺ buffers are parvalbumin (PV), calretinin (CR) and calbindin-D28k (CB). Even though this functional classification is still valid, some Ca²⁺ buffers might also have sensor functions (Schwaller, 2009).

The abundance and variety of calcium-binding proteins in the central nervous system (CNS) is surprising. Some of them are ubiquitous, like calmodulin, but others are exclusively expressed in specific neuronal populations. This latter group includes PV (Celio and Heizmann, 1981), CR (Rogers, 1987), CB (Jande et al., 1981), and neurocalcin (NC) (Terasawa et al., 1992), all members of a homologous family of Ca²⁺ binding receptors, with a domain called EF-hand, which is a helix-loop-helix motif where the ion binds (Nakayama and Kretsinger, 1994). These proteins have distinct functions in the CNS. For example, PV-positive interneurons in the hippocampus might be involved in social memory, as they present higher activity when mice meet for the first time than when they are familiar with each other (Deng et al., 2019). In the case of CR, GABAergic neurons expressing CR in the prefrontal cortex might modulate limbic activity and thus affect emotional and cognitive behavior (Saffari et al., 2019). Nowadays, the relationship between these proteins and different pathologies has also been extensively studied, including autism spectrum disorders (Adorjan et al., 2017) and Alzheimer’s disease (Zallo et al., 2018). Beyond the study of their function, these proteins have also been widely used for neuroanatomical studies (Celio, 1990; Résibois and Rogers, 1992; Weruaga et al., 2000; Alonso et al., 2001; Briñón et al., 2001), as their staining reveals not only the neuronal soma but also dendritic processes and long portions of the axon.

Secretagogin (SCGN) is a calcium-binding protein discovered in 2000 in the β-pancreatic cells from the islets of Langerhans (Wagner et al., 2000). It is a member of the sensor family of calcium-binding proteins, with six EF-hand domains and the ability to bind four Ca²⁺ ions simultaneously with a half maximal affinity of 25 μM approximately, which is a relatively low affinity compared with other calcium-binding proteins (Rogstam et al., 2007).

In 2001 SCGN was observed for the first time in human post-mortem tissues (Gartner et al., 2001), and in 2009, it was studied in the nervous system of mice and lemurs, both in adult and developing neurons (Mulder et al., 2009). Since its discovery, SCGN has been widely studied in the brain of different species. Some studies show SCGN distribution in the whole brain, for example in fishes (Basu et al., 2022), birds (Gáti et al., 2014) or rats (Maj et al., 2012), but most of them focus on a specific brain area. In addition to the studies mentioned above, SCGN expression has been analyzed in different species in the amygdala, (Mulder et al., 2010; Gyengesi et al., 2013; Hevesi et al., 2021), the olfactory bulb (Mulder et al., 2009; Kosaka and Kosaka, 2013; Ortiz-Leal et al., 2023), or the hippocampus (Attems et al., 2007; Tapia-González et al., 2020), among others.

However, the specific function of SCGN in the CNS is still unclear. Some authors suggest that it is involved in the release of stress hormone, as it is necessary for the secretion of adrenocorticotropic hormone (Romanov et al., 2015). At a cellular level, SCGN is expressed earlier than other calcium-binding proteins in migrating neurons, so it could play an important role in the formation of neuronal circuits (Mulder et al., 2010), and may be involved in processes such as neuronal remodeling, differentiation, and proliferation (Alpár et al., 2012).

According to pathological situations, the study of SCGN may be relevant in the Alzheimer’s disease field. SCGN might make neurons largely resistant to neurodegeneration, as in human hippocampus the colocalization between SCGN and Tau-positive neurons has rarely been observed (Attems et al., 2008). Moreover, in a transgenic mice model with a widespread Tau pathology, the expression of SCGN in the hippocampus is reduced (Attems et al., 2011) and it has been observed in rats that SCGN interacts with Tau protein (Maj et al., 2012). In addition to Alzheimer’s disease, a deficiency in SCGN might be a risk factor for autism spectrum disorders (Liu et al., 2023). It has recently been discovered that the interaction between SCGN and Doc2α (a protein related to autism spectrum disorders) plays an important role in neuronal morphology, glutamatergic transmission and different behaviors of mice, leading to abnormalities if such interaction is disrupted (Wang et al., 2023). Finally, it has also been observed that SCGN could help to compensate for sensory deprivation in the olfactory bulb of mice (Pérez-Revuelta et al., 2020). However, additional information is necessary to support or reject these hypotheses, and the anatomical distribution of this protein can provide useful clues.

A major peculiarity of the nervous tissue is its intrinsic heterogeneity. Whereas cells in other tissues are considered similar elements, neurons differ in their morphology, position, synaptic connections, and chemical content. The study of the expression of different proteins along the nervous system is a useful tool (Merkulyeva et al., 2016; Reuss et al., 2016), as knowing the specific distribution of proteins can lead to establishing their function. Regarding SCGN expression, it has revealed new cellular types in different regions and species. For instance, in the mouse main olfactory bulb, SCGN marks some juxtaglomerular cells that do not express other neurochemical classification markers (Mulder et al., 2009; Kosaka and Kosaka, 2013). Similarly, in the zebrafish retina, SCGN labels a novel subpopulation of amacrine interneurons (Dudczig et al., 2017). Finally, in the rat striatum, SCGN characterizes certain subpopulations of known striatal interneurons, but probably also other neuronal groups with unknown function and structure (Kosaka et al., 2017).

In conclusion, even though different research articles have reported SCGN distribution in some discrete regions of the CNS of healthy adult mice, a more exhaustive analysis is needed. Here, we present a detailed study of SCGN expression in the whole mouse brain using immunohistochemistry, which allows us to observe a characteristic distribution of immunopositive neurons.

Secretagogin-immunoreactive cells were observed in many different nuclei and areas, with no differences among animals. The main characteristics of these groups of cells throughout the brain are described. A figure with coronal schemata of the more representative nuclei and areas with positive somata can be found at the end of the results (see Figure 8). Also, at the end of this part, a summary table collects all the nuclei presenting SCGN-positive cells, a semiquantitative analysis of the area covered by cell somata and the figures where they can be found (Table 1).

Several studies have analyzed the expression of SCGN in different brain areas of mice (Mulder et al., 2010; Gyengesi et al., 2013; Kosaka and Kosaka, 2013, 2018), but none of them has studied the whole brain, leaving many regions undescribed until the present study. In addition, many of these studies were carried out in developing or young animals, while we have used adult mice. Multiple studies have shown that the expression of different calcium-binding proteins changes during development (Hendrickson et al., 1991; Alfahel-Kakunda and Silverman, 1997; Fujiwara et al., 1997), so the use of adult mice can supplement those results from developing animals and provide a broader view of SCGN expression. The Allen Brain Atlas for mouse provides Scgn gene expression by in situ hybridization¹ (Lein et al., 2007) and the areas where the expression levels are higher (such as the different layers of the olfactory bulb) perfectly agree with those where we found more positive cells, which makes our results more robust.

Secretagogin is widely distributed in the mouse brain, similarly like other sensor calcium-binding proteins. This is not surprising, as Ca²⁺ homeostasis has to be finely regulated (Braun, 1990). However, SCGN distribution in mice is different from other calcium-binding proteins, and its total absence in the cerebellum and many cortical areas is really striking. Comparing our results in mice with those previously obtained in the rat brain, in both animal models SCGN-positive cells are missing in the cerebellum (Maj et al., 2012). In addition to this, similarities in SCGN expression can be found in the olfactory bulb, amygdala, hippocampus, basal ganglia, paraventricular nucleus or the superior colliculus of both species (Maj et al., 2012; Tapia-González et al., 2020; Hevesi et al., 2021). On the other hand, we did not find SCGN-positive cells in the supraoptic or suprachiasmatic nuclei, nor in the caudate putamen or the habenular region in mice, although its presence has been described in these nuclei in the rat brain (Maj et al., 2012).

Secretagogin expression is abundant in areas like the olfactory bulb and the hippocampus not only in rodents, but also in other mammals, such as humans (Attems et al., 2012; Tapia-González et al., 2020), red foxes (Ortiz-Leal et al., 2023), and even in chickens (Gáti et al., 2014) and catfishes (Basu et al., 2022). Knowing that the olfactory bulbs are part of the archicortex (the most primitive domain of the cerebral cortex), and that the hippocampus is included in the paleocortex, the next oldest cortical region, SCGN could have appeared in the brain early in evolution and could remain present in these cortical areas but not in more recent ones, where other calcium-binding proteins are abundant. A preserved role of SCGN in evolution has been also suggested by other authors (Gáti et al., 2014). On the other hand, some studies have compared the expression of SCGN in different mammals, mainly in mice, rats and humans, showing differences in the pattern of expression in areas like the developing neocortex (Raju et al., 2018), the hippocampus (Tapia-González et al., 2020) or the cerebellum (Maj et al., 2012), which shows phylogenetic differences across mammalian species, as has been previously reported (Mulder et al., 2010). Even though it is difficult to identify the cause for these differences, we could think that SCGN is not playing an essential role if it is present in some animals but not in others. Further research is necessary to know the specific function of SCGN in different animal species.

The expression of SCGN is also very heterogeneous when comparing different cerebral nuclei. Some nuclei had many SCGN-positive cells, which makes SCGN an ideal marker to identify them. This particular distribution could be related to the fact that having the same protein in most of the cells allows the modulation of the firing rate, as it happens with PV in the reticular thalamic nucleus (Albéri et al., 2013). By contrast, other nuclei had few SCGN-positive cells, which may mean that they express other calcium-binding proteins and that these cells have different functions. It has been observed that, in rat and primate striatum, SCGN separates two PV-positive cell populations with different structures, physiology, and topography (Garas et al., 2016). Future research combining SCGN with different calcium-binding proteins in mice is needed to confirm if these different distributions can be related to distinct functions. However, the colocalization analysis is beyond the scope of this work on the SCGN distribution.

Secretagogin might be a key element for the correct functioning of the senses, as it has been suggested by other authors due to its distribution in different sensory brain areas (Maj et al., 2019). Indeed, in the olfactory system, SCGN is present in a great number of cells of the olfactory bulb, but it also appears in the amygdala and other nuclei related to olfaction, such as the magnocellular preoptic nucleus and the horizontal limb of the diagonal band (Záborszky et al., 1986). This reinforces the idea that SCGN plays an essential role in olfaction, as previously described (Kosaka and Kosaka, 2013; Gáti et al., 2014; Pérez-Revuelta et al., 2020). SCGN expression is also remarkable in the visual system as immunoreactive cells are present in the superior colliculus and lateral geniculate nucleus, as well as in different cells of the retina (Puthussery et al., 2010; Weltzien et al., 2014). Finally, SCGN might be related to the sense of touch, as it appears in the cuneiform nucleus, the area where the secondary neurons of the dorsal column-medial lemniscus pathway (which carries the information from the posture, vibratory sensitivity, and discrimination between two points) are located. In addition, some authors have related SCGN expression to neurons that modulate nociception in the spinal cord of mice, rats, and humans (Shi et al., 2012).

Finally, SCGN may play an important role as a regulatory and neuroprotective protein at the cellular level. It acts as a regulatory protein in the amygdala (Hevesi et al., 2021) and controls the release of the stress hormone in the paraventricular hypothalamic nucleus (Romanov et al., 2015). Given the high density of SCGN-positive cells in the hypothalamus, it may also control the release of other hormones, such as the growth hormone, since it is released from the arcuate nucleus, where the SCGN expression was very abundant and similar to that found in the paraventricular hypothalamic nucleus. Finally, it is known that different calcium-binding proteins can influence the susceptibility of neurons to neurodegeneration (Fairless et al., 2019), and considering the expression of SCGN in the hippocampus, it is not surprising that it plays a role in neuronal resistance in Alzheimer’s disease (Attems et al., 2008). The neuronal resistance may be related to the type of damage, as SCGN expression increases in olfactory-deprived mice, but it does not change in the PCD (Purkinje Cell Degeneration) mice, which show neurodegeneration of the olfactory bulb (Pérez-Revuelta et al., 2020).

From these results, we can conclude that SCGN is widely distributed in the adult mouse brain and could be used as a specific marker for certain nuclei. SCGN is expressed by different neuronal populations and, based on their distribution and morphology, they might be involved in several functions and with different specific roles. Therefore, we consider the study of SCGN to be a very promising field that can help us to properly understand the regulatory systems of Ca²⁺ levels in the CNS, as well as other interesting neuronal properties, such as neuroresistance or vulnerability.

The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

The animal study was approved by the Bioethics Committee of the University of Salamanca (reference number: #00613). The study was conducted in accordance with the local legislation and institutional requirements.

PT, DD, and JA conceived the study, designed the experiments, wrote the manuscript, and prepared the figures. PT, LP-R, ÁC-A, and CH-P performed the histological analysis. PT, JV, TC, and EW carried out the image analyses. PT was a major contributor to the writing of the manuscript and the organization and design of all the figures. All authors critically revised and approved the final manuscript.