UNASSIGNED: The amygdala is a noticeable bilateral structure in the medial temporal lobe and it is composed of at least 13 different nuclei and cortical areas, subdivided into the deep nuclei, the superficial nuclei, and the remaining nuclei which contain the central nucleus (CeA). CeA mediates the behavioral and physiological responses associated with fear and anxiety through pituitary-adrenal responses by modulating the liberation of the hypothalamic Corticotropin Releasing Factor/Hormone.
UNASSIGNED: Five dolphins of three different species, belonging to the family Delphinidae (three striped dolphins, one common dolphin, and one Atlantic spotted dolphin), were used for this study. For a precise overview of the CeA\'s structure, thionine staining and the immunoperoxidase method using calbindin D-28k were employed.
UNASSIGNED: CeA extended mainly dorsal to the lateral nucleus and ventral to the striatum. It was medial to the internal capsule and lateral to the optic tract and the medial nucleus of the amygdala.
UNASSIGNED: The dolphin amygdaloid complex resembles that of primates, including the subdivision, volume, and location of the CeA.

UNASSIGNED: The entorhinal cortex has been shown to be involved in high-level cognitive functions in terrestrial mammals. It can be divided into two main areas: the lateral entorhinal area (LEA) and the medial entorhinal area (MEA). Understanding of its structural organization in cetaceans is particularly important given the extensive evidence for their cognitive abilities. The present study describes the cytoarchitectural and immunohistochemical properties of the entorhinal cortex of the bottlenose dolphin (Tursiops truncatus, Montagu, 1821), perhaps the most studied cetacean species and a paradigm for dolphins and other small cetaceans.
UNASSIGNED: Four bottlenose dolphins\' entorhinal cortices were processed. To obtain a precise overview of the organization of the entorhinal cortex we used thionin staining to study its laminar and regional organization, and immunoperoxidase technique to investigate the immunohistochemical distribution of three most commonly used calcium-binding proteins (CBPs), calbindin D-28k (CB), calretinin (CR) and parvalbumin (PV). Entorhinal cortex layers thickness were measured, morphological and morphometric analysis for each layer were conducted and statistically compared.
UNASSIGNED: Six layers in both the LEA and MEA were identified. The main difference between the LEA and the MEA is observed in layers II and III: the neurons in layer II of the LEA were denser and larger than the neurons in layer II of MEA. In addition, a relatively cell-free zone between layers II and III in LEA, but not in MEA, was observed. The immunohistochemical distribution of the three CBPs, CB, CR and PV were distinct in each layer. The immunostaining pattern of CR, on one side, and CB/PV, on the other side, appeared to be distributed in a complementary manner. PV and CB immunostaining was particularly evident in layers II and III, whereas CR immunoreactive neurons were distributed throughout all layers, especially in layers V and VI. Immunoreactivity was expressed by neurons belonging to different morphological classes: All CBPs were expressed in non-pyramidal neurons, but CB and CR were also found in pyramidal neurons.
UNASSIGNED: The morphological characteristics of pyramidal and non-pyramidal neurons in the dolphin entorhinal cortex are similar to those described in the entorhinal cortex of other species, including primates and rodents. Interestingly, in primates, rodents, and dolphins, most of the CBP-containing neurons are found in the superficial layers, but the large CR-ir neurons are also abundant in the deep layers. Layers II and III of the entorhinal cortex contain neurons that give rise to the perforant pathway, which conveys most of the cortical information to the hippocampal formation. From the hippocampal formation, reciprocal projections are directed back to the deep layer of the entorhinal cortex, which distributes the information to the neocortex and subcortical area. Our data reveal that in the dolphin entorhinal cortex, the three major CBPs label morphologically heterogeneous groups of neurons that may be involved in the information flow between entorhinal input and output pathways.

Amyloid β (Aβ) oligomers are the most neurotoxic forms of Aβ, and Aβ(1-42) is the prevalent Aβ peptide found in the amyloid plaques of Alzheimer\'s disease patients. Aβ(25-35) is the shortest peptide that retains the toxicity of Aβ(1-42). Aβ oligomers bind to calmodulin (CaM) and calbindin-D28k with dissociation constants in the nanomolar Aβ(1-42) concentration range. Aβ and histidine-rich proteins have a high affinity for transition metal ions Cu2+, Fe3+ and Zn2+. In this work, we show that the fluorescence of Aβ(1-42) HiLyteTM-Fluor555 can be used to monitor hexa-histidine peptide (His6) interaction with Aβ(1-42). The formation of His6/Aβ(1-42) complexes is also supported by docking results yielded by the MDockPeP Server. Also, we found that micromolar concentrations of His6 block the increase in the fluorescence of Aβ(1-42) HiLyteTM-Fluor555 produced by its interaction with the proteins CaM and calbindin-D28k. In addition, we found that the His6-tag provides a high-affinity site for the binding of Aβ(1-42) and Aβ(25-35) peptides to the human recombinant cytochrome b5 reductase, and sensitizes this enzyme to inhibition by these peptides. In conclusion, our results suggest that a His6-tag could provide a valuable new tool to experimentally direct the action of neurotoxic Aβ peptides toward selected cellular targets.

The protein calbindin-D28k modulates calcium reabsorption in the kidney. Here, we aimed to study the influence of proliferation and apoptosis in different compartments of the kidney on the developmental function of calbindin. Using immunohistochemistry, we investigated the postnatal development of rats\' kidneys by using calbindin, proliferative cell nuclear antigen (PCNA), and apoptotic single-stranded DNA (ssDNA). In the neonatal stage (1-day and 1-week-old rats), calbindin showed a positive reaction in the distal convoluted tubule (DCT), a short nephron segment between the macula densa, collecting ducts, and tubules. Moreover, the localization of calbindin was restricted to immature nephrons and mesenchymal tissues. Furthermore, PCNA immunoreactivity was moderate in early-developed podocytes with no reactivity in other renal tubules. The ssDNA immunoreactivity was moderate in the undifferentiated nephron. Then, in the mature stage (3 and 6 weeks old), there was an intense calbindin reaction in DCT but a moderate reaction to PCNA and ssDNA in podocytes. A more intense calbindin reactivity was found in the adult stage (2- and 3-month-old rats) in DCT and collecting tubules. Therefore, in this study, calbindin localization showed an inverse relationship with PCNA and ssDNA of the nephron compartments, which might reflect the efficiency of bone-building and muscle contraction during animal development.

Early life stress (ELS) in developing children has been linked to physical and psychological sequelae in adulthood. In the present study, we investigated the effects of ELS on brain and behavioral development by establishing a novel ELS model that combined the maternal separation paradigm and mesh platform condition. We found that the novel ELS model caused anxiety- and depression-like behaviors and induced social deficits and memory impairment in the offspring of mice. In particular, the novel ELS model induced more enhanced depression-like behavior and memory impairment than the maternal separation model, which is the established ELS model. Furthermore, the novel ELS caused upregulation of arginine vasopressin expression and downregulation of GABAergic interneuron markers, such as parvalbumin (PV), vasoactive intestinal peptide, and calbindin-D28k (CaBP-28k), in the brains of the mice. Finally, the offspring in the novel ELS model showed a decreased number of cortical PV-, CaBP-28k-positive cells and an increased number of cortical ionized calcium-binding adaptors-positive cells in their brains compared to mice in the established ELS model. Collectively, these results indicated that the novel ELS model induced more negative effects on brain and behavioral development than the established ELS model.

Calbindin-D28k is a calcium binding protein that is highly expressed in the mammalian central nervous system. It has been reported that calbindin-D28k binds to and increases the activity of inositol Monophosphatase (IMPase). This is an enzyme that is involved in the homeostasis of the Inositol trisphosphate signalling cascade by catalysing the final dephosphorylation of inositol and has been implicated in the therapeutic mechanism of lithium treatment of bipolar disorder. Previously studies have shown that calbindin-D28k can increase IMPase activity by up to 250 hundred-fold. A preliminary in silico model was proposed for the interaction. Here, we aimed at exploring the shape and properties of the calbindin-IMPase complex to gain new insights on this biologically important interaction. We created several fusion constructs of calbindin-D28k and IMPase, connected by flexible amino acid linkers of different lengths and orientations to fuse the termini of the two proteins together. The resulting fusion proteins have activities 200%-400% higher the isolated wild-type IMPase. The constructs were characterized by small angle X-ray scattering to gain information on the overall shape of the complexes and validate the previous model. The fusion proteins form a V-shaped, elongated and less compact complex as compared to the model. Our results shed new light into this protein-protein interaction.

Amyloid β1-42 (Aβ(1-42)) oligomers have been linked to the pathogenesis of Alzheimer\'s disease (AD). Intracellular calcium (Ca2+) homeostasis dysregulation with subsequent alterations of neuronal excitability has been proposed to mediate Aβ neurotoxicity in AD. The Ca2+ binding proteins calmodulin (CaM) and calbindin-D28k, whose expression levels are lowered in human AD brains, have relevant roles in neuronal survival and activity. In previous works, we have shown that CaM has a high affinity for Aβ(1-42) oligomers and extensively binds internalized Aβ(1-42) in neurons. In this work, we have designed a hydrophobic peptide of 10 amino acid residues: VFAFAMAFML (amidated-C-terminus amino acid) mimicking the interacting domain of CaM with Aβ (1-42), using a combined strategy based on the experimental results obtained for Aβ(1-42) binding to CaM and in silico docking analysis. The increase in the fluorescence intensity of Aβ(1-42) HiLyteTM-Fluor555 has been used to monitor the kinetics of complex formation with CaM and with calbindin-D28k. The complexation between nanomolar concentrations of Aβ(1-42) and calbindin-D28k is also a novel finding reported in this work. We found that the synthetic peptide VFAFAMAFML (amidated-C-terminus amino acid) is a potent inhibitor of the formation of Aβ(1-42):CaM and of Aβ(1-42):calbindin-D28k complexes.

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The medial preoptic area, which plays an essential role in the control of sexual behavior in rats, contains a sexually dimorphic nucleus that consists of neurons expressing calbindin-D28 K (Calb) that is referred to as the CALB-SDN. The CALB-SDN is larger and contains more Calb neurons in males than in females. The physiological functions of the CALB-SDN are not fully understood; however, CALB-SDN neurons are activated during sexual behavior in males, suggesting that the male CALB-SDN is involved in regulation of sexual behavior. However, no information exists about the physiological functions of the female CALB-SDN. In the present study, we performed an immunohistochemical analysis of c-Fos, a neuronal activity marker, in the CALB-SDN of female and male rats that had copulated with conspecifics of the opposite sex to determine whether neurons of the female CALB-SDN are activated during copulation and whether the neuronal activity of the CALB-SDN differs between sexes. The numbers of c-Fos-immunoreactive cells with or without Calb-immunoreactivity (c-Fos+/Calb+ and c-Fos+/Calb- cells) were greater in the CALB-SDN of rats that had copulated than in rats that had not copulated in each sex. Although the number of Calb+ cells in the CALB-SDN was smaller in females than in males, the increase in the number of c-Fos+/Calb+ cells in the female CALB-SDN with copulation was comparable to that in the male CALB-SDN with copulation. The increase in the number of c-Fos+/Calb- cells in the CALB-SDN with copulation was more prominent in males than in females. These results suggest that CALB-SDN neurons are activated during copulation in both sexes. The patterns of neuronal activation in the CALB-SDN during copulation may differ between sexes.

The presence of the lateral cervical nucleus (LCN) in different mammals, including humans, has been established in a number of anatomical research works. The LCN receives its afferent inputs from the spinocervical tract, and conveys this somatosensory information to the various brain areas, especially the thalamus. In the present study, the organization of the calf and pig LCN was examined through the use of thionine staining and immunohistochemical methods combined with morphometrical analyses. Specifically, the localization of calbindin-D28k (CB-D28k) and neuronal nitric oxide synthase (nNOS) in the LCN was investigated using the immunoperoxidase method. Calf and pig LCN appear as a clearly defined column of gray matter located in the three cranial segments of the cervical spinal cord. Thionine staining shows that polygonal neurons represent the main cell type in both species. The calf and pig LCN contained CB-D28k-immunoreactive (IR) neurons of varying sizes. Large neurons are probably involved in the generation of the cervicothalamic pathway. Small CB-D28k-IR neurons, on the other hand, could act as local interneurons. The immunoreactivity for nNOS was found to be mainly located in thin neuronal processes that could represent the terminal axonal portion of nNOS-IR found in laminae III e IV. This evidence suggests that nitric oxide (NO) could modulate the synaptic activity of the glutamatergic spinocervical tracts. These findings suggest that the LCN of Artiodactyls might play an important role in the transmission of somatosensory information from the spinal cord to the higher centers of the brain.