The Co-Expression Pattern of Calcium-Binding Proteins with γ-Aminobutyric Acid and Glutamate Transporters in the Amygdala of the Guinea Pig: Evidence for Glutamatergic Subpopulations.

The amygdala has large populations of neurons utilizing specific calcium-binding proteins such as parvalbumin (PV), calbindin (CB), or calretinin (CR). They are considered specialized subsets of γ-aminobutyric acid (GABA) interneurons; however, many of these cells are devoid of GABA or glutamate decarboxylase. The neurotransmitters used by GABA-immunonegative cells are still unknown, but it is suggested that a part may use glutamate. Thus, this study investigates in the amygdala of the guinea pig relationships between PV, CB, or CR-containing cells and GABA transporter (VGAT) or glutamate transporter type 2 (VGLUT2), markers of GABAergic and glutamatergic neurons, respectively. The results show that although most neurons using PV, CB, and CR co-expressed VGAT, each of these populations also had a fraction of VGLUT2 co-expressing cells. For almost all neurons using PV (~90%) co-expressed VGAT, while ~1.5% of them had VGLUT2. The proportion of neurons using CB and VGAT was smaller than that for PV (~80%), while the percentage of cells with VGLUT2 was larger (~4.5%). Finally, only half of the neurons using CR (~53%) co-expressed VGAT, while ~3.5% of them had VGLUT2. In conclusion, the populations of neurons co-expressing PV, CB, and CR are in the amygdala, primarily GABAergic. However, at least a fraction of neurons in each of them co-express VGLUT2, suggesting that these cells may use glutamate. Moreover, the number of PV-, CB-, and CR-containing neurons that may use glutamate is probably larger as they can utilize VGLUT1 or VGLUT3, which are also present in the amygdala.

Dopaminergic and cholinergic modulation of the amygdala is altered in female mice with oestrogen receptor ß deprivation.

The amygdala is modulated by dopaminergic and cholinergic neurotransmission, and this modulation is altered in mood disorders. Therefore, this study was designed to evaluate the presence/absence of quantitative alterations in the expression of main dopaminergic and cholinergic markers in the amygdala of mice with oestrogen receptor ß (ERß) knock-out which exhibit increased anxiety, using immunohistochemistry and quantitative methods. Such alterations could either contribute to increased anxiety or be a compensatory mechanism for reducing anxiety. The results show that among dopaminergic markers, the expression of tyrosine hydroxylase (TH), dopamine transporter (DAT) and dopamine D2-like receptor (DA2) is significantly elevated in the amygdala of mice with ERß deprivation when compared to matched controls, whereas the content of dopamine D1-like receptor (DA1) is not altered by ERß knock-out. In the case of cholinergic markers, muscarinic acetylcholine type 1 receptor (AChRM1) and alpha-7 nicotinic acetylcholine receptor (AChRα7) display overexpression while the content of acetylcholinesterase (AChE) and vesicular acetylcholine transporter (VAChT) remains unchanged. In conclusion, in the amygdala of ERß knock-out female the dopaminergic and cholinergic signalling is altered, however, to determine the exact role of ERß in the anxiety-related behaviour further studies are required.

GABAergic and Glutamatergic Phenotypes of Neurons Expressing Calcium-Binding Proteins in the Preoptic Area of the Guinea Pig.

The mammalian preoptic area (POA) has large populations of calbindin (CB), calretinin (CR) and parvalbumin (PV) neurons, but phenotypes of these cells are unknown. Therefore, the question is whether neurons expressing CB, CR, and/or PV are GABAergic or glutamatergic. Double-immunofluorescence staining followed by epifluorescence and confocal microscopy was used to determine the coexpression patterns of CB, CR and PV expressing neurons with vesicular GABA transporters (VGAT) as specific markers of GABAergic neurons and vesicular glutamate transporters (VGLUT 2) as specific markers of glutamatergic neurons. The guinea pig was adopted as, like humans, it has a reproductive cycle with a true luteal phase and a long gestation period. The results demonstrated that in the guinea pig POA of both sexes, ~80% of CB+ and ~90% of CR+ neurons coexpress VGAT; however, one-fifth of CB+ neurons and one-third of CR+ cells coexpress VGLUT. About two-thirds of PV+ neurons express VGAT, and similar proportion of them coexpress VGLUT. Thus, many CB+, CR+ and PV+ neurons may be exclusively GABAergic (VGAT-expressing cells) or glutamatergic (VGLUT-expressing cells); however, at least a small fraction of CR+ cells and at least one-third of PV+ cells are likely neurons with a dual GABA/glutamate phenotype that may coexpress both transporters.

Expression of Calbindin, a Marker of Gamma-Aminobutyric Acid Neurons, Is Reduced in the Amygdala of Oestrogen Receptor ß-Deficient Female Mice.

Oestrogen receptor ß (ERß) knock-out female mice display increased anxiety and decreased threshold for synaptic plasticity induction in the basolateral amygdala. This may suggest that the γ-aminobutyric acid (GABA) inhibitory system is altered. Therefore, the immunoreactivity of main GABAergic markers-i.e., calbindin, parvalbumin, calretinin, somatostatin, α1 subunit-containing GABAA receptor and vesicular GABA transporter-were compared in the six subregions (LA, BL, BM, ME, CE and CO) of the amygdala of adult female wild-type and ERß knock-out mice using immunohistochemistry and quantitative methods. The influence of ERß knock-out on neuronal loss and glia was also elucidated using pan-neuronal and astrocyte markers. The results show severe neuronal deficits in all main amygdala regions in ERß knock-out mice accompanied by astroglia overexpression only in the medial, basomedial and cortical nuclei and a decrease in calbindin-expressing neurons (CB+) in the amygdala in ERß knock-out mice compared with controls, while other markers of the GABAergic system remain unchanged. Concluding, the lack of ERß led to failure in the structural integrity of the CB+ subpopulation, reducing interneuron firing and resulting in a disinhibitory effect over pyramidal function. This fear-promoting excitatory/inhibitory alteration may lead to the increased anxiety observed in these mice. The impact of neuronal deficits and astroglia overexpression on the amygdala functions remains unknown.

The evolutionary trajectories of the individual amygdala nuclei in the common shrew, guinea pig, rabbit, fox and pig: A consequence of embryological fate and mosaic-like evolution.

The amygdala primarily evolved as a danger detector that regulates emotional behaviours and learning. However, it is also engaged in stress responses as well as olfactory/pheromonal and reproductive functions. All of these functions are processed by a set of nuclei which are derived from different telencephalic sources (pallial and subpallial) and have a unique cellular structure and specific connections. It is unclear how these individual anatomical and functional units evolved to fit the amygdala to the specific needs of various mammals. Thus, this study provides quantitative data regarding volumes, neuron density and neuron numbers in the main amygdala nuclei of the common shrew, guinea pig, rabbit, fox and pig - species from across the mammalian phylogeny which differ in brain complexity and ecology. The results show that the volume of the amygdala and its individual nuclei scale with negative allometry relative to brain mass (an allometric coefficient below one). However, in relation to the whole amygdala volume, volumes and volumetric percentages of the lateral (LA) and basomedial (BM) nuclei scale with positive allometry, for the medial (ME) and lateral olfactory tract (NLOT) nuclei these parameters scale with negative allometry while the values of these parameters for the basolateral (BL), central (CE) and cortical (CO) nuclei scale with isometry. Moreover, density of neurons scales with strong negative allometry relative to both brain mass and amygdala volume with values of allometric coefficient below zero across studied species. This value for BL is significantly lower than that for the whole amygdala, for ME it is significantly higher while values for NLOT, CE, CO, LA and BM are quite similar to the value for whole amygdala. Finally, neuron numbers in the whole amygdala and its individual nuclei scale with negative allometry in relation to brain mass. However, in relation to the number of neurons in the whole amygdala, neuron numbers and percentages of neurons for LA and BM scale with positive allometry, for BL and NLOT they scale with negative allometry while the values of these parameters for CE, CO and ME scale with isometry. In conclusion, all of these results indicate that each of the nuclei studied displays a different and unique pattern of evolution in relation to brain mass or the whole amygdala volume. These patterns do not match with the various classical concepts of amygdala parcellation; however, in some way, they reflect diversity revealed by the expression of homeobox genes in various embryological studies.

Relative abundance of chemerin mRNA transcript and protein in pituitaries of pigs during the estrous cycle and early pregnancy and associations with LH and FSH secretion during the estrous cycle.

Adipokines such as chemerin affect metabolic status and reproductive function in many species. The hypothesis in the present study was that there were chemerin mRNA transcript and protein in the pituitary of pigs and that relative abundances fluctuate during the estrous cycle and early pregnancy. Chemerin is thought to modulate luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion during the estrous cycle. Changes in the relative abundance of chemerin mRNA transcript and protein in anterior (AP) and posterior (PP) pituitaries of pigs were investigated, for the first time in the present study, during four phases of the estrous cycle and four periods of early pregnancy. Chemerin protein was localized in gonadotrophs, thyrotrophs and somatotrophs during the estrous cycle and early gestation. Chemerin treatments affected both basal, GnRH- and/or insulin-induced LH and FSH production, with there being variations with phase of the estrous cycle when tissues were collected. These findings indicate chemerin may be produced locally in the pituitary and may affect female reproductive function by controlling the release of LH and FSH from AP cells.

Expression of chemerin receptors CMKLR1, GPR1 and CCRL2 in the porcine pituitary during the oestrous cycle and early pregnancy and the effect of chemerin on MAPK/Erk1/2, Akt and AMPK signalling pathways.

Studies on adipokines, substances that are produced in adipose tissue, indicate that they influence both metabolism and reproduction. Chemerin is a novel addition to the adipokine family. It is believed that chemerin receptors are expressed in different structures of the hypothalamic-pituitary-gonadal (HPG) axis, which are crucial for endocrine control of reproductive functions, including the pituitary. The aim of this study was to investigate the expression of chemerin receptors (CMKLR1, GPR1, CCRL2) genes and proteins in the porcine pituitary. The effect of chemerin on MAPK/Erk1/2, Akt and AMPK signalling pathways was also investigated. The anterior (AP) and posterior (PP) lobes of the pituitary were examined on days 2 to 3, 10 to 12, 14 to 16, and 17 to 19 of the oestrous cycle and on days 10 to 11, 12 to 13, 15 to 16, and 27 to 28 of pregnancy. This is the first study to demonstrate that CMKLR1, GPR1 and CCRL2 are expressed in the porcine AP and PP, which implies that this gland is sensitive to chemerin action. The expression of the studied chemerin receptors fluctuated during different phases of the cycle and early gestation, which could be related to changes in the endocrine status of female pigs. The study also revealed that CMKLR1 and CCRL2 proteins were present in gonadotrophs and thyrotrophs, whereas CCRL2 was also present in somatotrophs, during the cycle and early pregnancy. We observed that chemerin affected MAPK/Erk1/2, Akt and AMPK signalling pathways in the porcine AP. These results suggest that chemerin may participate in the regulation of reproductive functions at the level of the pituitary.

The amygdala of the common shrew, guinea pig, rabbit, fox and pig: five flavours of the mammalian amygdala as a consequence of clade-specific mosaic-like evolution.

The amygdala is a part of neural networks that contribute to the regulation of emotional behaviours and emotional learning, stress response, and olfactory, pheromonal and reproductive functions. All these various functions are processed by the three main functional systems, frontotemporal, autonomic and olfactory, which are derived from different telencephalic sources (claustrum, striatum and olfactory cortex) and are represented, respectively, by the basolateral complex (BLC), the central complex (CC) and corticomedial complex (CMC) of the amygdala. The question arises of how these three functional systems evolved during mammalian phylogeny to fit the amygdala to specific needs of various animals. In the present study, we provide quantitative information regarding the individual volumes and neuron numbers in the BLC, CC and CMC of the common shrew, guinea pig, rabbit, fox and pig, a series of animals arranged according to increasing size and complexity of the brain. The results show that, in this series of animals, the BLC underwent a gradual size increase in volume and number of neurons, whereas the CMC was gradually reduced with regard to both these measures. The CC was more or less conserved across studied species. For example, the volume of the amygdala in pigs is ~250 times larger than that in shrews and it also has almost 26 times as many neurons as the amygdala of shrews. However, the volumes of the BLC, CC and CMC were ~380, 208 and 148 times larger, respectively, in pigs than in shrews. The number of neurons in these three regions was ~38, 23 and 20 times greater, respectively, in pigs than in shrews. The results also show striking morphometric similarities of the amygdala in the guinea pig and rabbit as well as fox and pig. For example, the percentages of neurons in the fox and pig are 42.23% and 42.78%, respectively, for the BLC, 16.64% and 16.58%, respectively, for the CC, and 41.12% and 40.64%, respectively, for the CMC. In conclusion, our results indicate that the amygdala does not evolve as a single unit but, instead, the three main functional systems evolved independently, which suggests that brain structures with major functional links evolve together independently of evolutionary changes in other unrelated structures. The size progression of the BLC parallels the size progression of the neocortex with which it is strongly functionally linked, whereas the CMC is strongly connected to olfactory regions, and all these structures follow the same regression course. Remarkable morphometric similarity of the amygdala in the guinea pig and rabbit as well as in the fox and pig, however, suggest that there must also be another mechanism shaping the morphology of the amygdala and the brain during evolution. The gradual nature of size changes in the BLC and CMC support this hypothesis as well.

Expression of Chemerin and Its Receptors in the Porcine Hypothalamus and Plasma Chemerin Levels during the Oestrous Cycle and Early Pregnancy.

Chemerin (CHEM) may act as an important link integrating energy homeostasis and reproductive functions of females, and its actions are mediated by three receptors: chemokine-like receptor 1 (CMKLR1), G protein-coupled receptor 1 (GPR1), and C-C motif chemokine receptor-like 2 (CCRL2). The aim of the current study was to compare the expression of CHEM and its receptor (CHEM system) mRNAs (quantitative real-time PCR) and proteins (Western blotting and fluorescent immunohistochemistry) in the selected areas of the porcine hypothalamus responsible for gonadotropin-releasing hormone production and secretion: the mediobasal hypothalamus, preoptic area and stalk median eminence during the oestrous cycle and early pregnancy. Moreover, plasma CHEM concentrations were determined using ELISA. The expression of CHEM system has been demonstrated in the porcine hypothalamus throughout the luteal phase and follicular phase of the oestrous cycle, and during early pregnancy from days 10 to 28. Plasma CHEM levels and concentrations of transcripts and proteins of CHEM system components in the hypothalamus fluctuated throughout pregnancy and the oestrous cycle. Our study was the first experiment to demonstrate the presence of CHEM system mRNAs and proteins in the porcine hypothalamus and the correlations between the expression levels and physiological hormonal milieu related to the oestrous cycle and early pregnancy.

Ontogeny of Neurons Containing Calcium-Binding Proteins in the Preoptic Area of the Guinea Pig: Sexually Dimorphic Development of Calbindin.

This study characterizes for the first time the distribution and coexistence patterns of calbindin (CB), calretinin (CR), and parvalbumin (PV) in the female and male guinea pig preoptic area (POA) during brain development, using immunohistochemistry and quantitative real-time PCR techniques. The results show that the prenatal development of the guinea pig POA takes place in elevated levels of CB and CR immunoreactivity with the peak at embryonic day 50 (E50) and generally in newborns both these proteins reach an adult-like pattern of immunoreactivity, contrary to PV which appears later, peaks at postnatal day (PND) 10 (P10), and stabilizes at P20. CB and CR have also overlapping distributions which differed from that of PV, and much higher expressions at mRNA and protein levels. However, CB-positive (+), CR+ and PV+ neurons create in the guinea pig POA separate populations as CB and CR coexisted only in a small number of neurons and CB+ cells never coexpressed PV. Moreover, the density of CB+ neurons, contrary to CR+ and PV+ cells, is sexually dimorphic favoring males at all the examined stages. In conclusion, elevated levels of CR and CB at the time of intense cell migration, differentiation, myelination, and synaptogenesis in the guinea pig brain suggest that these proteins may be engaged in similar processes in the POA, while late onset of PV may be rather linked with POA maturation. As the population of CB+ cells in the POA is very large, its dimorphic development may have huge impact on the sexual differentiation of this brain region.

Ontogeny of calcium-binding proteins in the cingulate cortex of the guinea pig: The same onset but different developmental patterns.

This paper compared the density of calbindin D28k (CB), calretinin (CR) and parvalbumin (PV) containing neurons in prenatal, newborn and postnatal periods in the cingulate cortex (CC) of the guinea pig as an animal model. The distribution and co-distribution among calcium-binding proteins (CaBPs) was also investigated during the entire ontogeny. The study found that CB-positive neurons exhibited the highest density in the developing CC. The CC development in the prenatal period took place with a high level of CB and CR immunoreactivity and both of these proteins reached peak density during fetal life. The density of PV-positive neurons, in contrast to CB and CR-positive neurons, reached high levels postnatally. The observed changes of the CaBPs-positive neuron density in the developing CC coincide with developmental events in the guinea pig. E.g. the eyes opening moment may be preceded by elevated levels of CB and CR at E50, whereas high immunoreactivity of PV from P10 to P40 with a peak at P20 may indicate the participation of PV in enhancement of the inhibitory cortical pathway maturation.

Calcium-binding proteins expression in the septum and cingulate cortex of the adult guinea pig.

For the first time this study demonstrates the distribution pattern and expression of three neuroanatomical markers: calbindin D28k (CB), calretinin (CR) and parvalbumin (PV) in topographically connected brain regions - the septum (SE) and the cingulate cortex (CC). The co-existence among calcium-binding proteins (CaBPs) was also examined. The study was conducted on the adult guinea pig with the use of immunohistochemical and molecular biological techniques. Among the studied CaBPs, which occurred in both examined brain regions at transcript and protein levels, CB was the most expressed in the SE, while CR in the CC. CR, unlike CB and PV, showed higher immunoreactivity in the superficial layers (II-III) of the CC than in the deep ones (V-VI). Most of CB and PV-positive perikarya were detected in the deep layers of the CC. Some CC neurons contained both CB and PV, suggesting cooperation between these CaBPs in the CC. Co-localization between CB and CR in the CC was not observed.

Tyrosine hydroxylase-immunoreactivity and its relations with gonadotropin-releasing hormone and neuropeptide Y in the preoptic area of the guinea pig.

The present study examines the distribution of tyrosine hydroxylase (TH) immunoreactivity and its morphological relationships with neuropeptide Y (NPY)- and gonadoliberin (GnRH)-immunoreactive (IR) structures in the preoptic area (POA) of the male guinea pig. Tyrosine hydroxylase was expressed in relatively small population of perikarya and they were mostly observed in the periventricular preoptic nucleus and medial preoptic area. The tyrosine hydroxylase-immunoreactive (TH-IR) fibers were dispersed troughout the whole POA. The highest density of these fibers was observed in the median preoptic nucleus, however, in the periventricular preoptic nucleus and medial preoptic area they were only slightly less numerous. In the lateral preoptic area, the density of TH-IR fibers was moderate. Two morphological types of TH-IR fibers were distinguished: smooth and varicose. Double immunofluorescence staining showed that TH and GnRH overlapped in the guinea pig POA but they never coexisted in the same structures. TH-IR fibers often intersected with GnRH-IR structures and many of them touched the GnRH-IR perikarya or dendrites. NPY wchich was abundantly present in the POA only in fibers showed topographical proximity with TH-IR structures. Althoug TH-IR perikarya and fibers were often touched by NPY-IR fibers, colocalization of TH and NPY in the same structures was very rare. There was only a small population of fibers which contained both NPY and TH. In conclusion, the morphological evidence of contacts between TH- and GnRH-IR nerve structures may be the basis of catecholaminergic control of GnRH release in the preoptic area of the male guinea pig. Moreover, TH-IR neurons were conatcted by NPY-IR fibers and TH and NPY colocalized in some fibers, thus NPY may regulate catecholaminergic neurons in the POA.

Distribution of cocaine- and amphetamine-regulated transcript (CART), neuropeptide Y (NPY) and galanin (GAL) in the pterygopalatine ganglion of the domestic duck (Anas platyrhynchos f. domestica).

INTRODUCTION: Cocaine- and amphetamine-regulated transcript (CART), neuropeptide Y (NPY) and galanin (GAL) act as neurotransmitters and neuromodulators in both the central and peripheral nervous systems. Their presence has been found in different taxonomic groups, in particular in mammals. However, only few investigators have studied these neuropeptides in the class Aves (birds). The aim of the present study was to describe the distribution of CART, NPY and GAL in the pterygopalatine ganglion (PPG) of the domestic duck (Anas platyrhynchos f. domestica). MATERIAL AND METHODS: The experiment was conducted on 16 one-year-old domestic ducks of the Pekin breed of both sexes (8 males and 8 females). Frozen sections of the PPG were subjected to immunofluorescence staining using primary mouse monoclonal antibodies directed against CART and GAL and rabbit polyclonal antibody directed against NPY. Secondary antibodies were conjugated with Cy3 and FITC fluorochromes. RESULTS: CART, NPY, and GAL were present in the PPG of the domestic duck. The highest immunoreactivity (IR) in the ganglionic cells was found for CART in the majority (83-85%) of neurons of both superior (SPPG) and inferior (IPPG) PPG. CART-IR was also found in small aggregations of neurons on the medial surface of the Harderian gland, and on the course of the palatine branch of the facial nerve. CART-IR was also observed in the nerve fibers of these neurons' aggregations; however, it was low in comparison to the immunoreactivity of the perikarya. Immunoreactivity of NPY was found in ganglionic neurons, but above all in numerous fibers of the SPPG and IPPG and within aggregations on the surface of the Harderian gland. NPY-IR cells were distributed irregularly over the cross-sections of the tested aggregations, and constituted from 36% to 43% of the SPPG and from 37% to 40% of the IPPG of all cross-sectioned neurons. GAL-immunoreactive perikarya, distributed irregularly across the sections, were observed in the SPPG, where they constituted 61-65%, and in the IPPG, where they made up 50-57% of all neurons. All immunoreactive neurons were characterized by immunopositive neuroplasm and immunonegative cell nuclei. CONCLUSIONS: The presence of CART, NPY, and GAL in the PPG of the domestic duck suggests that these peptides may contribute to the secretory innervation of the glands of the mucosa of the palate and nasal cavity, the Harderian gland, and the lacrimal gland.

Cocaine- and amphetamine-regulated transcript and calcium binding proteins immunoreactivity in the subicular complex of the guinea pig.

In this study we present the distribution and colocalization pattern of cocaine- and amphetamine-regulated transcript (CART) and three calcium-binding proteins: calbindin (CB), calretinin (CR) and parvalbumin (PV) in the subicular complex (SC) of the guinea pig. The subiculum (S) and presubiculum (PrS) showed higher CART-immunoreactivity (-IR) than the parasubiculum (PaS) as far as the perikarya and neuropil were concerned. CART- IR cells were mainly observed in the pyramidal layer and occasionally in the molecular layer of the S. In the PrS and PaS, single CART-IR perikarya were dispersed, however with a tendency to be found only in superficial layers. CART-IR fibers were observed throughout the entire guinea pig subicular neuropil. Double-labeling immunofluorescence showed that CART-IR perikarya, as well as fibers, did not stain positively for any of the three CaBPs. CART-IR fibers were only located near the CB-, CR-, PV-IR perikarya, whereas CART-IR fibers occasionally intersected fibers containing one of the three CaBPs. The distribution pattern of CART was more similar to that of CB and CR than to that of PV. In the PrS, the CART, CB and CR immunoreactivity showed a laminar distribution pattern. In the case of the PV, this distribution pattern in the PrS was much less prominent than that of CART, CB and CR. We conclude that a heterogeneous distribution of the CART and CaBPs in the guinea pig SC is in keeping with findings from other mammals, however species specific differences have been observed.

The densities of calbindin and parvalbumin, but not calretinin neurons, are sexually dimorphic in the amygdala of the guinea pig.

In the amygdala, the calcium-binding proteins (calbindin, parvalbumin or calretinin) are useful markers of specific subpopulations of γ-aminobutyric acid (GABA) containing neurons. In the rat and monkey they together mark the vast majority of GABA-containing neurons in this brain region. As GABA involvement in the control of various behaviors in a sex-specific manner and sexual dimorphism of the GABAergic system itself were recently proven, the question is how much dimorphic may be various subpopulations of this system. Thus, the present study investigates for the first time the presence/absence of sexual dimorphism among neurons expressing calbindin (CB), parvalbumin (PV) and calretinin (CR) which form in the amygdala main subsets of GABAergic system. The results show that in the amygdala of the guinea pig the densities of CB and/or PV expressing neurons are sexually dimorphic with the female>male pattern of sex differences in the basolateral amygdala. In the medial and cortical amygdala respectively CB and PV values are also sexually dimorphic, favoring males. The densities of CR expressing neurons are in the amygdala of the guinea pig sexually isomorphic. In conclusion, the results of the present study provide an evidence that in the amygdala of the guinea pig the densities of neurons expressing CB and/or PV are sexually dimorphic what supports the idea that GABA participates in the mediation of sexually dimorphic functions, controlled by this brain area.

Calcium-binding proteins in the laterodorsal thalamic nucleus during development of the guinea pig.

The laterodorsal thalamic nucleus (LD) is often treated as a part of the anterior thalamic nuclei (ATN) because of its location and similar connectivity. Our previous studies have shown that distribution of three calcium-binding proteins, i.e. calbindin D28k (CB), calretinin (CR) and parvalbumin (PV), changes within the ATN during development of the guinea pig. The aim of this study is to examine the immunoreactivity pattern of these proteins in the LD in the guinea pig ontogeny. Brains from animals ranging from 40th embryonic day to 80th postnatal day were used in the study. Two methods were applied: a single-labelling immunoenzymatic method and double-labelling immunofluorescence. No changes of the distribution pattern of the substances were observed throughout the examined developmental stages. CB and CR were the most abundantly expressed proteins in perikarya of the LD. Numerous CB- and CR-immunoreactive cell bodies were found throughout the whole extent of the nucleus. In most of these cell bodies both proteins colocalized vastly. The highest immunoreactivity of the perikarya containing CB and CR was observed in the mediodorsal part of the LD and in its rostral portion. In regard to PV, single cell bodies were observed mostly in the dorsal part of the nucleus. PV did not colocalize with the other proteins. In summary, all the studied calcium-binding proteins were already present in the LD at prenatal developmental stages and the pattern of distribution remained virtually constant until adulthood. Thus, the LD differs considerably from the ATN in an aspect of neurochemical cell differentiation.

The cocaine- and amphetamine-regulated transcript, calbindin, calretinin and parvalbumin immunoreactivity in the medial geniculate body of the guinea pig.

The purpose of this study was to describe the distribution and colocalization of cocaine- and amphetamine-regulated transcript (CART) and three calcium-binding proteins (calbindin, calretinin and parvalbumin) in each main division of the medial geniculate body (MGB) in the guinea pig. From low to moderate CART immunoreactivity was observed in all divisions of the MGB, although in most of its length only fibers and neuropil were labeled. A small number of CART immunoreactive somata were observed in the caudal segment of the MGB. The central parts of all divisions contained a distinctly smaller number of CART immunoreactive fibers relative to their outer borders, where CART fibers formed patchy clusters. As a whole, the intense CART immunoreactive borders formed a shell around the weakly CART labeled core. Double-labeling immunofluorescence showed that CART did not colocalize with either calbindin, calretinin or parvalbumin, whose immunoreactivity was predominantly restricted to perikarya. The distribution pattern of calretinin was more similar to that of calbindin than to that of parvalbumin. Calretinin and calbindin exhibited higher immunoreactivity in the medial and dorsal divisions of the MGB, where parvalbumin staining was low. In general, although parvalbumin exhibited the weakest immunoreactivity of all studied Ca(2+) binding proteins, it was most highly expressed in the ventral division of the MGB. Our results indicate that CART could be involved in hearing, although its immunoreactivity in the medial geniculate complex was not as intense as in other sensory brain regions. In the guinea pig the heterogeneous and complementary pattern of calbindin, calretinin and parvalbumin is evident, however, the overlap in staining appears to be more extensive than that seen in other rodents.

Embryonic and postnatal development of calcium-binding proteins immunoreactivity in the anterior thalamus of the guinea pig.

Our recent studies have shown that the distribution of calretinin (CR) in the anterior thalamic nuclei (ATN) changes significantly during the development of the guinea pig. The present study was designed to reveal the distribution pattern of calcium-binding proteins, i.e. calbindin (CB) and parvalbumin (PV), as well as the colocalization pattern of all three proteins, including CR, in the ATN of guinea pigs ranging from the 40th embryonic day (E40) to the 80th postnatal day (P80). According to these patterns, CB appears exclusively in the perikarya of the anteromedial nucleus (AM) not before P20 and always colocalizes with CR. Moreover, CB and CR colocalize in fibers of thin bundles traversing the anteroventral nucleus (AV) since E50. The ATN also display CB-positive neuropil in all studied stages, especially a strong one in the ventral part of the AV. PV was not observed in the perikarya of the ATN in all the stages, but was abundantly present in the neuropil of the anterodorsal nucleus (AD). No colocalizations exist between PV and the rest of the studied proteins. In conclusion, our study reveals that the distribution of the studied proteins differs greatly. Nevertheless, the postnatal coexistence of CB and CR in the AM perikarya may indicate the cooperation of both of the proteins in some functions of the nucleus. Parvalbumin is limited mostly to the neuropil of the AD, suggesting different functions in comparison to CB and CR.

Distribution and chemical coding pattern of the cocaine- and amphetamine-regulated transcript (CART) immunoreactivity in the preoptic area of the pig.

This study provides a detailed description of cocaine-and amphetamine-regulated transcript (CART) distribution and the co-localization pattern of CART and gonadotropin releasing hormone (GnRH), somatostatin (SOM), neuropeptide Y (NPY), cholecystokinin (CCK), and substance P (SP) in the preoptic area (POA) of the domestic pig. The POA displays a low density of immunoreactive cells and rich immunoreactivity for CART in fibers. CART-immunoreactive (CART-IR) cell bodies were single and faintly stained, and located in the medial preoptic area (MPA) and the periventricular region of the POA. A high density of immunoreactive fibers was observed in the periventricular preoptic nucleus (PPN); a high to moderate density of fibers was observed in the MPA; but in the dorso-medial region of the MPA the highest density of fibers in the whole POA was observed. The lateral preoptic area (LPA) exhibited a less dense concentration of CART-immunoreactive fibers than the MPA. The median preoptic nucleus (MPN) showed moderate to low expression of staining fibers. In the present study, dual-labeling immunohistochemistry was used to show that CART-IR cell bodies do not contain any GnRH and SP. CART-positive fibers were identified in close apposition with GnRH neurons. This suggests that CART may influence GnRH secretion. Double staining revealed that CART-IR structures do not co-express any of the substances we studied, but a very small population of CART-IR fibers also contain SOM, CCK or SP.