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Calbindin-D28k (CB), parvalbumin (PV) and calretinin (CR) are calcium-binding proteins (CaBPs) considered to be markers for certain subpopulations of neurons in the central nervous system. The aim of this study was to describe the pattern of distribution of CB-, PV- and CR-immunoreactive elements in the rabbit corticomedial amygdaloid complex during the postnatal period. The time course of changes in CaBPs expression during maturation of the selected nuclei indicates their diversity. During the first month after birth, CaBPs expression stabilizes earliest in the anterior cortical and then in the medial nuclei. Later, during the second month of postnatal life, the posteromedial and posterolateral cortical nuclei maturate. The central nucleus requires a considerably longer time to reach maturity - about three months are needed to stabilize CaBPs expression in all its subdivisions. This nucleus also shows the most differentiated, time-dependent distribution of CaBPs immunoreactivity (especially CB), distinct in its divisions. The differences in the CaBPs immunoreactivity confirm previous reports concerning dissimilar origin and development, and also reflect the diversity of connectivity of the amygdaloid body - the collection of nuclei, considered as one functional integrity.

Claustrum is a telencephalic structure integrating information of various modalities. Proper functioning of this structure depends on the presence of a network of intrinsic connections. This includes GABA-ergic neuronal populations that also contain calcium-binding proteins (CaBPs). The goal of this study was to analyze qualitative and quantitative the 5-HT-containing fibers in the rat claustrum and to assess the relationships between these fibers and the populations of claustral neurons expressing CaBPs. We used the methods of immunocytochemistry and morphometry. The serotonergic fibers in the claustrum are heterogeneous, both with respect to their morphology and spatial distribution. Thin varicose fibers are more numerous and are homogeneously distributed within the claustrum. Remaining fibers were thicker and possessed larger varicosities. They were present mainly in the ventral part of the claustrum. Although the serotonergic fibers are found in the vicinity of claustral cells containing CaBPs, direct contacts between these fibers and cells are rare. Other mechanisms, including volume transmission, may possibly mediate serotonergic influences.

In the present study we wanted to check whether the expression of the c-Fos protein (the marker of cellular activity) appears in cells containing calcium-binding proteins (CaBPs) in animals exposed to the open field test. Eight adult Wistar rats were examined. In the first step the open field test was applied throughout 10 minutes. After perfusional fixation brains were frozen and cut on the cryostat in the coronal plane and stained with the standard immunohistochemical method. Sections were double stained for c-Fos and CaBPs: parvalbumin (PV), calbindin (CB), calretinin (CR). c-Fos positive cells were localized predominantly in layers II and III of the piriform cortex (PC). The double labeling study showed that neurons containing CaBPs are rarely c-Fos-immunoreactive. Often PV-positive and CB-positive fibers surround c-Fos-positive neurons in layers II and III in a form of a basket. It seems that cells containing CaBPs are not directly involved in the response to aversive stimuli but cells containing those calcium-binding proteins might influence directly c-Fos positive neurons of PC.

The endopiriform nucleus, further divided into dorsal and ventral parts, and the neighbouring pre-endopiriform (pEn) nucleus form a region of highly heterogeneous structure involved in numerous physiological and pathological processes. Nonpyramidal neurons of this region containing three neuropeptides-somatostatin (SOM), neuropeptide Y (NPY), and vasoactive intestinal peptide (VIP)-were examined in this study. Their colocalization with three calcium-binding proteins-parvalbumin (PV), calbindin D28k (CB), calretinin (CR), and with nitric oxide synthase (NOS), was investigated by qualitative and quantitative methods. The results are summarized as follows: (1) all studied substances are distributed in neurons of the entire region, (2) SOM-ir neurons constitute the most numerous neuropeptide-containing population, whereas NOS-ir neurons make up the largest population of all studied, (3) colocalizations are found in the endopiriform region (Enr) (SOM with CB, PV and NOS; VIP with CR; NPY with NOS and NOS with CR), (4) heterogeneity of the endopiriform region appears in the differences of cells' shape distributions of single-labeled (SOM-, CR-PV-ir) and double-labeled (SOM/CB-, SOM/PV-, NPY/NOS- and NOS/CR-ir) neurons, as well as in differentiated percentage values of SOM/NOS, NPY/NOS and VIP/CR double-labeled neurons in three studied parts; additionally, differences in distribution of immunoreactive neuropil elements between parts of the region are observed. Numerous regional differences concerning neuronal morphology and immunocytochemical characteristics justify further division of the endopiriform region into distinguished parts. Some immunocytochemical features of the neurons in studied region may contribute to the role in epileptogenesis.

The piriform cortex has been extensively studied due to its possible role in epileptogenic activity. Neurones containing calcium-binding proteins (CaBPs), as a component of inhibitory circuitry, seem to be critically involved in this pathological process. The aim of the present study was to characterise the pattern of distribution of CaBPs-immunoreactivity in the piriform cortex of the adult rabbit. It comprises labelled cells, fibres (often with varicosities) and terminals. It varies among the layers. Moreover, the distribution of the parvalbumin- and calretinin-immunoreactive fibres and terminals allows even further subdivision of the layer I into two sublayers. Calretinin-ir neurones are located in subpial (Ia) layer, while parvalbumin - as well as calbindin-D28k-ir ones are mainly located in the second and third layer. Cajal-Retzius-like neurones containing calretinin, Chandelier cells containing parvalbumin and basket cells containing calbindin D28k and parvalbumin can be distinguished among labelled subpopulations of CaBPs neurones. In general, the pattern of PV- CR- and CB-immunoreactivity is similar to that previously characterised in other mammals, i.e., rats, guinea pigs, hedgehog, and tenrecs. The pattern is organised in topographic fashion confirming the complexity of regulatory circuits in the rabbit piriform cortex.

The present paper describes parvalbumin, calbindin-D28k and calretinin immunoreactivity in the claustrum and endopiriform nucleus of adult rabbits. Studied neuronal populations are characterized by morphological heterogeneity. Four types were identified in each subpopulation of cells containing calcium binding proteins on the basis of the number of processes and their branching pattern. There were no spatial differences in the distribution of cells containing either parvalbumin or calbindin-D28k in the claustrum and endopiriform nucleus. Well documented presence of the various projective zones in the rabbit claustrum did not reflect the specific distribution of neurons containing calcium binding proteins, except those containing calretinin. Their localization may correspond with the limbic zone. We have found that the rabbit claustrum and endopiriform nucleus have different pattern of parvalbumin and calretinin immunoreactivity. The former was more intense in the claustrum and the distribution of cell types was significantly different from that in the endopiriform nucleus. Calretinin-positive cells were observed in the claustrum, while in the endopiriform nucleus they were scarce. The distribution of neither calbindin-D28k-ir neurons nor fibers allowed differentiation of claustrum and endopiriform nucleus. Significant differences between the claustrum and endopiriform nucleus observed in the rabbit might be related with ontogenetic as well as other (functional) factors.

An immunocytochemical double-staining method was applied in order to study the co-localisation of nitric oxide synthase (NOS) with three calcium-binding proteins, calbindin D28k (CB), calretinin (CR) and parvalbumin (PV) in the claustrum of the rat during the first 4 months of life (postnatal days: PO-P120). The co-localisation of NOS/PV and NOS/CB is reported. These neurons fall into the category of non-pyramidal cells. Double-labelled NOS/CB neurons are observed in the claustrum starting from P4, whereas double-labelled NOS/PV neurons are observed from P14 onwards. The percentages of double-labelled neurons increase in relation to the age. Double-labelled NOS/CB and NOS/PV neurons, although they do not constitute a numerous population, play an important role in the process of maturation of the claustrum. This is confirmed by the occurrence of these types of neurons at definite stages of maturation and by the increase in their number.

Immunohistochemical study of the cholinergic innervation of the hippocampal cells containing glutamic acid decarboxylase (GAD) and calcium binding proteins: parvalbumin (PV), calbindin D28k (CB) and calretinin (CR) was conducted on 5 adult rat brains. Analysis of sections with double immunostaining for vesicular acetylcholine transporter (VAChT; the marker of cholinergic cells, fibres and terminals) and respectively either GAD or PV, CB, CR, using confocal laser-scanning microscope shows that the intensive cholinergic innervations receive GAD, PV and CB-positive hippocampal cells. Cholinergic afferentiations of the CR-positive neurones are considerably fewer.