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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.

Our intention in the present study was to ascertain whether NO-producing cells in the basolateral complex (BLC) and paracapsular intercalated nerve cell groups (Ip) of the amygdala are activated in the open field (OF) test. The material consisted of 8 adult rat brains. The OF test was applied throughout 10 min and 90 min before the death of the animals. The brain sections were double stained using the antibodies against c-Fos (marker of neuronal activation) and against nitric oxide synthase (NOS -- marker of NO-producing cells). The neurons containing NOS and those revealing c-Fos activity constituted distinct populations within both the BLC and Ip but NOS-immunoreactive fibres often surrounded the c-Fos-immunoreactive neurons. Our results suggest that (1) neurons of the basolateral complex of the amygdala and paracapsular intercalated islands are involved but probably not crucial for the open field stress response and (2) NOS-immunoreactive cells in the BLC and Ip are not activated after OF exposure.

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.

Ventral tegmental area (VTA) is a heterogeneous group of dopaminergic cells which contains interfascicular (IF), parabrachial (PBP) and rostral linear (RLi) nuclei. Neurons of this area are involved in the regulation of motor and motivational aspects of behavior and reveal high neuronal plasticity. Among many various neurotransmitters and neuromodulators, nitric oxide (NO) is localized in this region. In the present study, we investigated morphology and distribution of nitric oxide synthase (NOS)-positive neurons in VTA and their colocalization with dopaminergic neurons. The study was performed on six adult Wistar rats. After perfusional fixation, the brains were cut, immunostained for tyrosine hydroxylase (TH) and NOS and studied by confocal laser microscopy. In each of the three studied nuclei of VTA we investigated three different neuronal populations. Numerous TH-immunoreactive (TH-ir) and NOS-immunoreactive (NOS-ir) neurons are present in the studied region. Among them, a considerable number showed coexistence of both neurotransmitters. The populations of TH-ir and NOS-ir neurons interact with each other as manifested by the presence of NOS-ir endings on TH-ir neurons and vice versa. Taking the above into account, it may be suspected that NO is involved in the modulation of dopaminergic transmission.

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 morphometric analysis of changes occurring in the rat and rabbit ventroposterolateral (VPL) nucleus of the thalamus during the postnatal development was performed using unbiased stereological methods. The materials used in the study included 30 Wistar rats and 32 New Zealand rabbits aged from P0 to P180 (P-postnatal day), which were divided into six and eight age groups, respectively. The following stereological parameters of VPL nucleus on the cresyl violet stained sections were determined: volume of the nucleus, numerical density and total number of neurons. The total number of neurons indicated that the development of VPL nucleus in both species ended within the third week of postnatal life. The volume of VPL nucleus increased gradually (by about 2.2 and 5 times in rats and rabbits, respectively) in comparison with the volume of the cerebral hemisphere during the development from P0 to adulthood. The numerical density of VPL neurons decreased rapidly at the beginning of postnatal life and stabilized by the end of the third week. In both species, the gradual increase in the volume of VPL nucleus and the simultaneous decrease in the neuronal density in the first week of postnatal life were mainly caused by changes in the neuropil volume. The total number of cells did not change remarkably during the first postnatal week. However, it decreased significantly during the second week. This decrease was probably due to the naturally occuring cell death. These results show that the most prominent qualitative and quantitative changes in VPL nucleus and its neurons occur during the first two weeks of postnatal life of rats and rabbits. Also, because the thalamocortical relay neurons completely acquire their physiological features, this the most critical period of time for their morphological maturation.

The morphological features of nitric oxide synthase (NOS)-containing neurons in the rat claustrum (Cl) were studied during the period of four months after birth. Forty-five animals divided into nine groups, according to survival period (P0, P4, P7, P10, P14, P21, P28, P60, P120) were used in the study. The immunocytochemical staining to neuronal NOS was performed and the material was studied both qualitatively and quantitatively using unbiased stereological methods. Our observations indicate that the process of maturation of NOS-immunoreactive (ir) neurons in Cl takes place during the early postnatal period. We report the increase of numerical density of immunoreactive neurons, changes in neuronal size, expressed by the decrease of the percentage of small neurons with simultaneous increase of the participation of medium-sized neurons and large neurons. In the whole studied period the prevalence of oval and fusiform neurons is observed. However, the increase of the proportion of multipolar neurons takes place. Round neurons are most characteristic in the youngest groups of animals and later become dominated by the developing subpopulations of ir neurons of other shapes. In the anterior, central and posterior parts of Cl, a similar pattern of maturation of NOS-ir neurons is observed. No prevalence of characteristically shaped neurons is observed in any part of Cl. The adult-like pattern of morphological features in the NOS-ir neuronal population in Cl is reached in the third postnatal week. The maturation of NOS-ir neurons in the claustrum is a dynamic process which is not stabilised at the moment of birth. It may be assumed that characteristic changes of the NOS-ir population of neurons may be influential on the physiological processes observed in Cl. These may in particular have some importance for the processes of synaptogenesis and establishing as well as refining of numerous claustral connections with the other structures of the central nervous system.

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.

The aim of the present paper is to describe the morphology and topography of the nuclei of the amygdaloid complex in the rabbit. In the current study we also investigated the intensity of the enzymatic reaction for acetylcholinesterase (AChE) in the amygdaloid complex and the morphology of its neurones. Material consisted of 5 brains of adult New Zealand rabbit, stained either with cresyl violet or for AChE activity. Although, as in other mammals, the rabbit amygdala consists of two main nuclear groups (corticomedial and basolateral), it reveals a peculiar morphology pattern, forming a transition structure between those observed in the cat and rat. Especially characteristic is the arrangement of the basolateral complex. Within that the ventromedial division of the lateral nucleus seems to be the largest, while its dorsolateral division--the smallest. The arrangement of the corticomedial complex in the rabbit is similar to both the cat and rat. In the rabbit the highest acetylcholinesterase activity is found in the basolateral nucleus and the nucleus of the lateral olfactory tract. The lowest AChE staining is observed in the cortical and medial nuclei, amygdalohippocampal and anterior amygdaloid areas and intercalated masses.

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.