Interleukin-1ß-immunoreactive neurons in the hippocampus and paraventricular nucleus of the hypothalamus after stress stimulation in aged versus adult rats.

It is believed that the impact of stress on interleukin-1ß (IL-1ß) depends on the ontogenetic age. This study examines the influence of acute or chronic exposure to forced-swim (FS) stress or high-light open-field (HL-OF) stimulation on the expression of IL-1ß. Double immunofluorescence staining was used to reveal the density of IL-1ß/NeuN (NeuN is a neuronal nuclear marker)-immunoreactive (-ir) cells in the hippocampal subfields CA1 and CA3, dentate gyrus (DG), and paraventricular nucleus (PVN) of the hypothalamus. Adult postnatal day 90 (P90) and aged (P720) rats were used in this experiment. The data showed a significant increase in the density of IL-1ß/NeuN-ir cells in the CA1, CA3, DG, and PVN in P720 nonstressed rats in relation to P90 control animals. Neither FS nor HL-OF acute stimulation caused alteration in the density of IL-1ß-ir neurons in any of the investigated structures in P90 and P720 rats in comparison with control groups. However, chronic FS caused a significant increase in CA3 and DG of P720 rats, and chronic HL-OF led to a significant increase in the density of IL-1ß-ir neurons in the PVN of P90 rats and in all hippocampal subfields of P720 animals. These results indicate that chronic HL-OF stimulation is a factor that induces changes in the number of IL-1ß-ir neurons in the PVN of adult rats, whereas both chronic FS and HL-OF are aggravating factors for the hippocampus of aged (P720) animals.

Nicotine-induced CREB and DeltaFosB activity is modified by caffeine in the brain reward system of the rat.

Coffee and nicotine consumption are frequently combined, indicating possible intensifying effect of caffeine on smoking behavior, although neurobiological background of this phenomenon remains unknown. We aimed at determining the effect of caffeine and nicotine, applied separately or simultaneously, on activation of six structures of the brain reward system: nucleus accumbens (NAc), ventral tegmental area (VTA), amygdala (Amg), hippocampus (Hip), medial prefrontal cortex (mPfr) and dorsal striatum (CdP) in the adult male Wistar rats. Activation of two transcription factors, the phosphorylated form of cyclic AMP-response element binding protein (pCREB) and DeltaFosB (ΔFosB) was assessed by immunohistochemistry after multiple-dose five-days psychostimulants administration followed by 20min and 24h survival, respectively. Nicotine evoked the highest increase of pCREB-immunoreactivity (-ir) in NAc, while caffeine exerted the weakest effect in mPfr and CdP. Nicotine/caffeine co-administration resulted in decrease of pCREB-ir in NAc and increase in Amg, compared with the effect of each psychostimulant used separately. Nicotine was the strongest psychostimulant activating ΔFosB-ir in Amg, whereas caffeine - in Hip. Nicotine/caffeine-exerted effect upon ΔFosB-ir in Amg was weaker, whereas in mPfr stronger, than nicotine-evoked effect in these structures. In summary, pCREB and ΔFosB activation is dependent on the type of stimulus, brain structure and functional context. Activation of both transcription factors is responsible for caffeine's modifying effect upon nicotine-related behaviors and must be taken into account while quitting cigarette smoking.

Comparison of the influence of two models of mild stress on hippocampal brain-derived neurotrophin factor (BDNF) immunoreactivity in old age rats.

The way hippocampal neurons function during stress in old age (critical times of life) is dependent on brain derived neurotrophin factor (BDNF). This study examined the influence of acute and chronic forced swim (FS) or high-light open field (HL­OF) stimulation on the density of BDNF immunoreactive (ir) neurons in the hippocampal pyramidal layers of CA1, CA2, CA3 regions and the granular layer of dentate gyrus (DG) in old (postnatal day 720; P720) Wistar Han rats. Our data showed that in comparison with non-stressed rats, acute FS caused a significant increase in the density of BDNF-ir neurons in CA2 and CA3, while acute HL-OF led to an increase in this factor in all hippocampal subfields with the exception of DG. However, the density of BDNF-ir cells remained unchanged after exposure to chronic FS or HL­OF in the hippocampal regions in relation to the control rats. These results indicate that acute FS or HL-OF proved to be a stressor that induces an increase in the density of BDNF-ir pyramidal neurons, which was probably connected with up-regulation of HPA axis activity and short­time memory processing of the stressful situation. Moreover, as far as the influence on BDNF-ir cells in hippocampus is concerned, chronic FS or HL-OF was not an aggravating factor for rats in the ontogenetic periods studied.

The effect of propofol on astro- and microglial reactivity in the course of experimental intracerebral haemorrhage in rats.

The glial cells play an important role in pathophysiology of the intracerebral haemorrhage (ICH). Thus the attempt at evaluating the possible influence of the propofol on the reactivity of astro- and microglial cells in the course of ICH was performed. 50 rats were divided into two groups depending on the applied anaesthesia. All animals were generally anaesthetized with fentanyl, dehydrobenzperidol and midazolam. No additional agents were given to the animals of the control group (group I). In the experimental group (group II), the animals received additionally intraperitoneally propofol in a dose of 50 mg/kg every thirty minutes. ICH was produced through infusion of the blood into the striatum. The astrocytic and microglial cells population was assessed on the 1, 3, 7, 14 and 21 days after producing a haematoma using antibodies anti-GFAP and OX42. The stereological analysis was applied to estimate the numerical density of immunoreactive cells and the distribution of their types. On the 14th and 21st days of observation the density of GFAP-immunoreactivity (ir) cells was significantly higher in group II than that in group I. There were no differences in percentage distribution of GFAP-ir astrocytes between group I and group II. On the 3rd, 14th and 21st days of observation the density of OX42-ir cells was higher in group II in comparison with group I. For the 7th, and 21st days of survival the percentage of the ameboid form of OX42-ir cells was significantly lower in group I than that in group II. The administration of propofol during anaesthesia in the animals with ICH has evoked an increase of the activation of the astro- and microglial cells.

The impact of two mild stressors on the nerve growth factor (NGF) immunoreactivity in the amygdala in aged rats compared to adult ones.

Nerve growth factor (NGF) seems to play an important role in the ageing limbic system in response to stress. This study aimed to explore the influence of acute and chronic exposure to high-light open field (HL-OF) or forced swim (FS) stressors on the density of NGF immunoreactive (ir) neurons in the amygdala central (CeA), medial (MeA), lateral (LA) and basolateral (BLA) nuclei in adult (postnatal day 90; P90) and aged (P720) rats. In comparison with non-stressed rats, neither acute nor chronic HL-OF produced significant changes in the density of NGF-ir neurons of studied nuclei in P90 and P720 rats. However, not acute but chronic FS was the factor inducing an increase in the density of NGF-ir neurons in the CeA of both age groups and in the LA of P720 rats. Despite the lack of change in the density of NGF-ir neurons between P90 and P720 non-stressed rats, there were significant age-related changes in NGF-ir cells in FS and/or HL-OF stressed rats in all the tested nuclei, with the exception of the LA. It may be concluded that as far as the influence on NGF-ir cells in amygdaloid nuclei is concerned, HL-OF did not constitute an aggravating factor for rats in the ontogenetic periods studied. Moreover, upregulation of NGF-ir neurons predominantly in CeA after chronic FS seems to be neuroprotective. Age-dependent changes in the density of NGF-ir neurons in stressed rats are probably caused by ageing processes and they may point to dysregulation of excitatory control exerted by the amygdala.

Exposure to mild stress and brain derived neurotrophin factor (BDNF) immunoreactivity in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei: Comparison between aged and adult rats.

It seems that age-dependent changes in stress response may be correlated with alterations in the hypothalamic brain derived neurotrophin factor (BDNF). Two hypothalamic nuclei, ie. paraventricular (PVN) and supraoptic (SON) are closely related to the stress response. Therefore, the aim of our study was to explore the influence of acute and chronic high-light open field (HL-OF) or forced swim (FS) stimulation on the density of BDNF immunoreactive (ir) neurons in the PVN and SON in adult (postnatal day 90; P90) and aged (P720) Wistar Han rats. Our data demonstrated that in comparison with non-stressed rats, neither acute nor chronic FS caused significant changes in the density of BDNF-ir neurons of PVN and SON in P90 or P720 rats. However, a significant increase in the density of BDNF-ir neurons in the SON of P90 and a decrease in the PVN of the P720 rats were noted after acute HL-OF. Despite the lack of change in the density of BDNF-ir neurons between P90 and P720 non-stressed and FS stressed rats, there was an age-dependent decrease in the BDNF-ir cells of HL-OF stressed rats, especially in the PVN. We may conclude that in terms of its influence on the BDNF-ir neurons in PVN and SON, acute HL-OF was the factor inducing changes in the density of BDNF-ir neurons in the hypothalamic nuclei of adult or aged rats. Furthermore, ageing involutional processes, which can impair hypothalamic-pituitary-adrenal axis functioning, may have been able to cause an age-related decrease in the BDNF-ir PVN and/or SON neurons in HL-OF stressed rats.

Analysis of calretinin immunoreactivity in the rat piriform cortex after open field stress during postnatal maturation.

In our study we used c-Fos protein to identify whether cells containing calretinin (CR) in the rat piriform cortex are engaged in the response to stress stimulation and to find out how this expression changes during maturation (PC). The material consisted of Wistar strain rats of between 0 and 120 days of age divided into 9 groups. Each group consisted of 5 experimental and 3 control rats. Animals from the experimental groups were exposed to the open field test throughout 10 minutes. The control animals were kept in a home cage. In all age-related control rats weak c-Fos immunoreactivity was observed. Our results showed that cells containing c-Fos following an acute open field test were observed predominantly in layers II and III of the PC just after birth. Their number then increased and stabilised on P30. We had already observed immature CR-ir cells at birth. In the 4th week of life these neurons achieved maturity. Their number increased to P90 and decreased in older animals. CR-ir neurons were localised mainly in layer II and to a lesser degree in layers III and I of the PC. Double immunostaining c-Fos/CR revealed that the level of co-localisation was low. Only small differences were observed between the anterior and posterior parts of the PC. In the anterior part a higher number of CR-ir neurons was found. The difference in the level of co-localisation between the anterior and posterior parts was age-related and differentiated. Our results may suggest that during maturation CR-ir neurons of the piriform cortex are not the main population engaged in response to the open field test.

The influence of mild stressors on neurons containing interleukin-1ß in the central (CeA) and medial (MeA) amygdala in the ageing process of rats.

Proinflammatory cytokine - interleukin 1ß (IL-1ß) plays an important role in stress reactions in the structures of limbic system. The impact of stress on IL-1ß may depend on the ontogenetic age. The study examined the influence of acute and chronic exposure to forced swim (FS) or high-light open-field (HL-OF) stressors on neurons containing IL-1ß. Double immunofluorescence staining was used to reveal the density of IL-1ß/NeuN (NeuN - a neuronal nuclear marker) - immunoreactive (ir) cells in the amygdaloid central (CeA) and medial (MeA) nuclei, which are closely involved in the regulation of emotional stressors and hypothalamic-pituitary-adrenal axis (HPA) activation. Adult (P90; P - postnatal day), middle-aged (P360), and aged (P720) male Wistar Han rats were used in these experiments. We observed an age-dependent increase in the basal density of IL-1ß/NeuN-ir cells in CeA and MeA in P90 vs. P360 and P360 vs. P720 rats. Neither acute nor chronic FS caused significant changes in the density of IL-1ß-ir neurons in any of the investigated nuclei in P90, P360, and P720 rats as compared with the non-stressed groups. However, chronic but not acute HL-OF caused a marked increase in the density of IL-1ß/NeuN-ir cells in the CeA and MeA of P360 rats and in MeA of the P720 animals. Moreover, chronic HL-OF led to an increase in the density of IL-1ß-ir neurons in relation to acute HL-OF in the CeA and MeA of both P360 and P720 rats. Our results may indicate the involvement of IL-1ß neurons in the development of ageing processes in CeA and MeA. Furthermore, our results point out that chronic HL-OF is an aggravating factor that induces an increase in the density of IL-1ß/NeuN-ir cells in the MeA and/or CeA of middle-aged and aged rats. The increase is possibly due to insufficient control of the HPA axis associated with involutional ageing processes and seems to be a common denominator of the ageing process and stress.

Qualitative and quantitative study of the postnatal development of the rabbit claustrum.

The morphometric analysis of changes occurring in the rabbit claustrum in the early postnatal period was performed by means of unbiased stereological methods. Material consisted of 40 animals aged from P2 to P180 (P-postnatal day) divided into eight groups. The volume of the claustrum, total number and numerical density of its neurons change very rapidly at the beginning of the postnatal life and stabilize by about the fourth week. From the 21st postnatal day distribution and morphology of neurons correspond to that in adults. Almost from the beginning of the postnatal life the rabbit claustrum is composed of three different parts: anterior, central and posterior. The anterior part is the largest and it consists mainly of fusiform, pyramidal and multipolar neurons. The central part is generally composed of oval neurons. The posterior part is the smallest; the distribution of its neuronal types is similar to that in the anterior one. Rapid morphological changes of the claustrum and its neurons occurring during the first postnatal month seem to point out that this structure is able to fulfill its physiological role after this critical period.

Developmental changes of synaptic proteins expression within the hippocampal formation of the rat.

A properly structured neuronal network, which is a prerequisite of a correctly functioning central nervous system, depends on the proper construction of synaptic terminals. There are some studies concerning development of hippocampal cells, however, the data about maturation of the synaptic terminals network within the hippocampal formation are only fragmentary. The study was performed on Wistar rats of various ages. The following synaptic proteins: synaptophysin, SNAP-25 and GAP-43 (a growth axon cone protein) were used to study the maturation of synaptic terminals. We have found that, at the time of birth, the hippocampal formation is very immature. Synaptogenesis begins on P4 and lasts till P10 or P14. The strongest immunoreactivity of all proteins is observed at the beginning of this period. Thereafter, immunoreactivity decreases and, after P14, it is similar to that of adult animals. On P60 and P90, the level of immunoreactivity increases again. In the pyramidal and granular cell layers, the formation of synaptic terminals and their maturation occur earlier in embryonal life, and are almost completely mature at birth.

Distribution of immunoreactivity of calcium-binding proteins in the rabbit piriform cortex.

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.

Evolution of microglial and astroglial response during experimental intracerebral haemorrhage in the rat.

Intracerebral haemorrhage is a strong stimulus for both microglial and astroglial activations. There are some important pathophysiological features during haemorrhage that do not occur in ischaemic or traumatic brain injuries, and may influence the dynamics and intensity of glial activation. Studies on the evolution of glial reaction may have practical importance to the introduction of new therapeutic methods for influencing the inflammatory reaction during haemorrhage. Microglial and astroglial responses to experimental intracerebral haematoma were studied in 50 adult rats for 5 minutes after injection of 100 microl autologous arterial blood into the striatum. The survival period varied from 1 to 21 days. Microglial-macrophage lineage cells were immunocytochemically stained with antibodies OX42, OX6 and ED1. The astrocytic population was studied by means of anti-GFAP staining. Changes in cellular morphology and intensity of staining were time-dependent reactions in both microglial and astroglial cells. Strong activation of microglial-macrophage lineage cells revealed with OX6-and OX42-immunoreactivity started during the first postoperative day. The complete pattern of activation for ED1-immunoreactivity was observed from the third postoperative day. At this stage, numerous phagocytic macrophages started to appear in the perihaematoma region. Morphological changes were most intensive during the second postoperative week. The astroglial (anti-GFAP) reaction was observed after the third postoperative day and proceeded less dynamically. The glial reaction gradually stopped but not completely during the period of observation. The early occurrence of glial activation, pattern of morphological changes and characteristic sequence of antigens expression indicate a very intense type of glial reaction. Evolution of glial response to haemorrhage reveals characteristic features. In our opinion, the initial phase of glial activation, comprising 72 hours after the occurrence of haemorrhage, is potentially the most promising period for influencing the extent of glial reaction with therapeutic agents.

Analysis of calcium binding protein immunoreactivity in the claustrum and the endopiriform nucleus of the rabbit.

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.

Cholinergic innervation and calretinin-immunoreactive neurones in the hippocampus during postnatal development of the rat brain.

Immunohistochemical study of the cholinergic innervation of the hippocampal calretinin-containing cells was conducted on 28 rat brains of postnatal ages: P0, P4, P7, P14, P21, P30 and P60. Sections with double immunostaining for vesicular acetylcholine transporter (VAChT; the marker of cholinergic cells, fibres and terminals) and calretinin were analysed using confocal laser-scanning microscope. Obtained data demonstrate that during development as well as in adult species calretinin-containing neurones in the rat hippocampus form sparse synaptic contact with VAChT-ir terminals. It seems probable that cholinergic innervation is not crucial for the functioning of CR-ir cells--probably they remain under the greater influence of a system other than the cholinergic system.

Parvalbumin containing neurons of the piriform cortex in open field stress -- a developmental study in the rat.

In our study we used c-Fos protein (as a marker of cellular activity) to identify whether cells containing parvalbumin (PV) in the piriform cortex (PC) are engaged in the response to stress stimulation and to discover how this expression changes during maturation. The material consisted of Wistar rats of postnatal (P) ages between 0 and 120 days divided into 9 groups: P0, P4, P7, P10, P14, P21, P30, P90, P120. Each group consisted of 5 experimental and 3 control animals. Rats of the experimental groups were exposed to the "open field test" throughout 10 minutes. The control animals were kept in a home cage. Our results showed that c-Fos activity in the open field test was observed in layers II and III of PC after birth. It then increased and stabilised on P30. In the second week of life PV-positive cells were also observed in those layers. These achieved maturity in the 4th week of life. After this time basket-like structures appeared but the level of PV/c-Fos co-localisation was low. Only small differences were observed between the anterior and posterior parts of PC. In the anterior part a higher number of PV-positive neurons, neuropil threads, and basket-like structures and a larger degree of PV/c-Fos co-localisation were observed. Our results suggested that during maturation PV cells are not directly activated in response to stress stimuli but PV neurons via their numerous endings influence the activation of c-Fos-positive cells predominantly in the anterior part of PC.

Cholinergic innervation of parvalbumin- and calbindin-containing neurones in the hippocampus during postnatal development of the rat brain.

Immunohistochemical study of the cholinergic innervation of the parvalbumin- and calbindin-containing cells in the hippocampus was conducted on 30 rat brains of various postnatal ages: P0, P4, P7, P14, P21, P30, P60 and P180. Sections with double immunostaining for vesicular acetylcholine transporter (VAChT; the marker of cholinergic cells, fibres and terminals) and parvalbumin (PV) or calbindin (CB) were analysed using confocal laser-scanning microscope. Obtained data demonstrate that the pattern of cholinergic innervation of calbindin- and parvalbumin-immunoreactive hippocampal neurones shows some differences. During development as well as in the adult species cholinergic terminals preferentially innervate CB-containing neurones, while cholinergic terminals on PV-containing cells were observed rarely. Cholinergic endings on the CB-ir neurones are localised both on their somata and dendrites, whereas on PV-ir cells they form synaptic contact predominantly with processes. In spite of the unquestionable cholinergic influence particularly on CB-ir cells, the number of cholinergic endings suggests that this input seems not to be crucial for the activity of the studied cell populations.

Developmental pattern of calbindin D28k protein expression in the rat striatum and cerebral cortex.

We examined the protein expression of the calcium-binding protein calbindin D28k in two developing rat brain structures, the striatum and the cerebral cortex. The relative protein concentration level was quantified by means of the Western blotting method and densitometric scanning. 32 Wistar rats were used, divided according to survival period (P0-P120-postnatal days). Observations of the calbindin D28k protein expression in the rat striatum and the cerebral cortex revealed an increase in band color intensity between P0 and P10. The intensity of protein staining in older groups of animals stabilised at a similar level and in the P28 and P120 groups we observed a decrement of calbindin expression in the striatum. Calbindin D28k stabilises the intracellular calcium level, preventing calcium-induced apoptotic cell death in neurons. Thus, changes in calbindin D28k expression might be related to its neuroprotective role in differentiation and synaptogenesis during the postnatal development of the brain.

The influence of open field exposure on neurons containing nitric oxide synthase in the basolateral complex and paracapsular intercalated nerve cells of the rat amygdala.

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.

Correlation between dopaminergic phenotype and expression of calretinin in the midbrain nuclei of the opossum (Monodelphis domestica): an immunohistological study.

We investigated distribution and morphology of neurons of the midbrain nuclei: the ventral tegmental area (VTA), substantia nigra (SN) and periaqueductal gray (PAG) of the adult grey short-tailed opossums that were double immunolabeled for the presence of calretinin (CR) and/or tyrosine hydroxylase (TH). The majority of TH-immunopositive neurons and fibers were located in the VTA, SN, and only scarce population of small neurons expressing TH was present in the PAG. In the SN 80 percent of TH-expressing neurons had large cell bodies, and only a small fraction had small perikarya. In the PAG populations of large and medium sized neurons were equal and 20 percent of neurons had small perikarya. Much scarcer population of TH-immunoreactive neurons in the PAG consisted of large or small neurons in its dorsal part (PAGd) and almost exclusively small neurons in the ventral part (PAGv). Distribution of neurons expressing TH and their types in the opossum are similar to those in rodents. The majority of CR-immunolabeled neurons were found in the VTA. In its subdivision, the parabrachal pigmented nucleus (PBP) cells expressing CR were approximately 28 percent more numerous than cells expressing TH. In spite of that, only 42 percent of TH-expressing neurons coexpressed CR. The high degree of colocalization TH and CR was observed in the SN. We propose that a higher percentage of TH/CR colocalization, which is observed in the opossums SN, may give them the ability to adapt to changes in their motor functions.