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.

Changes in NGF/c-Fos double staining in the structures of the limbic system in juvenile and aged rats exposed to forced swim test.

This study aimed to investigate the influence of acute (a single 15 min) and chronic (15 min daily for 21 days) exposure to forced swim (FS) test on nerve growth factor (NGF)/c-Fos cells in hypothalamic paraventricular (PV) and supraoptic (SO) nuclei, the central (CeA) and medial (MeA) amygdaloid nuclei and CA3-hippocampus in juvenile (P28) and aged (P360) rats. The double-immunofluorescence (-ir) method was used to detect NGF-ir and c-Fos-ir cells. The amount of NGF/c-Fosir cells in relation to all NGF-ir cells is shown as a percentage. In the acute FS test an increase in NGF/c-Fos-ir cells (P<.05) was observed in all studied structures of juvenile rats and in the PV and SO of the aged individuals. After chronic FS stress, the NGF/c-Fos-ir ratio remained unaltered (except in the SO) in P28, but it increased (P<.05) in all investigated regions in P360 compared with the controls. The findings may reflect the state of molecular plasticity within the limbic hypothalamicpituitary- adrenocortical (HPA) axis in both age groups, yet the phenomenon of habituation in NGF/c-Fos-ir after chronic FS exposure was observed only in juvenile animals.

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 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.

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.

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.

Hippocampal interleukin-1beta in the juvenile and middle-aged rat: response to chronic forced swim or high-light open-field stress stimulation.

It is postulated that stress differentially affects interleukin-1beta (IL-1beta) during ontogenetic life. This study examined the influence of chronic exposure to forced swim (FS) stress or high-light open-field (HL-OF) stress on interleukin-1beta (IL-1beta). The total level of IL-1beta protein was assessed by Western blot analysis of hippocampal extracts. Double immunofluorescence staining was used to reveal the percentage of IL-1beta/NeuN (NeuN - neuronal marker) cells in the CA1, CA3 and dentate gyrus (DG) hippocampal subfields. Juvenile (P28; P - postnatal day) and middle-aged (P360) rats were used in the experiment. The research showed no significant differences in IL-1beta protein levels between P28 and P360 non-stress rats. However, a substantial increase in the percentage of IL-1beta-ir neurons in the CA1, CA3 and DG in P360 rats was observed. Chronic FS had no significant influence on IL-1beta expression in the hippocampus or on the percentage of IL-1beta-ir neurons in CA1, CA3 and DG hippocampal subfields in either age group. During HL-OF, the IL-1beta level was significantly increased in the hippocampus of P28 and P360 rats, whereas a marked increase in the percentage of IL-1beta-ir neurons in the CA1, CA3 and DG hippocampal areas occurred only in P360 animals. These results indicate that chronic HL-OF stimulation was the factor inducing changes in the IL-1beta protein levels in P28 and P360 rats and in the percentage of IL-1beta/NeuN-ir cells in the hippocampus of P360 animals.

Do two models of acute and chronic stress stimulation influence the amount of nerve growth factor (NGF) and its receptor TrkA in the hippocampal neurons of middle aged rats?

Our study aimed to explore the influence of two different stressors: acute (once for 15 min) and chronic (15 min daily for 21 days) exposure to high light open field (HL-OF) or forced swim (FS) on the density of nerve growth factor (NGF) and tyrosine kinase A (TrkA) immunoreactive neurons in the hippocampal CA1 and CA3 pyramidal cell layers and dentate gyrus (DG) granule cell layer in middle aged (360 days old; P360; P, postnatal day) rats. In contrast to non-stressed animals, acute HL-OF stimulation resulted in an increase (p<0.001) in the density of NGF-ir cells in CA1, CA3, DG, whereas chronic HL-OF produced no changes in all hippocampal regions. The rats which underwent acute and chronic FS tests showed no statistically significant differences in the density of NGF-ir containing cells in the CA1, CA3, and DG subfields compared with control rats. Except for DG, where after 21 days of FS the density of TrkA-ir neurons was found to increase (p<0.05) in comparison to unstressed rats, no changes were noted in the density of TrkA-ir in the studied hippocampal structures as a result of acute and chronic HL-OF or FS exposure. These results indicate that acute HL-OF stress stimulation was the only factor inducing changes in the density of NGF-ir containing neurons in the hippocampal CA1, CA3, and DG of middle aged rats. In respect of the density of NGF-ir and TrkA-ir cells in the hippocampal structures, prolonged exposure to HL-OF or FS stressors did not constitute an aggravating factor for rats in the studied ontogenetic period.

Effects of chronic forced swim stress on hippocampal brain-derived neutrophic factor (BDNF) and its receptor (TrkB) immunoreactive cells in juvenile and aged rats.

A type of stress stimulation and age are claimed to affect the expression of brain-derived neurotrophic factor (BDNF) and its receptor - tyrosine kinase B (TrkB) in the hippocampal regions differentially. This study aimed to explore the influence of chronic (15 min daily for 21 days) forced swim stress (FS) exposure on the BDNF and TrkB containing neurons in the hippocampal CA1, CA3 pyramidal cell layers and dentate gyrus (DG) granule cell layer in juvenile (P28) and aged (P360) rats. An immunofluorescence (-ir) method was used to detect BDNF-ir and TrkB-ir cells. Under chronic FS exposure, in the group of juvenile rats a significant decrease in the density of BDNF immunoreactive neurons was observed in CA1 and DG (P less than <0.001), unlike CA3, where it remained unaltered just as the density of TrkB-ir cells in CA1 and DG, but in CA3 the number of TrkB-ir cells was found to grow (P less than 0.05) in comparison with control groups. After chronic FS exposure of aged (P360) rats, the density of BDNF-ir and TrkB-ir cells did not decline in any of the subregions of the hippocampus. In all subfields of the hippocampus, the denseness of BDNF-positive neurons was significantly higher in P360 stressed group, compared with P28 stressed group, but the density of TrkB-ir fell more markedly in P360 than in P28. In conclusion, chronic FS stress influenced the number of BDNF and TrkB immunoreactive neurons only in juvenile animals. The age of rats tested in the chronic forced swim test was a decisive factor determining changes in the density of BDNF-ir and TrkB-ir in the hippocampal structures.