Neuroscience.2015 Apr;290:346-356 

The effect of mild stress stimulation on the nerve growth factor (NGF) and tyrosine kinase receptor A (TrkA) immunoreactivity in the paraventricular nucleus (PVN) of the hypothalamus and hippocampus in aged versus adult rats.

Ewa Badowska-Szalewska, Rafał Krawczyk, Beata Ludkiewicz, Janusz Moryś.

Department of Anatomy and Neurobiology, Medical University of Gdańsk, Dębinki st. 1, 80-211 Gdańsk, Poland

 

Abstract

Ontogenetic life and stress can have different effects on the nerve growth factor (NGF) and its tyrosine kinase receptor A (TrkA) in the structures of the limbic system. This study aimed to explore the influence of two different stressors, acute and chronic exposure to forced swim (FS) stress or high-light open-field (HL-OF) stress, on cells containing NGF and TrkA . Immunofluorescence staining was used to reveal the density of NGF and TrkA immunoreactive (ir) cells in the paraventricular nucleus (PVN) of the hypothalamus or hippocampal subfields CA1, CA3 and dentate gyrus (DG) in adult (postnatal day 90; P90) and aged (P720) rats. The data revealed that neither acute nor chronic FS caused any alteration in NGF-ir and TrkA-ir cells in any of the structures investigated in P90 and P720 rats. However, a significant increase in NGF-ir was detected in the CA1 and CA3 after acute but not after chronic HL-OF in both age groups. The TrkA-ir remained unchanged after exposure to HL-OF in the PVN and hippocampus. Despite the lack of change in the density of NGF-ir and TrkA-ir cells between P90 and P720 non-stressed rats, a significant age-related decrease in NGF-ir and TrkA-ir cells in the PVN of FS and HL-OF stressed rats was noted. However, in the hippocampus, an age-related decrease in NGF-ir or TrkA-ir cells was observed in all rats except acute FS stressed rats. The changes are possibly associated with involutionally aging processes caused by insufficient control of hypothalamic-pituitary-adrenal (HPA) axis functioning in P720 rats and may contribute to disturbance in NGF signaling.

KEYWORDS: aging; limbic system; nerve growth factor (NGF); stress; tyrosine kinase receptor A (TrkA)

PMID: 25644424

 

Supplementary

The paraventricular nucleus (PVN) of the hypothalamus is the primary nucleus involved in the central regulation of hypothalamic-pituitary-adrenal (HPA) axis activity, as well as in the hypothalamic response to stimulatory and inhibitory inputs [1]. Hippocampal structures have a close functional relationship with PVN [2]. Due to its involvement in the termination of the HPA axis response to stress, the hippocampus is also one of the major components of the stress circuit [3] regulating the HPA axis in an inhibitory manner [2]. The biology of hippocampal and PVN neurons is closely connected with the nerve growth factor (NGF) [4]. The action of NGF is initiated by neurotrophin interaction with the high-affinity of tyrosine kinase receptor A (TrkA) [5]. TrkA is responsible for most of the biological properties of the NGF on neurons [6]. NGF and TrkA play an important role in the structures of the nervous system at different ontogenetic stages [7]. Multiple functions are suggested for endogenous NGF signalling: induction of enhanced neuronal growth and dendritic arborization, neuronal differentiation, facilitating neuronal survival and modulating synaptic plasticity [8]. By being involved in neuroendocrine secretion [9] and by taking part in modulating the HPA axis activity [4], NGF and TrkA could participate in stress response [10].

The response to stressors changes throughout the ontogenetic period of life [11]. This is caused by alterations in HPA axis functioning, which induce changes in the release of stress hormones [12]. Unfortunately, there is no agreement on the changes or lack of changes in the NGF and/or TrkA during the ageing process in the structures of the limbic system both in normally aged animals [13, 14] and in aged animals that underwent stress stimulation [10]. In addition, little is known about ageing-related changes in the NGF and TrkA immunoreactivity (-ir)  in the hypothalamus or hippocampus under mild stress models. We hypothesized that exposure to acute (15 min once) and chronic (15 min for 21 days) forced swim (FS) or high-light open-field (HL-OF) (which were chosen as different aversive stimuli to provoke emotional responses in rats) would change the density of NGF-ir and TrkA-ir neurons in PVN and hippocampal structures (pyramidal layer of CA1, CA3 and granular layer of dentate gyrus) in aged rats ( postnatal day 720; P720) in comparison with adult (P90) Wistar rats.

Immunohistochemical studies have shown the presence of NGF and TrkA immunoreactive cells in the PVN of the hypothalamus and hippocampal pyramidal layers of the CA1, CA3 as well as in the granular layer of the dentate gyrus (DG) in adult (P90) and aged (P720) non-stressed rats. Moreover, age-related changes in the density of both NGF-ir and TrkA-ir cells in the PVN and in all hippocampal subfields were not observed (Figures 1-2). We can assume that NGF-ir and TrkA-ir, observed in non-stressed rats, make the PVN and hippocampal neurons function effectively and also counteract the neurodegeneration processes (e.g. prevention of glutamate excitotoxicity, glucose deprivation, anoxia, nitric oxide cytotoxicity or the Ca2+ influx) at least in adult rats. It is known that in the senescence, the neurodegeneration processes and deficiencies connected with them are significantly amplified. The fact that the number of NGF-ir and TrkA-ir cells in P720 rats versus P90 rats did not change may indicate that either their effectiveness is sufficient or, as cannot be excluded, the mechanisms for the adequate production of NGF and TrkA dysregulate with age.

 

ebs fig1

 

In our research, the adult (P90) and aged (P720) rats that underwent acute or chronic exposure to FS stimulation showed no significant disparities in the density of NGF-ir and TrkA-ir cells in the PVN (Figures 1A, 2A) and hippocampal CA1, CA3, and DG areas (Figures 1B-D; 2B-D). Swimming is a psychophysical stressor (muscular activity, novelty, water), that rats might encounter in their natural environment [15], so swimming in the FS test could be a typical situation for them. It is possible that natural stressors may provoke evolutionally developed compensatory homeostatic mechanisms (e.g. antioxidant processes) [16, 17], which might prevent changes in the density of NGF-ir and TrkA-ir cells in the PVN and hippocampal subfields. We believe that FS used in our study (due to its naturalistic character and relatively low aversion) was not an aggravating factor (rats “coping” with it) for either adult or aged rats with regard to its impact on the NGF-ir and TrkA-ir cells in the PVN and hippocampal CA1, CA3 and DG. 

 ebs fig2 

An open field is a factor in which the rat can engage in an active exploration of an unfriendly novelty [18]. In our study, in which high-intensity light was used as an additional stimulus in the open field area (HL-OF), it was noted that neither acute nor chronic HL-OF caused significant changes in the density of NGF-ir or TrkA-ir cells in the PVN of adult and aged rats (Figures 1A, 2A). However, in the hippocampus of both P90 and P720 rats, a marked increase in NGF-ir (higher in P90) but not in TrkA-ir was detected in CA1 and CA3 after acute HL-OF but not after chronic HL-OF (Figures 1B-C, 2B-C).

Unlike FS, HL-OF stressor was purely psychological in nature. Two components are responsible for the aversion to acute HL-OF: “open field” – new, unknown potentially dangerous surrounding without the possibility to escape and “bright light” – rats are nocturnal and tend to avoid brightly lit places [19]. We can assume that acute HL-OF was effective in evoking a neuronal response in the CA1 and CA3 in adult and aged rats. A marked increase in the density of NGF-ir in the pyramidal cells of CA1 and CA3 in adult and aged rats during acute HL-OF could be due to the higher (in relation to FS) activation of the HPA axis. Indirectly, it is supported by the fact, that the density of the c-Fos-ir cells (c-Fos is a marker of neuronal/trans-synaptic activations among others in the stress-related circuitry) in PVN, which is the site where HPA axis activation is initiated, was highest in the rats that were under the effect of acute HL-OF in both age groups [20]. The activation might affect the functioning of the neuroendocrine response system in relation to acute HL-OF, which may have resulted in the increased secretion of glucocorticoids. This glucocorticoids hypersecretion could act on the hippocampal neurons and cause an increase in the density of NGF-ir in the CA1 and CA3 (Figure 1B-C). Due to the fact that NGF may play a functional role in stress-coping responses, an increase in NGF-ir after acute HL-OF seems to be neuroprotective both for adult and aged rats, because it could counteract the deleterious effect of stress hormones. No change in NGF-ir after chronic HL-OF may indicate that the processes described above has not continued for a long time. We can assume that the outcome of chronic exposure to HL-OF was an adaptive response of NGF-ir neurons (habituation) in the CA1 and CA3.

The strong expression of TrkA-ir which was observed in our study implies that this receptor may play a role in signal transduction mechanisms linked to NGF. The lack of any significant change in the density of TrkA-ir in the PVN and hippocampal areas of adult and aged rats treated with acute or chronic HL-OF (Figure 2A-D) may suggest that the stress did not affect the alterations in the density of cells containing TrkA-ir. In addition, our results may indicate that regulation of NGF and TrkA immunoreactivity did not appear simultaneously. We assume that the TrkA level in the TrkA-ir cells might have been sufficient for neuroprotective NGF-TrkA signaling during acute HL-OF. 

Despite the lack of changes in the density of NGF-ir and TrkA-ir cells between P90 and P720 non-stressed rats, a significant age-related (P90 vs. P720) decrease in the density of NGF-ir and TrkA-ir cells in the PVN was observed both in FS and HL-OF stressed rats (Figures 1A, 2A). However, in the hippocampus, an age-related decrease in the density of NGF-ir cells was found in the CA1 and CA3 of chronic FS stressed rats, and in the CA3 and DG of acute HL-OF stressed animals (Figure 1B-D). At the same time, there was no age-related decrease in TrkA-ir cells in the hippocampal areas, with the  exception of the CA3 after chronic HL-OF (Figure 2B-D).

An age-related decrease in NGF-ir and TrkA-ir cells, especially in the PVN in the groups exposed to stressful procedures, was found to indicate greater susceptibility to stress during ageing. Moreover, it may point to the reduction of the protective effects of NGF on neurons in these structures because of the weakened ability to synthesis NGF or TrkA in response to stress. The reason for these changes might have been a disturbance in the regulation of the HPA axis due to the ageing process. Changes in NGF-ir may cause the disruption of synthesis and secretion of hormones, enzymes or neurotransmitters present in the PVN. We believe that the age-dependent decrease in the density of NGF-ir or TrkA-ir cells may contribute to disturbance to NGF signaling. This disturbance might lead to atrophic changes in hippocampal neurons, which may impair their functioning and might influence certain neurodegenerative processes.

 

The importance of this study is four-fold. Firstly, no age-related (P90 versus P720) changes in the basal density of NGF-ir and TrkA-ir neurons in the PVN and hippocampal CA1, CA3 and DG has been observed. It is probably connected with the involvement of the proteins in important but age-different neuronal functions.

Secondly, our results have shown that FS was not an aggravating factor for adult and aged rats in the impact on the density of NGF-ir and TrkA-ir cells in the PVN and CA1, CA3 and DG. Presumably this occurs because this natural stimulation is able to activate the homeostatic mechanisms counteracting the changes in NGF-ir or TrkA-ir cells in the structures.

Thirdly, it was noted that acute but not chronic HL-OF caused an increase in the density of NGF-ir cells in the hippocampal CA1 and CA3 of adult and aged rats. This could be related to the neuroprotective role of NGF-ir neurons in response to acute HL-OF stimulation. The lack of changes after chronic HL-OF may indicate an adaptive response of NGF-ir hippocampal neurons.

Fourthly, an age-dependent (P90 versus P720) decrease in the density of cells containing NGF-ir and/or TrkA-ir in FS and/or HL-OF stressed rats, particularly in the PVN was demonstrated. The changes are possibly associated with involutionally ageing processes caused by insufficient control of HPA axis functioning in P720 rats and may contribute to the disturbance of NGF signaling.

 

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Acknowledgements: This study was supported by funds from the Polish Committee of Scientific Research (Research Project No. N401 011 31/0168).

 

Contact:

Ewa Badowska-Szalewska, Ph.D.

Department of Anatomy and Neurobiology, Medical University of Gdańsk, Dębinki st. 1, 80-211 Gdańsk, Poland.

e-mail: ewabadowska@gumed.edu.pl

 

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