Effect of repeated restraint on homotypicstress-induced nitric oxide synthases expressionin brain structures regulating HPAaxis

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Effect of repeated restraint on homotypic

stress-induced nitric oxide synthases expression in brain structures regulating HPA axis

Anna G¹dek-Michalska, Joanna Tadeusz, Paulina Rachwalska, Jadwiga Spyrka, Jan Bugajski

Department of Physiology, Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12, PL 31-343 Kraków, Poland

Correspondence:Anna G¹dek-Michalska, e-mail: gadek@if-pan.krakow.pl

Abstract:

Background: Restraint stress (RS) markedly increases interleukin 1-b (IL-1b) generation in brain structures involved in hypothalamic-pituitary adrenocortical (HPA) axis regulation. The IL-1b-induced transient stimulation of HPA axis activity was par- allel in time and magnitude to respective changes in regulation of HPA activity. In the present experiment the expression of neuron al and inducible nitric oxide synthase (nNOS and iNOS) were investigated in prefrontal cortex, hippocampus and hypothalamus in response to acute restraint stress in control and prior repeatedly restrained rats.

Methods: Experiments were performed on male Wistar rats which were exposed to 10 min restraint stress or restrained twice a day for 3 days, and 24 h after the last stress period exposed to homotypic stress for 10 min. After rapid decapitation at 0, 1, 2 and 3 h after cessation of stress, trunk blood was collected and prefrontal cortex, hippocampus and hypothalamus were excised and frozen.

Interleukin-1b, adrenocorticotropic hormone (ACTH) and corticosterone (CORT) levels were determined in plasma using commer- cially available kits and neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in brain structure samples were analyzed by western blot procedure.

Results: Prior repeated restraint stress enhanced the acute restraint stress induced increase in IL-1b levels in all three structures ex- amined. Restraint stress for 10 min moderately decreased nNOS level in prefrontal cortex in control rats, augmented this level in hippocampus and markedly increased nNOS level in hypothalamus. Restraint itself significantly decreased iNOS level in prefrontal cortex, while it enhanced iNOS level in hippocampus and hypothalamus. Prior restraint stress for 3 days enhanced the nNOS level in prefrontal cortex and hippocampus and did not substantially affect nNOS levels response in hypothalamus. Repeated restraint stress considerably augmented the iNOS levels in both prefrontal cortex, hippocampus and hypothalamus induced by followed homotypic stress.

Conclusion: These results indicate that during restraint stress nNOS regulate formation of low amount of NO and the high-output generation of NO is effected by inducible isoform of nitric oxide synthase. Prior repeated stress significantly enhances the homotypic stress-induced nNOS and iNOS responses.

Key words:

repeated restraint, homotypic stress, neuronal nitric oxide synthase, inducible nitric oxide synthase, brain structures, prefrontal cortex, hippocampus, hypothalamus

Pharmacological Reports 2012, 64, 13811390 ISSN 1734-1140

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nervous system (CNS) and subserves interneuronal sig- naling [15, 28, 31, 43]. Many reviews have been pub- lished in the last years on the roles of NO in many as- pects of CNS function, including neuroendocrinology of stress systems [2, 3, 11, 14, 20, 26, 37]. Nitric oxide is a free radical gas synthesized from arginine and oxygen by two constitutive enzyme isoforms, nitric oxide synthase neuronal (nNOS) and endothelial (eNOS).The third type, inducible (iNOS) is rarely present normally and can be expressed mainly in microglia in the CNS during immunological challenge and stress [26, 31].

NO functions in the mammalian CNS like a con- ventional neurotransmitter with the ability to spread extremely rapidly through cell membranes and can operate much more effectively than a transmitter [15].

The neuronal NO synthase is highly expressed by cells of the hypothalamic paraventricular nucleus (PVN). In the rodent brain the magnocellular neurons located within hypothalamic supraoptic (SON) and paraventricular nuclei (PVN), contain high levels of nNOS mRNA and protein [6, 9]. Brain nNOS exists in particulate and soluble forms and the differential subcellular localization of nNOS may contribute to its di- verse functions. Neuronal NOS is involved in a vari- ety of synaptic events and is involved in modulating physiological functions and in a number of human diseases [4, 18, 42, 43]. In neurons nNOS is located mainly at the post-synaptic terminal, where it is acti- vated by increased intracellular levels of calcium. Be- cause of its diffusible nature and its ability to freely cross cell membranes, NO can act in an autocrine and paracrine manners also on targets relatively distant from its source of generation. The endothelial and neuronal NO synthase regulate formation of low amount of NO. The high-output generation of NO is mediated by inducible isoform (iNOS).

NO initiates stimulation of central brain structures by activation of CRH release [12, 23, 35, 38] by inter- leukin (IL-1b) in the PVN. In the PVN of the hypo- thalamus NO inhibits sympathetic outflow through increased GABA release. NO potentiates GABA-ergic synaptic inputs to spinally projecting PVN neurons through a cGMP protein kinase G pathway and central nNOS is involved in restraint stress-induced responses [24, 34]. In experimental conditions NO can either enhance or suppress secretion of a number of cytokines including IL-1. Cyclic AMP is a potent dual

NO, although it may also show either no or inhibitory modulation [44].

The general response to stress involves NO production due to induced expression of iNOS by stress mediators [30] and thus iNOS and NO are implicated in the patho- physiology of inflammation caused by stress. In recent years, inflammation has been implicated in chronic psy- chiatric disorders [18]. Acute phase proteins and cytoki- nes such as IL-1b and IL-6, levels are high in the serum of depressed patients. IL-1b may mediate some of the be- havioral responses, that occur during stress, including depressive-like behaviors, while NO induced by synthe- sis after stress exposure cause depression-like and an- xiety-like behaviors in rats [14]. Antidepressant-like activity in stressed mice may be related to the inhibition of plasma iNOS activity [7]. In the stress axis NOS sub- types particularly nNOS are distributed in the hippocampus, pituitary and adrenal gland axis [5, 10, 27, 32].

Cytokines are capable of modulating stress responses in the CNS mediated by different transmitter systems [16, 17, 21, 30].

We recently found that restraint stress markedly in- creased IL-1b generation in brain structures involved in regulation of HPA axis and plasma IL-1b during adaptation of HPA response to prolonged stress. The IL-1b-induced transient stimulation of HPA axis activity was parallel in time and magnitude to respective changes of NOS in prefrontal cortex and hypothalamus – the brain structures involved in regulation of HPA axis activity [13, 28].

The aim of the present study was to determine the nNOS and iNOS activity responses in brain structures related to acute restraint stress in control rats and in rats prior repeatedly restrained twice a day for 3 days.

A possible relations of NOS system response in brain structures and HPA axis response expressed by plasma ACTH, corticosterone and IL-1b levels were also investigated.

Materials and Methods

Animals

Experiments were carried out on male Wistar rats housed in groups of 5 per cage under standard conditions with an arti- ficial 12-h light/dark cycle (lights on from 7.00 to 19.00),

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a constant temperature 22 ± 2°C and food and tap water freely available. The rats were allowed 1 week of habituation period before the experimental session.

All the procedures were carried out in accordance with the requirements of Local Bioethics Commission for Animal Experiments at the Institute of Pharmacol- ogy, Polish Academy of Sciences, Kraków, Poland.

Experimental procedures

Experiments were performed on 4 groups of rats in which the changes in plasma levels of IL-1b, ACTH and corticosterone were determined and nNOS and iNOS content in prefrontal cortex, hippocampus and hypothalamus were measured.

The first group consisted of control non- stressed animals. In the second group the effect of restraint stress for 10 min on the above mentioned parameters was determined 0, 1, 2 and 3 h after the restraint period. The restraint was performed with metal tubes with ample holes for ventilation. In the third group, the animals were restrained for 10 min twice a day at 8 a.m. and 4 p.m. repeatedly for 3 consecutive days (3D RS 10 min) and decapitated 24 h after the last restraint. NOS levels in brain structures as well as IL-1b, ACTH and corticosterone in plasma were con- sidered as reference control values for changes in rats treated additionally with 10 min RS 24 h after prior restraint for 3 days. The fourth group consisted of 3D RS 10 min rats which were subjected to 10 min restraint stress 24 h after the last restraint period and rapidly decapitated 0, 1, 2 and 3 h later.

Trunk blood was collected in the presence of EDTA (for plasma corticosterone and IL-1b determinations) or EDTA and aprotinin (for ACTH immunoassay).

After decapitation, the brains were rapidly removed from the skulls and prefrontal cortex, hippocampus and hypothalamus were excised on ice-cold glass plates. The tissues were immediately frozen on dry ice and stored at –70°C. Protein extracts were pre- pared according to G¹dek-Michalska et al. [13]. Total corticosterone, ACTH and IL-1b levels were meas- ured in plasma using commercially available kits.

Western blot

The equal amounts of denaturated proteins (10 µg) were loaded into each lane, run on 7.5% SDS- polyacrylamide gels and then transferred onto a nitro- cellulose membranes.

The membranes were blocked with 5% non-fat dry milk and then incubated overnight at 4°C with the following primary antibodies: rabbit anti-NOS1, anti- NOS2 or anti-b-actin polyclonal antibody (1:400, Santa Cruz Biotechnology). b-Actin levels were used for normalization of nNOS and iNOS bands. After in- cubation, the membranes were washed four times with TBST and finally incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1 : 10,000, Santa Cruz Biotechnology). Afterwards, the membranes were washed with TBST, the blots were visualized by ECL (Lumi-Light^PlusWestern Blot- ting Kit, Roche) and evaluated with a luminescent image analyzer (Fujifilm LAS-4000). The optical density of appropriate bands was quantified by densitometric analysis of blots using Image Gauge V4.0 Software (Fujifilm) and normalized to b-actin levels. All the val- ues are expressed as a percentage of controls.

Statistical analysis

All results are expressed as the means ± standard error of the mean (SEM) where n = 10–12 rats per group.

Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software Inc.). Data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple range test. Groups that are significantly different from control are indicated in the figures as: + p < 0.05, ++ p < 0.01, +++ p < 0.001, and significant difference from 3D RS 10 min group is shown as: * p < 0.05, ** p < 0.01, *** p < 0.001.

Results

Stress-induced nNOS and iNOS responses in prefrontal cortex

In rats restrained for 10 min nNOS and iNOS protein levels in prefrontal cortex, hippocampus and hypothalamus were determined immediately after stressed termination and 1, 2 and 3 h later. In prefrontal cortex nNOS level did not markedly alter (+14.9% to –12.4%) compared to its level in control rats in respective time intervals. Restraint for 10 min markedly lowered iNOS levels in 1–3 h after its termination by 22.5, 36 and 14.3%, respectively (Fig. 1).

Repeated restraint, homotypic stress, nNOS, iNOS, HPA axis

Anna G¹dek-Michalska et al.

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Repeated restraint two times a day for 10 min per 3 days alone resulted in a marked increase in nNOS and iNOS level in prefrontal cortex measured 24 h after the last restraint, by 117.5 and 138%, respectively. In prior stressed rats during 3 days subsequent homotypic stress for 10 min significantly increased nNOS level in prefrontal cortex immediately after termination of restraint and during consecutive 3 h compared to respective responses in rats restrained for 10 min (Fig. 2). By contrast, iNOS level in these rats significantly increased at 0 and 1 h after the 10 min restraint stress, over both the levels induced by 3 day repeated stress (278%) and ca. 190% over control level in non-stressed rats (Fig. 3).

Stress-induced nNOS and iNOS levels in hippocampus

In the hippocampus, restraint stress for 10 min significantly increased the nNOS protein level (by 26%) 2 h

after termination of stress and markedly decreased iNOS level (by 22%) 3 h after termination of restraint.

Both iNOS and nNOS izoenzymes expression increased to their maximum levels, 1 h and 2 h after cessation of restraint then decreased to either control or 25% below the control level, respectively (Fig. 4).

Prior repeated restraint alone for 3 days slightly increased nNOS level in hippocampus (13%). Homo- typic stress, that followed repeated stress, significantly enhanced the nNOS level of the termination of restraint and abolished the restraint alone-induced significant increase of nNOS 2 h after stress cessation (Fig. 5). Restraint for 10 min markedly increased iNOS level in the hippocampus 1 h after termination of stress and moderately decreased by 28% this level 3 h after cessation of restraint compared to respective control levels. Repeated restraint for 3 days moderately increased iNOS level in that brain structure (24%) above the control level in non-stressed rats. In prior repeatedly stressed rats homotypic restraint

ures 111, 810 rats per group were used; + p < 0.05 vs. non-stressed control group. Bottom panels show the representative immunoblot

showing the expression of nNOS and iNOS in prefrontal cortex sentative immunoblot showing the expression of nNOS and iNOS in prefrontal cortex

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stress for 10 min induced a significant and persistent increase in the hippocampus iNOS level immediately after termination of restraint and 1 h, 2 h and 3 h later by 53, 61 and 33%, respectively (Fig. 6).

Stress-induced nNOS and iNOS levels in the hypothalamus

Restraint of rats for 10 min induced significant en- hancement of nNOS level in the hypothalamus immediately after cessation of restraint and 1 and 3 h later, compared to respective control levels. That stress evoked a moderate alterations of iNOS level (13.4 – 18.3%) in the hypothalamus at 0–3 h following termination of restraint (Fig. 7).

Repeated restraint alone for 3 days resulted in a moderate increase of hypothalamus nNOS level (18%) over control level in non stressed rats. Subse- quent restraint for 10 min induced moderately lower nNOS response compared to nNOS response in control rats in each time-point examined (Fig. 8).

Fig. 5.Effect of prior restraint for 10 min two times per day for 3 days on 10 min; RS-induced nNOS in hippocampus; + p < 0.05 vs. non- stressed control group. Bottom panels show the representative immunoblot showing the expression of nNOS and iNOS in hippocampus Fig. 4.nNOS and iNOS content in hippocampus in rats restrained for 10 min; + p < 0.05 vs. non-stressed control group. Bottom panels show the representative immunoblot showing the expression of nNOS and iNOS in hippocampus

Fig. 3.Effect of prior restraint for 10 min two times per day for 3 days on 10 min restraint stress-induced iNOS content in prefrontal cortex; + p <

0.05 and +++ p < 0.001 vs. non-stressed control group, ** p < 0.01 vs.

restraint for 10 min two times per day for 3 days on 10 min restraint (3D RS 10 min). Bottom panels show the representative immunoblot showing the expression of nNOS and iNOS in hippocampus

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Fig. 7.nNOS and iNOS content in hypothalamus in rats restrained for 10 min; ++ p < 0.01 and +++ p < 0.001 vs. non-stressed control group. Bottom panels show the representative immunoblot showing the expression of nNOS and iNOS in hippocampus

RS 10 min). Bottom panels show the representative immunoblot showing the expression of nNOS and iNOS in hippocampus

Fig. 9.Effect of prior restraint for 10 min two times per day for 3 days on 10 min RS-induced iNOS in hypothalamus; ++ p < 0.01 and +++ p < 0.001 vs. non-stressed control group, ** p < 0.01 vs. restraint for 10 min two times per day for 3 days on 10 min restraint (3D RS 10 min). Bottom panels show the representative immunoblot showing the expression of nNOS and iNOS in hippocampus

iNOS in hippocampus

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Restraint for 10 min did not markedly altered hypothalamic iNOS levels (+13.4 to –18.3%) compared with levels in control non-stressed rats (Fig. 9). Re- peated restraint itself for 3 days markedly (38.1%) increased the hypothalamic iNOS levels compared to the levels in non-stressed rats. Subsequent 10 min re-

straint of stress-pretreated group further significantly enhanced hypothalamic iNOS activity 0, 1 and 2 h after cessation of restraint (by 102.7, 73.6 and 74.6%, respectively) (Fig. 9).

Stress-induced plasma IL-1b, ACTH and corticosterone responses

Acute restraint stress for 10 min considerably in- creased plasma IL-1b, ACTH and corticosterone lev- els immediately after restraint termination by ca. 4, 5 and 9.5 times their control values, respectively. The elevated IL-1b and ACTH levels returned almost to- tally to their control values 1 h after stress termination and corticosterone level after 2 h (Fig. 10). Prior repeated restraint stress for 3 days moderately impaired the homotypic stress-induced elevation of IL-1b level at the time of stress termination and significantly en- hanced plasma IL-1b level 2 h later (600%) compared with control, prior non-stressed rats. Plasma IL-1b, ACTH and corticosterone levels in rats stressed two times a day for 10 min per 3 days 24 h after the last restraint without additional 10 min restraint, did not dif- fer substantially from the corresponding levels in control non-stressed rats.

The pattern of changes in plasma ACTH and corticosterone levels induced by acute restraint in prior stressed rats were similar in time and magnitude to those after acute 10 min restraint alone (Fig. 11).

Discussion

Acute restraint stress for 10 min markedly increased IL-1b level in prefrontal cortex and hippocampus im- mediately after termination of stress, decreased these levels 1 h later and gradually increased 3 h after stress termination. In hypothalamus, a marked increase of these levels appeared after cessation of stress. Prior repeated restraint stress enhanced the acute restraint stress induced increase in IL-1b levels in all three structures examined [13].

In our experiment, under resting conditions both nNOS and iNOS were detected in brain structures, although it is widely considered that in normal conditions nNOS is expressed in the brain only in occasional neurons, while iNOS is not detected [22]. In prefrontal cortex acute restraint stress induced distinct alterations in

Fig. 11.Effect of prior restraint for 10 min two times per day for 3 days on 10 min RS-induced plasma levels of IL-1b, ACTH and corticosterone; ++ p < 0.01 and +++ p < 0.001 vs. non-stressed control group Fig. 10. Effect of restraint stress (RS) on plasma levels of interleukin-1b (IL-1b), ACTH and corticosterone; + p < 0.05 and +++ p < 0.001 vs. non-stressed control group

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decrease 2 h later and approaching the control level 3 h after stress termination. However, only the decrease in iNOS levels 1 h and 2 h after disruption of restraint was significant (23 and 36%, respectively).

These changes in nNOS and iNOS levels in prefrontal cortex may suggest an exhaustion of nNOS, and particularly iNOS generation effected by an acute, short-lasting stress. The marked decline, particularly of iNOS level, caused by acute stress was almost compensated in the third hour after stress termination, probably by an increased generation of izoenzyme peptide biosynthesis. A longer period of acute restraint lasting 2 h induced changes in gene expression of nNOS and significant increase in nNOS mRNA in brain areas related to stress, the medial parvocellular part of PVN and medial amygdaloid nucleus [6, 19, 39]. In the hippocampus we observed a marked transient increase in nNOS level 2 h after restraint termination. Inducible NOS activity slightly increased 1 h earlier, before the gradual decrease 2 h and 3 h later, compared with the levels in control non-stressed rats.

Single restraint caused acute changes in NO system in the amygdala complex and delayed modifications in the hippocampal formation [8, 25].

In the hypothalamus, the increase of nNOS was significant and relatively more pronounced than in the prefrontal cortex or the hippocampus. The restraint stress- induced iNOS level in the hypothalamus slightly dif- fered from the respective control level in the whole ob- servation period. Our results suggest a more pronounced participation of NO generated by iNOS than nNOS in the prefrontal cortex response to acute restraint stress.

The NOS/NO systems adapt to various forms of acute and chronic stress by modulating several mediators, such as neurotransmitters, cytokines, NO, and pros- taglandins, that can affect their complex interactions at the brain and systemic levels [2, 20, 31, 33, 41]. Nitric oxide mediates in the CRH release stimulated by IL-1 in both basal and stress conditions [16, 23, 40].

Prior repeated restraint, two times a day for 10 min per 3 days itself, induced slight increase in nNOS level and significant increase in iNOS levels in prefrontal cortex. Our results indicate much stronger involvement of iNOS than nNOS system in NO generation in prefrontal cortex during repeated restraint for 3 days. It is known that exposure to predominantly emotional and systemic stressors causes long term

last prior repeated restraint significantly increased nNOS level in prefrontal cortex 1 h after termination of restraint, but it did not alter this level later compared to the levels in untreated controls. By contrast, homotypic stress considerably enhanced iNOS level compared to the levels in both control untreated and prior repeatedly restrained rats.

These results indicate that prefrontal cortex is able to a rapid and high induction of NO generation by iNOS in the first 3 h after termination of homotypic stress in prior repeatedly restrained rats. In the hippocampus, nNOS activity significantly increased 2 h after disruption of restraint for 10 min, while iNOS level decreased progressively at 2 h and 3 h. Repeated restraint stress for 3 days itself slightly increased nNOS and iNOS level in that structure. Homotypic stress applied 24 h after the last prior restraint induced a transient, significant increase in nNOS activity immediately after its termination and lineary vanished later. Homotypic stress in repeatedly restrained rats considerably enhanced iNOS level compared with corresponding levels in both untreated controls and prior restrained rats. Therefore, in the hippocampus like in the prefrontal cortex, prior repeated restraint strongly intensified iNOS activity. In the hypothalamus, repeated restraint for 3 days itself markedly increased iNOS level and the followed homotypic stress considerably augmented this level 0–3 h after restraint termination.

Acute restraint stress considerably increased plasma IL-1b,ACTH and corticosterone levels com- pared with untreated controls. The changes in IL-1b and ACTH levels disappeared 1 h after stress termination and corticosterone level returned to the control value 1 h later, due to a direct peripheral stimulation of adrenal cortex, including NOS/NO system [5, 10, 29, 36] in addition to ACTH. Prior repeated restraint did not markedly alter the pattern of changes in ACTH and corticosterone response to following homotypic stress but significantly increased plasma IL-1b level 2 h after stress termination. This suggests that repeated stress sensitizes plasma IL-1b response which does not correlate with ACTH and corticosterone response.

In conclusion, prior repeated restraint stress for 10 min during 3 days transiently sensitized nNOS activity in the prefrontal cortex and the hippocampus

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and strongly and persistently enhanced iNOS activity induced by a subsequent homotypic stress in prefrontal cortex, hippocampus and hypothalamus. The mechanisms and the exact sites responsible for strong sensitization of iNOS activity in these brain structures by acute homotypic stress need further elucidation.

Acknowledgments:

This research was supported by grant: POIG

01.01.02-12-004/09-00 financed by European Regional Development Fund as well as by the statutory funds from the Institute of Pharmacology, Polish Academy of Sciences.

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Received:July 30, 2012; in the revised form: October 4, 2012;

accepted:October 15, 2012.