Neonatal shaking brain injury changes psychological stress-induced neuronal activity in adult male rats

Journal Pre-proof Neonatal shaking brain injury changes psychological stress-induced neuronal activity in adult male rats Shuichi Ueda (Conceptualization) (Methodology) (Data curation) (Writing - review and editing), Tsuyoshi Yamaguchi (Investigation), Ayuka Ehara (Investigation) (Software)

PII:

S0304-3940(20)30014-8

DOI:

https://doi.org/10.1016/j.neulet.2020.134744

Reference:

NSL 134744

To appear in:

Neuroscience Letters

Received Date:

21 November 2019

Revised Date:

24 December 2019

Accepted Date:

6 January 2020

Please cite this article as: Ueda S, Yamaguchi T, Ehara A, Neonatal shaking brain injury changes psychological stress-induced neuronal activity in adult male rats, Neuroscience Letters (2020), doi: https://doi.org/10.1016/j.neulet.2020.134744

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Neonatal shaking brain injury changes psychological stress-induced neuronal activity in adult male rats

Shuichi Ueda*, Tsuyoshi Yamaguchi, and Ayuka Ehara Department of Histology and Neurobiology, Dokkyo Medical University School of

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Medicine, Mibu, Tochigi, Japan

*

Corresponding author: Shuich Ueda, MD, PhD, Department of Histology and

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Neurobiology, Dokkyo Medical University School of Medicine, Tochigi 321-0293,

Tel: +81-282-87-2124

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Email: [email protected]

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Japan

Highlights

We made a new model for SBI in neonatal rats.

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Repeated SBI in neonates changed stress-induced neuronal activity as adults.

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These areas include mPFC, amygdala, vBNST, and ventral subiculum.

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Altered neuronal activity may contribute to behavioral outcomes in adults.

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Abstract

Neonatal shaking brain injury (SBI) leads to increases in anxiety-like behavior and altered hormonal responses to psychological stressors as adults. These abnormalities are hypothesized to be due to a change in sensitization in neuronal circuits as a consequence

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of neonatal SBI. We examined the effects of neonatal SBI on neuronal activity in the anxiety- and/or stress-related areas of adult rats using Fos immunohistochemistry. Exposure to a novel elevated plus maze (EPM) resulted in a marked increase in Fos expression in the parvocellular (PVNp) and magnocellular parts of the paraventricular nucleus and the ventral part of the bed nucleus of the stria terminalis (vBNST) of

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shaken rats (S group) compared to non-shaken control rats (C group). On the contrary, Fos expression was significantly lower in the medial nucleus of the amygdala and the

ventral subiculum (vS) of S group rats than C group rats exposed to EPM. Although we

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found no significant correlation in the number of Fos-expressing cells in the vBNST and

PVNp in the C group rats, these numbers were significantly correlated in the S group

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rats. Furthermore, in the S group rats, but not in the C group rats, the number of

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Fos-expressing cells in the vBNST was inversely correlated with that in the vS. Interestingly, previous neuronal tracing studies have demonstrated direct projections

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from the vS to the vBNST and from the vBNST to the PVNp. The present data suggest that neonatal SBI can alter neuronal activity in anxiety- and/or stress-related neuronal

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circuits.

Keywords: Shaken baby syndrome, Animal model, Fos, Hippocampus, Bed nucleus of the stria terminalis

1. Introduction A large number of clinical studies suggest that children exposed to early adverse 2

experiences are at increased risk for the development and persistence of mental disorders, such as depression, anxiety disorders, panic disorders, and posttraumatic stress disorder [1, 2]. Violent shaking of the body with or without impact has been recognized as a dangerous form of child physical abuse. Infants subjected to excessive acceleration-deceleration of the head may develop shaking brain injury (SBI) with

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intracranial and retinal hemorrhages, known as “shaken baby syndrome” [3]. Although the mortality rate of shaken baby syndrome is estimated to be 20-25%, survivors show a higher prevalence of mental disorders, such as depression, anxiety, psychosis, and

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posttraumatic stress disorder [4, 5].

The sensitization of neuronal circuits as a consequence of abusive early life stress (ELS)

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may represent the underlying biological substrate of increased vulnerability to

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subsequent stress, and may lead to the development of depression and anxiety [1]. Several animal models of ELS, especially rodent models, have contributed to addressing

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challenges to these problems. Maternal separation is a well-characterized study model of rodent ELS [6,7]. Prolonged maternal separation during the postnatal period in rats

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can produce long-lasting increases in anxiety-like behavior and stress reactivity [6, 8].

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Because rodent models of neonatal SBI usually show large-sized hemorrhages that create apparent hematomas and thus high mortality, the long-term outcomes have received little attention [9 - 12]. Recently, as a novel model for physical child abuse, we developed a new experimental rodent model for neonatal SBI [13]. In this model, transient microhemorrhages (MHs) were observed in the gray matter of the cerebral cortex and hippocampus. These rats also show anxiety-like behavior and abnormal 3

hormonal responses to psychological stress as adults [13, 14]. Furthermore, adrenocorticoid receptors are decreased in the hippocampus of male adult rats of this model [14]. Using a highly sensitive iron histochemical staining method, we have demonstrated the leakage of free iron and iron uptake by cells that surround MHs [13, 14]. In this model, therefore, MHs in the hippocampus and medial prefrontal cortex

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(mPFC), brain structures that are involved in anxiety- and stress-related neuronal circuits, seem to cause iron-induced superoxide production and mitochondrial dysfunction, which may lead to long-lasting dysfunction of these regions. Limbic

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modulation of stress responses occurs predominantly through direct and indirect inputs

to the paraventricular nucleus of the hypothalamus (PVN). These areas include the

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mPFC, amygdaloid complex (AMY), bed nucleus of the stria terminalis (BNST), and

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ventral subiculum (vS) [15].

In the present study, to elucidate whether neonatal SBI changes psychological

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stress-induced neuronal activity in adult rats, we examined the effects of exposure to a novel elevated plus maze (EPM) on Fos expression in stress- and/or anxiety-related

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rats.

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areas (mPFC, BNST, PVN, AMY, and vS) and compared the results with non-SBI adult

2. Materials and methods 2.1 Animals and experimental design All animal experimental procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were 4

approved by the Animal Welfare Committee of Dokkyo Medical University School of Medicine. Timed pregnant female Sprague-Dawley rats were purchased from Japan Charles River Lab. Inc. (Tsukuba, Japan) and housed under controlled conditions of temperature (22 ± 2°C) and humidity (50-60%) and a regular 12-h light-dark cycle with ad libitum access to food and water. The date of birth was considered postnatal day 0

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(P0). The shaking apparatus and the procedure for SBI were reported in our previous paper [13]. Briefly, male P3-14 rat pups were anesthetized with 2% isoflurane in air, shaken for 60 s, and allowed to rest for 60 s; these steps were repeated five times per

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day (S group). The control (C group) pups were placed in the shaking apparatus without

shaking for the same experimental time under anesthesia. After shaking, all pups were

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subsequently returned to their dam until weaning (P21). Pups were then placed in a cage

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with 2-3 animals per cage until 8-10 weeks (P8W-P10W) of age when they were

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analyzed.

2.2 EPM exposure, tissue preparation, and immunohistochemistry

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The procedures for the EPM exposure were described elsewhere [14]. Male adult

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offspring (P8W-P10W) were exposed to a novel EPM for 15 min. Two hours after the exposure, each rat was deeply anesthetized with sodium pentobarbital (10 mg/100 g body weight) and transcardially perfused with physiological saline, followed by a mixture of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4, 800 ml). The following Fos immunohistochemical procedures were based on our previous paper [16]. The brain was frozen in powdered dry ice, and serial frontal sections (30 m thick, 5

180-m intervals) were cut with a cryostat. These serial sections were divided into six groups. After pre-incubation in 1% H2O2, the first series of sections were washed in 0.1 M phosphate-buffered saline (PBS, pH 7.4), and then incubated in rabbit polyclonal antibody against Fos (Oncogene Research Product, Cambridge, MA, USA; 1:5000) for 72 h at 4°C. After washing with PBS, the sections were incubated with biotinylated goat

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anti-rabbit IgG (Vector, Burlingame, CA, USA) for 2 h at room temperature. Sections were then incubated with avidin-biotin peroxidase complex (ABC, Vectastain,

Burlingame, CA, USA; 1:200). The sections were then reacted with 20 mg

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3,3’-diaminobenzidine in 100 ml Tris-HCl buffer (pH 7.4) containing 20 l 30% H2O2 for 15 min at room temperature. Between each step, the sections were washed in 0.1 M

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PBS. The stained sections were mounted on gelatin-coated glass slides and treated with

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0.05% OsO4. The adjacent series of sections were stained with cresyl violet. Similar to the previous experiments [14], the present experiments were performed

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between 9 am and 11 am.

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2.3 Evaluation of Fos immunohistochemistry

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Similar to our previous paper [16], the sampling areas included the mPFC including the infralimbic cortex (IL) and the prelimbic cortex (PL) at the level of Bregma 3.26 mm (atlas of Paxinos and Watson [17]) (Fig. 1); the ventral part of the BNST (vBNST) at the level of Bregma 0.12 mm [18]; the PVN including the magnocellular part (PVNm) and the parvocellular part (PVNp) at the level of Bregma −1.8 mm (Fig. 2); the amygdaloid complex including the anterior part of the basolateral nucleus of the amygdala (BLA), 6

the central nucleus of the amygdala (Ce), and the medial nucleus of the amygdala (Me) at the level of Bregma −3.00 mm (Fig. 3); and vS at the level of Bregma −6.0 mm (Fig. 4). The IL and PL were further divided into two areas; the superficial area including layers I, II, and III (sIL and sPL, respectively) and the deep area including layers IV, V, and VI (dIL and dPL, respectively). The BLA was divided into two parts:

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the medial part of the BLA (mBLA) and the lateral part of the BLA (lBLA). Modified stereological counting using Stereo Investigator (Micro Bright Field Japan, Tokyo, Japan) was used to count the total number of Fos immunoreactive (Fos-IR) cells in the

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sampling areas (sIL, dIL, sPL, and dPL: 0.25 mm2 ; mBLA and lBLA: 0.16 mm2 ; vBNST, Me: 0.21 mm2 ; Ce: 0.16 mm2 ; PVNm: 0.04 mm2 ; PVNp: 0.08 mm2 ; vS: 0.12

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mm2). Each nuclear area was identified in the adjacent cresyl violet-stained sections.

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The present study focused on the altered neuronal activity following exposure to the novel EPM in adult rats exposed to SBI as neonates. Thus, the rats were divided into

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four experimental groups, control rats without EPM exposure (C−), control rats with EPM exposure (C+), shaken rats without EPM exposure (S−), and shaken rats with

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EPM exposure (S+).

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In the present rat model, the appearance of MHs was usually bilateral, but MHs were sometimes observed unilaterally [13]. Thus, we counted Fos-IR cells on both sides individually.

2.4 Statistical analysis Data are presented as the mean ± standard error of the mean. All statistical 7

measurements were carried out using Stat View (Abacus Concepts, Inc., Berkeley, CA, USA). To compare the mean number of Fos-IR cells per sample area, two-factorial (way) analysis of variance (ANOVA) was performed for each region in each group (C vs. S) and for exposure to EPM (no exposure vs. exposure) as the main factor. Bonferroni-Dunn's F post-hoc comparison was subsequently used to determine the

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sources of variance underlying the effects of group and exposure, as well as individual pairwise group comparison based on a 0.05 level of significance. The Pearson's

correlation coefficient test and linear regression were used to examine the

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non-parametric correlation between values. Differences with P value less than 0.05 were

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considered significant.

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3. Results

After exposure to the novel EPM, induction of Fos-IR cells was observed throughout

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the brain, and their distribution was similar to that reported in previous studies [19, 20]. Table 1 shows the summary of two-factor ANOVA data. Two-factor ANOVA revealed a

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significant exposure effect in all examined areas except the lBLA, and a significant group effect in the PVNm, PVNp, and vS. A significant interaction between group and

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exposure effects (group × exposure effects) was observed in the vS (Table 1). In the mPFC, post-hoc analyses showed that exposure of the C group to the EPM (C+) significantly increased the number of Fos-IR cells in the sIL (C− : 25.60 ± 3.91, C+ : 52.33 ± 2.52, P = 0.0015), dIL (C− : 44.66 ± 7.77, C+ : 97.00 ± 3.16, P < 0.0001), sPL (C− : 36.20 ± 7.55, C+ : 85.50 ± 3.62, P = 0.0004), and dPL (C− : 57.20 ± 8

10.04, C+ : 112.50 ± 10.69, P = 0.0005). Exposure of the S group to the EPM (S+) significantly increased the number of Fos-IR cells in the sIL (S− : 16.75 ± 1.49, S+ : 59.00 ± 6.21, P < 0.0001), dIL (S− : 49.25 ± 4.97, S+ : 96.37 ± 9.04, P = 0.0005), sPL (S− : 35.00 ± 8.97, S+ : 101.00 ± 8.74, P < 0.0001), and dPL (S− : 51.75 ± 5.85, S+ : 101.25 ± 7.59, P = 0.0015) (Fig. 1).

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In the vBNST, post-hoc analyses revealed that the number of Fos-IR cells was significantly higher in the S+ (76.37 ± 7.79) than in the S− (52.80 ± 3.87, P = 0.017) and C+ (56.83 ± 6.27, P = 0.017) (Fig. 2).

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In the PVN, post-hoc analyses showed that exposure of both the C and S groups to the EPM (C+ and S+) significantly increased the number of Fos-IR cells in the PVNp

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(C− : 20.91 ± 1.93, C+ : 40.75 ± 3.0, P = 0.0034, S− : 25.62 ± 5.36, S+ : 60.0 ±

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8.08, P < 0.0001). The number of Fos-IR cells was significantly higher in the PVNm of S+ than in the PVNm of S− (S− : 13.91 ± 2.21, S+ : 22.9 ± 2.76, P = 0.14). In

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contrast, no significant difference was observed between the PVNm of C− and C+ (C− : 8.91 ± 1.20, C+ : 13.91, P = 0.13). Furthermore, the number of Fos-IR cells was

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significantly different in the PVNm (P = 0.011) and PVNp (P = 0.006) of the C+

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compared to the S+ groups (Fig. 2). In the AMY, exposure to the EPM significantly increased the number of Fos-IR cells in the Me of the C (C− : 22.83 ± 2.58, C+ : 75.37 ± 8.59, P < 0.0001) and S (S− : 25.80 ± 2.92, S+ : 53.00 ± 8.60, P = 0.021) groups, and in the mBLA of the S (S− : 21.00 ± 1.00, S+ : 30.87 ± 2.48, P = 0.081) groups, whereas the same procedure significantly decreased the number of Fos-IR cells in the Ce of the C (C− : 49.30 ± 9

5.09, C+ : 31.62 ± 2.93, P = 0.003) and S (S− : 42.83 ± 3.55, S+ : 24.00 ± 3.45, P = 0.0061) groups (Fig. 3). In the hippocampal regions, the numbers of Fos-IR cells in the dorsal hippocampus were comparable regardless of group (C or S) or exposure effect (data not shown). In contrast, in the vS of the ventral hippocampus, exposure of the C group to the EPM

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(C+) significantly increased the number of Fos-IR cells in the vS (C− : 15.57 ± 1.49, C+ : 53.37 ± 2.55, P < 0.0001), and exposure of the S group to the EPM (S+)

significantly increased the number of Fos-IR cells in the vS (S− : 14.66 ± 1.60, S+ :

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39.66 ± 2.29, P < 0.0001). The numbers of Fos-IR cells in the vS of S+ were

between C− and S− (P = 0.78) (Fig. 4).

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significantly lower than in the vS of C+ (P < 0.0001), although we found no difference

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Furthermore, we examined whether the numbers of Fos-expressing cells in the vBNST were correlated with those in the PVNp or vS of the S and C groups. Calculation of the

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Pearson's correlation coefficient indicated a significant positive association between Fos-expressing cells in the vBNST and PVNp of the S group (r = 0.783, P = 0.0053),

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and a significant negative association between Fos-expressing cells in the vBNST and

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vS of the S group (r = −0.740, P = 0.0199) (Table 2). No other correlations with Fos-expressing cells between the areas in the S and C groups were found.

4. Discussion In the present study, we examined Fos expression following psychological stress to assess the change in sensitization in the anxiety- and/or stress-related regions between 10

male adult rats with and without neonatal SBI. The EPM assesses the fear and/or anxiety of open spaces that rodents naturally display. Thus, the EPM is widely used to study hormonal changes, especially changes in corticosterone (CORT) and adrenocorticotrophic hormone, and to define the brain regions and mechanisms underlying anxiety behaviors [21 - 23]. For these reasons, novel EPM exposure was

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used for the psychological stress. The main finding in the present study was that neonatal SBI altered neuronal activity in the PVNp, PVNm, vBNST, Ce, Me, mBLA, and vS in male adult rats following novel EPM exposure.

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In the present study, exposure to the EPM significantly increased Fos expression in the PVN of both the C and S groups. These results were in accordance with previous

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studies [24, 25]. Furthermore, Fos expression in the PVN was higher in the S+ group

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than in the C+ group, indicating higher activation of PVN neurons in the S+ group. Neurons in the PVN, especially in the PVNp, mainly consist of corticotropin-releasing

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hormone-containing neurons, which represent the origin of a final common pathway in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis that constitutes the

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major neuroendocrine stress response system [1, 15]. The present results strongly

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support our previous hormonal study in which we demonstrated that S rats exposed to a novel EPM showed long-lasting hypersecretion of CORT and adrenocorticotrophic hormone in serum as adults [14]. A positive relationship between plasma CORT levels and anxiety-like behaviors has been reported in rodents [23]. The S+ group rats also show increased anxiety-like behaviors in open-field, EPM, and light/dark transition tests [13, 14]. 11

Psychological and/or emotional stressors manifest widespread activation in the limbic forebrain, including the mPFC, AMY, BNST, and hippocampus, and correspond to a broad array of behavioral changes including fear and anxiety [26]. Among these structures, neurons in the BNST only directly innervate the HPA effector neurons within the PVN [15, 26]. Although the BNST can be subdivided into at least 16 subregions

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[27], an ibotenate lesion study revealed that the posterior BNST is involved in inhibition of the HPA axis, whereas the anteroventral BNST is involved in excitation of the HPA axis [28]. In agreement with previous reports [18, 28], we found that the neurons in the

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vBNST that correspond to the anteroventral BNST neurons described by Choi et al. [28]

were activated by the psychological stressor (EPM). Our data further demonstrated a

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positive correlation in Fos expression between the vBNST and the PVNp of the S group,

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but not the C group. In the vBNST, a small group of glutamatergic neurons is scattered among a dominant population of GABAergic cells, and the two cell types may exert

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opposite influences on anxiety-like behavior [27, 29]. A recent optogenetic study [30] also clearly demonstrated a role for both types of neurons in anxiety-like behavior in

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mice. Furthermore, the activity of the vBNST is largely dependent on the input activity

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from outside the BNST [29, 31]. Noradrenergic neurons from the A1 and A2 medullary cell group and the locus coeruleus [32], AMY [15, 33], and upstream limbic cortical regions such as the vS [26, 34] and mPFC (PL and IL) [15, 26] innervate the vBNST. Double labeling and excitotoxin lesion studies have demonstrated a disynaptic relay from the vS to the PVN. The vS is one of the output regions of the ventral hippocampus, and excitatory monosynaptic transmission from CA1 pyramidal neurons to the 12

subiculum has been reported [35]. Glutamatergic neurons in the vS project to GABAergic neurons in the vBNST that directly innervate the PVN, indicating an inhibitory influence of the ventral hippocampus on the HPA [26, 33]. Mineralcorticoid receptors (MRs) in the ventral hippocampus have been postulated to be involved in anxiolytic mechanisms [36]. Recent extensive studies demonstrate that CORT has a

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rapid and nongenomic effect on the cellular excitability of CA1 pyramidal neurons [review; 37]. This effect of CORT is responsible for enhancement of glutamate

transmission in the CA1 pyramidal neurons through membrane-located MRs [38]. In the

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present study, Fos expression in the vS was decreased in the S+ compared with the C+ rats. Furthermore, a negative correlation for Fos expression was only observed between

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the vS and vBNST. Rats exposed to SBI as neonates show lower expression of mRNA

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for MRs in the hippocampus as adults than non-SBI rats [14]. Taken together, the present data indicate that the decreased MRs in the hippocampus of these model rats, at

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least in part, led to the decreased activity in the vS neurons, and these neurons participate in increasing PVN activity through the vBNST. In the present study, we

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could not determine what type of cells were activated in the vBNST, but our on-going

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study will determine what cells were activated. Networks between the limbic forebrain (AMY and mPFC) and BNST also regulate HPA axis activation during emotionally stressful experiences. In the AMY, GABAergic neurons in the Ce and Me also innervate GABAergic vBNST neurons. In the mPFC, two disynaptic pathways from the PL and IL to the vBNST have been proposed to account for the different modulation of emotional stress-induced HPA output [26]. 13

Further detailed neuroanatomical studies will be needed. In conclusion, neonatal SBI caused persistent brain activity changes in adults in specific regions that react to psychological stress exposure. These areas have been implicated in neuroendocrine and behavioral changes that promote coping with stress. The development of maladaptive states following neonatal SBI may be related to to

stress-related

psychiatric

illness.

The

present

functional

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susceptibility

neuroanatomical data provide useful information to elucidate mechanisms of the anxiety-prone state in children who were abused by shaking.

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Credit authors statement Writing-Reviewing and Editing

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Shuichi Ueda: Conceptualization, Methodology, Data curation, Tsuyoshi Yamaguchi: Investigation, Staining

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Acknowledgements

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Ayuka Ehara: Investigation, Staining, Software

The authors are grateful to Ms. Shukuko Minami for technical assistance and Ms. Fusae

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Terauchi for assistance with manuscript preparation.

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19204-19207.

Figure 1. (A) A representative photomicrograph of a Nissl-stained section showing the mPFC. Areas in the PL and IL in which cells were counted are delineated. (B, C) Photomicrographs showing sections that were immunohistochemically stained for Fos in the PL of C− (B1), C+ (B2), S− (B3), and S+ (B4), and the IL of C− (C1),

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C+ (C2), S− (C3), and S+ (C4). (D) Quantification of Fos-IR cells in the mPFC of C-

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(gray), C+ (pink), S− (black), and S+ (red). *P < 0.05 vs. C−, **P < 0.05 vs. S−.

Figure 2. (A, C) Representative photomicrographs of Nissl-stained sections showing

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the BNST (A) and PVN (C). Areas in the vBNST and PVNp in which cells were counted are delineated. (B, D) Photomicrographs showing sections that were

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immunohistochemically stained for Fos in the vBNST of C− (B1), C+ (B2), S− (B3),

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and S+ (B4), and the PVN of C− (C1), C+ (C2), S− (C3), and S+ (C4). (D) Quantification of Fos-IR cells in the vBNST, PVNm, and PVNp of C− (gray), C+ (pink), S− (black), and S+ (red). ac: anterior commissure. **P < 0.05 vs. S−, #P < 0.05 vs. C+.

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Figure 3. (A) A representative photomicrograph of a Nissl-stained section in the AMY. Areas in the Ce, Me, and BLA in which cells were counted are delineated. (B, C, D)

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Photomicrographs showing sections that were immunohistochemically stained for Fos in the Ce of C− (B1), C+ (B2), S− (B3), and S+ (B4); the Me of C− (C1), C+

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(C2), S− (C3), and S+ (C4); and the BLA of C− (D1), C+ (D2), S− (D3), and S+ (D4). (E) Quantification of Fos-IR cells in the Ce, Me, mBLA, and lBLA of C− (gray), C+ (pink), S− (black), and S+ (red). *P < 0.05 vs. C−, **P < 0.05 vs. S−, #P < 0.05 vs. C+.

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Figure 4. (A) A representative photomicrograph of a Nissl-stained section in the vS. The area in the vS in which cells were counted is delineated. (B) Photomicrographs

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showing sections that were immunohistochemically stained for Fos in the vS of C−

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(B1), C+ (B2), S− (B3), and S+ (B4). (C) Quantification of Fos-IR cells in the vS of C− (gray), C+ (pink), S− (black), and S+ (red). *P < 0.05 vs. C−, **P < 0.05 vs. S−, #P < 0.05 vs. C+.

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Table 1. Summary of the data of two-factorial ANOVA

regions

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Brain

group effect

exposure effect

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p

F value

exposure effects p

F value

value

p F value

value

p=

F(1,20) =

p<

F(1,20) =

p=

0.002

0.967

50.027

0.0001*

1.629

0.216

F(1,21) =

p=

F(1,21) =

p<

F(1,21) =

p=

0.385

0.541

28.934

0.0001*

0.015

0.902

F(1,19) =

p=

F(1,19) =

p<

F(1,19) =

p=

sPL

0.325

0.575

48.600

0.0001*

1.522

0.232

dPL

F(1,19) =

p=

F(1,19) =

p<

F(1,19) =

p=

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F(1,20) =

value

group x

sIL

dIL

23

Me mBLA lBLA

vS

0.095

0.761

F(1,20) =

p=

F(1,20) =

p=

F(1,20) =

p=

3.970

0.060

6.774

0.0170*

0.929

0.346

F(1,38) =

p=

F(1,38) =

p=

F(1,38) =

p=

7.204

0.011*

8.719

0.0050*

0.878

0.354

F(1,38) =

p=

F(1,38) =

p<

F(1,38) =

p=

6.064

0.018*

31.046

0.0001*

2.234

0.143

F(1,28) =

p=

F(1,28) =

p=

F(1,28) =

p=

2.906

0.099

19.171

0.0002*

0.013

0.909

F(1,23) =

p=

F(1,23) =

p<

F(1,23) =

p=

1.624

0.215

27.407

0.0001*

2.768

1.097

F(1,23) =

p=

F(1,23) =

p=

F(1,23) =

p=

0.027

0.871

9.703

0.0049*

0.343

0.563

F(1,23) =

p=

F(1,23) =

p=

F(1,23) =

P=

0.014

0.906

1.448

0.2410

0.981

0.332

F(1,26) =

p=

F(1,26) =

F(1,26) =

p=

11.097

0.003*

204.497

8.519

0.007*

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P < 0.05

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* significant difference

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Table 2. Pearson's correlation coefficient analysis

Compared regions

Fisher's r to z

P

C group

0.439

P = 0.292

S group

0.783

P = 0.005

vBNST,

vBNST,

PVNp

vS

24

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Ce

0.0001*

-p

PVNp

30.978

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PVNm

0.386

lP

vBNST

0.787

p<

0.0001*

0.083

P = 0.850

S group

-0.74

P = 0.019

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-p

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C group

25