Expression of Dynorphin and Kappa-Opioid Receptors in the Bed Nucleus of the Stria Terminalis: Focus on Adolescent Development

2.1. Kappa-Opioid Receptors

Moderate expression of KORs was seen in the BNSTLP and BNSTLD subnuclei (Figure. 3). Consistent with its known role as a presynaptic receptor, KOR immunostaining was found outlining the neuronal soma. In addition, we observed a few KOR-immunoreactive neurons in the BNSTMPM of the preadolescent animals. Nonetheless, the most intense KOR-immunostaining was present in the post-adolescent rats, with somewhat predominant expression in the BNSTLP but not in BNSTLD of females (Figure 3). However, the BNSTMPM was the nucleus with the highest KOR expression in the BNST.

On the other hand, we found that the overall KOR expression was markedly in-creased during the adolescent period in all investigated nuclei. In fact, we registered a mild prevalence in the KOR-expression in the BNSTMPM of preadolescent females (Figure 3) while after the puberty such a difference in immunostaining was not observed. In the preadolescent period we also noted that distinct KOR-immunoreactivity was present in the neuronal perikarya in the BNSTMPM which was not found in the adults.

Densitometric analysis of KOR immunoreactivity in the BNSTLD revealed significant effects of both age and sex on gray level intensity values, which are inversely proportional to receptor expression (Figure. 4A). Specifically, lower mean gray values reflect higher KOR expression, whereas higher gray values indicate lower expression levels. Two-way ANOVA showed a highly significant main effect of age (F(1,116) = 31.23, p < 0.0001) and a significant main effect of sex (F(1,116) = 6.521, p = 0.0120), while the interaction between these factors was not significant (F(1,116) = 0.032, p = 0.859), indicating independent effects of age and sex on KOR expression.

Mean gray intensity values demonstrated a progressive decrease with age, consistent with increased receptor expression in older animals. In 20-day-old males, the mean value was 143.31 ± 1.51, significantly higher than in 60-day-old males (133.79 ± 1.62; mean difference: 9.51 ± 2.49; p < 0.01), indicating a higher KOR expression in the adult group. Similarly, 20-day-old females had a mean of 139.13 ± 1.87, compared to 128.99 ± 2.00 in 60-day-old females (mean difference: 10.14 ± 2.49; p < 0.001). The most pronounced difference was between 20-day-old males and 60-day-old females (mean difference: 14.32 ± 2.49; p < 0.0001), further emphasizing the age-related increase in expression and the higher overall expression in females. Although not all pairwise comparisons reached significance, the main effect of sex supports a general trend of a higher KOR expression in females. Collectively, these findings indicate that KOR expression in the BNSTLD increases with age and is moderately influenced by sex, with lower gray values in older and female animals reflecting stronger immunoreactive signal.

Densitometric analysis of KOR immunoreactivity in the BNSTLP revealed a statistically significant effect of age on gray level intensity values (Figure. 4B). In particular, two-way ANOVA showed a highly significant main effect of age (F(1,116) = 50.79, p < 0.0001), whereas the main effect of sex (F(1,116) = 1.614, p = 0.2065) and the interaction between age and sex (F(1,116) = 0.4575, p = 0.5001) were not statistically significant. These findings suggest that age is the dominant factor influencing KOR expression in the BNSTLP.

The mean gray intensity values decreased with age, reflecting an age-dependent increase in KOR expression. In 20-day-old males, the mean gray value was 135.35 ± 1.04, compared to 122.53 ± 1.37 in 60-day-old males, i.e. a statistically significant difference of 12.82 ± 2.32 (p < 0.0001). Similarly, 20-day-old females had a mean of 132.15 ± 2.08, significantly higher than 121.55 ± 1.88 in 60-day-old females (mean difference: 10.60 ± 2.32, p < 0.0001). The most evident difference was observed between 20-day-old males and 60-day-old females (mean difference: 13.80 ± 2.32, p < 0.0001), further confirming the substantial increase in expression with age. Although a modest difference was also found between 60-day-old males and 20-day-old females (mean difference: –9.63 ± 2.32, p < 0.001), no other pairwise comparisons reached statistical significance. These data demonstrate a clear and robust age-dependent increase in KOR expression in the BNSTLP, as indicated by significantly lower gray values in older animals. Despite a tendency for higher expression in females, sex was not a statistically significant factor in determining KOR levels in this region.

Quantitative analysis of KOR expression in the BNSTMPM, a posterior part of the medial BNST, revealed a significant age-related modulation, as assessed by two-way ANOVA (Figure 4C). Among the main effects, age showed a highly significant influence [F(1,36) = 42.64, p < 0.0001], accounting for 50.75% of the total variation in the data. In contrast, sex did not reach statistical significance (p = 0.0580), nor did the age × sex interaction (p = 0.2209). These results suggest that receptor expression in this subregion is predominantly determined by the developmental stage, rather than by sex or by the interaction between the two.

Descriptive data support this conclusion that 20-day-old males exhibit the highest mean optical density (168.9 ± 3.45 SEM), corresponding to the lowest KOR expression, while the receptor availability was comparatively higher in 60-day-old males (140.5 ± 2.27 SEM) and 60-day-old females (137.9 ± 2.75 SEM). 20-day-old females had a moderately high mean value (157.2 ± 5.36 SEM), suggesting intermediate levels of KOR supression. Post-hoc Tukey’s multiple comparisons confirmed that KOR expression was significantly increased, i.e., lower mean values, in 60-day-old males and females compared to 20-day-old males. Specifically, significant differences were detected between 20-day-old males and 60-day-old males (p < 0.0001), 20-day-old males and 60-day-old females (p < 0.0001), and 20-day-old females and 60-day-old females (p < 0.01). Interestingly, a moderate but significant difference was also observed between 60-day-old males and 20-day-old females (p < 0.05), indicating some sex-related variations in early development.

KOR expression in the BNSTMPM increases significantly with age, reflecting a developmental upregulation of the receptor availability that occurs in both sexes. While sex differences were not consistently significant across all comparisons, the overall pattern suggests that juvenile animals, especially 20-day-old males, exhibit the lowest receptor expression, potentially indicating a period of functional suppression of KOR signaling during early development. This may have important implications for understanding the developmental regulation of stress and affective circuits governed by the DYN/KOR system within the extended amygdala.

4. Materials and Methods

4.1. Experimental Animals

The experiments were carried out on preadolescent Wistar rats (20 days-old; n = 12) and postadolescent (60 days-old; n = 12) of both sexes (equal number = 6 for males and females) weighing 25-40 g for preadolescent and 200-250 g for postadolescent, respectively. The experimental procedures were in keeping with the ethical guidelines of the EU Directive 2010/63/EU for the protection of animals used for scientific purposes, and the protocol was approved by the Bulgarian Food Safety Agency (Approval Protocol Nr. 411 of 16.12.2024). All efforts were made to minimize the number of animals used and their suffering.

4.2. Tissue Preparation

The animals were deeply anesthetized with an intraperitoneal injection of sodium pentobarbital (40 mg/kg; Supelco Inc., USA) and then transcardially perfused first with 0.05 M phosphate-buffered saline (PBS), followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. After perfusion, the brains of the animals were quickly removed, trimmed at the level of the BNST and then postfixed in the same fixative overnight at 4°C. Thereafter, the tissue pieces were processed for paraffin embedding. Afterwards, the paraffin blocks were cut into 6 µm thick sections which were collected sequentally and mounted on poly-L-lysine coated glass slides (Sigma-Aldrich, St. Louis, MO, USA). The first section of each series was routinely stained with hematoxylin and eosin (H&E) for histological study and the remaining sections were used for immunohistochemistry (IHC) on the same specimen slide.

4.3. Immunohistochemical Procedure

Immunohistochemical detection of Dynorphin A and Kappa opioid receptor-1 (KOR-1) was performed on paraffin-embedded brain sections following standard deparaffinization procedures. For antigen retrieval, the sections were incubated in 0.01 M citrate buffer, pH 6.0 and heated in a water bath at 95°C for 20 minutes (WB-4MS model). After cooling, the slides were rinsed in Tris-buffered saline with 0.05% Tween-20 (TBST) buffer, pH 7.6. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide in distilled water for 10 minutes at room temperature. To minimize non-specific background staining, sections were pretreated with Super Block solution (ScyTek Laboratories, USA) for 10 minutes, followed by three short washes in TBST. Biotin blocking was subsequently carried out using the Biotin Blocking Kit (Cat. No. BBK120, ScyTek) in two sequential steps (Part A and Part B), each applied for 15 minutes with an intervening wash. Primary antibodies were diluted in Tris-based antibody diluent (ATG125, ScyTek) and applied overnight at 4°C in a humid chamber. The following antibodies were used: rabbit polyclonal anti-Dynorphin A (1:100, Antibodies, A283838) and rabbit polyclonal anti-KOR-1 (1:200, Elabscience, E-AB-64902). Thereafter, sections were incubated with the UltraTek Biotinylated Secondary Reagent and the HRP-conjugated detection reagent (UltraTek HRP Anti-Polyvalent Detection System, Cat. No. AFN600, ScyTek Laboratories, USA). Visualization of the immunoreactive signal was performed using the DAB Chromogen/Substrate Kit (ScyTek Laboratories). Finally, the slides were dehydrated through a graded series of ethanol, cleared, and coverslipped.

4.4. Antisera Specificity Tests

We applied both positive and negative controls to test the specificity of the antibodies used in this study. For immunoreaction specificity testing, omission of the specific primary antibodies by their replacement with PBS or non-immune serum, at the same dilution as the primary antiserum, was performed and no specific immunostaining was observed under these conditions. In addition, the specificities of the antibodies were controlled by preabsorption of the primary antisera for 2 h at room temperature or for 24 h at 4^⸰C with the respective synthetic antigen at a concentration of 20 or 200 μg/ml and the preabsorbed antisera were substituted for the non-absorbed antisera in the immunohistochemical procedure. Preabsorbed antibodies failed to stain any brain tissues. The antibodies were further characterized with tissue from regions known to contain the antigen. Immunolabeled sections of various brain regions known to contain the antigen were used as positive controls.

4.1. Image Analysis and Statistics

After immunostaining, the specimens were examined and photographed on an Olympus research microscope. We used the standardized nomenclature system provided by atlases of the rat brain based on selections of histological sections [3, 53], and the Bota and Swanson [54] table for comparison of two atlas parcellation schemes. Photomicrographs were taken from each BNST subnucleus, both from the left and right side to increase the number of measured neurons. At least 30 neurons from the BNSTMPM were assessed.

The digital images were obtained with an Olympus VS 120 slide scanner with a microscope acquisition system (Olympus VS-ASW Image Acquisition software). Then the color images were converted into black and white images and we measured the intensity of grey in the grayscale (from 0 - black to 255 - white). Thus, the binary images represented the analyzed reaction and were further used for measurements with the ImageJ software.

Quantification of DYN-positive fibers was carried out on high-resolution digital images acquired under constant microscope and camera settings. The analysis was performed using ImageJ software (NIH, USA). Within each BNST subnucleus, a fixed-size region of interest (ROI) was delineated, and a binary threshold was applied to isolate immunopositive fibers. The density of DYN-positive fibers was calculated as the percentage of the total ROI area occupied by the DAB-labeled signal (% area).

The expression of the KOR was evaluated in specific subdivisions of the BNST, including the BNSTLD, BNSTLP, and BNSTMPM subnuclei. High-resolution digital images of immunolabeled brain sections were acquired under identical exposure conditions using a light microscope equipped with a calibrated camera system. Grayscale intensity was measured within manually delineated ROIs using standardized settings in ImageJ (NIH, USA). Pixel intensity values ranged from 0 (black, maximal signal) to 255 (white, no signal). Since signal intensity is inversely proportional to protein expression, lower grayscale values were interpreted as reflecting higher levels of KOR immunoreactivity.

Quantitative data were analyzed using Prism 9 (GraphPad Software). Normality of distribution was confirmed using the Shapiro–Wilk test. Data were subjected to two-way analysis of variance (ANOVA) with age and sex as independent factors, followed by Tukey’s multiple comparisons test for pairwise group comparisons. All results are presented as mean ± standard error of the mean (SEM). Statistical significance was accepted at p < 0.05.

5. Conclusions

In conclusion, the adolescent dynamics of DYN/KOR system observed in several BNST subnuclei may help us explain the well-known phenomenon of increased vulnerability for developing an addiction to drugs and alcohol use in adolescents. Indeed, the increased expression of both DYN and KOR and/or the dysregulation of this system in the BNSTLD and BNSTLP which are associated with drug-seeking behavior might underlie the formation of abstinence and could be considered the morphological substrate of such addiction hypothesis. Moreover, the reported sex differences in neural mechanisms mediating substance-related attitudes in adolescent development could further explain some notable sex differences in addiction and anxiety.