0.840) values were comparable in the two genotypes (data not shown). *3.2. Pharmacological AVP Deficiency*

**Figure 2.** Wistar rats, treated with SR49059, a vasopressin (V) 1aR antagonist. The 7–8-day-old Wistar rat pups were treated intraperitoneally with a 3, 10 or 30 mg/kg V1aR antagonist 30 min before a 10 min maternal separation. The 30 mg/kg dose significantly reduced the emitted ultrasonic vocalization both in terms of (**a**) the number of calls and (**b**) the duration without changes in (**c**) adrenocorticotropin (ACTH, fmol/mL), but an elevation in (**d**) corticosterone (pmol/mL) levels. *n* = 12–14. \* *p* < 0.05 vs. control. There was no difference between the groups in the latency of the righting reflex as well as the negative geotaxis (Table 1). **Figure 2.** Wistar rats, treated with SR49059, a vasopressin (V) 1aR antagonist. The 7–8-day-old Wistar rat pups were treated intraperitoneally with a 3, 10 or 30 mg/kg V1aR antagonist 30 min before a 10 min maternal separation. The 30 mg/kg dose significantly reduced the emitted ultrasonic vocalization both in terms of (**a**) the number of calls and (**b**) the duration without changes in (**c**) adrenocorticotropin (ACTH, fmol/mL), but an elevation in (**d**) corticosterone (pmol/mL) levels. *n* = 12–14. \* *p* < 0.05 vs. control.

Righting 2.333 ± 0.343 2.361 ± 0.230 2.758 ± 0.397 2.212 ± 0.187 Neg.geo. 19.861 ± 2.687 20.750 ± 2.984 18.212 ± 2.735 16.485 ± 2.929

Righting 1.303 ± 0.156 2.279 ± 0.669 1.947 ± 0.303 2.103 ± 0.266 Neg.geo. 9.194 ± 0.963 8.487 ± 0.880 9.528 ± 1.215 10.100 ± 1.673

Righting 2.133 ± 0.218 2.267 ± 0.325 2.033 ± 0.195 2.250 ± 0.300 Neg.geo. 11.967 ± 1.961 13.970 ± 2.707 15.267 ± 3.010 18.667 ± 2.567

**0 3 10 30**

**Table 1.** Righting reflex and negative geotaxis values.

Neg.geo. 8.296 ± 1.301 7.125 ± 0.900 No significant alterations were discovered. Righting = righting reflex; Neg.geo. = negative geotaxis.

**Antagonist Treatment Time (s) Doses (mg/kg)**

V1aR + V1bR Righting 1.741 ± 0.282 2.750 ± 0.552

V1aR

V1bR

V2R



No significant alterations were discovered. Righting = righting reflex; Neg.geo. = negative geotaxis.

#### 3.2.2. V1bR Antagonist 3.2.2. V1bR Antagonist

The V1bR antagonist treatment decreased the MS-USV number of calls (F(3,43) = 5.719; *p* = 0.002; Figure 3a) and duration (F(3,43) = 4.470; *p* = 0.008; Figure 3b), accompanied by reduced ACTH levels (F(3,19) = 3.008; *p* = 0.056; Figure 3c) without changes in corticosterone levels (F(3,19) = 1.230; *p* = 0.326; Figure 3d). The V1bR antagonist treatment decreased the MS-USV number of calls (F(3,43) = 5.719; *p* = 0.002; Figure 3a) and duration (F(3,43) = 4.470; *p* = 0.008; Figure 3b), accompanied by reduced ACTH levels (F(3,19) = 3.008; *p* = 0.056; Figure 3c) without changes in corticosterone levels (F(3,19) = 1.230; *p* = 0.326; Figure 3d).

**Figure 3.** Wistar rats, treated with SSR149415, a a vasopressin (V) 1bR antagonist. The 7–8-day-old Wistar rat pups were treated intraperitoneally with 3, 10 or 30 mg/kg of the V1bR antagonist 30 min before a 10 min maternal separation. All doses significantly reduced the emitted ultrasonic vocalization both in terms of (**a**) the number of calls and (**b**) the duration and reduced (**c**) adrenocorticotropin (ACTH, fmol/mL) levels without affecting the (**d**) corticosterone (pmol/mL) values. *n*  = 10–13. \* *p* < 0.05. \*\* *p* < 0.01 vs. control. **Figure 3.** Wistar rats, treated with SSR149415, a a vasopressin (V) 1bR antagonist. The 7–8-day-old Wistar rat pups were treated intraperitoneally with 3, 10 or 30 mg/kg of the V1bR antagonist 30 min before a 10 min maternal separation. All doses significantly reduced the emitted ultrasonic vocalization both in terms of (**a**) the number of calls and (**b**) the duration and reduced (**c**) adrenocorticotropin (ACTH, fmol/mL) levels without affecting the (**d**) corticosterone (pmol/mL) values. *n* = 10–13. \* *p* < 0.05. \*\* *p* < 0.01 vs. control.

There was no difference between the groups in the latency of the righting reflex and the negative geotaxis (Table 1) [24]. There was no difference between the groups in the latency of the righting reflex and the negative geotaxis (Table 1) [24].

The 3 mg/kg V2R antagonist enhanced MS-USV (number of calls: F(3,35) = 4.891; *p* = 0.006; Fisher post hoc control vs. 3 mg/kg: *p* = 0.041; Figure 4a; duration: F(3,35) = 4.935; *p* = 0.006; Fisher post hoc control vs. 3 mg/kg: *p* = 0.057; Figure 4b), while the higher doses had no effect on MS-USV. Both stress hormone levels were higher 45 min after a single 30 mg/kg V2R antagonist treatment compared with the control injection group (ACTH: F(3,35) = 13.321; *p* = 0.000; Fisher post hoc control vs. 30 mg/kg: *p* = 0.000; Figure 4c; corticosterone:

F(3,35) = 8.363; *p* = 0.000; Fisher post hoc control vs. 30 mg/kg: *p* = 0.000; Figure 4d).

3.2.3. V2R Antagonist

#### 3.2.3. V2R Antagonist

The 3 mg/kg V2R antagonist enhanced MS-USV (number of calls: F(3,35) = 4.891; *p* = 0.006; Fisher post hoc control vs. 3 mg/kg: *p* = 0.041; Figure 4a; duration: F(3,35) = 4.935; *p* = 0.006; Fisher post hoc control vs. 3 mg/kg: *p* = 0.057; Figure 4b), while the higher doses had no effect on MS-USV. Both stress hormone levels were higher 45 min after a single 30 mg/kg V2R antagonist treatment compared with the control injection group (ACTH: F(3,35) = 13.321; *p* = 0.000; Fisher post hoc control vs. 30 mg/kg: *p* = 0.000; Figure 4c; corticosterone: F(3,35) = 8.363; *p* = 0.000; Fisher post hoc control vs. 30 mg/kg: *p* = 0.000; Figure 4d). *Brain Sci.* **2021**, *11*, x FOR PEER REVIEW 8 of 14

**Figure 4.** Wistar rats, treated with SR121463B, a a vasopressin (V) 2R antagonist. The 7–8-day-old Wistar rat pups were treated intraperitoneally with a 3, 10 or 30 mg/kg V2R antagonist 30 min before a 10 min maternal separation. The 3 mg/kg dose significantly enhanced the emitted ultrasonic vocalization in terms of (**a**) the number of calls, with a similar tendency in (**b**) the duration and 30 mg/kg elevated (**c**) adrenocorticotropin (ACTH, fmol/mL) and (**d**) corticosterone (pmol/mL) levels. *n* = 9–11; \* *p* < 0.05, \*\* *p* < 0.01 vs. control. **Figure 4.** Wistar rats, treated with SR121463B, a a vasopressin (V) 2R antagonist. The 7–8-day-old Wistar rat pups were treated intraperitoneally with a 3, 10 or 30 mg/kg V2R antagonist 30 min before a 10 min maternal separation. The 3 mg/kg dose significantly enhanced the emitted ultrasonic vocalization in terms of (**a**) the number of calls, with a similar tendency in (**b**) the duration and 30 mg/kg elevated (**c**) adrenocorticotropin (ACTH, fmol/mL) and (**d**) corticosterone (pmol/mL) levels. *n* = 9–11; \* *p* < 0.05, \*\* *p* < 0.01 vs. control.

the negative geotaxis (Table 1). There was no difference between the groups in the latency of the righting reflex or the negative geotaxis (Table 1).

There was no difference between the groups in the latency of the righting reflex or

#### 3.2.4. V1aR + V1bR Antagonists The combination of V1aR and V1bR antagonists effectively reduced MS-USV (num-3.2.4. V1aR + V1bR Antagonists

ber of calls: F(1,15) = 10.440; *p* = 0.006; Figure 5a; duration: F(1,15) = 15.616; *p* = 0.001; Figure 5b) without any effect on the stress hormones (ACTH: F(1,15) = 0.008; *p* = 0.931; Figure 5c; corticosterone: F(1,15) = 0.001; *p* = 0.982; Figure 5d). The same dose of the V1aR antagonist induced 34.3% and 26.8% nonsignificant reductions in the MS-USV number of calls and duration, respectively, while in the case of the V1bR-antagonist, 51.5% and 54.3% significant reductions were visible. The combination induced a 57.1% reduction in the MS-USV number of calls and a 68.5% reduction in duration. The combination of V1aR and V1bR antagonists effectively reduced MS-USV (number of calls: F(1,15) = 10.440; *p* = 0.006; Figure 5a; duration: F(1,15) = 15.616; *p* = 0.001; Figure 5b) without any effect on the stress hormones (ACTH: F(1,15) = 0.008; *p* = 0.931; Figure 5c; corticosterone: F(1,15) = 0.001; *p* = 0.982; Figure 5d). The same dose of the V1aR antagonist induced 34.3% and 26.8% nonsignificant reductions in the MS-USV number of calls and duration, respectively, while in the case of the V1bR-antagonist, 51.5% and 54.3% significant reductions were visible. The combination induced a 57.1% reduction in the MS-USV number of calls and a 68.5% reduction in duration.

There was no difference between the groups in the latency of the righting reflex or the negative geotaxis (Table 1). *Brain Sci.* **2021**, *11*, x FOR PEER REVIEW 9 of 14

**Figure 5.** Wistar rats, treated with SR49059 (a vasopressin (V) 1aR) + SSR149415 (V1bR) antagonists. The 7–8-day-old Wistar rat pups were treated intraperitoneally with a mixture of 10 + 10 mg/kg V1aR + V1bR antagonists 30 min before a 10 min maternal separation. The combination significantly reduced the emitted ultrasonic vocalization both in terms of (**a**) the number of calls and (**b**) the duration without affecting the (**c**) adrenocorticotropin (ACTH, fmol/mL) or (**d**) corticosterone (pmol/mL) levels. *n* = 9–8. \*\* *p* < 0.01 vs. control. **Figure 5.** Wistar rats, treated with SR49059 (a vasopressin (V) 1aR) + SSR149415 (V1bR) antagonists. The 7–8-day-old Wistar rat pups were treated intraperitoneally with a mixture of 10 + 10 mg/kg V1aR + V1bR antagonists 30 min before a 10 min maternal separation. The combination significantly reduced the emitted ultrasonic vocalization both in terms of (**a**) the number of calls and (**b**) the duration without affecting the (**c**) adrenocorticotropin (ACTH, fmol/mL) or (**d**) corticosterone (pmol/mL) levels. *n* = 9–8. \*\* *p* < 0.01 vs. control.

#### There was no difference between the groups in the latency of the righting reflex or *3.3. Correlations*

Figure 6c).

the negative geotaxis (Table 1). *3.3. Correlations* As could have been expected, the number of calls and duration of MS-USV positively correlated with each other in all experimental series. Interestingly, the same was true for ACTH and corticosterone correlation, except in the case of Brattleboro animals, where there was no correlation at all (data not shown). In the Brattleboro strain, the AVP content of the hypophysis showed a significant positive correlation with the MS-USV number of calls (r = 0.556; *p* = 0.017; Figure 6a) and duration (r = 0.541; *p* = 0.020). Moreover, in this As could have been expected, the number of calls and duration of MS-USV positively correlated with each other in all experimental series. Interestingly, the same was true for ACTH and corticosterone correlation, except in the case of Brattleboro animals, where there was no correlation at all (data not shown). In the Brattleboro strain, the AVP content of the hypophysis showed a significant positive correlation with the MS-USV number of calls (r = 0.556; *p* = 0.017; Figure 6a) and duration (r = 0.541; *p* = 0.020). Moreover, in this case, the serum ACTH level also showed positive correlation with the number of emitted MS-USVs (r = 0.491; *p* = 0.038; Figure 6b). Interestingly, similar ACTH and MS-USV number of calls correlation was detected after V1bR antagonist treatment (r = 0.424; *p* = 0.044; Figure 6c).

case, the serum ACTH level also showed positive correlation with the number of emitted MS-USVs (r = 0.491; *p* = 0.038; Figure 6b). Interestingly, similar ACTH and MS-USV number of calls correlation was detected after V1bR antagonist treatment (r = 0.424; *p* = 0.044;

**Figure 6.** The most important correlations. In Brattleboro pups (both di/+ and di/di genotypes), (**a**) the hypophysis vasopressin (AVP) content positively correlated with the emitted number of ultrasonic vocalizations. A similar positive correlation was observable between the serum ACTH levels and the ultrasonic vocalization number both in (**b**) the Brattleboro pups and (**c**) after a vasopressin (V) 1bR antagonist treatment. **Figure 6.** The most important correlations. In Brattleboro pups (both di/+ and di/di genotypes), (**a**) the hypophysis vasopressin (AVP) content positively correlated with the emitted number of ultrasonic vocalizations. A similar positive correlation was observable between the serum ACTH levels and the ultrasonic vocalization number both in (**b**) the Brattleboro pups and (**c**) after a vasopressin (V) 1bR antagonist treatment.

#### **4. Discussion 4. Discussion**

Our results confirmed that genetic AVP deficiency already had an anxiolytic effect during the early postnatal age, which was not influenced by the mild stress of an ip saline injection. The positive correlation between the pituitary AVP content and MS-USV further confirmed the participation of this neuropeptide in the separation-induced vocalization. Pharmacological analysis showed that a high dose (30 mg/kg) of the V1aR antagonist and all studied doses of the V1bR antagonist reduced MS-USV, while the V2R antagonist elevated it in the smallest studied dose (3 mg/kg). The number of MS-USVs correlated positively with the ACTH levels in the case of the V1bR antagonist only, similar to the Brattleboro rats. None of the studied interventions influenced the latency of the righting reflex Our results confirmed that genetic AVP deficiency already had an anxiolytic effect during the early postnatal age, which was not influenced by the mild stress of an ip saline injection. The positive correlation between the pituitary AVP content and MS-USV further confirmed the participation of this neuropeptide in the separation-induced vocalization. Pharmacological analysis showed that a high dose (30 mg/kg) of the V1aR antagonist and all studied doses of the V1bR antagonist reduced MS-USV, while the V2R antagonist elevated it in the smallest studied dose (3 mg/kg). The number of MS-USVs correlated positively with the ACTH levels in the case of the V1bR antagonist only, similar to the Brattleboro rats. None of the studied interventions influenced the latency of the righting reflex negative geotaxis, suggesting that they were without any sedative side effects.

negative geotaxis, suggesting that they were without any sedative side effects. As anxiety is a stress-related disorder, drugs influencing the HPA axis were the focus of interest for its treatment. Indeed, the first selective and orally active nonpeptide antagonist of V1bRs (SSR149415) in adult rodent models had anxiolytic and antidepressant-like effects [25]. We also found a strong anxiolytic effect of the V1bR antagonist in rat pups with all the studied doses (3, 10 or 30 mg/kg), which confirmed our previous results with 10 [6] and 30 mg/kg [5]. In a previous experiment, the same V1bR antagonist in the same doses showed only a tendency to reduce MS-USV [26]. However, the authors used 9–11 day-old animals (for age-dependent MS-USV, see Figure 1 in [6]) and 5 min of measurement, which might be responsible for the reduced sensitivity of their assay. Similarly, the tendency seen with another V1bR-antagonist, TASP0233278 [27], might be also attributed to their less sensitive assay, as they also used 5 min of measurement and older animals (21–30 g in contrast to our 16–20 g animals). In another study on mice, the shorter record-As anxiety is a stress-related disorder, drugs influencing the HPA axis were the focus of interest for its treatment. Indeed, the first selective and orally active nonpeptide antagonist of V1bRs (SSR149415) in adult rodent models had anxiolytic and antidepressantlike effects [25]. We also found a strong anxiolytic effect of the V1bR antagonist in rat pups with all the studied doses (3, 10 or 30 mg/kg), which confirmed our previous results with 10 [6] and 30 mg/kg [5]. In a previous experiment, the same V1bR antagonist in the same doses showed only a tendency to reduce MS-USV [26]. However, the authors used 9–11-day-old animals (for age-dependent MS-USV, see Figure 1 in [6]) and 5 min of measurement, which might be responsible for the reduced sensitivity of their assay. Similarly, the tendency seen with another V1bR-antagonist, TASP0233278 [27], might be also attributed to their less sensitive assay, as they also used 5 min of measurement and older animals (21–30 g in contrast to our 16–20 g animals). In another study on mice, the shorter recording time (5 min) might also have been responsible for the possible small difference between wild type (WT) and V1bR knockout (KO) animals [28]. However, in

these V1bR KO animals, the repeated MS was not able to induce any increase in the number of MS-USV, in contrast to their WT littermates, supporting some anxiolytic role of this VR subtype.

In our hands, the positive correlation between MS-USV and stress hormones, in the case of V1bR antagonist treatment, confirmed that this VR subtype is able to influence anxiety through the regulation of stress hormones. Indeed, although SSR149415 can penetrate into the brain, its half-life is relatively short (about 60 min) [24]. Thus, it is more likely that it acts on the pituitary V1b receptors influencing the HPA axis. Interestingly, it was not the end hormone of the axis (corticosterone in rodents), but the pituitary component where ACTH was implicated in this phenomenon. This was in line with the results found in the Brattleboro rats (see Figure 6b and [5,6]), where ACTH levels did not go parallel with the corticosterone levels. Although it is hard to separate the effect of ACTH from the effect of its downstream molecules (e.g., glucocorticoids, mineralocorticoids or adrenal androgens produced in the adrenal cortex), based upon ACTH administration, many extra-adrenal effects of ACTH have been suggested (e.g., cardiovascular, metabolic, motivational or memory influencing [29]). Moreover, chronic ACTH administration in rats induced depression-like behavioral changes [30]. ACTH-producing tumors were also associated with mood swings [31]; however, in this case, the role of other factors could not be entirely excluded. Additionally, other pathways may also contribute to the V1bRinduced anxiolysis, as previous studies showed altered V1bR protein levels in the rat hypothalamus in connection with anxiolytic treatment [32].

On the other hand, several data speak in favor of the role of V1aRs in anxiety [33]. In adult rodents, both the genetic (KO mice) and pharmacological (VR antagonism) blockade of the V1aRs showed anxiolytic effects [34]. Furthermore, in adult rats, overexpression of the V1aR gene in the lateral septum increased anxiety-related behavior [35]. Our data confirmed the anxiolytic effect of V1aR antagonism in pups, but only in the highest used dose (30 mg/kg). A 10 min observation period was sufficient to reveal the anxiolytic role of another V1aR antagonist (JNJ-17308616) in 11-day-old Sprague Dawley rat pups, too [36]. Interestingly, in this study, anxiolysis was detected only in the highest (100 mg/kg) dose, when JNJ-17308616 may influence the V2Rs as well. At the periphery, V1aRs might induce vasodilatation, confirmed also by lower blood pressure in the V1aR KO mice [37]. The drop in blood pressure might be stressful; thus, it was not surprising that the highest dose of the V1aR antagonist (30 mg/kg) stimulated the HPA axis. However, this stress could hardly explain the anxiolysis. Thus, we can conclude that, in our hands, V1aR antagonists should have a central effect. Although SR49059 cannot cross the blood–brain barrier in adults [38], the increased permeability of the blood–brain barrier [39] in pups can make its anxiolytic effect possible.

The decrease in the MS-USV number and duration after combined V1aR and V1bR antagonism was higher than after any antagonist treatment alone (for exact numbers, see Section 3.2.3), without any correlation with the stress hormones. Thus, it seems that during the early postnatal period, both V1bRs and V1aRs are involved in the development of anxiety, most probably through a central brain target other than the HPA axis.

Based upon a previous finding that a V1R antagonist was able antagonize AVP administration-induced MS-USV in 8–9-day-old Sprague Dawley rat pups [40], we did not truly expect an anxiolytic effect from the V2R antagonist. In our hands, even 3 mg/kg of the V2R antagonist was anxiogenic, while 30 mg/kg was highly stressful. As V2Rs are important in saltwater homeostasis, their antagonism may induce an imbalance, leading to the appearance of anxiety and high levels of stress hormones. Once again, the HPA axis parameters were clearly separated from the MS-USV behavioral measure.

The relevance of our study is supported by the presence of AVP already in rat embryos (first appearance of its binding at embryonic day 16) [40–42]. At birth, the hypothalamic level of AVP is comparable to adult levels, with the same ligand selectivity and affinity. In the brains of rat pups, VR1 [42] as well as V2R subtypes [43] were found, while V2Rs were expressed at the periphery [44].

We observed interesting strain differences. First, the length of an MS-USV bout (duration/number of calls) was substantially longer in the Brattleboro (87.335 ± 5.685 ms/bout) than in the Wistar rat strain (18.995 ± 2.142 ms/bout), despite similar MS-USV frequencies (no differences were found in this parameter between the treatment groups). The Brattleboro pups (17.768 ± 0.327 g) were smaller than the age-matched Wistar rats (19.718 ± 0.242 g), suggesting possible developmental differences. However, our previous study showed that the number of MS-USV calls was higher, not lower, in a heavier pup [5]. Further, in the controls, the corticosterone values were higher in the Brattleboro than in the Wistar pups. This is consistent with our previous results [6,45]. Moreover, an earlier study found that Long Evans animals (the origin of the Brattleboro strain) were more stress reactive than the Wistar strain [46]. This different stress sensitivity of the two strains can—at least partly—explain the observed strain differences.

#### **5. Conclusions**

All in all, we confirmed the involvement of both V1bRs and V1aRs in the anxiolytic effect of AVP without the contribution of V2Rs. HPA axis changes can only partly contribute to the observed anxiolysis. Taking into consideration the possible side effects, a mixed V1aR/V1bR antagonist might be more beneficial than either antagonist alone.

**Author Contributions:** Conceptualization, D.Z.; methodology, S.Z., who also developed and validated the software for USV detection and analysis; formal analysis, B.T., A.F. and D.Z.; investigation, B.T., A.F., E.S. and D.Z.; resources, D.Z.; writing—original draft preparation, B.T. and D.Z.; writing review and editing, all co-authors; visualization, B.T. and D.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Research, Development and Innovation Office (NKFIH) of Hungary, grant no. K120311 and K131406 to D.Z. and PD-115730 and K-129215 to S.Z.s.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, in accordance with the European Communities Council Directive recommendations for the care and use of laboratory animals (2010/63/EU) and was approved by the Animal Welfare and Ethics Committee of Institute of Experimental Medicine (protocol code 22.1/3895/003/2009 and date of approval: 07/10/2009 and protocol code PEI/001/38-4/2013 and date of approval: 06/03/2013).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** We are thankful to Éva Dobozi for her help in the radioimmunoassay.

**Conflicts of Interest:** The authors declare no conflict of interest. The funding agency had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

