Systemic Interplay of BDNF and Serotonin Pathways Defines Behavioral and Molecular Responses to Midbrain 5-HT7 Overexpression and Chronic Ethanol Consumption methods
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2. Materials and Methods
Male C57BL/6 mice (8-10 weeks; 25 ± 1 g, n = 65) were obtained from the SPF (Specific Pathogen-Free) vivarium of the Federal Research Center, Institute of Cytology and Genetics SB RAS (Novosibirsk, Russia) (supported by the basic research projects No. FWNR-2026-0028 and RFMEFI62117X0015). After weaning, mice were housed in groups of five per cage under standard conditions (14:10 h light/dark, 20-22 °C, humidity 45-50%, food/water ad libitum). All surgeries were performed under appropriate anesthesia, and every effort was made to minimize animal suffering.
2.3. Stereotaxic Microinjections
Mice were anesthetized by intraperitoneally (i.p.) administered a solution (1 mL/kg) of 2,2,2-Tribromoethanol (T48402-25G, Sigma-Aldrich, Darmstadt, Germany) in 2-Methyl-2-butanol (240486, Sigma-Aldrich, Darmstadt, Germany). The AAV vectors (carrying pAAV-Syn-HTR7-EGFP or pAAV-Syn-EGFP plasmids) were unilaterally injected into the raphe nuclei area using following coordinates: AP-3; L-2; DV-4; Angle = 38°; Rotation = 40°, according to the mouse brain atlas [ ] and our previous study [ ]. 1 uL of the viral vector (1 × 10 9 viral particles/µL) were microinjected into site at the rate of 0.2 µL/min using a Hamilton Syringe. The syringe was left in place for 5 min and then raised slowly. Histological validation of injection targeting was performed in a separate validation animal as described in our previous article [ ]; representative EGFP expression/spread and th...
2.4. Experimental Design
The study used a 2 × 2 factorial design: AAV vector (EGFP control vs. 5-HT7-EGFP) × drinking regimen (water vs. 10% ethanol). On day 0, mice received stereotaxic injections of the genetic construct; from day 1 they had single-bottle access to 10% ( v / v ) ethanol or water for 5 weeks. C57BL/6 mice are known to exhibit a strong preference for ethanol over water when given a choice between two bottles across a broad range of ethanol concentrations [, ]. Chronic alcohol exposure continued until the animals were decapitated, followed by dissection of brain regions on ice, including the prefrontal cortex, hippocampus, hypothalamus, and the midbrain raphe nuclei area.
2.5. Behavioral Tests
The open field test was conducted using a circular arena (40 cm in diameter) surrounded by a white plastic wall (25 cm in height) and illuminated from below through a matt semi-transparent floor by two 12 W halogen lamps positioned 40 cm beneath the arena floor. Each mouse was placed near the wall, and its behavior was recorded for 5 min using a digital video camera (Sony, Japan) located 80 cm from the arena. The arena was cleaned after each test session. Video footage was analyzed frame by frame using custom EthoStudio software 2.0. Horizontal locomotor activity (path length) was automatically measured.
2.5.2. Novel Object Recognition Test (NOR)
This test relies on differential exploration time of novel versus familiar objects. It was conducted over three days, following previously described procedures [, ]. The same circular arena as in the open field test was used. On the first day, each mouse was habituated to the empty arena for 10 min. On the second day, two identical, previously unfamiliar objects were placed symmetrically relative to the center of the arena, and the mouse was allowed to explore them for 10 min. On the third day, one of the familiar objects was replaced with a novel object differing in shape, material, or color, and the exploration time of both objects was recorded for 10 min. Object exploration was defined as the mouse approaching, sniffing, or making physical contact with the object using its snout or forepaws. Recognition memory was assessed by calculating the ratio of time spent exploring the novel...
2.5.3. Dark-Light Box Test (DLB)
This test was conducted using a plastic chamber (20 × 40 × 27 cm), painted white and divided into two compartments-light and dark-by an opaque partition with a 7 × 7 cm opening. The light compartment had a translucent plastic floor illuminated from below by two 12 W halogen lamps. Mice were placed in the light compartment, facing the opening. Over a 5 min session, the following parameters were recorded: the duration of time spent in the light compartment, the proportion of the light area explored, and the number of entries into the light compartment from the dark.
2.5.4. Forced Swim Test (FST)
Each mouse was placed in a transparent glass cylinder (30 cm in diameter and height) filled with water at 25 °C. After a 2 min habituation period, the animal's mobility was recorded automatically for 4 min using custom EthoStudio software. The program measured the rate of change in the animal's silhouette, defined as the number of pixels associated with the animal that changed between consecutive frames [ ].
3.2. Expression of 5-HT Brain System Key Elements
As expected, AAV injection produced an approximately 100-fold increase in Htr7 mRNA in the midbrain compared with AAV-Syn-EGFP controls (F 1,29 = 124.50, p < 0.0001), but did not affect Htr1a, Htr2a, Tph2, or Slc6a4 mRNA levels in this region (all p > 0.05). No detectable changes in Htr7 mRNA were observed in the frontal cortex, hippocampus, or hypothalamus (all p > 0.05). At the protein level, Western blotting with commercial anti-5-HT 7 antibodies yielded a 5-HT 7 -immunoreactive signal that showed an ethanol-related increase in the midbrain (F 1,20 = 4.66, p = 0.043) and frontal cortex (F 1,20 = 5.17, p = 0.034); however, given the lack of validated 5-HT 7 antibody specificity (Ref. [ ]) this signal cannot be interpreted as a reliable measure of 5-HT 7 receptor abundance and may reflect cross-reactivity. Consistently, viral 5-HT 7 overexpression did not produce a detectabl...
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Evidence quotes (8)
Male C57BL/6 mice (8-10 weeks; 25 ± 1 g, n = 65) were obtained from the SPF (Specific Pathogen-Free) vivarium of the Federal Research Center, Institute of Cytology and Genetics SB RAS (Novosibirsk, Russia) (supported by the basic research projects No. FWNR-2026-0028 and RFMEFI62117X0015). After weaning, mice were housed in groups of five per cage under standard conditions (14:10 h light/dark, 20-22 °C, humidity 45-50%, food/water ad libitum). All surgeries were performed under appropriate anesthesia, and every effort was made to minimize animal suffering.
Mice were anesthetized by intraperitoneally (i.p.) administered a solution (1 mL/kg) of 2,2,2-Tribromoethanol (T48402-25G, Sigma-Aldrich, Darmstadt, Germany) in 2-Methyl-2-butanol (240486, Sigma-Aldrich, Darmstadt, Germany). The AAV vectors (carrying pAAV-Syn-HTR7-EGFP or pAAV-Syn-EGFP plasmids) were unilaterally injected into the raphe nuclei area using following coordinates: AP-3; L-2; DV-4; Angle = 38°; Rotation = 40°, according to the mouse brain atlas [ ] and our previous study [ ]. 1 uL of the viral vector (1 × 10 9 viral particles/µL) were microinjected into site at the rate of 0.2 µL/min using a Hamilton Syringe. The syringe was left in place for 5 min and then raised slowly. Histological validation of injection targeting was performed in a separate validation animal as described in our previous article [ ]; representative EGFP expression/spread and the targeting schematic are provided in. Brains from the main experimental cohort were used for protein/RNA analyses, precluding histological verification in all animals.
The study used a 2 × 2 factorial design: AAV vector (EGFP control vs. 5-HT7-EGFP) × drinking regimen (water vs. 10% ethanol). On day 0, mice received stereotaxic injections of the genetic construct; from day 1 they had single-bottle access to 10% ( v / v ) ethanol or water for 5 weeks. C57BL/6 mice are known to exhibit a strong preference for ethanol over water when given a choice between two bottles across a broad range of ethanol concentrations [, ]. Chronic alcohol exposure continued until the animals were decapitated, followed by dissection of brain regions on ice, including the prefrontal cortex, hippocampus, hypothalamus, and the midbrain raphe nuclei area.
The open field test was conducted using a circular arena (40 cm in diameter) surrounded by a white plastic wall (25 cm in height) and illuminated from below through a matt semi-transparent floor by two 12 W halogen lamps positioned 40 cm beneath the arena floor. Each mouse was placed near the wall, and its behavior was recorded for 5 min using a digital video camera (Sony, Japan) located 80 cm from the arena. The arena was cleaned after each test session. Video footage was analyzed frame by frame using custom EthoStudio software 2.0. Horizontal locomotor activity (path length) was automatically measured.
This test relies on differential exploration time of novel versus familiar objects. It was conducted over three days, following previously described procedures [, ]. The same circular arena as in the open field test was used. On the first day, each mouse was habituated to the empty arena for 10 min. On the second day, two identical, previously unfamiliar objects were placed symmetrically relative to the center of the arena, and the mouse was allowed to explore them for 10 min. On the third day, one of the familiar objects was replaced with a novel object differing in shape, material, or color, and the exploration time of both objects was recorded for 10 min. Object exploration was defined as the mouse approaching, sniffing, or making physical contact with the object using its snout or forepaws. Recognition memory was assessed by calculating the ratio of time spent exploring the novel object to the total time spent exploring both objects.
This test was conducted using a plastic chamber (20 × 40 × 27 cm), painted white and divided into two compartments-light and dark-by an opaque partition with a 7 × 7 cm opening. The light compartment had a translucent plastic floor illuminated from below by two 12 W halogen lamps. Mice were placed in the light compartment, facing the opening. Over a 5 min session, the following parameters were recorded: the duration of time spent in the light compartment, the proportion of the light area explored, and the number of entries into the light compartment from the dark.
Each mouse was placed in a transparent glass cylinder (30 cm in diameter and height) filled with water at 25 °C. After a 2 min habituation period, the animal's mobility was recorded automatically for 4 min using custom EthoStudio software. The program measured the rate of change in the animal's silhouette, defined as the number of pixels associated with the animal that changed between consecutive frames [ ].
As expected, AAV injection produced an approximately 100-fold increase in Htr7 mRNA in the midbrain compared with AAV-Syn-EGFP controls (F 1,29 = 124.50, p < 0.0001), but did not affect Htr1a, Htr2a, Tph2, or Slc6a4 mRNA levels in this region (all p > 0.05). No detectable changes in Htr7 mRNA were observed in the frontal cortex, hippocampus, or hypothalamus (all p > 0.05). At the protein level, Western blotting with commercial anti-5-HT 7 antibodies yielded a 5-HT 7 -immunoreactive signal that showed an ethanol-related increase in the midbrain (F 1,20 = 4.66, p = 0.043) and frontal cortex (F 1,20 = 5.17, p = 0.034); however, given the lack of validated 5-HT 7 antibody specificity (Ref. [ ]) this signal cannot be interpreted as a reliable measure of 5-HT 7 receptor abundance and may reflect cross-reactivity. Consistently, viral 5-HT 7 overexpression did not produce a detectable change in this anti-5-HT 7 immunoreactive signal in any analyzed region. No significant ethanol × overexpression interactions were detected for the anti-5-HT 7 signal or other serotonin-related proteins in the midbrain (all p > 0.05) ( c,d).
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