Lack of striatal-enriched protein tyrosine phosphatase affected the serotonin system, behavior, and brain morphology in mice methods
Aim. Evidence-backed execution summary for Lack of striatal-enriched protein tyrosine phosphatase affected the serotonin system, behavior, and brain morphology in mice methods from Lack of striatal-enriched protein tyrosine phosphatase affected the serotonin system, behavior, and brain morphology in mice.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Materials and methods
reagent used in the protocol.
- Use
- (A) Scheme of the CRISPR/Cas9 system applied to mouse Ptpn5 gene; (B) amino acid sequence of mouse STEP protein encoded by the Ptpn5 gene. Dark and light gray lettering indicates the exonal structure. Green highlighted sequence is the translation start of the STEP46 isoform and the antigen sequence for the anti-STEP...
CRISPR system design and preparation of microinjections components
reagent used in the protocol.
- Use
- Single guide RNAs (sgRNAs) were designed with the Benchling online tool ( https://benchling.com ) ( ) using the scoring method described previously ( ). Genotyping primers were created using the Primer Blast tool ( https://www.ncbi.nlm.nih.gov/tools/primer-blast ) ( ). The sgRNAs were synthetized with the HiScribe&#...
Generation of Ptpn5 knockout mice
reagent used in the protocol.
- Use
- In vitro fertilization was performed as described earlier ( ). Oocytes and spermatozoa were taken from C57BL/6 females (4-6 weeks old) and C57BL/6 males (3 months old), respectively. Oocytes were microinjected with a solution containing 100 ng/µL ssODN ( ), 25 ng/µL sgRNA, and an equimolar concentrat...
Experimental design
reagent used in the protocol.
- Use
- Experiment 4. Home cage activity was registered and the operant wall paradigm was carried out over 3 days. Two days later, the animals were euthanized by carbon dioxide asphyxiation followed by decapitation. The frontal cortex, hippocampus, striatum, and midbrain were rapidly dissected, frozen in liquid nitrogen, an...
5-HT receptor functional activity
reagent used in the protocol.
- Use
- The functional activity of the 5-HT 1A receptor was estimated by quantifying the hypothermic response obtained after acute administration of the 5-HT 1A agonist 8-OH-DPAT (1 mg/kg, i.p.) (, ). The body temperature was measured by means of a KJT thermocouple (Hanna Instruments, Singapore) with Cooper Constantan Rect...
Protein quantification with Western blot analysis
reagent used in the protocol.
- Use
- For the assessment of protein levels, the homogenate was prepared for Western blot analysis as described earlier ( ). The extracts (20 µg per lane) were resolved on a 10% sodium dodecyl sulfate (SDS) polyacrylamide gel and blotted onto a nitrocellulose membrane. The antibody used for target protein detection an...
Biomolecular techniques
reagent used in the protocol.
- Use
- Brain structures including the frontal cortex, hippocampus, striatum, and midbrain were homogenized in 300 µL of Tris-HCl buffer (50 mM, pH 7.6) at 4°C using a mechanical homogenizer (Z359971, Sigma-Aldrich, USA). Aliquots of the homogenate were used for 5-HT and 5 - HIAA content and TPH2 activity assays w...
Tryptophan hydroxylase 2 activity assay
reagent used in the protocol.
- Use
- TPH2 activity was assessed using the modular chromatographic analysis system described above and following a previously reported method ( ). The substrate of the reaction of L-tryptophan was present in the mixture in a concentration of 0.4 mM. Enzymatic activity was calculated as the amount of synthesized 5-hydroxyt...
Key enzymes of serotonergic system
Effects of the Ptpn5 gene knockout on the TPH2 protein level in the frontal cortex (FC), hippocampus (HC), striatum (ST), and midbrain (MB) of mice, presented as the percentage of the GAPDH protein level. * p < 0.05 compared to the wild type (seven to eight animals per group). Groups were compared with t -test for i...
- Use
- Effects of the Ptpn5 gene knockout on the TPH2 protein level in the frontal cortex (FC), hippocampus (HC), striatum (ST), and midbrain (MB) of mice, presented as the percentage of the GAPDH protein level. * p < 0.05 compared to the wild type (seven to eight animals per group). Groups were compared with t -test for i...
Methods
Utilizing the CRISPR/Cas9 system, we cleaved the PTP-domain-encoding sequence from the Ptpn5 gene of C57BL/6 mice. The resulting strain ( Ptpn5 KO) demonstrated STEP protein absence and ERK1/2 hyperphosphorylation (STEP substrate) in the brain. We performed behavioral phenotyping, structural magnetic resonance imagi...
- Use
- Utilizing the CRISPR/Cas9 system, we cleaved the PTP-domain-encoding sequence from the Ptpn5 gene of C57BL/6 mice. The resulting strain ( Ptpn5 KO) demonstrated STEP protein absence and ERK1/2 hyperphosphorylation (STEP substrate) in the brain. We performed behavioral phenotyping, structural magnetic resonance imagi...
Social interaction test
The test followed the "resident-intruder" paradigm. A juvenile Balb/c male (4 weeks old) was introduced to the home cage of the tested male. During 10 min, social interactions were registered with the EthoStudio software. Social behavior was evaluated as the total duration of social contacts (intru...
- Use
- The test followed the "resident-intruder" paradigm. A juvenile Balb/c male (4 weeks old) was introduced to the home cage of the tested male. During 10 min, social interactions were registered with the EthoStudio software. Social behavior was evaluated as the total duration of social contacts (intru...
Materials and methods
STEP is a protein tyrosine phosphatase; its catalytic domain (PTP-sequence) is located at amino acids positions 470-480 and is encoded in exons 12 and 13. To generate a mouse strain with a functional STEP knockout, we removed the PTP sequence from the Ptpn5 gene using the CRISPR/Cas9 system and applied the pre...
- Use
- STEP is a protein tyrosine phosphatase; its catalytic domain (PTP-sequence) is located at amino acids positions 470-480 and is encoded in exons 12 and 13. To generate a mouse strain with a functional STEP knockout, we removed the PTP sequence from the Ptpn5 gene using the CRISPR/Cas9 system and applied the pre...
Materials and methods
(A) Scheme of the CRISPR/Cas9 system applied to mouse Ptpn5 gene; (B) amino acid sequence of mouse STEP protein encoded by the Ptpn5 gene. Dark and light gray lettering indicates the exonal structure. Green highlighted sequence is the translation start of the STEP46 isoform and the antigen sequence for the anti-STEP...
- Use
- (A) Scheme of the CRISPR/Cas9 system applied to mouse Ptpn5 gene; (B) amino acid sequence of mouse STEP protein encoded by the Ptpn5 gene. Dark and light gray lettering indicates the exonal structure. Green highlighted sequence is the translation start of the STEP46 isoform and the antigen sequence for the anti-STEP...
CRISPR system design and preparation of microinjections components
Single guide RNAs (sgRNAs) were designed with the Benchling online tool ( https://benchling.com ) ( ) using the scoring method described previously ( ). Genotyping primers were created using the Primer Blast tool ( https://www.ncbi.nlm.nih.gov/tools/primer-blast ) ( ). The sgRNAs were synthetized with the HiScribe&#...
- Use
- Single guide RNAs (sgRNAs) were designed with the Benchling online tool ( https://benchling.com ) ( ) using the scoring method described previously ( ). Genotyping primers were created using the Primer Blast tool ( https://www.ncbi.nlm.nih.gov/tools/primer-blast ) ( ). The sgRNAs were synthetized with the HiScribe&#...
CRISPR system design and preparation of microinjections components
Sequences utilized in the CRISPR system and genotyping primers.
- Use
- Sequences utilized in the CRISPR system and genotyping primers.
Experimental design
Experiment 1. The mice were tested in the marble burying test (MBT) and, on the next day, in the novel object recognition (NOR) test for three subsequent days. Then, the behavior was analyzed for the three subsequent days in the social-interaction test, elevated plus-maze (EPM) test, and three-chambered social appro...
- Use
- Experiment 1. The mice were tested in the marble burying test (MBT) and, on the next day, in the novel object recognition (NOR) test for three subsequent days. Then, the behavior was analyzed for the three subsequent days in the social-interaction test, elevated plus-maze (EPM) test, and three-chambered social appro...
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Social interaction test
The test followed the "resident-intruder" paradigm. A juvenile Balb/c male (4 weeks old) was introduced to the home cage of the tested male. During 10 min, social interactions were registered with the EthoStudio software. Social behavior was evaluated as the total duration of social contacts (intruder's head and body sniffing).
Generation of Ptpn5 knockout mice
In vitro fertilization was performed as described earlier ( ). Oocytes and spermatozoa were taken from C57BL/6 females (4-6 weeks old) and C57BL/6 males (3 months old), respectively. Oocytes were microinjected with a solution containing 100 ng/µL ssODN ( ), 25 ng/µL sgRNA, and an equimolar concentration of Alt-R HiFi Cas9 Nuclease V3 (IDT, Coralville, IA, USA) into the cytoplasm. After the microinjection, embryos were cultivated overnight, and 2-cell stage embryos were transferred into the oviducts of pseudopregnant mothers (females of the CD-1 strain) ( ).
Experimental design
Experiment 2. The behavior of mice was evaluated in the open field (OF) test, in the forced swim test (FST) on the next day, and in the tail suspension test (TST) after 1 day of rest.
Experimental design
Experiment 3. The mice's performance was assessed in the OF, in the Morris water maze (MWM) on the next 5 days, and in the rotarod test after 2 days of rest. After 1 day of rest, the functional activity of the 5-HT 1A receptor was measured; the next day, the functional activity of the 5-HT 2A receptor was studied; and after 2 days of rest, the functional activity of the 5-HT 7 receptor was evaluated.
Elevated plus-maze test
The test was carried out in the apparatus made of gray plastic consisting of four arms connected perpendicularly (closed and open, 30 × 6 cm each) with a central area (6 × 6 cm) ( ). The closed arms were bordered with 20-cm-high walls. The device was elevated by 60 cm above the floor and dimly illuminated with diffuse lighting (100 lx) of a halogen lamp (25 W) placed under the device. The animal's movements were automatically traced with a 3D sensor Kinect 1 connected to the PC through a USB-2 port. During the 5 min of testing, the sensor automatically detected the animal's movement in the open as well as the closed arms. The total path (m), time (%) spent in the center, and open and closed arms were automatically computed by the EthoStudio software. Stretch poses and head dips from the open arms were counted by an experienced rater. The arena was cleaned with we...
Experimental design
Experiment 4. Home cage activity was registered and the operant wall paradigm was carried out over 3 days. Two days later, the animals were euthanized by carbon dioxide asphyxiation followed by decapitation. The frontal cortex, hippocampus, striatum, and midbrain were rapidly dissected, frozen in liquid nitrogen, and stored at -80°C for further assay of monoamine content, TPH2 activity, proteins, and gene expression. The selected brain regions exhibit the most abundant expression of Ptpn5 RNA ( ) and are involved in the 5-HT system regulated processes: mood regulation, decision-making, and depressive and anxiety-related behavior; midbrain is home for 5-HT nuclei.
Behavioral testing
The OF test was carried out to assess the locomotor and exploratory activity. The test took place at the circular arena, 60 cm in diameter ( ), as described earlier ( ). Movement of the mouse was automatically traced for 5 min with a digital camera. The total distance traveled (m) and time (%) spent in the center of the arena were automatically evaluated by the EthoStudio software ( ). The number of vertical postures (rearing) and the number of grooming episodes were marked by experienced rater blinded to the experimental group assignment. After each test, the arena was cleaned with wet (H 2 O 2 ) and dry napkins.
Forced swim test
In the FST and TST, mice were tested for depressive-like behavior. The FST was performed in a clear, cylindrical glass reservoir ( h = 30 cm, d = 15 cm) half filled with water ( T = 25°С) and illuminated from beneath. A mouse was carefully placed into the water for 6 min. The first 2 min of the test are adaptive and were not analyzed. For the latter 4 min, the total immobility time was evaluated by an experienced rater, and depressive-like behavior was determined in correspondence to this parameter.
Measurement outputs
What raw and processed outputs should exist?
Ptpn5 gene knockout did not affect the serotonin transporter expression in the frontal cortex, hippocampus, midbrain, or striatum ( ).
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
The test followed the "resident-intruder" paradigm. A juvenile Balb/c male (4 weeks old) was introduced to the home cage of the tested male. During 10 min, soc...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
(A) Scheme of the CRISPR/Cas9 system applied to mouse Ptpn5 gene; (B) amino acid sequence of mouse STEP protein encoded by the Ptpn5 gene. Dark and light gray lettering indicate...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Experiment 1. The mice were tested in the marble burying test (MBT) and, on the next day, in the novel object recognition (NOR) test for three subsequent days. Then, the behavio...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
Experiments were carried out on adult 2-month-old male mice of the C57BL/6-Ptpn5_KO ( Ptpn5 knockout mice) and C57BL/6 (wild type) inbred strains, 24 ± 1 g of weight.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Ptpn5 gene knockout did not affect the serotonin transporter expression in the frontal cortex, hippocampus, midbrain, or striatum ( ).; The test followed the "resident-intruder" paradigm. A juvenile Balb/c male (4 weeks old) was introduced to the home cage of the tested male. During 10 min, soc...; (A) Scheme of the CRISPR/Cas9 system applied to mouse Ptpn5 gene; (B) amino acid sequence of mouse STEP protein encoded by the Ptpn5 gene. Dark and light gray lettering indicate...; Experiment 1. The mice were tested in the marble burying test (MBT) and, on the next day, in the novel object recognition (NOR) test for three subsequent days. Then, the behavio....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Experiments were carried out on adult 2-month-old male mice of the C57BL/6-Ptpn5_KO ( Ptpn5 knockout mice) and C57BL/6 (wild type) inbred strains, 24 ± 1 g of weight. In th...; On day 5, the retention test took place. The platform was removed and a mouse was placed at the center of the pool. Results were averaged over the three attempts. A statisticall...; Home cage behavior monitored parameters: locomotor activity, sleep duration, and food and water consumption were analyzed with repeated-measures ANOVA with factors "Genoty...; Repeated-measures ANOVA with the Fisher post-hoc analysis was likewise used to analyze the behavior of mice in the learning phase of the MWM test with factors "Genotype...
from paperReporting output
Report representative outputs alongside summary comparisons for Ptpn5 gene knockout did not affect the serotonin transporter expression in the frontal cortex, hippocampus, midbrain, or striatum ( )., The test followed the "resident-intruder" paradigm. A juvenile Balb/c male (4 weeks old) was introduced to the home cage of the tested male. During 10 min, soc..., (A) Scheme of the CRISPR/Cas9 system applied to mouse Ptpn5 gene; (B) amino acid sequence of mouse STEP protein encoded by the Ptpn5 gene. Dark and light gray lettering indicate..., Experiment 1. The mice were tested in the marble burying test (MBT) and, on the next day, in the novel object recognition (NOR) test for three subsequent days. Then, the behavio....
inferred from protocolStructured statistical methods
Experiments were carried out on adult 2-month-old male mice of the C57BL/6-Ptpn5_KO ( Ptpn5 knockout mice) and C57BL/6 (wild type) inbred strains, 24 ± 1 g of weight. In th...; On day 5, the retention test took place. The platform was removed and a mouse was placed at the center of the pool. Results were averaged over the three attempts. A statisticall...; Home cage behavior monitored parameters: locomotor activity, sleep duration, and food and water consumption were analyzed with repeated-measures ANOVA with factors "Genoty...; Repeated-measures ANOVA with the Fisher post-hoc analysis was likewise used to analyze the behavior of mice in the learning phase of the MWM test with factors "Genotype...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
The test followed the "resident-intruder" paradigm. A juvenile Balb/c male (4 weeks old) was introduced to the home cage of the tested male. During 10 min, social interactions were registered with the EthoStudio software. Social behavior was evaluated as the total duration of social contacts (intruder's head and body sniffing).
In vitro fertilization was performed as described earlier ( ). Oocytes and spermatozoa were taken from C57BL/6 females (4-6 weeks old) and C57BL/6 males (3 months old), respectively. Oocytes were microinjected with a solution containing 100 ng/µL ssODN ( ), 25 ng/µL sgRNA, and an equimolar concentration of Alt-R HiFi Cas9 Nuclease V3 (IDT, Coralville, IA, USA) into the cytoplasm. After the microinjection, embryos were cultivated overnight, and 2-cell stage embryos were transferred into the oviducts of pseudopregnant mothers (females of the CD-1 strain) ( ).
Experiment 2. The behavior of mice was evaluated in the open field (OF) test, in the forced swim test (FST) on the next day, and in the tail suspension test (TST) after 1 day of rest.
Experiment 3. The mice's performance was assessed in the OF, in the Morris water maze (MWM) on the next 5 days, and in the rotarod test after 2 days of rest. After 1 day of rest, the functional activity of the 5-HT 1A receptor was measured; the next day, the functional activity of the 5-HT 2A receptor was studied; and after 2 days of rest, the functional activity of the 5-HT 7 receptor was evaluated.
The test was carried out in the apparatus made of gray plastic consisting of four arms connected perpendicularly (closed and open, 30 × 6 cm each) with a central area (6 × 6 cm) ( ). The closed arms were bordered with 20-cm-high walls. The device was elevated by 60 cm above the floor and dimly illuminated with diffuse lighting (100 lx) of a halogen lamp (25 W) placed under the device. The animal's movements were automatically traced with a 3D sensor Kinect 1 connected to the PC through a USB-2 port. During the 5 min of testing, the sensor automatically detected the animal's movement in the open as well as the closed arms. The total path (m), time (%) spent in the center, and open and closed arms were automatically computed by the EthoStudio software. Stretch poses and head dips from the open arms were counted by an experienced rater. The arena was cleaned with wet (H 2 O 2 ) and dry napkins after each test.
Experiment 4. Home cage activity was registered and the operant wall paradigm was carried out over 3 days. Two days later, the animals were euthanized by carbon dioxide asphyxiation followed by decapitation. The frontal cortex, hippocampus, striatum, and midbrain were rapidly dissected, frozen in liquid nitrogen, and stored at -80°C for further assay of monoamine content, TPH2 activity, proteins, and gene expression. The selected brain regions exhibit the most abundant expression of Ptpn5 RNA ( ) and are involved in the 5-HT system regulated processes: mood regulation, decision-making, and depressive and anxiety-related behavior; midbrain is home for 5-HT nuclei.
The OF test was carried out to assess the locomotor and exploratory activity. The test took place at the circular arena, 60 cm in diameter ( ), as described earlier ( ). Movement of the mouse was automatically traced for 5 min with a digital camera. The total distance traveled (m) and time (%) spent in the center of the arena were automatically evaluated by the EthoStudio software ( ). The number of vertical postures (rearing) and the number of grooming episodes were marked by experienced rater blinded to the experimental group assignment. After each test, the arena was cleaned with wet (H 2 O 2 ) and dry napkins.
In the FST and TST, mice were tested for depressive-like behavior. The FST was performed in a clear, cylindrical glass reservoir ( h = 30 cm, d = 15 cm) half filled with water ( T = 25°С) and illuminated from beneath. A mouse was carefully placed into the water for 6 min. The first 2 min of the test are adaptive and were not analyzed. For the latter 4 min, the total immobility time was evaluated by an experienced rater, and depressive-like behavior was determined in correspondence to this parameter.
Machine-readable layer
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"text": "Experiment 3. The mice's performance was assessed in the OF, in the Morris water maze (MWM) on the next 5 days, and in the rotarod test after 2 days of rest. After 1 day of rest, the functional activity of the 5-HT 1A receptor was measured; the next day, the functional activity of the 5-HT 2A receptor was studied; and after 2 days of rest, the functional activity of the 5-HT 7 receptor was evaluated."
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"datePublished": "2026",
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"identifier": "10.3389/fpsyt.2025.1730197"
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"item": "https://replicatescience.com/experiments/lack-of-striatal-enriched-protein-tyrosine-phosphatase-affected-the-serotonin-system-behavior-and-brain-morphology-in-mice-methods-vitalii-moskaliuk-pmc12848919/lack-of-striatal-enriched-protein-tyrosine-phosphatase-affected-the-serotonin-sy"
}
]
}
]