TrkB regulates hippocampal neurogenesis and governs sensitivity to antidepressive treatment methods
Aim. Evidence-backed execution summary for TrkB regulates hippocampal neurogenesis and governs sensitivity to antidepressive treatment methods from TrkB regulates hippocampal neurogenesis and governs sensitivity to antidepressive treatment.
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mouse
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Ablation of TrkB impairs proliferation and neurogenesis
reagent used in the protocol.
- Use
- Reduced neurogenesis in the absence of TrkB could be caused by increased cell death, decreased proliferation, or a combination of both processes. We first examined programmed cell death using TdT-mediated dUTP nick end labeling (TUNEL) assay and immunohistochemistry for activated caspase 3. At all time points examin...
Activation of TrkB in response to BDNF facilitates proliferation in vitro
reagent used in the protocol.
- Use
- Our finding that the TrkB hGFAP but not the TrkB Syn mice displayed impaired hippocampal neurogenesis suggested a cell autonomous requirement for TrkB in NPCs. To further examine their intrinsic properties, we cultured NPCs from the DG in serum-free conditions. In the presence of epidermal growth factor (EGF) and ba...
TrkB is required for induced proliferation and neurogenesis by antidepressants and voluntary exercise
reagent used in the protocol.
- Use
- Reduction in hippocampal volume has been observed in animal models of stress (; ), which may be reversed or prevented by chronic AD treatment ( ). Similarly, reduction of hippocampal volume has been reported in some studies of human patients with major depression (; ) and post-traumatic stress disorder (;; ). Al...
TrkB is required for behavioral improvement induced by ADs and exercise
reagent used in the protocol.
- Use
- Next we examined depression-like behavior in the control and TrkB hGFAP mice by using the tail-suspension test (TST), a paradigm of inescapable stress ( ). All mice were tested 48 hours after the last dose of AD or saline to exclude the acute effects on behaviors that do not correlate with prior duration of drug tre...
Histology and Quantitative Analysis
reagent used in the protocol.
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- Mice were intra-cardially perfused with PBS followed by 4% (w/v) paraformaldehyde (PFA) in PBS, and the dissected brains postfixed in 4% PFA at 4 degree. In situ hybridization, TUNEL assay, X-gal, Nissl, and immuno-staining were performed as previously described (; ). Detailed methods for these assays and the stere...
Protein Analysis
reagent used in the protocol.
- Use
- Total protein was extracted and measured for western blotting and BDNF ELISA assay. Total protein from the supernatant was used for western blotting as described ( ). BDNF level was measured using a BDNF ELISA kit (Promega) as per manufacturer's instructions.
Neurosphere cultures
reagent used in the protocol.
- Use
- Neurosphere culture was established and maintained as described ( ) with some modifications. Briefly, DG of adult mice were dissected ( ) and digested in 0.1% trypsin-EDTA (Sigma) followed by mechanical trituration until smooth. Cells were plated at a density of 20 cells/ul in complete growth medium, consisting of m...
FACS
reagent used in the protocol.
- Use
- For FACS isolation of GFP positive or negative cells, primary cells were prepared from the DG of Nestin-GFP transgenic mice at the age of postnatal day 2, 15 and 2 months following the procedure for establishing neurosphere culture. GFP positive and negative fractions of the live cells (PI negative) were analyzed an...
hGFAP-Cre activity in hippocampal progenitor cells
We next enriched NPCs from 2-month old mice by culturing primary cells from the DG in neurosphere forming media ( ). Upon X-gal staining, only neurospheres isolated from the DG of R26 hGFAP, but not those from the R26 Syn or control mice stained positive for β-gal activity, indicating that recombination only o...
- Use
- We next enriched NPCs from 2-month old mice by culturing primary cells from the DG in neurosphere forming media ( ). Upon X-gal staining, only neurospheres isolated from the DG of R26 hGFAP, but not those from the R26 Syn or control mice stained positive for β-gal activity, indicating that recombination only o...
TrkB is required for behavioral improvement induced by ADs and exercise
Despite the lack of responses to ADs and exercise in the behavioral paradigms of NSFT and TST, the saline-treated TrkB hGFAP mice performed similarly compared to the control mice, suggesting relatively normal depression and anxiety-like behaviors at the basal level. To further investigate this finding, we examined a...
- Use
- Despite the lack of responses to ADs and exercise in the behavioral paradigms of NSFT and TST, the saline-treated TrkB hGFAP mice performed similarly compared to the control mice, suggesting relatively normal depression and anxiety-like behaviors at the basal level. To further investigate this finding, we examined a...
Specific ablation of TrkB in adult NPCs is sufficient to block sensitivity to AD
As described above, hGFAP-Cre mediated ablation of TrkB resulted in a smaller DG that we attribute to the lack of TrkB signaling in early postnatal NPCs to sustain rapid proliferation required for normal structural development. To bypass this essential phase of DG postnatal morphogenesis, we subjected TrkB flox/flox...
- Use
- As described above, hGFAP-Cre mediated ablation of TrkB resulted in a smaller DG that we attribute to the lack of TrkB signaling in early postnatal NPCs to sustain rapid proliferation required for normal structural development. To bypass this essential phase of DG postnatal morphogenesis, we subjected TrkB flox/flox...
TrkB and the behavioral efficacy of antidepressants
There has been considerable evidence that genetic ablation of BDNF or TrkB may interfere with the normal function of the adult brain, depending on the regions affected. Conditional knockout animals lacking TrkB in forebrain neurons (with broader recombination than TrkB hGFAP and TrkB Syn ) have impaired spatial lear...
- Use
- There has been considerable evidence that genetic ablation of BDNF or TrkB may interfere with the normal function of the adult brain, depending on the regions affected. Conditional knockout animals lacking TrkB in forebrain neurons (with broader recombination than TrkB hGFAP and TrkB Syn ) have impaired spatial lear...
FACS
For FACS isolation of GFP positive or negative cells, primary cells were prepared from the DG of Nestin-GFP transgenic mice at the age of postnatal day 2, 15 and 2 months following the procedure for establishing neurosphere culture. GFP positive and negative fractions of the live cells (PI negative) were analyzed an...
- Use
- For FACS isolation of GFP positive or negative cells, primary cells were prepared from the DG of Nestin-GFP transgenic mice at the age of postnatal day 2, 15 and 2 months following the procedure for establishing neurosphere culture. GFP positive and negative fractions of the live cells (PI negative) were analyzed an...
Figures
(C) RT-PCR detection of TrkB and BDNF transcripts in FACS sorted Nestin-GFP positive cells and DG derived neurospheres. NS, neurosphere.
- Use
- (C) RT-PCR detection of TrkB and BDNF transcripts in FACS sorted Nestin-GFP positive cells and DG derived neurospheres. NS, neurosphere.
Figures
(E) RT-PCR detection of TrkB and G3PDH transcripts in FACS sorted Nestin-GFP positive cells from the DG of Control and TrkB hGFAP mice. Note the absence of TrkB in the TrkB hGFAP mice.
- Use
- (E) RT-PCR detection of TrkB and G3PDH transcripts in FACS sorted Nestin-GFP positive cells from the DG of Control and TrkB hGFAP mice. Note the absence of TrkB in the TrkB hGFAP mice.
Figures
(D) RT-PCR detection of TrkB and G3PDH transcripts in FACS sorted Nestin-GFP positive and negative cells from the DG of TrkB flox/flox; Nestin-CreER mice treated with tamoxifen (TrkB Nestin ) or vehicle (control) at 1 month old.
- Use
- (D) RT-PCR detection of TrkB and G3PDH transcripts in FACS sorted Nestin-GFP positive and negative cells from the DG of TrkB flox/flox; Nestin-CreER mice treated with tamoxifen (TrkB Nestin ) or vehicle (control) at 1 month old.
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Figures
(A) Representative images of Nissl stained DG sections from control, TrkB hGFAP and TrkB Syn mice at P15. The decreases in DG size and granular layer thickness were only observed in TrkB hGFAP mice. Red lines and circles highlight the length of the granular layer, and the size of single cell, respectively. Scale bars, 1mm (hippocampus) and 10um (DG).
Ablation of TrkB in early postnatal NPCs impairs DG morphogenesis
The conditional knockout animals, either carrying the hGFAP-Cre (TrkB hGFAP ) or the Syn-Cre (TrkB Syn ) are viable at birth and have normal survival rates (up to 15 months recorded) ( ). The TrkB hGFAP mice displayed a significant reduction in the volume of the DG granular layer ( ) that first became measurable at P10. The volume reduction stabilized at around 30% after the initial postnatal weeks and persisted throughout adulthood. This abnormality was not caused by changes in cell density or cell size (; p>0.2), but rather was a result of decrease in the number of granule neurons, evidenced by a significant reduction in the thickness of DG granular layer (; n=6 for each, F 2,15 =5.477, p=0.0164). The number and morphology of glial cells were not appreciably affected in the TrkB hGFAP mice ( and not shown). The reduction of volume was most prominently observed in the hippocampus a...
Ablation of TrkB impairs proliferation and neurogenesis
We next assessed proliferation by measuring the number of cells that incorporate the DNA synthesis marker 5-bromo-2'-deoxyuridine (BrdU). P15 mice were treated with pulses of BrdU and sacrificed 24 hours after the first injection. Within the area encompassing the inner granular layer and SGZ, the number of BrdU positive cells in the TrkB hGFAP mice displayed a 48% reduction compared to control mice (; n=7-9 for each; F 2,21 =78.39, p<0.0001; p<0.001 for post hoc test of control to TrkB hGFAP comparison). Similarly, the number of cells positive for Ki67, an endogenous marker for actively cycling cells, was decreased by 51% in the SGZ of the TrkB hGFAP mice (; n=7-9 for each; F 2,21 =51.64, p<0.0001; p<0.001 for post hoc test of control to TrkB hGFAP comparison). Again, the numbers of BrdU or Ki67 positive cells were unaffected in the DG of the TrkB Syn mice (; p>0....
Activation of TrkB in response to BDNF facilitates proliferation in vitro
Our finding that the TrkB hGFAP but not the TrkB Syn mice displayed impaired hippocampal neurogenesis suggested a cell autonomous requirement for TrkB in NPCs. To further examine their intrinsic properties, we cultured NPCs from the DG in serum-free conditions. In the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), DG NPCs formed neurospheres, which expressed TrkB receptor that could be activated by exogenous BDNF ( ). Although maintaining adult DG-derived primary neurospheres in media containing BDNF (50 ng/ml) for 7-10 days did not increase the frequency of primary neurosphere formation (not shown), there was a significant increase in the size of the neurospheres, suggesting that activation of TrkB facilitates the expansion of neurosphere-forming cells (; n=4 for each group, p<0.05 for post hoc test of with to without BDNF comparison in co...
TrkB is required for induced proliferation and neurogenesis by antidepressants and voluntary exercise
Reduction in hippocampal volume has been observed in animal models of stress (; ), which may be reversed or prevented by chronic AD treatment ( ). Similarly, reduction of hippocampal volume has been reported in some studies of human patients with major depression (; ) and post-traumatic stress disorder (;; ). Although the cellular mechanism is unclear in humans, animal studies have demonstrated that chronic exposure to various types of ADs induces DG proliferation and neurogenesis ( ), thereby potentially contributing to the recovery of volume loss. In this context, we examined whether chronic AD treatment can restore neurogenesis in TrkB hGFAP mice. We thus treated control, TrkB hGFAP and TrkB Syn mice with daily injections of the serotonin reuptake inhibitor fluoxetine (10 µg/g) or the tricyclic imipramine (20 µg/g) for 21 days (n=7-29 for each group, ). As previ...
TrkB is required for induced proliferation and neurogenesis by antidepressants and voluntary exercise
In rodents, voluntary exercise, such as wheel-running behavior has been demonstrated to robustly induce neurogenesis, much in the same fashion as AD treatment ( ). Less is known, however, about the underlying mechanism of this AD-like effect of exercise. To determine whether TrkB is also required for this process, we subjected control and TrkB hGFAP mice to 6 weeks of wheel-running (n=8-16 for each group, ). Both the numbers of Ki67 positive and Doublecortin positive cells increased in control runners, compared to control sedentary animals (: F 1,36 =13.64, p=0.0007 for exercise;: F 1,36 =16.01, p=0.0003; p<0.001 for post hoc test of both comparisons). A significant increase of BDNF protein level was also observed in the hippocampus of runners (66.44 ± 3.65 pg/mg in runners; 45.83 ± 2.99 pg/mg in sedentary controls; n=3 for each; p<0.05). The TrkB hGFAP mice, despite...
TrkB is required for behavioral improvement induced by ADs and exercise
To determine whether the lack of neurogenic response in the TrkB hGFAP mice was coupled with general insensitivity to chronic ADs and exercise, we compared the depression and anxiety-like behaviors in control and TrkB hGFAP mice. First, mice treated with fluoxetine, imipramine, or saline for 21 days were examined in the novelty-suppressed feeding test (NSFT), a conflict paradigm in which the latency to feed in a novel environment is used as an indicator of anxiety level ( ). In agreement with the general capacity of chronic ADs to ease anxiety, 24 hours after the last dose, control mice receiving fluoxetine or imipramine displayed significantly shorter latency to feed compared to saline-treated control mice (; F 2,100 =8.022, p=0.0006 for treatment; p<0.001 for post hoc test both AD-to-saline comparisons). The TrkB hGFAP mice, on the contrary, were insensitive to the effects of eithe...
TrkB is required for behavioral improvement induced by ADs and exercise
Next we examined depression-like behavior in the control and TrkB hGFAP mice by using the tail-suspension test (TST), a paradigm of inescapable stress ( ). All mice were tested 48 hours after the last dose of AD or saline to exclude the acute effects on behaviors that do not correlate with prior duration of drug treatment, or clinical responses. Control runners and AD treated control mice showed decreased immobility (a state of "behavioral despair"), compared to sedentary or saline treated control mice, respectively (: F 1,41 =9.082, p=0.0044 for exercise, post hoc p<0.01;: F 2,100 =20.03, p<0.0001 for AD, post hoc p<0.01 for fluoxetine, p<0.001 for imipramine). The TrkB hGFAP mice again failed to display any appreciable response to either treatment (: F 2,100 =4.233, p=0.0172 for genotype;: F 1,41 =9.082, p=0.0044 for genotype).
Measurement outputs
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(D) Comparative analysis on the size and density of DG granular neurons from control and TrkB hGFAP mice. Note the absence of difference in either category.
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
(F) Quantitative analysis revealed a 30% reduction in DG volume in the BDNF hGFAP mice at the age of 2 months. N=8-10 for each.
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Germline homozygous TrkB knockout animals die shortly after birth. To investigate the role of TrkB in postnatal stages, conditional knockout animals were generated by crossing m...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
We next enriched NPCs from 2-month old mice by culturing primary cells from the DG in neurosphere forming media ( ). Upon X-gal staining, only neurospheres isolated from the DG...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
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Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
(C) At P15 days, TrkB hGFAP, but not TrkB Syn mice had thinner granular layer, demonstrated a decrease in neuronal number.
from paperScoring or quantification
Quantify the primary readouts for this experiment: (D) Comparative analysis on the size and density of DG granular neurons from control and TrkB hGFAP mice. Note the absence of difference in either category.; (F) Quantitative analysis revealed a 30% reduction in DG volume in the BDNF hGFAP mice at the age of 2 months. N=8-10 for each.; Germline homozygous TrkB knockout animals die shortly after birth. To investigate the role of TrkB in postnatal stages, conditional knockout animals were generated by crossing m...; We next enriched NPCs from 2-month old mice by culturing primary cells from the DG in neurosphere forming media ( ). Upon X-gal staining, only neurospheres isolated from the DG....
from paperStatistical comparison
(C) At P15 days, TrkB hGFAP, but not TrkB Syn mice had thinner granular layer, demonstrated a decrease in neuronal number. Data was shown as percentage of control. N=6 for each...; (B-C) Proliferation in the DG was decreased in TrkB hGFAP, but not TrkB Syn mice, evidence by a reduction in Ki67 positive (C), or BrdU positive cells (B and C). Scale ba...; The conditional knockout animals, either carrying the hGFAP-Cre (TrkB hGFAP ) or the Syn-Cre (TrkB Syn ) are viable at birth and have normal survival rates (up to 15 months reco...; Reduced neurogenesis in the absence of TrkB could be caused by increased cell death, decreased proliferation, or a combination of both processes. We first examined programmed ce...
from paperReporting output
Report representative outputs alongside summary comparisons for (D) Comparative analysis on the size and density of DG granular neurons from control and TrkB hGFAP mice. Note the absence of difference in either category., (F) Quantitative analysis revealed a 30% reduction in DG volume in the BDNF hGFAP mice at the age of 2 months. N=8-10 for each., Germline homozygous TrkB knockout animals die shortly after birth. To investigate the role of TrkB in postnatal stages, conditional knockout animals were generated by crossing m..., We next enriched NPCs from 2-month old mice by culturing primary cells from the DG in neurosphere forming media ( ). Upon X-gal staining, only neurospheres isolated from the DG....
inferred from protocolStructured statistical methods
(C) At P15 days, TrkB hGFAP, but not TrkB Syn mice had thinner granular layer, demonstrated a decrease in neuronal number. Data was shown as percentage of control. N=6 for each...; (B-C) Proliferation in the DG was decreased in TrkB hGFAP, but not TrkB Syn mice, evidence by a reduction in Ki67 positive (C), or BrdU positive cells (B and C). Scale ba...; The conditional knockout animals, either carrying the hGFAP-Cre (TrkB hGFAP ) or the Syn-Cre (TrkB Syn ) are viable at birth and have normal survival rates (up to 15 months reco...; Reduced neurogenesis in the absence of TrkB could be caused by increased cell death, decreased proliferation, or a combination of both processes. We first examined programmed ce...
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Evidence quotes (8)
(A) Representative images of Nissl stained DG sections from control, TrkB hGFAP and TrkB Syn mice at P15. The decreases in DG size and granular layer thickness were only observed in TrkB hGFAP mice. Red lines and circles highlight the length of the granular layer, and the size of single cell, respectively. Scale bars, 1mm (hippocampus) and 10um (DG).
The conditional knockout animals, either carrying the hGFAP-Cre (TrkB hGFAP ) or the Syn-Cre (TrkB Syn ) are viable at birth and have normal survival rates (up to 15 months recorded) ( ). The TrkB hGFAP mice displayed a significant reduction in the volume of the DG granular layer ( ) that first became measurable at P10. The volume reduction stabilized at around 30% after the initial postnatal weeks and persisted throughout adulthood. This abnormality was not caused by changes in cell density or cell size (; p>0.2), but rather was a result of decrease in the number of granule neurons, evidenced by a significant reduction in the thickness of DG granular layer (; n=6 for each, F 2,15 =5.477, p=0.0164). The number and morphology of glial cells were not appreciably affected in the TrkB hGFAP mice ( and not shown). The reduction of volume was most prominently observed in the hippocampus and DG, and did not appear to be a secondary result of broader developmental defect, as the TrkB hGFAP mice had normal body and brain size at all ages examined ( ). We also measured the volumes of other anatomical regions, such as the striatum, in 2-month old TrkB hGFAP mice and found normal volume (...
We next assessed proliferation by measuring the number of cells that incorporate the DNA synthesis marker 5-bromo-2'-deoxyuridine (BrdU). P15 mice were treated with pulses of BrdU and sacrificed 24 hours after the first injection. Within the area encompassing the inner granular layer and SGZ, the number of BrdU positive cells in the TrkB hGFAP mice displayed a 48% reduction compared to control mice (; n=7-9 for each; F 2,21 =78.39, p<0.0001; p<0.001 for post hoc test of control to TrkB hGFAP comparison). Similarly, the number of cells positive for Ki67, an endogenous marker for actively cycling cells, was decreased by 51% in the SGZ of the TrkB hGFAP mice (; n=7-9 for each; F 2,21 =51.64, p<0.0001; p<0.001 for post hoc test of control to TrkB hGFAP comparison). Again, the numbers of BrdU or Ki67 positive cells were unaffected in the DG of the TrkB Syn mice (; p>0.2 for control to TrkB Syn comparisons).
Our finding that the TrkB hGFAP but not the TrkB Syn mice displayed impaired hippocampal neurogenesis suggested a cell autonomous requirement for TrkB in NPCs. To further examine their intrinsic properties, we cultured NPCs from the DG in serum-free conditions. In the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), DG NPCs formed neurospheres, which expressed TrkB receptor that could be activated by exogenous BDNF ( ). Although maintaining adult DG-derived primary neurospheres in media containing BDNF (50 ng/ml) for 7-10 days did not increase the frequency of primary neurosphere formation (not shown), there was a significant increase in the size of the neurospheres, suggesting that activation of TrkB facilitates the expansion of neurosphere-forming cells (; n=4 for each group, p<0.05 for post hoc test of with to without BDNF comparison in control cells). Primary neurospheres derived from the DG of adult TrkB hGFAP mice, when plated at equal density, displayed reduction in both the number (; n=4 for each group, p<0.005) and size (; p<0.005 for post hoc test of control to TrkB hGFAP comparison without BDNF; F 1,12 =102.2, p<0.0001 for...
Reduction in hippocampal volume has been observed in animal models of stress (; ), which may be reversed or prevented by chronic AD treatment ( ). Similarly, reduction of hippocampal volume has been reported in some studies of human patients with major depression (; ) and post-traumatic stress disorder (;; ). Although the cellular mechanism is unclear in humans, animal studies have demonstrated that chronic exposure to various types of ADs induces DG proliferation and neurogenesis ( ), thereby potentially contributing to the recovery of volume loss. In this context, we examined whether chronic AD treatment can restore neurogenesis in TrkB hGFAP mice. We thus treated control, TrkB hGFAP and TrkB Syn mice with daily injections of the serotonin reuptake inhibitor fluoxetine (10 µg/g) or the tricyclic imipramine (20 µg/g) for 21 days (n=7-29 for each group, ). As previously established, compared with mice receiving daily saline injections, both drugs induced substantial Ki67 immuno-reactivity in the DG of control mice (; F 2,98 =40.48, p<0.0001 for treatment; p<0.001 for post hoc test of both AD-to-saline comparisons). The induction of proliferation was echoed b...
In rodents, voluntary exercise, such as wheel-running behavior has been demonstrated to robustly induce neurogenesis, much in the same fashion as AD treatment ( ). Less is known, however, about the underlying mechanism of this AD-like effect of exercise. To determine whether TrkB is also required for this process, we subjected control and TrkB hGFAP mice to 6 weeks of wheel-running (n=8-16 for each group, ). Both the numbers of Ki67 positive and Doublecortin positive cells increased in control runners, compared to control sedentary animals (: F 1,36 =13.64, p=0.0007 for exercise;: F 1,36 =16.01, p=0.0003; p<0.001 for post hoc test of both comparisons). A significant increase of BDNF protein level was also observed in the hippocampus of runners (66.44 ± 3.65 pg/mg in runners; 45.83 ± 2.99 pg/mg in sedentary controls; n=3 for each; p<0.05). The TrkB hGFAP mice, despite normal running distance (5.96 ± 0.41 km/day in controls, 5.48 ± 0.34 km/day in TrkB hGFAP, p>0.2) as well as elevation of BDNF level in runners (60.33 ± 4.43 pg/mg in runners; 40.94 ± 1.37 pg/ml in sedentary controls; n=3 for each; p<0.05), did not show any increase in prolifer...
To determine whether the lack of neurogenic response in the TrkB hGFAP mice was coupled with general insensitivity to chronic ADs and exercise, we compared the depression and anxiety-like behaviors in control and TrkB hGFAP mice. First, mice treated with fluoxetine, imipramine, or saline for 21 days were examined in the novelty-suppressed feeding test (NSFT), a conflict paradigm in which the latency to feed in a novel environment is used as an indicator of anxiety level ( ). In agreement with the general capacity of chronic ADs to ease anxiety, 24 hours after the last dose, control mice receiving fluoxetine or imipramine displayed significantly shorter latency to feed compared to saline-treated control mice (; F 2,100 =8.022, p=0.0006 for treatment; p<0.001 for post hoc test both AD-to-saline comparisons). The TrkB hGFAP mice, on the contrary, were insensitive to the effects of either AD (; F 2,100 =10.49, p<0.0001 for genotype). Similarly, TrkB hGFAP mice exposed to 6 weeks of running showed no improvement in the NSFT, whereas the control runners displayed clear decrease in latency compared to sedentary animals (; F 1,41 =15.09, p=0.0004 for genotype; p<0.05 for post hoc tes...
Next we examined depression-like behavior in the control and TrkB hGFAP mice by using the tail-suspension test (TST), a paradigm of inescapable stress ( ). All mice were tested 48 hours after the last dose of AD or saline to exclude the acute effects on behaviors that do not correlate with prior duration of drug treatment, or clinical responses. Control runners and AD treated control mice showed decreased immobility (a state of "behavioral despair"), compared to sedentary or saline treated control mice, respectively (: F 1,41 =9.082, p=0.0044 for exercise, post hoc p<0.01;: F 2,100 =20.03, p<0.0001 for AD, post hoc p<0.01 for fluoxetine, p<0.001 for imipramine). The TrkB hGFAP mice again failed to display any appreciable response to either treatment (: F 2,100 =4.233, p=0.0172 for genotype;: F 1,41 =9.082, p=0.0044 for genotype).
Machine-readable layer
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"text": "(A) Representative images of Nissl stained DG sections from control, TrkB hGFAP and TrkB Syn mice at P15. The decreases in DG size and granular layer thickness were only observed in TrkB hGFAP mice. Red lines and circles highlight the length of the granular layer, and the size of single cell, respectively. Scale bars, 1mm (hippocampus) and 10um (DG)."
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"name": "Ablation of TrkB in early postnatal NPCs impairs DG morphogenesis",
"text": "The conditional knockout animals, either carrying the hGFAP-Cre (TrkB hGFAP ) or the Syn-Cre (TrkB Syn ) are viable at birth and have normal survival rates (up to 15 months recorded) ( ). The TrkB hGFAP mice displayed a significant reduction in the volume of the DG granular layer ( ) that first became measurable at P10. The volume reduction stabilized at around 30% after the initial postnatal weeks and persisted throughout adulthood. This abnormality was not caused by changes in cell density or cell size (; p>0.2), but rather was a result of decrease in the number of granule neurons, evidenced by a significant reduction in the thickness of DG granular layer (; n=6 for each, F 2,15 =5.477, p=0.0164). The number and morphology of glial cells were not appreciably affected in the TrkB hGFAP mice ( and not shown). The reduction of volume was most prominently observed in the hippocampus a..."
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"name": "Ablation of TrkB impairs proliferation and neurogenesis",
"text": "We next assessed proliferation by measuring the number of cells that incorporate the DNA synthesis marker 5-bromo-2'-deoxyuridine (BrdU). P15 mice were treated with pulses of BrdU and sacrificed 24 hours after the first injection. Within the area encompassing the inner granular layer and SGZ, the number of BrdU positive cells in the TrkB hGFAP mice displayed a 48% reduction compared to control mice (; n=7-9 for each; F 2,21 =78.39, p<0.0001; p<0.001 for post hoc test of control to TrkB hGFAP comparison). Similarly, the number of cells positive for Ki67, an endogenous marker for actively cycling cells, was decreased by 51% in the SGZ of the TrkB hGFAP mice (; n=7-9 for each; F 2,21 =51.64, p<0.0001; p<0.001 for post hoc test of control to TrkB hGFAP comparison). Again, the numbers of BrdU or Ki67 positive cells were unaffected in the DG of the TrkB Syn mice (; p>0...."
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"text": "Our finding that the TrkB hGFAP but not the TrkB Syn mice displayed impaired hippocampal neurogenesis suggested a cell autonomous requirement for TrkB in NPCs. To further examine their intrinsic properties, we cultured NPCs from the DG in serum-free conditions. In the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), DG NPCs formed neurospheres, which expressed TrkB receptor that could be activated by exogenous BDNF ( ). Although maintaining adult DG-derived primary neurospheres in media containing BDNF (50 ng/ml) for 7-10 days did not increase the frequency of primary neurosphere formation (not shown), there was a significant increase in the size of the neurospheres, suggesting that activation of TrkB facilitates the expansion of neurosphere-forming cells (; n=4 for each group, p<0.05 for post hoc test of with to without BDNF comparison in co..."
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"name": "TrkB is required for induced proliferation and neurogenesis by antidepressants and voluntary exercise",
"text": "Reduction in hippocampal volume has been observed in animal models of stress (; ), which may be reversed or prevented by chronic AD treatment ( ). Similarly, reduction of hippocampal volume has been reported in some studies of human patients with major depression (; ) and post-traumatic stress disorder (;; ). Although the cellular mechanism is unclear in humans, animal studies have demonstrated that chronic exposure to various types of ADs induces DG proliferation and neurogenesis ( ), thereby potentially contributing to the recovery of volume loss. In this context, we examined whether chronic AD treatment can restore neurogenesis in TrkB hGFAP mice. We thus treated control, TrkB hGFAP and TrkB Syn mice with daily injections of the serotonin reuptake inhibitor fluoxetine (10 µg/g) or the tricyclic imipramine (20 µg/g) for 21 days (n=7-29 for each group, ). As previ..."
},
{
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"position": 6,
"name": "TrkB is required for induced proliferation and neurogenesis by antidepressants and voluntary exercise",
"text": "In rodents, voluntary exercise, such as wheel-running behavior has been demonstrated to robustly induce neurogenesis, much in the same fashion as AD treatment ( ). Less is known, however, about the underlying mechanism of this AD-like effect of exercise. To determine whether TrkB is also required for this process, we subjected control and TrkB hGFAP mice to 6 weeks of wheel-running (n=8-16 for each group, ). Both the numbers of Ki67 positive and Doublecortin positive cells increased in control runners, compared to control sedentary animals (: F 1,36 =13.64, p=0.0007 for exercise;: F 1,36 =16.01, p=0.0003; p<0.001 for post hoc test of both comparisons). A significant increase of BDNF protein level was also observed in the hippocampus of runners (66.44 ± 3.65 pg/mg in runners; 45.83 ± 2.99 pg/mg in sedentary controls; n=3 for each; p<0.05). The TrkB hGFAP mice, despite..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "TrkB is required for behavioral improvement induced by ADs and exercise",
"text": "To determine whether the lack of neurogenic response in the TrkB hGFAP mice was coupled with general insensitivity to chronic ADs and exercise, we compared the depression and anxiety-like behaviors in control and TrkB hGFAP mice. First, mice treated with fluoxetine, imipramine, or saline for 21 days were examined in the novelty-suppressed feeding test (NSFT), a conflict paradigm in which the latency to feed in a novel environment is used as an indicator of anxiety level ( ). In agreement with the general capacity of chronic ADs to ease anxiety, 24 hours after the last dose, control mice receiving fluoxetine or imipramine displayed significantly shorter latency to feed compared to saline-treated control mice (; F 2,100 =8.022, p=0.0006 for treatment; p<0.001 for post hoc test both AD-to-saline comparisons). The TrkB hGFAP mice, on the contrary, were insensitive to the effects of eithe..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "TrkB is required for behavioral improvement induced by ADs and exercise",
"text": "Next we examined depression-like behavior in the control and TrkB hGFAP mice by using the tail-suspension test (TST), a paradigm of inescapable stress ( ). All mice were tested 48 hours after the last dose of AD or saline to exclude the acute effects on behaviors that do not correlate with prior duration of drug treatment, or clinical responses. Control runners and AD treated control mice showed decreased immobility (a state of \"behavioral despair\"), compared to sedentary or saline treated control mice, respectively (: F 1,41 =9.082, p=0.0044 for exercise, post hoc p<0.01;: F 2,100 =20.03, p<0.0001 for AD, post hoc p<0.01 for fluoxetine, p<0.001 for imipramine). The TrkB hGFAP mice again failed to display any appreciable response to either treatment (: F 2,100 =4.233, p=0.0172 for genotype;: F 1,41 =9.082, p=0.0044 for genotype)."
}
],
"tool": [
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"name": "hGFAP-Cre activity in hippocampal progenitor cells"
},
{
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"name": "TrkB is required for behavioral improvement induced by ADs and exercise"
},
{
"@type": "HowToTool",
"name": "Specific ablation of TrkB in adult NPCs is sufficient to block sensitivity to AD"
},
{
"@type": "HowToTool",
"name": "TrkB and the behavioral efficacy of antidepressants"
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"name": "FACS"
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"@type": "HowToSupply",
"name": "Ablation of TrkB impairs proliferation and neurogenesis"
},
{
"@type": "HowToSupply",
"name": "Activation of TrkB in response to BDNF facilitates proliferation in vitro"
},
{
"@type": "HowToSupply",
"name": "TrkB is required for induced proliferation and neurogenesis by antidepressants and voluntary exercise"
},
{
"@type": "HowToSupply",
"name": "TrkB is required for behavioral improvement induced by ADs and exercise"
},
{
"@type": "HowToSupply",
"name": "Histology and Quantitative Analysis"
},
{
"@type": "HowToSupply",
"name": "Protein Analysis"
},
{
"@type": "HowToSupply",
"name": "Neurosphere cultures"
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{
"@type": "HowToSupply",
"name": "FACS"
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"@type": "ScholarlyArticle",
"headline": "TrkB regulates hippocampal neurogenesis and governs sensitivity to antidepressive treatment",
"datePublished": "2008",
"author": [
{
"@type": "Person",
"name": "Yun Li"
},
{
"@type": "Person",
"name": "Bryan W. Luikart"
},
{
"@type": "Person",
"name": "Shari Birnbaum"
},
{
"@type": "Person",
"name": "Jian Chen"
},
{
"@type": "Person",
"name": "Chang-Hyuk Kwon"
},
{
"@type": "Person",
"name": "Steven G. Kernie"
},
{
"@type": "Person",
"name": "Rhonda Bassel-Duby"
},
{
"@type": "Person",
"name": "Luis F. Parada"
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"identifier": "10.1016/j.neuron.2008.06.023"
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