| Objectives:Depression is closely related to cholinergic nervous system abnormalities.The evidence has been shown that cholinergic M receptor antagonists have a rapid improvement in depression-like symptoms,and the role of cholinergic nervous system in pathogenesis of depression is not understood.The 5-hydroxytryptamine(5-HT)deficiency is dominant hypothesis that is currently recognized as the pathogenesis of depression.The mechanism underlying the interaction between cholinergic and 5-HT energy systems in depression is not yet clear.The present study was designed to induce depressive-like behavior in rats by specifically damaging the horizontal branch of cholinergic neurons in the basal ganglia of the rat,and to inhibit specifically pathway from the diagonal band of broca cholinergic neurons(HDB)to the dentate gyrus(DG)through DREADD-hM4 Di.Here specific damage of the cholinergic neurons of HDB in Pet1-CreER/Flox-lmx1b-/-mice was evaluated and the role of the interaction between 5-HT and cholinergic neurons in the depression-like behavior of depression was explored.Experiment 1:Effect of specific damage to cholinergic neurons in HDB on depressionlike behavior in SD rats or in wild-type C57 miceMethods:Adult male Sprague-Dawley rats were randomly divided into three groups: blank control group,sham-operated PBS group,and neuroimmune toxin saporin(192 IgG-Saporin)group.Brain stereotaxic experiments were performed on rats in the sham-operated PBS group and the neuroimmunotoxin saporin group,respectively.The sham-operated PBS group was located in the nucleus horizontal branch of the oblique angle(AP: +0.48 mm,ML: ±0.8 mm,DV:-8.0 mm)Nuclei were microinjected with 0.5 μl PBS,and the neuroimmunotoxin saporin group was found in the nucleus branch of the oblique angle band(AP: +0.48 mm,ML: ±0.8 mm,DV:-8.0mm).Injection of 0.5 μg/μl of neuroimmunotoxin saporin was performed.The blank control group was not treated.After 2 weeks,all rats were subjected to behavioral experiments such as sugar water preference,forced swimming,open field experiments,and elevated cross labyrinth experiments.Finally,brain slices of SD rats were sacrificed and perfused for immunofluorescence staining to detect the number of ChAt-positive cells in the horizontal branch of the oblique angle band and the changes of pyramidal cells and granular cell thickness in the hippocampus.Adult male wild-type C57 mice were randomly divided into three groups: blank control group,sham-operated PBS group,neuroimmune toxin saporin group.Brain stereotaxic experiments wereperformed on wild-type C57 mice in the sham-operated PBS group and the neuroimmune toxin saporin group,respectively.The sham-operated PBS group was in the horizontal branch of the oblique angle band(AP: +0.74 mm,ML: ±0.625 mm,DV:-4.75 mm)nuclei,and horizontal branch of the neuroimmunotoxin saporin in the oblique nuclear zone(AP: +0.74 mm,ML: ±0.625 mm,DV:-4.75 mm).The group was given a microinjection of 0.25 μg/μl neurotoxin saporin,and the blank control group was not treated.After 2 weeks,all mice were subjected to behavioral experiments such as sugar water preference,open field experiments,novelty discrimination experiments,tail suspension experiments,and forced swimming.After the behavioral experiments were completed,mice were sacrificed and brain tissue sections were fixed for immunofluorescence staining.ChAT-positive cells and hippocampal pyramidal cells and granular cell thickness were observed.Results:Three groups of SD rats were tested for sugar-water preference,forced-swimming test,open field test,and elevated plus-maze test.The results showed that the neuroimmune toxins that specifically damage the horizontal branch of cholinergic neurons with nuclear level Compared with the control group,the percentage of sugar water preference of the grass group rats was significantly lower(P<0.01),the immobility time of forced swimming was significantly prolonged(P<0.01),and the number of stringing sessions and the number of erections in the open field experiment were significantly reduced(P<0.01).P<0.05),but there was no statistically significant difference between the sham-operated PBS group and the blank control group.These results demonstrate that cholinergic neurons that specifically damage the horizontal branch of the oblique nucleus can cause depressive-like behavior in SD rats.The results of immunofluorescence staining showed that the number of ChAT-positive cells in the nucleus branch of the oblique angle band of the neuroimmunotoxin saporin group was significantly reduced(P<0.01),and the neuroimmunotoxin saporin was compared with the rats of the sham-operated PBS group.The thickness of pyramidal cells and granulosa cells in the hippocampus of rats was significantly reduced.Three groups of wild-type C57 mice were subjected to sugar-water preference test,forced swimming test,open field test,elevated plus-maze test,new thing discrimination test,and tail suspension test.Compared with the control group,the percentage of the sugar water preference of the neuroimmune toxin saurin group C57 mice of the alkaliergic neurons was significantly lower(P<0.01),and the immobility time of forced swimming was significantly prolonged(P<0.01).The exercise time was significantly prolonged(P<0.01),and the number of threadings in the open field experiment was significantly reduced(P<0.01).There was no statistically significant difference between the sham-operated PBS group and the blank control group.These results demonstrate that cholinergic neurons that specifically damage the horizontal branch of the nucleus can causedepressive-like behavior in wild-type C57 mice.The results of immunofluorescence staining showed that the number of ChAT-positive cells in the nucleus oblique branch of the wild-type C57 mice was significantly decreased(P<0.01).The thickness of pyramidal cells and granulosa cells in the hippocampus of wild-type C57 mice was significantly decreased in neuroimmune toxin saporin group.Experiment 2:Effect of Chemically Inhibiting Cholinergic Projection from HDB to DG Zone on Depressive Behavior of SD RatsMethods:Adult male Sprague-Dawley rats were randomly divided into three groups: blank control group,sham operation group,and inhibition group.The blank control group received no treatment.The sham-operated group was injected with pAAV-ChAT-mini-at the horizontal oblique branch(HDB)of the bilateral oblique angle(AP: +0.48 mm,ML: ±0.8 mm,DV:-8.0mm).A mixture of TK-promoter-EGFP-2A-Cre and pAAV-hSyn-DIO-hM4D(Gi)-mCherry adeno-associated virus,each volume was 1 μl,injection time 5 min,needle retention 5 min,and then bilateral DG(AP:-3.8 mm,ML: ±1.6 mm,DV:-3.75 mm)embedded stereotactic sleeve;Inhibition group also injected with pAAV-ChAT-mini-TK-promoter-EGFP-2A-Cre and pAAVhSyn-DIO-hM4D(Gi)-mCherry adeno-associated virus mixture,viral injection volume and time were the same as in the sham group,but also bilateral DG(AP:-3.8 mm,ML: ± 1.6 Mm,DV:-3.75mm)embedded three-dimensional positioning sleeve.In the sham-operated group and the inhibitory group,SD rats were post-infected with penicillin for 3 days.After 3 weeks of viral transfection,0.5 μl 1 mM CNO was injected into the DG region of the inhibitory group.After 30 minutes,all rats were subjected to an elevated cross-maze.Experiment,forced swimming experiment,sugar-water preference experiment,open-field experiment test.The final sacrificed rats were sacrificed and the brain tissue sections were taken for immunofluorescence staining to observe the transfection of the virus.Results:After injection of 0.5 μl CNO in hippocampal DG area,the cholinergic projection of DG area to HDB area was inhibited chemically.The immobility time of forced swimming in the inhibition group was significantly prolonged(P<0.01).And the number of erections was decreased(P<0.05).In the open field experiment,the time for the rats to go out of the first latent period was reduced(P<0.05),but there was no statistically significant difference between the rats in the shamoperated group and the blank control group.The results suggest that chemical genetics can cause depression-like behavior in rats by inhibiting cholinergic projection in the HDB to the DG region.Experiment 3:The effect of specific damaging oblique nuclear nuclear cholinergic neurons on depression-like behavior in Pet1-CreER/Flox-lmx1b-/-miceMethods:Selecting Pet1-CreER/Flox-lmx1b-/-mice,this mouse selectively inactivates the expression of Lmx1 b in the raphe nuclei of adult mice by inducing the Cre-LoxP system,with 5-HT levels compared to normal mice.A drop of 60%.After mating and breeding with the Pet1-creER/Flox-lmx1 b +/-genotype mice,the mice were labeled for trimming at 1 month after birth,the samples were lysed,DNA was amplified by PCR,agarose gel electrophoresis was performed,and the bands were compared.Pet1-CreER/Flox-lmx1b-/-mice.Adult male Pet1-CreER/Flox-lmx1b-/-mice were randomly divided into three groups: blank control group,sham operation group,neuroimmune toxin saporin group.Brain stereotaxic experiments were performed on the Pet1-CreER/Flox-lmx1b-/-mice in the sham-operated group and the neuroimmunotoxin saporin group.The sham-operated group was in the nucleus horizontal branch(AP: +0.74 mm,ML:(± 0.625 mm,DV:-4.75 mm)nuclei were injected microinjected with0.25 μl PBS,neuroimmunotoxin saporin group was in the horizontal branch of the oblique angle band(AP: +0.74 mm,ML: ± 0.625 mm,DV:-4.75 Mm)Nuclei were microinjected with 0.25μg/μl of the neuroimmune toxin saporin,and the blank control group was not treated.After 2weeks,all mice were subjected to behavioral experiments such as sugar water preference,open field experiments,novelty discrimination experiments,tail suspension experiments,and forced swimming.At the end of the behavioral experiment,brain tissues were perfused and fixed in Pet1-CreER/Flox-lmx1b-/-mice.Immunofluorescence staining was performed on the sections to observe the changes of ChAT-positive cells and hippocampal pyramidal cells and granular cell thickness.Results:Three groups of Pet1-CreER/Flox-lmx1b-/-mice were tested for glucose-preference,forced-swimming,tail-suspended,open-field,and elevated-plus-maze tests.Analysis of ethological data of Pet1-CreER/Flox-lmx1b-/-mice by cholinergic neurons revealed no statistically significant difference,but the number of erections was reduced in open field experiments.Immobility time is slightly longer.The results of immunofluorescence staining showed that the number of ChATpositive cells in the horizontal branch of the basal ganglia of Pet1-CreER/Flox-lmx1b-/-mice in the neuroimmune toxin saporin group was significantly reduced(P<0.05),and was associated with the sham group Pet1.Compared to creER/Flox-lmx1b-/-mice,there was no significant change in the pyramidal cell and granule cell thickness in the neuronal immunotoxin saporin group Pet1-CreER/Flox-lmx1b-/-mouse hippocampus,suggesting that Pet1-CreER/Flox-lmx1b-/-mice were damaged.Creur/Flox-lmx1b-/-mice have a hypothalamic horizontal cholinergic neuron that does not produce depression-like behavior that may be related to neurogenesis in the hippocampus.Conclusion: This project demonstrates that the specific destruction of the HDB with cholinergic neurons and the selective inhibition of the cholinergic projection pathway from the HDB region to the DG region can lead to produce of depression-like behavior.Lesions of HDB in5-HT-deficient mice failed to produce the depression like symptoms,which may be related to neurogenesis in the hippocampus of 5-HT-deficient mice. |
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