Electroacupuncture promotes proliferation of amplifying neural progenitors and preserves quiescent neural progenitors from apoptosis to alleviate depressive-like and anxiety-like behaviours

Abstract

The present study was designed to investigate the effects of electroacupuncture (EA) on depressive-like and anxiety-like behaviours and neural progenitors in the hippocampal dentate gyrus (DG) in a chronic unpredictable stress (CUS) rat model of depression. After being exposed to a CUS procedure for 2 weeks, rats were subjected to EA treatment, which was performed on acupoints Du-20 (Bai-Hui) and GB-34 (Yang-Ling-Quan), once every other day for 15 consecutive days (including 8 treatments), with each treatment lasting for 30 min. The behavioural tests (i.e., forced swimming test, elevated plus-maze test, and open-field entries test) revealed that EA alleviated the depressive-like and anxiety-like behaviours of the stressed rats. Immunohistochemical results showed that proliferative cells (BrdU-positive) in the EA group were significantly larger in number compared with the Model group. Further, the results showed that EA significantly promoted the proliferation of amplifying neural progenitors (ANPs) and simultaneously inhibited the apoptosis of quiescent neural progenitors (QNPs). In a word, the mechanism underlying the antidepressant-like effects of EA is associated with enhancement of ANPs proliferation and preserving QNPs from apoptosis.

Figure 1

Animal groups and schematic representations of the behaviour testing procedure. The rats were randomly divided into three groups (n = 8 per group): Normal (a naïve unchallenged group without any stress and any treatment), Model (rats which were exposed to 4-week CUS), and EA (model rats which received EA treatment in the last two weeks of the CUS procedure). For the latter 2 groups, 4-week CUS with/without 2-week EA treatment was performed. The forced swimming test (FST) was performed at the end of every week. The elevated plus-maze (EPM) test and the open field test (OFT) were performed before the CUS, at the end of week 2, week 3, and week 4. For analysis of cell proliferation, another batch of the rats, which was also divided into three groups and subjected to the same procedure, received a single injection of BrdU (200 mg kg1 i.p.) on the first day of the week 5 and was sacrificed (S) 24 h after BrdU administration.

Figure 2

Stressed rats (Model group) show significantly depressive-like and anxiety-like behaviours after 2-week CUS exposure. (a) The immobility time and (b) the climbing time in forced swimming test are expressed in seconds. (c) Percentage of open-arm entries and (d) percentage of time spent in open arms during a 5 min test on the elevated plus-maze. (e) The number of rearing during the open field test. The results are expressed as the mean ± SEM. *P < 0.05 and **P < 0.01, versus Normal group.

Figure 3

EA alleviates the CUS-induced depressive-like and anxiety-like behaviours. One- or two-week EA significantly decreases the immobility time (a) and increases the climbing time (b) in the forced swimming test. (c) One-week EA treatment significantly elevates the open-arm entries but not two-week EA treatment. (d) Stressed rats spend more time in the open arms in the elevated plus-maze test after one-week EA treatment but not two-week EA treatment. (e) One-week EA treatment reverses the declined rearing number of the stressed rats in the open field test. The results are expressed as the mean ± SEM. ^# P < 0.05 and ^## P < 0.01, versus Model group.

Figure 4

Proliferation study. EA increases the proliferative cells in the DG of the stressed rats. (a) Representative microphotographs showing BrdU-positive cells (administered 24 h before killing) in DG of rats in different groups. The red square in the schematic drawing indicates the regions of microphotographs shown in (a). Scale bar corresponds to 100 μm. Bar gram in panel (b) depicts (mean ± SE) the quantification of the proliferation study showing the number of BrdU-positive cells in 3 experimental groups. n = 5, *P < 0.05, and **P < 0.01, versus Normal group; ^# P < 0.05, versus Model group.

Figure 5

EA reverses the inhibition of ANPs proliferation in the DG of the stressed rats. (a) Representative triple-stained sections in the DG: BrdU (red), GFAP (green), and Hoechst (blue). The translucent square in the Normal figure is magnified in (b) to point out the division of quiescent neural progenitors (QNPs) and amplifying neural progenitors (ANPs). The BrdU⁺/GFAP⁻ cells (black arrows) are considered the proliferating ANPs and those BrdU⁺/GFAP⁺ cells (white arrows) the proliferating QNPs. Scale bar represents 100 μm. Bar gram in panel (c) depicts (mean ± SE) the quantification of the dividing cells belonging to different subtypes of NPs in the whole DG. The proliferations of QNPs and ANPs are both decreased by CUS, and EA upregulates the ANP proliferation in stressed rats. n = 5 and *P < 0.05, versus Normal group; ^# P < 0.05, versus Model group.

Figure 6

EA relieves the apoptosis of QNPs in DG of the stressed rats. ((a)–(h)) Representative GFAP (red), Sox2 (green), and Hoechst (blue) triple-stained sections show that typical cells are undergoing apoptosis or not in different types. The Hoechst staining is below the merged triple staining in every figure to show the typical apoptotic morphology of the nucleus (pyknosis, deep into dense) or normal nucleus (without any nuclear condensation and fragmentation). (a) A normal QNP (arrow). (b) An apoptotic QNP (arrow). (c) A normal ANP (arrow). (d) An apoptotic ANP (arrow). (e) A normal astrocyte (arrow). (f) An apoptotic astrocyte (arrow). (g) Normal granular cells in GCL (arrows). (h) Apoptotic granular cells in GCL (arrows). Bar gram in panel (i) depicts (mean ± SE) the quantification of the different types of the apoptotic cells in the whole DG. EA protects the stressed rats when they exhibits an antiapoptotic effect on the QNPs in DG. n = 5 and ^# P < 0.05, versus Model group.