13-cis-retinoic acid suppresses hippocampal cell division and hippocampal-dependent learning in mice

Abstract

The active component of the acne drug Accutane is 13-cis-retinoic acid (RA), and it is highly teratogenic for the developing central nervous system. Very little is known, however, regarding the effect of this drug on the adult brain. Regions of the brain that may be susceptible to RA are those that continue to generate new neurons. In the adult mouse, neurogenesis is maintained in the hippocampus and subventricular zone. This report demonstrates that a clinical dose (1 mg/kg/day) of 13-cis-RA in mice significantly reduces cell proliferation in the hippocampus and the subventricular zone, suppresses hippocampal neurogenesis, and severely disrupts capacity to learn a spatial radial maze task. The results demonstrate that the regions of the adult brain where cell proliferation is ongoing are highly sensitive to disruption by a clinical dose of 13-cis-RA.

Fig. 1.

Experimental paradigms of BrdUrd labeling. (A) Mice were injected with 1 mg/kg 13-cis-RA or vehicle daily for 1-6 weeks. After this period, the mice were injected with BrdUrd at 50 mg/kg every 2 h over a 6-h period. The brains were then perfused, sectioned, and immunostained, and the number of BrdUrd-labeled cells was counted in every 12th section in the hippocampal formation and SVZ. (B) The mice were injected with BrdUrd at 50 mg/kg every 2 h over a 6-h period, and the mice were allowed to survive for another 3 weeks with continued daily RA injections. The animals were then killed and processed as described above and stained for BrdUrd or double labeled for BrdUrd and the neuronal marker Neu-N.

Fig. 2.

The decline in hippocampal and SVZ cell proliferation after long-term RA treatment. Seven days of exposure to 13-cis-RA resulted in no statistically significant difference in the average number of BrdUrd-positive cells between the experimentally treated and control animals in the hippocampus (A). However, by 21 days of treatment (B), a significant decline was evident, which had reached a reduction of 41% by 42 days (C). This effect was not specificto the hippocampus as indicated by a decline in proliferation in the SVZ (D), although this decline was not to the same degree. The decline in BrdUrd labeling in the hippocampal formation is exemplified in control (E) and 13-cis-RA-treated mice (F) for 42 days. Note that different y axis scales were used between the hippocampus (A-C) and SVZ (D). (A) Control = 467 ± 47, 13-cis-RA = 559 ± 91. (B) Control = 395 ± 20; 13-cis-RA = 285 ± 20, t = 4.59, P = 0.004 compared to control. (C) Control = 481 ± 38, noninjected = 478 ± 33. tRA = 284 ± 21, t = 4.55, P = 0.010 compared to control (vehicle injected). 13-cis-RA = 299 ± 21, t = 4.21, P = 0.014 compared to control (vehicle injected). (D) Control = 1721 ± 54, noninjected = 1,676 ± 55. tRA = 1342 ± 62, t = 4.55, P = 0.010 compared to control (vehicle injected). 13-cis-RA = 1,362 ± 54, t = 4.68, P = 0.009 compared to control (vehicle injected).

Fig. 3.

The decline in number of BrdUrd-positive cells remaining in the hippocampus and hippocampal SGZ after 21 days of exposure to 13-cis-RA. If proliferating cells are labeled with BrdUrd and 13-cis-RA treatment is instigated and continued over 21 days, then a significant reduction in the number of BrdUrd-positive cells is evident both throughout the hippocampal formation (A) and SGZ (B), illustrated in representative sections (C and D) with labeled cells marked with arrows. (A) Control = 383 ± 42; 13-cis-RA = 203 ± 15, t = 4.07, P = 0.015. (B) Control = 131 ± 22; 13-cis-RA = 56 ± 9, t = 3.15, P = 0.035.

Fig. 4.

The decline in neurogenesis resulting from 21 days of 13-cis-RA treatment. Neurogenesis, as determined by the number of BrdUrd and Neu-N double-positive cells, is significantly reduced by RA treatment (A), illustrated in representative sections of control Neu-N (B), BrdUrd (C), and Neu-N/BrdUrd merged (D) and RA-treated Neu-N (E), BrdUrd (F), and Neu-N/BrdUrd merged (G). Double-labeled cells are tagged with asterisks. (A) Control = 63 ± 2.2; 13-cis-RA = 41 ± 3, t = 4.79, P = 0.009.

Fig. 5.

The influence of 13-cis-RA exposure on radial maze performance after 28 days of exposure to 13-cis-RA (RA). This treatment regime dramatically decreases the ability of mice to perform a spatial 8-arm radial maze test (five daily trials) (16) as shown by numbers of errors made (A) and number of new arms visited during the first eight choices (B) (chance level is 5.3). n = 10 per group, and errors bars are mean ± SE. •, control; ○, RA treated. The statistical significance of the errors for each trial in A is as follows: er2: t = 4.21, P < 0.0018; er3: t = 4.62, P < 0.0009; er4: t = 3.40, P < 0.0067; and er5: t = 7.29, P < 0.0001. The significance of the new entries for each trial in B is as follows: ne2: t = 0.49, P = 0.6314; ne3: t = 2.78, P = 0.0195; ne4: t = 2.29, P = 0.0451; and ne5: t = 5.23, P = 0.0004.

Fig. 6.

The plasma concentration of 13-cis-RA after a single injection of RA at 1 mg/kg and the entry of RA into the hippocampus to bind to cellular RA-binding protein I (CRABPI). (A) The plasma RA concentration quickly reached 2.2 μg/ml after 20 min and was down to 0.3 μg/ml after 2 h(n = 3). (B) The movement of RA into hippocampal cells could be shown by separating hippocampal CRABPI by isoelectric focusing (which runs at a pI of 4.8) and eluting the RA bound to this. •, hippocampus without RA; ○, hippocampus plus RA.