Female rats learn trace memories better than male rats and consequently retain a greater proportion of new neurons in their hippocampi

Abstract

Learning increases the survival of new cells that are generated in the hippocampal formation before the training experience, especially if the animal learns to associate stimuli across time [Gould E, Beylin A, Tanapat P, Reeves A, Shors TJ (1999) Nat Neurosci 2:260-265]. All relevant studies have been conducted on male rats, despite evidence for sex differences in this type of learning. In the present study, we asked whether sex differences in learning influence the survival of neurons generated in the adult hippocampus. Male and female adult rats were injected with one dose of bromodeoxyuridine (BrdU; 200 mg/kg), to label one population of dividing cells. One week later, half of the animals were trained with a temporal learning task of trace eyeblink conditioning, while the other half were not trained. Animals were killed 1 day after training (12 days after the BrdU injection). Hippocampal tissue was stained for BrdU and a marker of immature neurons, doublecortin. Both sexes learned to emit the conditioned eyeblink response during the trace interval. As a consequence, more new neurons remained in their hippocampi than in sex-matched controls. In individual animals, the number of surviving cells correlated positively with asymptotic performance; those that expressed more learned responses retained more new neurons. However, animals that learned very well retained even more new cells if they required many trials to do so. Because females emitted more learned responses than males did, they retained nearly twice as many new cells per unit volume of tissue. This effect was most evident in the ventral region of the hippocampal formation. Thus, sex differences in learning alter the anatomical structure of the hippocampus. As a result, male and female brains continue to differentiate in adulthood.

Fig. 1.

Experimental design. (A) Male and female rats were surgically implanted with electrodes to measure the electromyographic response (EMG) of the eyelid. Afterward, animals were injected with 1 dose of bromodeoxyuridine (BrdU), which labels 1 population of dividing cells. Females were cycling normally and injected during estrus. One week later, males and females were trained with trace eyeblink conditioning each day for 4 days. One day later, trained and untrained animals were killed to assess the number of new cells in the dentate gyrus of the hippocampus (12 days after the BrdU injection). (B) During trace conditioning, a CS of white noise was paired with a US (periorbital stimulation), which elicits an eyeblink response. The 2 conditioning stimuli were separated by a trace interval or temporal gap of 500 ms. As an animal learns that the 2 stimuli are associated, it blinks during the trace interval and in anticipation of the US. Eyeblinks were detected by an increase in the magnitude of the EMG. Those that occurred during the trace interval were considered CRs. The electrophysiological record shows an example of a CR that occurred just before the onset of the US.

Fig. 2.

Learning increased the survival of new cells in the hippocampus. (A) Learning during trace conditioning increased the number of new cells that survived after training (* indicates that the effect of training on cell number was significant: P < 0.05). The number of BrdU-labeled cells in the dentate gyrus (GCL/SGZ and hilus) of male and female animals that learned (n = 14) is shown in contrast to the number of new cells remaining in animals that were not trained (n = 17). (B) The number of BrdU-labeled cells that were present in an animal after training correlated positively and significantly with the period during training when the animal learned to emit the CR during the trace interval. The asymptotic trial was used to estimate the trial when an animal's conditioned response rate was 95% or more of its asymptote. The asymptotic trial did not differ between male and female animals that learned (P > 0.05).

Fig. 3.

Females learned better than males and rescued a greater proportion of new neurons. (A) Both sexes acquired the conditioned response (P < 0.05), but females emitted more CRs than males did across trials of training (P < 0.05). More females (7 out of 8) reached a learning criterion than did males (7 out of 10). (B) The volume of the dentate gyrus was calculated to assess the density of new cells that were generated and maintained in the male versus female hippocampus with and without training. Overall, the density of BrdU-labeled cells in the dentate gyrus (GCL/SVZ and hilus) was higher in trained than untrained animals, irrespective of sex. However, the percent increase was greater in trained females (34%) than in trained males (17%). The density of new cells in untrained females was lower than that in untrained males (significant differences are noted with an *). (C and D) The adult-generated cells were labeled with BrdU (brown) and counterstained with cresyl violet (purple). Representative images from a male and female hippocampus were magnified (1,000×) with an optical microscope. (E) The vast majority of new cells (≈80%) differentiated into neurons by the end of training (12 days after the BrdU injection). These cells expressed BrdU (green) and DCX (red), which is expressed in immature neurons. Representative images of a new cell expressing DCX alone (Left), BrdU alone (Middle), and both DCX and BrdU (Right) were obtained with a confocal scanning microscope.

Fig. 4.

Learning increased the density of new cells that survived in the ventral hippocampus of females. (A and B) The hippocampal formation was arbitrarily divided into the dorsal (A) and the ventral (B) regions. Cell layers were stained with cresyl violet, illustrating the GCL and the hilus. Images were obtained with an optical microscope (20×). (C) Training increased the density of new cells that resided in the ventral region of the female hippocampus (P < 0.05). The density of new cells in the ventral GCL of untrained females was lower than that in untrained males. (* indicates significant posthoc tests). (D) The level of responding that an animal achieved by the end of training (“asymptote”) correlated with the density of new cells that survived in the ventral GCL, especially in females.