Research reportEffects of active shock avoidance learning on hippocampal neurogenesis and plasma levels of corticosterone
Introduction
The dentate gyrus (DG) of the hippocampal formation remains capable of generating new neurons during adulthood. The formation of these new cells depends on rapidly dividing progenitors residing in the subgranular zone (SGZ). Upon migration into the hippocampal granule cell layer (GCL), these cells differentiate into a neuronal phenotype [4], [41], form mossy fibers [20], receive input from other cells [35] and ultimately become functional granule neurons [55].
Although adult neurogenesis has received much attention during the last decade, the function of newly generated hippocampal neurons is still unclear. Since, the hippocampus plays a key role in learning and memory (reviewed by [21], [26]), it has been hypothesized that newly formed neurons are involved in one or more aspects of learning [16]. This hypothesis is supported by the observation that factors known to facilitate learning performance, such as estrogens [22], [48], running wheel activity [45], [54] or environmental enrichment [5], [12], also have a stimulatory effect on adult neurogenesis [24], [51], [54]. Aging, on the other hand, or high glucocorticoid levels, which can have a negative impact on learning [1], [36], [40], reduce hippocampal neurogenesis [2], [27].
Several studies have been performed to investigate the connection between learning and neurogenesis. Experimental data on progenitor proliferation or survival of newly formed neurons exist for Morris water maze (MWM) learning and trace eyeblink conditioning (TEC), but the results are still rather controversial [9], [10], [14], [30], [39], [54], possibly caused by differences in species, gender and timing of BrdU injections [17]. In view of the fact that so far only the hippocampus-dependent and not the hippocampus-independent versions of MWM learning and TEC have been found to be able to induce changes in hippocampal neurogenesis, it has been postulated that only learning tasks that are dependent on the hippocampus have the capability to affect neurogenesis [14].
Surprisingly, most of the work concerning learning and neurogenesis has been limited to only two specific learning tasks, MWM and TEC. Because of the differential results that were obtained using these two learning tasks, it is interesting to investigate whether an essentially different type of learning, such as active shock avoidance (ASA), is able to induce changes in hippocampal neurogenesis. ASA is a classical Pavlovian conditioning task that involves non-declarative memory. Acquisition of this task is not supposed to be strictly dependent on the hippocampus, since there is an overlap between the conditioned and the unconditioned stimulus, meaning that no temporal gap between both stimuli has to be bridged by the hippocampus [50], [58], [60]. If the hypothesis was correct that only hippocampus-dependent learning tasks can influence neurogenesis, ASA acquisition should not have an effect on hippocampal neurogenesis. Yet, multiple studies have demonstrated that the hippocampal formation, although not necessary for acquisition of ASA, is certainly affected by this task [44], [53], [56], which emphasizes the importance of investigating the effects of ASA training on hippocampal neurogenesis.
In the present study, rats were trained in the ASA task during 1 or 4 days. Two different training protocols were used, because we aimed to investigate whether a short 1-day protocol had a different effect on hippocampal neurogenesis than a more prolonged training of 4 days, which requires repeated activation of the hippocampus. Twenty-four hours after the last training session, animals were sacrificed and brains were analyzed for progenitor proliferation in the SGZ with Ki-67 immunocytochemistry and for survival of newly formed granule cells, which was achieved by staining for the thymidine analogue BrdU that had been injected one week before training. In addition, since ASA is supposed to be a stressful learning task and because several studies have reported that stress has a negative impact on adult neurogenesis [7], [11], [15], [42], [52], blood samples were taken during the experiment to determine plasma levels of corticosterone (CORT).
Section snippets
Animals and housing
Fifty-seven male Wistar rats (circa 300 g, bred in our own facility) were housed individually, had free access to food and water and were kept in climate rooms with a 12/12 h light–dark cycle (lights on at 8:00 a.m.). All procedures concerning animal care and treatment were in accordance with the regulations of the ethical committee for the use of experimental animals of the University of Groningen (DEC 2719).
ASA training
In both experiments, ASA testing was performed during the light phase of the animals,
Learning performance
Animals were trained in a shuttle box for either 1 or 4 days and the number of active avoidances was recorded. Since in both experiments a considerable number of animals hardly displayed any active avoidance, it was decided to divide the animals in two groups: responders and non-responders. An animal was considered to be a responder if it was able to display four successive active avoidances. This criterion was reached by 50% of the animals that were trained for 1 day and by 60% of the rats
Discussion
In order to gain more insight into the connection between hippocampal neurogenesis and learning, particularly hippocampus-independent associative learning, rats were trained in a 1- or a 4-day version of the ASA task. Progenitor proliferation and survival of newly generated granule cells were not changed after both ASA training paradigms. Blood samples taken throughout the 4-day experiment revealed no changes in basal CORT levels. Although trained animals as well as habituated controls showed
Acknowledgements
We thank Barbara Biemans and Theo Dinklo for their assistance with setting up the shuttle box and Jan Bruggink for his contribution to the corticosterone assays.
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