Prolactin stimulates precursor cells in the adult mouse hippocampus

In the search for ways to combat degenerative neurological disorders, neurogenesis-stimulating factors are proving to be a promising area of research. In this study, we show that the hormonal factor prolactin (PRL) can activate a pool of latent precursor cells in the adult mouse hippocampus. Using an in vitro neurosphere assay, we found that the addition of exogenous PRL to primary adult hippocampal cells resulted in an approximate 50% increase in neurosphere number. In addition, direct infusion of PRL into the adult dentate gyrus also resulted in a significant increase in neurosphere number. Together these data indicate that exogenous PRL can increase hippocampal precursor numbers both in vitro and in vivo. Conversely, PRL null mice showed a significant reduction (approximately 80%) in the number of hippocampal-derived neurospheres. Interestingly, no deficit in precursor proliferation was observed in vivo, indicating that in this situation other niche factors can compensate for a loss in PRL. The PRL loss resulted in learning and memory deficits in the PRL null mice, as indicated by significant deficits in the standard behavioral tests requiring input from the hippocampus. This behavioral deficit was rescued by direct infusion of recombinant PRL into the hippocampus, indicating that a lack of PRL in the adult mouse hippocampus can be correlated with impaired learning and memory

PRL deficiency results in hippocampal learning defects.

<p>(<b><i>A</i></b>) The PRL null mice spent significantly less time in the novel arm of a Y-maze compared to their wild-type littermates (*p<0.05; PRL^−/−<i>n</i> = 8 animals and PRL^+/+<i>n</i> = 9 animals). (<b><i>B</i></b>) In the reversal version of the water maze, male and female PRL^−/− and PRL^+/+ mice were trained over a period of three days to locate the hidden platform. Prior to the fourth day of training the hidden platform was moved to a novel location and the mice were assessed on their ability to adapt and learn the new platform location. On day 4 the PRL^−/− mice took significantly longer to adapt to the novel location than the PRL^+/+ mice (Student’s t-test *p<0.05; <i>n</i> = 6 PRL^+/+ and 7 PRL^−/− animals).</p

The effects of PRL deficiency on exploratory and anxiety behaviors.

<p>(<b><i>A</i></b>) In the open field test no significant differences were observed in the frequency, total duration, or latency of first occurrence in the inner zone nor in the total average distance moved between the genotypes (<i>n</i> = 43 animals). (<b><i>B</i></b>) Mice were also tested in the Porsolt forced swim test. The time that the mice spent immobile on the second day was measured and no significant difference between the PRL^−/−, PRL^−/+ and PRL^+/+ mice was observed (<i>n</i> = 43).</p

The effects of PRL-deficiency on hippocampal precursor number <i>in vivo</i>.

<p>(<b><i>A</i></b>) No difference was observed in proliferation (BrdU-positive cells; <b><i>A</i></b>), nor in the generation of new neurons (DCX-positive cells; <b><i>B</i></b>) in the hippocampus of PRL^−/− mice compared to wild-type littermates (<i>n</i> = 6 animals per genotype). (<b><i>C</i></b>) A representative image of BrdU-positive (green) and DCX-positive staining in the PRL^−/− hippocampus.</p

The effects of PRL-deficiency on hippocampal precursor number <i>in vitro</i>.

<p>A large decrease in hippocampal-derived neurospheres was observed in PRL^−/− mice compared to wild-type females (***p<0.001; <i>n</i> = 3 PRL ^+/+ and 6 PRL^−/− animals) and males (***p<0.001; <i>n</i> = 9 animals each). There was also a significant decrease in hippocampal-derived neurosphere number in PRL^+/− compared to wild-type females (***p<0.001; <i>n</i> = 3 PRL ^+/+ and 5 PRL^+/− animals) and males (***p<0.001; <i>n</i> = 6 PRL ^+/+ and 7 PRL^+/− animals).</p

The precursor deficit in PRL-deficient mice cannot be rescued by the addition of PRL or KCl-induced depolarizarion.

<p>(<b><i>A</i></b>) The addition of 2 ng/ml PRL to cultures of hippocampal cells from adult PRL^+/+ mice resulted in a significant increase in neurosphere numbers (*p<0.05; <i>n</i> = 6 animals). However, the addition of exogenous PRL to cultures derived from PRL^−/− or PRL^−/+ hippocampi resulted in no increase in neurosphere number (not significant; <i>n</i> ≥ 6). (<b><i>B</i></b>) The PRL^−/−, PRL^+/− and PRL^+/+ hippocampus could all be activated by KCl-induced depolarization <i>in vitro</i> to generate approximately twice as many neurospheres (p<0.01; <i>n</i> = 11 animals per group). (<b><i>C</i></b>) This activation,while significant in the PRL−/− hippocampus was not enough to bring the neurosphere number back to those generated from a PRL wild-type hippocampus. (<b><i>D</i></b>) The addition of purified Wnt3a to primary hippocampal cells from wild-type mice resulted in a significant increase in neurosphere number at concentrations of 5 ng/ml and 10 ng/ml Wnt3a, with the most significant increase observed at 10 ng/ml (*p<0.05, **p<0.01; <i>n</i> = 3 experiments). (<b><i>E</i></b>) The PRL^−/− hippocampus produced a lower number of neurospheres and the addition of Wnt3a to these cells was unable to rescue this deficit (*p<0.05; <i>n</i> = 4 PRL^+/+ and 5 PRL^−/− animals).</p