New research in male rats suggests that the timing and duration of testosterone exposure may influence whether newly born brain cells survive to maturity. The study, published in Neuroscience, indicates that testosterone may be most effective at boosting the survival of young neurons during the later stages of their development, particularly within a part of the hippocampus involved in spatial learning and memory. These findings offer new insight into how male sex hormones support brain plasticity in adulthood.
Adult neurogenesis is the biological process by which the mature brain generates new functional nerve cells, or neurons, from dividing stem cells. This process takes place largely inside the hippocampus, a seahorse-shaped structure located deep within the brain’s temporal lobe that is essential for learning, memory formation, and stress regulation. Specifically, new cells are born along a thin band of tissue called the subgranular zone, located within a subregion of the hippocampus known as the dentate gyrus.
Once these precursor cells divide, they migrate into the neighboring granule cell layer and begin extending communication fibers to other parts of the hippocampus. Over several weeks, the surviving cells mature into functional neurons that are highly excitable and adaptable, helping the brain form new circuits and store new memories. However, roughly half of all newly generated cells in the hippocampus naturally die off within the first month before they can fully integrate into the brain’s network.
Hormones are among the major internal factors that regulate how many of these young cells survive. Testosterone, the primary male sex hormone, naturally declines as men age, and low circulating levels of the hormone are associated with an elevated risk of age-related memory decline and dementia, although results from clinical trials testing testosterone treatment for memory in older men have been mixed. In the body and brain, testosterone can act directly on androgen receptors or be converted by enzymes into other active hormones, such as estradiol, a form of estrogen, or dihydrotestosterone, a potent androgen.
“Growing clinical evidence has shown that testosterone can improve or sustain memory in aging men, potentially reducing the risk and severity of age-related memory loss, including Alzheimer’s disease,” senior study author Mark D. Spritzer, a professor of biology at Middlebury College, told PsyPost. “One mechanism by which testosterone may be having these effects is by acting as a neuroprotectant in the brain. The hippocampus is a region of the brain that is essential for learning and memory, and it is an area that degrades with age.”
Previous experiments in male rats have shown that removing the testes, and with them the main source of testosterone, does not change the rate at which new hippocampal cells are born, but it substantially reduces how many of those new cells survive. Restoring testosterone through daily injections for 30 consecutive days reliably rescues these young neurons. In contrast, several studies using shorter treatment windows of 15 to 21 days found no benefit for cell survival, while a recent experiment by the same research group showed that a brief five-day burst of testosterone given between days 11 and 15 after cell birth did improve survival.
This pattern raised questions about whether testosterone needs to build up over a full month to protect new neurons, or if young cells are simply more sensitive to the hormone during specific developmental stages. Between 11 and 30 days after birth, maturing neurons go through a sensitive window where they begin forming active connections, or synapses, with other brain regions. During this same timeframe, chemical signals from a neurotransmitter called GABA shift from exciting the young cells to inhibiting them, a transition that promotes proper maturation.
“We had previously shown that testosterone injections can improve levels of adult neurogenesis (proliferation and maturation of new neurons) within the hippocampus,” Spritzer explained. “However, we did not know whether or not specific periods of neuron development were more strongly influenced by testosterone. Therefore, the current experiment involved giving rats testosterone during different stages of neural development to assess the effects on adult neurogenesis. The ultimate goal is to better understand how testosterone influences the brain and cognition.”
To resolve the contradictions in earlier work, Spritzer, lead author Tatum W. Hamilton, and their colleagues at Middlebury College designed an experiment to test how different windows of testosterone exposure affect neuron survival at 30 days of age. They directly compared a full 30-day treatment against two 15-day treatments covering either the first half or the second half of the cells’ 30-day maturation period.
The researchers worked with 43 adult male Sprague-Dawley rats that were approximately 60 days old at the start of the study. All rats were housed individually to control for social variables, as prior work showed that testosterone’s effects on the brain are easier to detect in individually housed animals. One week after arriving at the laboratory, all of the rats underwent surgical castration to remove their natural source of testosterone, allowing the scientists to precisely control hormone levels through injections.
Eleven days after surgery, every rat received a single abdominal injection of bromodeoxyuridine, a synthetic compound commonly known as BrdU. When cells divide, they incorporate BrdU into their newly synthesized DNA, which permanently tags the exact batch of cells born during the brief window after the injection. By waiting 30 days and then counting how many BrdU-tagged cells remain in the brain, scientists can measure the survival rate of that specific generation of cells.
Starting the day after the BrdU injection, the rats were divided into six groups of six to eight animals each. Half of the groups received daily injections of testosterone propionate at a high physiological dose of 0.500 milligrams per rat; earlier work by the same group found that a lower dose of 0.125 milligrams had no effect on new neuron survival. The other half received inert sesame oil injections as a comparison control. These injections were administered during one of three time windows following the BrdU tag: an early period (days 1 to 15), a late period (days 16 to 30), or the full period (days 1 to 30).
On day 31, the researchers collected blood samples to verify hormone levels and extracted the rats’ brains for microscopic analysis. They sliced the entire hippocampus into thin sections and used chemical staining techniques to make the BrdU-tagged cells visible under a light microscope. A researcher who was blinded to which treatment each rat received counted the labeled cells across the granule cell layer and subgranular zone, as well as in an adjacent area called the hilus to check whether any changes were specific to the neurogenic zone.
The team also divided their cell counts between the dorsal (top/front) and ventral (bottom/back) portions of the dentate gyrus, because these two zones serve different functions. The dorsal hippocampus is primarily involved in spatial navigation and memory, whereas the ventral hippocampus mainly regulates stress and anxiety. Finally, using a laser-scanning confocal microscope and fluorescent markers, the researchers examined 50 randomly selected BrdU-tagged cells per rat to see how many also contained NeuN, a nuclear protein found in mature neurons.
Blood tests confirmed that rats receiving testosterone during the late period and the full 30-day period had elevated serum testosterone concentrations at the end of the experiment, averaging about 5.7 nanograms per milliliter, compared to less than 0.3 nanograms per milliliter in the oil-treated groups and the early-period testosterone group, which had stopped receiving hormone injections 16 days earlier.
Across the entire dentate gyrus, rats treated with testosterone had higher numbers of surviving BrdU-labeled cells in the neurogenic zone than rats given oil. Planned statistical comparisons between the hormone and oil groups within each time window showed that only the full 30-day testosterone treatment produced a statistically significant increase across the whole structure. Rats given testosterone during the late window (days 16 to 30) showed a trend toward higher cell survival across the whole dentate gyrus that fell just short of statistical significance, while early treatment (days 1 to 15) had no effect.
“The results suggest that testosterone may have an additive effect, which is a novel finding,” Spritzer noted. “Namely, longer exposure to testosterone increased neurogenesis to a greater degree.”
When the researchers examined the dorsal and ventral regions separately, a more specific pattern emerged. In the dorsal dentate gyrus, testosterone increased cell survival in both the full 30-day group and the late 16-to-30-day group, while the early 1-to-15-day group showed no benefit over oil injections. In the ventral dentate gyrus, testosterone had no effect on cell survival during any of the three treatment windows, although rats injected during the early period unexpectedly had fewer surviving cells in this region than rats injected during the late or full periods regardless of whether they received hormone or oil.
Fluorescent microscopy confirmed that roughly 79 to 87 percent of the surviving BrdU-tagged cells had matured into NeuN-positive neurons across all groups, regardless of whether the rats received testosterone or oil. Because the hormone did not alter the proportion of new cells that became neurons, the higher BrdU counts can be read as a greater number of surviving new neurons. No treatment differences were found in the overall tissue volume of the dentate gyrus or in cell counts within the neighboring hilus region.
Together, these results suggest that maturing neurons in the dorsal hippocampus are especially responsive to testosterone when they are between two and four weeks old. Because the dorsal hippocampus governs spatial learning, the authors note that this regional sensitivity aligns with past behavioral experiments showing that testosterone improves spatial working memory in male rats. The hormone may help preserve new cells during this late window by blocking programmed cell death, boosting supportive growth proteins like brain-derived neurotrophic factor, or aiding the transition of GABA signals as young neurons wire into existing brain circuits.
“We demonstrated that testosterone enhances adult neurogenesis, with the effects being strongest during the later stages of neuronal development,” Spritzer said. “These results suggest that testosterone may be a useful therapeutic target for helping to sustain neuroplasticity, among men in particular.”
There are a few things to keep in mind when interpreting these findings. First, the experiment was conducted in surgically castrated young adult rats housed in social isolation, which does not mirror the gradual, partial hormone decline experienced by aging men in daily life. Second, the blood and brain tissue were collected 24 hours after the final testosterone injection for the full and late groups, but 16 days after the final injection for the early group. The authors raise the possibility that this gap helps explain why early treatment showed no benefit, but note that this explanation would require new cells to die off at a steady rate throughout development, which earlier research does not support.
“This was preclinical research conducted with rats, so it cannot be extrapolated directly to humans,” Spritzer cautioned. “However, it is suggestive that testosterone may have therapeutic value. Of importance was that we used a high physiological dose of testosterone, and the effects of dose levels have not been considered carefully in past human clinical studies involving testosterone.”
Spritzer also pointed out that boosting cell survival without limit is not necessarily the goal. “One challenge with neurogenesis research is the assumption that more is always better,” he added. “There is likely an optimal level of adult neurogenesis for best brain functioning.”
In addition, the overall statistical analysis did not show that testosterone’s effect differed across the three time windows, so the conclusions about timing rest on separate comparisons within each window, each involving only six to eight rats per group. The study also did not directly measure the molecular pathways inside the cells, so it remains uncertain whether testosterone acted through androgen receptors, estrogen receptors, or downstream growth factors during that 16-to-30-day window. Nor did it test whether the extra neurons translated into better learning or memory. Future research will need to test these specific molecular mechanisms and examine whether similar timing effects apply to older animals or females.
“Now that we know more about the stages of neural development that are influenced by testosterone, assessing the molecular mechanism will be important,” Spritzer said. “Testosterone is metabolized in the brain and likely has many effects on neuronal development, which should be explored.”
The study, “Effects of testosterone upon the survival of new neurons in the dentate gyrus of adult male rats,” was authored by Tatum W. Hamilton, Ezra J. LeMole, Grace K. Nadecki, Jordan M. Gardner, Quinn P. Kennedy, Kathryn A. Blek, and Mark D. Spritzer.