Main Research Findings

1) Both acute and chronic treatment with pregnenolone was found to reduce schizophrenic-like behavior in dopamine transporter knockout mice.

Selected Data
1) The research team of Wong et al investigated the effects of pregnenolone on locomotion, sensorimotor gating, and cognitive function in dopamine transporter knockout mice and wild-type mice. Locomotor activity, rearing, and stereotypy were assessed following drug administration, revealing potential modulations in movement and behavior. The mice, both male and female, were generated by breeding heterozygous DAT mice on a congenic C57BL/6J background, and genotyping was conducted using polymerase chain reaction. The mice were housed in pathogen-free conditions at 22°C with 55% humidity under a 12-hour light/dark cycle, with food and water provided ad libitum [1].
Haloperidol (HAL) and clozapine (CLZ) were dissolved in minimal amounts of 0.1 M HCl and glacial acetic acid, respectively, before dilution in distilled water. Pregnenolone was dissolved in 0.5% sodium dodecyl sulfate (SDS) and then resuspended in peanut oil, which was also used as the vehicle. All injections were administered intraperitoneally (i.p.) or subcutaneously (s.c.) in a volume of 5 ml/kg. Mice were acclimated for 30 minutes before being injected with either vehicle, 0.2 mg/kg HAL, 2.0 mg/kg CLZ, or 30–60 mg/kg pregnenolone, and then returned to the apparatus for a 120-minute monitoring period. Locomotion was assessed as total distance traveled, rearing was measured as vertical movement, and stereotypical activity was recorded as consecutive beam breaks within one second [1].
PPI testing was then conducted using SR-LAB startle chambers. After an initial 10-minute habituation period, mice underwent startle trials with a 40 ms burst of 120 dB white noise. Pre-pulse trials included a 20 ms pre-pulse at 4, 8, or 12 dB above the background noise (64 dB), followed by the 120 dB startle stimulus 100 ms later. Null trials consisted of only the background noise. The PPI response was calculated using the formula: %PPI=(1−prepulse trials startle-only trials)×100. Prior to testing the mice were intraperitoneally injected with a vehicle, 30 mg/kg of pregnenolone, or 60 mg/kg of pregnenolone.
Mice received daily subcutaneous injections of 60 mg/kg pregnenolone for 14 days before being tested in an object recognition memory task. Twenty-four hours after the final injection the mice were exposed to two identical objects in an acrylic test chamber for 10 minutes. Short-term memory was assessed 20 minutes after training, long-term memory was assessed after 24 hours, and remote memory was assessed after 14 days. The objects were chosen for similarity in dimensions and complexity, and the testing area was cleaned with 70% ethanol between trials [1].
The time each mice spent interacting with each object was recorded and analyzed using JWatcher software by an observer blinded to treatment and genotype. Preference scores for the objects were calculated as follows: Preference Score = (Time with novel object − Time with familiar object)/Total time spent with both objects. A positive score indicated a preference for the novel object, a negative score signified a preference for the familiar object, and a score near zero reflected no preference.
To evaluate social memory, mice were subjected to the social transmission of food preference test after 14 days of pregnenolone administration. The tester mice were food-deprived for 16–18 hours before exposure to a demonstrator mouse that had consumed a flavored diet for 30 minutes. After interacting with the demonstrator mouse for 20 minutes, tester mice were given access to both the familiar and a novel flavored diet. Short term memory was tested immediately after this exposure, while long term and remote memory were assessed at 24 hours and 14 days, respectively [1].
The familiar diet was flavored with 1% ground oregano, while the novel diets used for short term, long term, and remote memory tests were flavored with 1% ground thyme, marjoram, or cumin, respectively. Food consumption was measured, and preference scores were calculated as follows: Preference Score = (Amount of familiar diet consumed − Amount of novel diet consumed)/Total amount of both diets consumed. A positive score indicated a preference for the familiar diet, a negative score showed preference for the novel diet, and a score near zero suggested no preference.
Locomotor activity, rearing, and stereotyping were assessed following drug administration, revealing potential modulations in movement and behavior. PPI testing evaluated sensorimotor gating, and object recognition memory tasks examined different stages of memory. The social transmission of food preference test assessed socially transmitted dietary preferences as an indicator of social memory. These findings contribute to the understanding of the role of pregnenolone in modulating behavioral and cognitive functions, potentially implicating its therapeutic relevance in neuropsychiatric disorders [1].
2) The research team of Ramirez et al studied the relationship between levels of hypothalamic pregnenolone levels and the precedence of metabolic diseases and resulting cognitive degeneration. For the purpose of this study C57BL/6J mice from Janvier Labs were utilized, including genetically modified lines. Mice with a floxed allele for Stard1 (Starflox/flox) were crossed with POMC-Cre or AgRP-Cre mice to create POMCStarKO and AgRPStarKO mice, respectively. Additionally, Insulin receptor floxed mice were crossed with POMC-Cre mice to generate POMCInsrKO mice. Control mice were Cre-negative, floxed littermates, all on a C57BL/6 background. Body weight and daily food intake were monitored using a precision scale. Blood glucose levels were measured via tail vein sampling, glucose tolerance tests, and insulin sensitivity tests, that were performed on overnight and 6-hour fasted mice, respectively [2].
Prior to behavioral testing, all mice underwent a basic sensorimotor assessment to ensure similar capabilities for all assigned behavioral tasks. This assessment included 10 standardized tests. Key tests included balance, defined as latency to fall from a wooden beam, tail elevation, scored on position, and the presence or absence of piloerection. Reflex tests, such as paw withdrawal defined as the reaction to pressure on the hind limb and ear withdrawal to touch, were also conducted. Negative geotaxis assessed climbing ability, while visual acuity tested the ability of the animal to respond to a pencil. Additionally, scope response measured forepaw extension when lowered to a surface.
Finally, the olfactory capacity was assessed by presenting different odors such as water, a high-fat diet, green tea, and bedding, and measuring the amount of time each mouse spent sniffing the object. Mice typically showed habituation to repeated odors and dishabituation when a new odor was presented. All tests were video recorded for analysis [2].
The first behavioral test conducted was the Barnes maze that is used to assess hippocampal-dependent spatial memory. It consists of a circular platform with 20 holes, one of which hides an escape chamber. Mice are trained for five days to locate the escape hole under high-intensity light. On the test day, the escape chamber is removed, and mice are evaluated based on the latency to find the target hole, the time spent in the target quadrant, and the total distance traveled.
Next,the novel object recognition test (NORT) was completed in order to measure recognition memory by assessing a mouse’s ability to distinguish between a familiar and a novel object. Mice explore two identical objects during training and are then tested with one novel object 2 hours later. Discrimination indices are calculated based on the time spent exploring each object, with video tracking used for analysis. A similar protocol is used for odor discrimination in which mice are trained to explore two identical odors and later tested with a novel odor. The time spent with each odor is used to calculate a discrimination index [2].
The open field test evaluates general activity of the animals by measuring the total distance traveled and time spent in the center of the arena, while the dark-light box test assesses anxiety by measuring the time spent in a brightly lit area. The elevated plus maze also measures anxiety based on a rodent’s preference for closed arms. For depression-like behavior, the tail suspension test records immobility time when mice are suspended by their tails. Finally, the marble burying test evaluates anxiety or obsessive-compulsive behavior by recording how many marbles mice bury in a bedding-filled arena. All behavioral tasks were video recorded and analyzed using SMART v3.0 software to minimize experimenter interference and ensure accurate data collection [2].

Discussion
1) The research team of Wong et al examined the effects of pregnenolone on locomotor, rearing, and stereotypical activities, by analyzing data collected across a 30-minute baseline period (0–30 min) and a post-injection period (31–150 min). Two-way ANOVA revealed significant genotype effects for locomotion, rearing, and stereotypy, but no significant treatment effects or interactions. Baseline activities were found to be significantly higher in dopamine transporter knockout than wild-type mice. In the post-injection period, significant genotype and treatment effects were observed across all activities, with significant genotype by treatment interactions [1].
Wild type mice showed no response to pregnenolone at either 30 or 60 mg/kg doses, whereas dopamine transporter knockout mice exhibited significantly decreased activity at 30 mg/kg compared to vehicle-treated knockout mice. The 60 mg/kg dose further reduced locomotor and stereotypical hyperactivities relative to 30 mg/kg. Additionally, the 60 mg/kg dose reduced all hyperactivities of dopamine transporter knockout mice to wild type vehicle control levels. These results suggest that administration of pregnenolone reduces hyperactivity in dopamine transporter knockout mice in a dose-dependent manner while exerting minimal effects on wild type mice [1].
To compare the effects of pregnenolone to standard antipsychotics, 0.2 mg/kg of HAL and 2.0 mg/kg of CLA were tested. A two-way ANOVA for the 30-minute baseline period found significant genotype effects on locomotion, rearing, and stereotypy, but no significant genotype by treatment interactions. As expected, baseline activities were higher in dopamine transporter knockout mice than wild type mice. Post-injection analysis revealed significant genotype and treatment effects across all activities, with significant genotype by treatment interactions.
For locomotion, HAL and CLZ significantly reduced activity in both genotypes. While antipsychotics suppressed locomotion in wild type mice more than 60 mg/kg pregnenolone, they had comparable effects in dopamine transporter knockout mice. Both HAL and CLZ significantly decreased rearing in wild type and dopamine transporter knockout mice, while the 60 mg/kg dose of pregnenolone suppressed rearing in dopamine transporter knockout mice but not in wild type mice. Regarding stereotypy, both HAL and CLZ reduced activity in wild type mice, whereas treatment with pregnenolone had no significant effect. In dopamine transporter knockout mice, all three drugs significantly reduced stereotypy to wild type vehicle control levels. These findings suggest that 60 mg/kg of pregnenolone effectively suppresses dopamine transporter knockout hyperactivity to levels similar to HAL and CLZ but without affecting wild type mice [1].

Figure 1: Changes in A) cumulative distance traveled, B) cumulative vertical activity, C) cumulative stereotypical activity, D) cumulative distance traveled post-injection, E) cumulative vertical activity post-injection, and F) cumulative stereotypical activity post-injection, in response to treatment with pregnenolone, HAL, and CLZ.
The study also investigated whether pregnenolone could rescue the prepulse inhibition deficit in dopamine transporter knockout mice. Overall, there were no significant genotype differences in null activity percentages. A two-way ANOVA showed significant genotype effects on startle responses, with dopamine transporter knockout mice exhibiting increased responses, although treatment effects and interactions were not significant. A mixed-design ANOVA revealed significant genotype and treatment effects, as well as a significant genotype by treatment interaction.
Bonferroni-corrected comparisons confirmed PPI dependence on prepulse intensity across all conditions. Administration of pregnenolone in 30 or 60 mg/kg doses did not affect wild type mice. However, in dopamine transporter knockout mice, 30 mg/kg of pregnenolone improved the 4 dB response while responses to 8 and 12 dB prepulses remained deficient. The 60 mg/kg dose fully restored PPI in dopamine transporter knockout mice across all intensities, significantly improving PPI relative to administration of a vehicle and 30 mg/kg pregnenolone. These results indicate that 60 mg/kg of pregnenolone normalizes PPI in dopamine transporter knockout mice [1].

Figure 2: Changes in A) amplitude of startle responses and B) PPI in dopamine transporter knockout mice and wild type mice treated with pregnenolone.
To assess long-term effects, both dopamine transporter knockout mice and wild type mice were treated with 60 mg/kg pregnenolone for 14 days and tested in the novel object recognition test. A mixed-design ANOVA found significant genotype and treatment effects, with a significant genotype by treatment interaction. There was a significant test-day effect but no interactions. During training, neither genotype nor treatment affected object preference. During testing, wild type vehicle-treated mice consistently preferred the novel object across three test days, and this preference remained with pregnenolone treatment. Vehicle-treated dopamine transporter knockout mice displayed no preference across test days, scoring significantly lower than wild type mice [1].
Pregnenolone-treated dopamine transporter knockout mice exhibited significant preference for the novel object across all test days, with scores comparable to wild type mice. One-sample t-tests confirmed that vehicle- and pregnenolone-treated wild type groups and pregnenolone-treated dopamine transporter knockout mice had significant preference scores. To rule out exploration differences, object contacts were counted. A repeated measures ANOVA found a significant test-day effect but no main effects or interactions. Bonferroni corrections revealed no differences across test days, indicating that dopamine transporter knockout deficiencies were not due to reduced object exploration. These results suggest that 14-day of treatment with pregnenolone normalizes novel object recognition deficits in dopamine transporter knockout mice [1].
Finally, the effects of 14-day treatment with pregnenolone on social memory were examined using the social transmission of food preference test. A mixed-design ANOVA revealed significant genotype and treatment effects, with a significant genotype by treatment interaction. No significant test-day effects or interactions were found. Wild type mice preferred the familiar diet across all test days, regardless of treatment. Vehicle-treated dopamine transporter knockout mice showed no preference, scoring significantly lower than wild type controls. Dopamine transporter knockout mice treated with pregnenolone demonstrated a preference for the familiar diet across all test days, with scores comparable to wild type mice. One-sample t-tests confirmed significant preference scores, except for vehicle- and pregnenolone-treated wild type mice during long-term memory testing [1].

Figure 3: Changes in A) preference score for object, B) preference score for diet, and C) total number of contacts made during training, short term memory testing, long term memory testing, and remote memory testing.
To rule out motivational differences, food consumption was analyzed. A repeated measures ANOVA found no significant main effects or interactions, indicating similar motivation between genotypes. These results suggest that 14 days of treatment with pregnenolone normalizes social memory deficits in dopamine transporter knockout mice. Overall, these findings demonstrate that pregnenolone reduces hyperactivity and rescues deficits in PPI, novel object recognition, and social memory in dopamine transporter knockout mice, with minimal effects on wild type mice. The results suggest that pregnenolone has the potential to act as a therapeutic agent for disorders associated with dopamine dysfunction [1].
2) The research team of Ramirez et al investigated the relationship between obesity, type 2 diabetes and cognitive decline by isolating the effects of metabolic alterations from cognitive dysfunction. C57Bl/6J mice were fed a high-fat, high-sucrose western diet for just four days. This short-term dietary intervention did not result in notable metabolic changes, such as obesity or systemic glucose disturbances, which were ruled out as confounding factors in the study.
The researchers tested the effects of the 4 day Western diet on spatial and recognition memory using the Barnes maze test and the novel object recognition test. The short-term Western diet did not interfere with spatial memory, as the mice performed comparably to control (chow-fed) mice in the Barnes maze and no cognitive impairments were observed in spatial memory. However, the Western diet was found to significantly impair recognition memory in the novel object recognition test, as the Western diet-fed mice spent less time exploring the novel object compared to chow-fed mice. This cognitive decline occurred without affecting exploration or locomotor behavior, suggesting that the observed memory deficits were specific to recognition memory rather than general activity or exploration [2].
Given that the hippocampus plays a crucial role in memory formation and flexibility, the study also investigated the impact of the Western diet on synaptic plasticity, particularly long-term potentiation. Long term potentiation is a form of synaptic plasticity strongly associated with memory function. Previous studies have shown that energy-dense diets can reduce the magnitude of long term potentiation in mice. To examine this further, the research team recorded field potentials in the CA1 region of hippocampal slices from C57Bl/6J mice fed either chow or a Western diet for 4 days. The results showed that while baseline synaptic properties, such as input-output relationships and paired-pulse facilitation, were similar between the two groups, acute Western diet feeding significantly reduced the field excitatory postsynaptic potential slope after theta-burst stimulation, indicating that hippocampal long term potentiation was impaired in mice fed a Western diet [2].
Next, in order to determine whether the hypothalamic pregnenolone deficiency contributed to the cognitive impairment, the researchers administered pregnenolone intracerebroventricularly, adjacent to the ARC, of the Western diet-fed mice following the novel object recognition test training phase. This treatment was found to normalize recognition memory in the mice, as shown by improved performance in the novel object recognition test. These results link the action of pregnenolone in the hypothalamus to the restoration of cognitive function, specifically recognition memory, suggesting that the reduction in hypothalamic pregnenolone biosynthesis plays a key role in the cognitive deficits observed in mice fed a high-fat, high-sucrose diet [2].

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

Citations

[1] Wong P, Chang CC, Marx CE, Caron MG, Wetsel WC, Zhang X. Pregnenolone rescues schizophrenia-like behavior in dopamine transporter knockout mice. PLoS One. 2012;7(12):e51455. doi: 10.1371/journal.pone.0051455. Epub 2012 Dec 11. PMID: 23240026; PMCID: PMC3519851.

[2] Ramírez S, Haddad-Tóvolli R, Radosevic M, Toledo M, Pané A, Alcolea D, Ribas V, Milà-Guasch M, Pozo M, Obri A, Eyre E, Gómez-Valadés AG, Chivite I, Van Eeckhout T, Zalachoras I, Altirriba J, Bauder C, Imbernón M, Garrabou G, Garcia-Ruiz C, Nogueiras R, Soto D, Gasull X, Sandi C, Brüning JC, Fortea J, Jiménez A, Fernández-Checa JC, Claret M. Hypothalamic pregnenolone mediates recognition memory in the context of metabolic disorders. Cell Metab. 2022 Feb 1;34(2):269-284.e9. doi: 10.1016/j.cmet.2021.12.023. PMID: 35108514; PMCID: PMC8815774.

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