Allopregnanolone mediates the exacerbation of Tourette-like responses by acute stress in mouse models

Tourette syndrome (TS) is a neuropsychiatric disorder characterized by multiple tics and sensorimotor abnormalities, the severity of which is typically increased by stress. The neurobiological underpinnings of this exacerbation, however, remain elusive. We recently reported that spatial confinement (SC), a moderate environmental stressor, increases tic-like responses and elicits TS-like sensorimotor gating deficits in the D1CT-7 mouse, one of the best-validated models of TS. Here, we hypothesized that these adverse effects may be mediated by neurosteroids, given their well-documented role in stress-response orchestration. Indeed, SC increased the levels of progesterone, as well as its derivatives 5α-dihydroprogesterone and allopregnanolone, in the prefrontal cortex (PFC) of D1CT-7 mice. Among these steroids, however, only allopregnanolone (5–15 mg/kg, IP) dose-dependently exacerbated TS-like manifestations in D1CT-7, but not wild-type littermates; these effects were countered by the benchmark anti-tic therapy haloperidol (0.3 mg/kg, IP). Furthermore, the phenotypic effects of spatial confinement in D1CT-7 mice were suppressed by finasteride (25–50 mg/kg, IP), an inhibitor of the main rate-limiting enzyme in allopregnanolone synthesis. These findings collectively suggest that stress may exacerbate TS symptoms by promoting allopregnanolone synthesis in the PFC, and corroborate previous clinical results pointing to finasteride as a novel therapeutic avenue to curb symptom fluctuations in TS.

aliquots of the homogenate were used for protein determination by a modified Lowry protein assay method (DC protein assay, Bio-Rad Laboratories, Hercules, CA). Equal amounts of proteins were separated on a 4-15% Criterion TGX Precast Gel (Bio-Rad Laboratories) by electrophoresis and transferred to a polyvinylidene difluoride membrane using the Trans-Blot Turbo Transfer system (Bio-Rad Laboratories).
After washing, membranes were incubated with HRP-conjugated secondary antibodies.
Antibody binding was detected using Clarity ECL substrate (Bio-Rad Laboratories) and proteins were analyzed by the ChemiDoc Touch system and the Image Lab software Each cage was mounted on a piezoelectric accelerometric platform connected to an analogue-digital converter. The response to each stimulus was recorded as 65 consecutive 1-ms readings. A dynamic calibration system was used to ensure comparable sensitivities across chambers. The startle testing protocol featured a 70-dB background white noise, and consisted of a 5-min acclimatization period, followed by three consecutive blocks of pulse, prepulse+pulse and "no-stimulus" trials. During the first and the third block, mice received only five pulse-alone trials of 115 dB. Conversely, in the second block mice were exposed to a pseudorandom sequence of 50 trials, consisting of 12 pulse-alone trials, 30 trials of pulse preceded by 73, 76 or 82 dB prepulses intensities (ten for each level of prepulse loudness) and eight no-stimulus trials, where only background noise was delivered. Intertrial intervals were selected randomly between 10 and 15 s. Sound levels were assessed using an A Scale setting. Percent prepulse inhibition was calculated with the following formula: The first and last blocks of 5 pulse-alone bursts were excluded from the calculation. As no interaction between prepulse levels and treatment were found in the statistical analysis, % prepulse inhibition values were collapsed across prepulse intensity to represent average % prepulse inhibition.

Analysis of locomotor behaviors. Spontaneous locomotor responses to novel
environments were tested for 20-30 min in a square force plate actometer (side: 28 cm; height: 30 cm). Each of the four force plate actometers consisted of 4 force transducers placed at the corners of each loading plate. Transducers were sampled 100 times/s, yielding a 0.01 s temporal resolution, a 0.2 g force resolution, and a 2 mm spatial resolution. Custom software directed the timing and data-logging processes via a USB-1208FS interface (Measurement Computing, Norton, MA). Additional algorithms were used to extract macrobehavioral variables. Distance traveled, rotation bias, velocity, thigmotaxis, stride length and stride rate were all analyzed as previously described 7 .
Distance traveled was calculated as the sum of the distances between coordinates of the location of the center of force recorded every 0.50 s over the recording session.
Rotation bias was calculated by summing the locomotor turn direction over time using the center of the actometer floor as a reference point. Velocity was defined as the distance coved by a run divided by the duration of that run and expressed as mm/s (equivalent to the product of stride length and stride rate). Distance to the nearest wall was used as an index of thigmotaxis. Stride length was obtained by dividing the distance covered by a run by the number of strides in that run for each subject. Stride rate was calculated for each trot by dividing the number of half strides by 2 to obtain the number of strides and then dividing the number of strides by the duration of the trot to yield stride rate expressed in Hz.