An analog of psychedelics restores functional neural circuits disrupted by unpredictable stress

This animal study (n=76) tested the rescue effects of a single dose of the ibogaine-analog tabernanthalog (10 mg/kg) administered after mild exposure to unpredictable mild stress in mice and found that it restored deficits in dendritic spine structural dynamics, neuronal activities, and the bottom-up processing of novel contextual information.

Abstract

“Introduction: Psychological stress affects a wide spectrum of brain functions and poses risks for many mental disorders. However, effective therapeutics to alleviate or revert its deleterious effects are lacking. A recently synthesized psychedelic analog tabernanthalog (TBG) has demonstrated anti-addictive and antidepressant potential. Whether TBG can rescue stress-induced affective, sensory, and cognitive deficits, and how it may achieve such effects by modulating neural circuits, remain unknown.

Methods/Results: Here we show that in mice exposed to unpredictable mild stress (UMS), administration of a single dose of TBG decreases their anxiety level and rescues deficits in sensory processing as well as in cognitive flexibility. Post-stress TBG treatment promotes the regrowth of excitatory neuron dendritic spines lost during UMS, decreases the baseline neuronal activity, and enhances whisking-modulation of neuronal activity in the somatosensory cortex. Moreover, calcium imaging in head-fixed mice performing a whisker-dependent texture discrimination task shows that novel textures elicit responses from a greater proportion of neurons in the somatosensory cortex than do familiar textures. Such differential response is diminished by UMS and is restored by TBG.

Discussion: Together, our study reveals the effects of UMS on cortical neuronal circuit activity patterns and demonstrate that TBG combats the detrimental effects of stress by modulating basal and stimulusdependent neural activity in cortical networks.”

Authors: Ju Lu, Michelle Tjia, Brian Mullen, Bing Cao, Kacper Lukasiewicz, Sajita Shah-Morales, Sydney Weiser, Lindsay P. Cameron, David E. Olson, Lu Chen & Yi Zuo

Summary

Psychological stress affects a wide spectrum of brain functions and poses risks for many mental disorders. A recently synthesized psychedelic analog, tabernanthalog (TBG), decreases anxiety levels and rescues deficits in sensory processing as well as cognitive flexibility in mice exposed to unpredictable mild stress.

Introduction

Stress, often caused by unpredictable and unpleasant events and circumstances, pervades modern life and affects the brain at several levels, including synaptic, circuit, and behavioral levels. Chronic stress elevates anxiety and aggression, impairs sensory processing, and deteriorates decision-making and cognitive flexibility.

The pleiotropic effects of stress pose a significant challenge to finding effective therapies, and psychedelics have attracted much research interest as potential treatments for mental illnesses.

A new psychedelic, tabernanthalog, was synthesized and found not to induce the head-twitch response in mice, a rodent behavioral proxy for hallucinations. It exhibits encouraging anti-addictive and antidepressant potential, but its effects on the stressed brain are unknown.

Experimental animals

Thy1-GFP-M and C57BL/6J mice were used in the experiments. They were kept on a 12 h light/dark cycle and were randomly assigned to experimental groups.

Elevated plus maze (EPM)

We used a custom-made plexiglass EPM with four arms and elevated 50 cm from the ground by sturdy metal posts. We used a video-tracking system controlled by Bonsai and custom-written Python 3.6 and Matlab R2019a programs to quantify mouse behavior.

Four-choice odor discrimination and reversal

We modified the protocol described previously [42] and used a four-chamber arena with white ceramic ramekins to present odor stimuli and food reward. The arena was wiped with 70% ethanol between animals.

The mouse was food restricted starting 5 days before the testing day, and was habituated to the arena and ramekins for 1 h on pre-training day 1.

On pre-training day 2, the mouse learned to dig in the media to find buried food rewards. The amount of wood shavings was gradually increased, and the mice completed shaping within 1 h.

On the testing day, the mouse was subjected to a four-choice discrimination session followed by a reversal session. It learned which odor was associated with the buried cereal reward by sniffing and chewing the wood shavings. The mouse was returned to the arena center after each trial, and the ramekins were rearranged and re-baited if necessary. The session criterion was the same as above.

Whisker-dependent texture discrimination (WTD)

The WTD test was performed on free-moving mice. The mice were habituated to the testing chamber for 2 days, then were presented with two columns, one with the same texture as before and the other with a novel texture.

The WTD test was performed on head-fixed mice using the Neurotar mobile home cage. The mouse was habituated to head-fixation on the empty MHC and went through 4 phases: free exploration (5 min), encoding (15 min), resting (10 min), and testing (15 min).

Cranial window implantation and virus injection

We performed cranial window implantation and virus injection on mice around postnatal day 30 according to established protocols with slight modifications.

A circular piece of the skull was removed with a trephine and a high-speed micro-drill, and a custom-made stainless-steel head plate was secured on the skull with dental cement. The mice received antibiotics and analgesics preemptively and daily for 2 more days.

In vivo spine imaging and image analysis

In vivo two-photon imaging of dendritic spines was performed on a 2P microscope equipped with a 16 NA = 0.8 water immersion objective and an ultrafast 2P laser operating at 940 nm. Stacks of images were acquired with a Z-step size of 1 m at 12 zoom.

In vivo wide-field Ca imaging and image analysis

The awake mouse was head-fixed over a custom-built flat rotating disk and imaged with a pair of photographic lenses in tandem coupled to a scientific cMOS camera. A profile view of the mouse was collected concurrently with an infrared camera.

Wide-field Ca imaging data were first processed with pySEAS, an independent component analysis (ICA) filtering method, and then recombined for subsequent data analysis.

To define whisking episodes, we used a grid-based optic flow algorithm to calculate the motion magnitude across grid points, and then used the average motion magnitude of all grid points to represent the magnitude of whisking. The cross-correlation between whisking magnitude and individual pixel’s dF/F0 was calculated using Python.

In vivo 2P Ca imaging and image analysis

Imaging was performed using a 2P microscope, a resonant scanner, and an ultrafast 2P laser operating at 940 nm. Calcium images were taken at 150 – 200 m beneath the pial surface at 30 fps and were motion-corrected and down-sampled to 10 fps by average every three consecutive images.

Ca transients were detected using the Matlab function peakfinder, and the denoised dF/F0 was obtained by setting to zeros the values of the trace below 2 standard deviation of baseline.

Synchronous Ca events were detected by binarizing each neuron’s denoised dF/F0 and then summing the binarized Ca traces of all neurons. We then constructed surrogate population Ca traces and found the 95th percentile value of all surrogate population Ca traces as the synchrony threshold.

We calculated the average touch response of a neuron to each texture by subtracting the average baseline dF/F0 from the average touch dF/F0 for each interaction. We used receiver operating characteristic (ROC) analysis to identify neurons responding to either novel or familiar texture, or both. We classified neurons as either novel texture-selective (NTS) or familiar texture-selective (FTS) based on the area under the ROC curve for discrimination on the basis of decision variable (DV).

In vitro electrophysiology

Mice were anesthetized with isoflurane, decapitated, and their brains were quickly removed and transferred to ice-cold cutting solution. After cutting, the brain slices were immediately transferred to 32 – 34 °C artificial cerebrospinal fluid (ACSF), pH 7.3, 300 mOsm.

Patch-clamp recordings were performed at room temperature on PV+ INs in S1BF L2/3 to measure resting membrane potential, active membrane properties, and action potential discharges. Rheobases, input resistances, and cell excitability were measured using a Multiclamp 700B amplifier, Digidata 1440A, and pClamp10 software.

Quantifications and statistical analyses

We used a two-sided unpaired t-test and one-way ANOVA followed by post hoc Tukey’s multiple comparisons to analyze behavioral and imaging data. The statistical tests were performed with GraphPad Prism 8.4 (GraphPad Software, San Diego, CA).

Post-stress treatment with a single dose of TBG rescues stress-induced behavioral deficits

We used unpredictable mild stress (UMS) to simulate real-life stress and conducted behavioral tests one day after the termination of UMS. We found that UMS mice had higher anxiety levels and lost the capability to distinguish between two textures. We assessed cognitive flexibility with a four-choice odor discrimination and reversal task. UMS mice took significantly more trials to learn the new odor-reward contingency than control mice. We injected TBG (10 mg/kg) into mice immediately after 7d UMS to evaluate its effects on stress-induced behavioral deficits. We found that TBG alleviated stress-induced anxiety, restored the novel texture preference in WTD, and normalized cognitive flexibility in the 4-choice task.

Post-stress TBG treatment promotes dendritic spine regrowth on cortical PyrNs

Ketamine boosts dendritic spine formation and rescues stress-induced behavioral deficits, and TBG promotes dendritic growth and spine formation like classical psychedelics. TBG almost doubled spine formation within a day but did not alter spine elimination. We found that TBG rapidly promotes spine formation and selectively restores lost spines in the post-stress cortex, and that 49.1% of new spines formed within 1-day after TBG treatment survived over 2 days, and 29.1% of them survived over 12 days.

We used wide-field and 2P calcium imaging to examine cortical neural activities. We found that the whole-field Caactivity correlated well with whisking in control mice, and that the whisking-modulation of CaWF was reduced by stress, but was partially rescued by TBG.

We performed 2P Ca imaging of L2/3 PyrNs in awake, head-fixed mice with cellular resolution and found that overall Ca activity was significantly higher in UMS mice, but that post-stress TBG treatment restored the Ca transient size to control level.

TBG rescues the excitability of parvalbumin-expressing inhibitory interneurons (PV+ INs) in the stressed brain

Fast-spiking PV+ INs are the main source of inhibitory inputs onto PyrNs, and their intrinsic excitability is decreased in mice subjected to restraint stress. TBG increases the intrinsic excitability of PV+ INs, which may contribute to the altered baseline activity and synchrony of excitatory neurons in the S1BF.

TBG restores the neuronal population with novel texture-specific response lost in the stressed brain

We studied TBG’s effect on neuronal activities during sensory tasks in head-fixed mice using the Neurotar mobile home cage. We found that TBG restored neuronal activity during both periods back to the control level.

We found that in control mice, 32% of neurons were novel texture-selective, while 6% of neurons were familiar texture-selective. After stress, the percentage of neurons that responded to the novel texture was dramatically reduced, but was partially recovered by TBG.

Discussion

A single dose of TBG (10 mg/kg) administered after stress can rescue deficits in dendritic spine structural dynamics, neuronal activities, and behavior. The efficacy of TBG at the lower dosage suggests that it has a broader therapeutic window than originally thought.

Chronic stress is thought to be due to allostatic overload. TBG may increase the allostatic capacity of the brain by promoting dendritic spine formation, which may help the brain cope better with subsequent behavioral tasks.

Human and rodent studies have demonstrated that psychological stress adversely affects sensory processing. We found that stress diminished the novelty-dependent response of S1BF neurons during texture interactions, which may reflect the top-down modulation of sensory information.

Cortical L2/3 neurons receive inputs from multiple regions, including the thalamus, the posterior medial thalamus, and the ventral posteromedial nucleus. Their activity is modulated by both somatosensory and motor signals, and TBG likely acts on both local and distant circuits.

The study shows that TBG promotes spine formation on L5 PyrNs, which is consistent with the spinogenesis-promoting effect of psychedelics such as LSD, DOI, and DMT, as well as non-psychedelic N,N-dimethylaminoisotryptamine (isoDMT) analogs, observed in vitro.

TBG may promote spine formation and behavior by activating 5-HT2A receptors in cortical L5 PyrNs and middle-layer PV+ INs, but the pharmacological targets for TBG’s therapeutic effects remain to be elucidated.

While TBG does not produce behavioral responses characteristic of serotonergic hallucinogens in rodents, it promotes rapid spine formation and slightly elevates their rate survival, leading to more newly formed spines being consolidated into the neuronal network.