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Title: Psilocybin induces rapid and persistent growth of dendritic spines in frontal cortex in vivo
Authors: Ling-Xiao Shao1, Clara Liao2, Ian Gregg1, Neil K. Savalia3, Kristina Delagarza1, Alex C. Kwan1,2,3,4
1Department of Psychiatry,
2Interdepartmental Neuroscience Program,
3Medical Scientist Training Program,
4Department of Neuroscience,
Yale University School of Medicine, New Haven, Connecticut, 06511, USA
Correspondence: alex.kwan@yale.edu
Keywords: dendrites; neural plasticity; structural remodeling; serotonergic psychedelic; hallucinogen;
antidepressant
Abstract
Psilocybin is a serotonergic psychedelic with untapped therapeutic potential. Here we chronically imaged
apical dendritic spines of layer 5 pyramidal neurons in mouse medial frontal cortex. We found that a single
dose of psilocybin led to ~10% increases in spine density and spine head width. Synaptic remodeling occurred
quickly within 24 hours and was persistent 1 month later. The results demonstrate structural plasticity that may
underpin psilocybin’s long-lasting beneficial actions.
Main Text
Serotonergic psychedelics are compounds that produce an atypical state of consciousness characterized by
altered perception, cognition, and mood. It has long been recognized that these compounds may have
therapeutic potential for neuropsychiatric disorders including depression, obsessive-compulsive disorder, and
addiction1. Among psychedelics, psilocybin is recently shown to relieve depression symptoms rapidly and with
sustained benefits for several months2, 3. The therapeutic effects may arise from the ability of psilocybin to
induce neural adaptations. The expressions of genes involved in synaptic plasticity are elevated after
administration of serotonergic psychedelics in rats4, 5. In neuronal cultures, bath application of serotonergic
psychedelics induces transient increases in spine size6 and proliferation of dendritic branches7. A recent study
showed that an analogue of ibogaine, a psychedelic with differing molecular targets from psilocybin, increases
spine formation rate in mice8. Thus, there is only limited evidence linking serotonergic psychedelics to
structural plasticity in vivo. Importantly, the time scale in which synaptic remodeling may occur in the
mammalian brain is unknown.
To test the potency and dose dependence of psilocybin in mice, we measured the head-twitch response, a
classic assay for characterizing psychedelic compounds in rodents. We observed that mice would exhibit high-
frequency headshakes intermittently after administration of psilocybin. We characterized 82 C57BL/6J mice
with 5 doses of psilocybin (0, 0.25, 0.5, 1, 2 mg/kg, i.p.; range = 7-10 per sex per dose). A sharp rise of elicited
head-twitch responses occurred at 1 mg/kg (Fig. 1a, b), consistent with a prior report9. Thus, we chose to use
1 mg/kg – the inflection point of the dose-dependence curve – to assess psilocybin’s effect on structural
plasticity.
In the body, psilocybin is dephosphorylated to psilocin, an agonist of 5-HT2A receptors that are densely
expressed in apical dendrites of pyramidal neurons in the medial frontal cortex of primates and rodents10, 11.
We therefore hypothesize that psilocybin may modify the dendritic architecture in the medial frontal cortex. We
used chronic two-photon microscopy to track apical dendritic spines in the cingulate/premotor (Cg1/M2) region
of Thy1GFP mice, in which a sparse subset of layer 5 pyramidal neurons express GFP12 (Fig. 1c, d). We
imaged before and after administering psilocybin (1 mg/kg, i.p.) or saline at 2-day intervals and then again 1
month later for a total of 7 imaging sessions (Fig. 1e, f). In total, we tracked 1,820 dendritic spines on 161
branches from 12 animals. Spine morphology was analyzed blind to experimental conditions using
standardized procedures13. Our results indicate that a single dose of psilocybin induces a significant elevation
in spine density (+7±2% on Day 1, +12±3% on Day 7; main effect of treatment, P=0.011, mixed-effects model
to account for variations across dendrites and mice; Fig. 1g–i) and increase in the width of spine heads
(+11±2% on Day 1, and +5±1% on Day 7; main effect of treatment, P=0.013; Fig. 1j–l, Supplementary Fig.
1). Details for all statistical tests including sample sizes are provided in Supplementary Table 1.
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Figure 1: Psilocybin increases the density and size of dendritic spines in the mouse medial frontal cortex.
(a) Head-twitch responses as a function of dose, tested on 82 C57BL/6J mice. (b) Time course of head-twitch
responses after administrating psilocybin (1 mg/kg, i.p.), averaged from 2 males and 2 female C57BL/6J mice.
Line, moving average. (c) Imaging setup. (d) Fixed coronal section from Thy1GFP mice. (e) Timeline of experiment.
(f) Example field of view. (g) Effects of psilocybin or saline treatment on spine density, plotted as fold-change from
baseline value on Day -3. Mean ± SEM. (h, i) Similar to (g), plotted separately for females and males. (j – l) Similar
to (g – i) for spine head width. Sample sizes and details of the ANOVA models are provided in Supplementary
Table 1.
Increased spine density could be due to higher formation rate, lower elimination rate, or both. To distinguish
between the possibilities, we leveraged the longitudinal data set to determine the turnover rates of dendritic
spines. In females, the spine formation increased by 8±2% after psilocybin (7±1% on Day -1, 15±2% on Day 1;
Fig. 2a, b). Likewise, the spine formation rate was higher by 4±2% in males after psilocybin (6±1% on Day -1,
10±2% on Day 1). By contrast, there was no change in the elimination rate of spines (Fig. 2c). A key question
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was whether the new spines formed after psilocybin administration would persist, because nascent dendritic
spines can take 4 days to mature into functional synapses14. For this reason, we imaged 34 days after
psilocybin administration and observed that a fraction of the psilocybin-induced new spines remained stable
(Fig. 2d). Altogether, these results demonstrate that a single dose of psilocybin induces rapid and long-lasting
dendritic remodeling in layer 5 pyramidal neurons in the mouse medial frontal cortex.
Figure 2. Psilocybin elevates the formation rate of dendritic spines. (a) Example field of view. Purple
arrowhead, stable spine. Green arrowhead, new spine. (b) Effects of psilocybin or saline treatment on the
formation rates of dendritic spines for female and male mice, plotted as difference from baseline value on Day -1.
Mean ± SEM. (c) Similar to (b) for elimination rates. (d) Fraction of spines newly formed on Day 1 that remained
stable on Day 7 and Day 34 for female and male mice. Filled circles, individual dendritic segments. Sample sizes
and details of the ANOVA models are provided in Supplementary Table 1.
To further support the conclusions, we tried to replicate the findings in another cohort of animals using a
different approach. We administered Thy1GFP mice with psilocybin (1 mg/kg, i.p.) or saline, sacrificed them 24
hours later, and imaged coronal brain sections using confocal microscopy. We expanded analyses to 6 areas
of the brain, including 2 zones that encompass apical and basal dendrites and 3 regions of the frontal cortex:
Cg1/M2, prelimbic/infralimbic (PrL/IL), and primary motor cortex (M1) (Fig. 3a–c). The results, consisting of
23,226 dendritic spines counted on 1,885 branches from 12 animals, reaffirmed the ability of psilocybin to
promote the growth of new dendritic spines in Cg1/M2 in female mice (spine density: 0.46±0.02 versus 0.50
±0.01 μm-1; Fig. 3d). Effects of psilocybin on spine density were more pronounced in female animals
(treatment x sex, P = 0.013, two-way ANOVA; Fig. 3d), echoing the trend observed in the two-photon imaging
data (Fig. 1h–i, k–l). We did not detect differences in spine protrusion length and spine head width (Fig. 3e, f),
which may be due to the across-subjects design, as it has less power than the within-subjects design of the
chronic imaging experiment. There were select morphological differences in PrL/IL and M1 (Fig. 3g–n;
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Supplementary Fig. 2). Psilocybin also had significant impact on basal dendrites in Cg1/M2 (Fig. 3o–r).
Overall, the two sets of data converge to indicate that psilocybin promotes the rapid growth of new dendritic
spines in layer 5 pyramidal neurons in the medial frontal cortex.
Figure 3. Region-specific effects of psilocybin. (a) Stitched confocal image of a coronal brain section from a
Thy1GFP mouse. (b) Magnified images showing apical and basal dendritic segments. (c) Images of apical dendrites
in Cg1/M2. (d) Effects of psilocybin and saline on spine density for apical dendrites in Cg1/M2. Open circles,
individual dendritic segments. Gray line, mean ± SEM. (e) Similar to (d) for spine protrusion length. (f) Similar to
(d) for spine head width. (g – j) Similar to (c – f) for PrL/IL. (k – n) Similar to (c – f) for M1. (o – r) Similar to (c – f)
for basal dendrites in Cg1/M2. Sample sizes and details of the ANOVA models are provided in Supplementary
Table 1.
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The regional specificity of the psilocybin-induced structural plasticity is notable, because prior studies indicated
that the cingulate cortex is essential for head-twitch responses in rodents15. The colocalization of head-twitch
responses (which reflect hallucinogenic potency) and dendritic plasticity (which is typically involved in
therapeutic effects) in the same brain region adds to the debate over whether the hallucinogenic effects of
serotonergic psychedelics are dissociable from the therapeutic effects16, 17. By demonstrating that psilocybin
can promote structural plasticity in the mammalian brain in vivo, our study suggests that dendritic remodeling
may be a mechanism shared by compounds with rapid antidepressant effects. For instance, ketamine similarly
increases spine density by elevating spine formation rate in the medial frontal cortex18, 19. However, still
unknown is how drugs with disparate molecular targets may yield comparable circuit-level modifications20.
Elucidating the mechanisms will be crucial towards unraveling the neurobiology of rapid-acting
antidepressants.
Methods
Animals
All experiments were performed on males and females. Thy1GFP (Tg(Thy1-EGFP)MJrs/J, Stock No.007788)
transgenic mice and C57BL/6J (Stock No. 000664) mice were obtained from Jackson Laboratory. For head-
twitch response, 6 to 10-week-old C57BL/6J mice were used. For two-photon imaging, Thy1GFP mice
underwent surgery when they were 6 to 8-week-old and then were used for imaging ~2 weeks later. For
confocal imaging, 8 to 12-week-old Thy1GFP mice were used. Mice were group housed (2 – 5 mice per cage)
under controlled temperature in a 12hr light–dark cycle (7:00 AM to 7:00 PM) with free access to food and
water. Animal care and experimental procedures were approved by the Institutional Animal Care & Use
Committee (IACUC) at Yale University.
Head-twitch response
Head-twitch response was evaluated using 40 male and 42 female C57BL/6J mice. Upon arrival, animals
habituated at the housing facility for >2 weeks before behavioral testing. Behavioral testing took place between
10:00 AM and 4:00 PM. Animals were weighed and injected intraperitoneally with saline or psilocybin (0.25,
0.5, 1, or 2 mg/kg). We measured head-twitch response in groups of two animals: after injections, the two
animals were immediately placed into separate chambers, made by inserting a plastic divider to halve an open-
field-activity box (12” W x 6” H x 10” D). The box was within a sound attenuating cubicle with a built-in near-
infrared light source and a white light source (interior: 28” W x 34” H x 22” D, Med Associates Inc.). Videos
were recorded by a high-speed (213 fps), near-infrared camera (Genie Nano M1280, Teledyne Dalsa)
mounted overhead above the open-field-activity box. Typical recordings were 30 minutes long and, for a
subset of mice (2 males and 2 females), extended to >150 minutes. Between each measurement, the open-
field activity box was thoroughly cleaned with 70% ethanol. The videos were scored for head twitches by an
experienced observer blind to the experimental conditions.
Surgery
Prior to surgery, the mouse was injected with carprofen (5 mg/kg, s.c.; 024751, Henry Schein Animal Health,)
and dexamethasone (3 mg/kg, i.m.; 002459, Henry Schein Animal Health). During surgery, the mouse was
anesthetized with isoflurane (3 – 4% for induction and 1 – 1.5% for the remainder of surgery) and fixed in a
stereotaxic apparatus (David Kopf Instruments). The body of the mouse rested on a water-circulating heating
pad (Stryker Corp) set to 38 °C. Petrolatum ophthalmic ointment (Dechra) was used to cover the animal’s
eyes. The hair on the head was shaved, and the scalp was wiped and disinfected with ethanol pad and
betadine. An incision was made to remove the skin and the connective tissue above the skull was removed.
Subsequently, a dental drill was used to make a ~3-mm-diameter circular craniotomy above the right medial
frontal cortex (center position: +1.5 mm anterior-posterior, AP; +0.4 mm medial-lateral, ML; relative to bregma).
Artificial cerebrospinal fluid (ACSF, containing (in mM): 135 NaCl, 5 HEPES, 5 KCl, 1.8 CaCl2, 1 MgCl2; pH
7.3) was used to irrigate the exposed dura above brain. A two-layer glass window was made from two round 3-
mm-diameter, #1 thickness glass coverslip (64-0720 (CS-3R), Warner Instruments), bonded by UV-curing
optical adhesive (NOA 61, Norland Products). The glass window was carefully placed over the craniotomy and,
while maintaining a slight pressure, adhesive (Henkel Loctite 454) was used to secure the glass window to the
surrounding skull. A stainless steel headplate was affixed on the skull with C&B Metabond (Parkell) centered
on the glass window. Carprofen (5 mg/kg, s.c.) was given to the mouse immediately after surgery and on each
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of the following 3 days. The mouse would recover for at least 10 days after the surgery before the start of
imaging experiments.
Two-photon imaging
The two-photon microscope (Movable Objective Microscope, Sutter Instrument) was controlled by ScanImage
2020 software21. The laser excitation was provided by a tunable Ti:Sapphire femtosecond laser (Chameleon
Ultra II, Coherent) and focused onto the mouse brain with a water-immersion 20X objective (XLUMPLFLN,
20x/0.95 N.A., Olympus). The laser power measured at the objective was ≤ 40 mW. To image GFP-expressing
dendrites, the laser excitation wavelength was set at 920 nm, and a 475 – 550 nm bandpass filter was used to
collect the fluorescence emission. During an imaging session, the mouse was head fixed and anesthetized
with 1 – 1.5% isoflurane. Body temperature was controlled using a heating pad and DC Temperature Controller
(40-90-8D, FHC) with rectal thermistor probe feedback. Each imaging session did not exceed 2 hours. We
imaged apical tuft dendrites at 0 – 200 µm below the dura. To target Cg1/M2 region, we imaged within 0 – 400
µm of the midline as demarcated by the sagittal sinus. Multiple fields of view were imaged in the same mouse.
For each field of view, 10 – 40-µm-thick image stacks were collected at 1 µm steps and at 1024 × 1024 pixels
at 0.11 µm per pixel resolution.
For longitudinal imaging, we would return to the same fields of view across imaging sessions by locating and
triangulating from a landmark on the left edge of the glass window. Each mouse was imaged on days -3, -1, 1,
3, 5 and 7 relative to the day of treatment. A subset of mice (2 males and 2 females) was imaged additionally
on day 34. On the day of treatment (day 0), there was no imaging, and the mouse was injected with either
psilocybin (1 mg/kg, i.p.) or saline (10 mL/kg, i.p.). After injection, the mouse was placed in a clean cage under
normal room lighting to visually inspect for head-twitch responses for 10 minutes, before returning the mouse
to its home cage.
Confocal imaging
Each mouse was injected with either psilocybin (1 mg/kg, i.p.) or saline (10 mL/kg, i.p.). At 24 hr after injection,
the mouse was deeply anesthetized with isoflurane and transcardially perfused with phosphate buffered saline
(PBS, P4417, Sigma-Aldrich) followed by paraformaldehyde (PFA, 4% in PBS). The brains were fixed in 4%
PFA for 24 hours at 4 °C, and then 50-µm-thick coronal brain slices were sectioned using a vibratome
(VT1000S, Leica) and placed on slides with coverslip with mounting medium. The brain slices were imaged
with a confocal microscope (LSM 880, Zeiss) equipped with a Plan-Apochromat 63x/1.40 N.A. oil objective for
dendritic spine imaging and a Plan-Apochromat 20x/0.8 N.A. objective for stitching images of an entire brain
slice.
Analysis of the imaging data
Analyses of the two-photon and confocal imaging data were mostly similar, with an additional pre-processing
step for motion correction of the two-photon imaging data using the StackReg plug-in22 in ImageJ23. Structural
parameters such as spine head width and spine protrusion length were quantified based on a standardized
protocol13, 18. Briefly, if a protrusion extended for >0.4 µm from the dendritic shaft, a dendritic spine was
counted. The head width of a dendritic spine was measured as the width at the widest part of the head of the
spine. The protrusion length of a dendritic spine referred to the distance from its root at the shaft to the tip of
the head. The line segment tool in ImageJ was used to measure the distances. Change in spine density, spine
head width and spine protrusion length across imaging sessions were shown as fold-change from the value
measured on the first imaging session (day -3) for each dendritic segment. The spine formation rate was
calculated as the number of dendritic spines newly formed between two consecutive imaging sessions divided
by the total number of dendritic spines observed in the first imaging session. The spine elimination rate was
calculated as the number of dendritic spines lost between two consecutive imaging sessions divided by the
total number of dendritic spines observed in the first imaging session. To assess the long-term dynamics of the
spine formation and elimination rates across imaging sessions, we calculated the difference from the baseline
rate, which was the spine formation or elimination rate of the same dendritic segment before psilocybin and
saline injection (i.e., from day -3 to day -1). To quantify the persistence of newly formed spines, we calculated
the number of dendritic spines newly formed on day 1 that are still present on day 7 and day 34, and divided by
the total number of newly formed dendritic spines on day 1.
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Statistics
Sample sizes and statistical analyses for each experiment are listed in Supplementary Table 1. Sample sizes
were selected based on previous experiments reported in related publications18, 24. Animals were randomly
assigned in the saline and psilocybin groups. No animals were excluded from data analysis. GraphPad Prism 8
and R were used for statistical analysis. In the figures, data are presented as the mean ± SEM per dendritic
branch.
For in vivo two-photon imaging, dendritic spine scoring was performed while blind to treatment and time.
Longitudinal measurements of dendrite structure were analyzed with mixed effects models for repeated
measures using the lme4 package in R. Linear mixed effects models were preferred to the commonly used
repeated measures analysis of variance (ANOVA) due to fewer assumptions being made about the underlying
data (e.g., balanced sampling, compound symmetry). Separate mixed effects models were created for each of
five dependent variables: fold-change in spine density, fold-change in average spine head width, fold-change
in average spine protrusion length, spine formation rate, and spine elimination rate. Each model included fixed
effects for treatment (psilocybin vs. saline), sex (female vs. male), and time (Day 1, 3, 5, and 7) as factors,
including all second and higher-order interactions between terms. Importantly, variation within mouse and
dendrite across days was accounted by including random effects terms for dendrites nested by mice. Visual
inspection of residual plots revealed no deviations from homoscedasticity or normality. P-values were
calculated by likelihood ratio tests of the full model with the effect in question against the model without the
effect in question. Post hoc t-tests were used to contrast psilocybin and saline groups per day, with and without
splitting the sample by sex, applying Bonferroni correction for multiple comparisons. Spine persistence from
two-photon imaging was analyzed with separate repeated measures ANOVAs for male and female mice, using
fixed effects of treatment (psilocybin vs. saline), time (day 7 vs. day 34), and their interaction as independent
predictors within dendrite.
For confocal imaging data, stricter blinding procedures involved one person performing imaging, another
person scrambling the image file names, and a third person performing dendritic structural measurements blind
to sex, treatment, and brain region. Data were unblinded after all of the measurements were completed. For
each brain region in the confocal dataset (Cg1/M2, PrL/IL, and M1), separate two-way ANOVAs were
constructed for apical and basal dendrites using spine density, spine head width, or spine protrusion length as
the dependent variable. Treatment (psilocybin vs. saline), sex (female vs. male), and their interaction were
included as independent predictors. Post hoc t-tests were used to contrast psilocybin and saline groups within
sex, applying Bonferroni correction for multiple comparisons.
Data availability
The data that support the findings of this study will be made publicly available at https://github.com/Kwan-Lab.
Code availability
The code used to analyze the data in this study will be made publicly available at https://github.com/Kwan-Lab.
Acknowledgements
We thank B. Kelmendi and C. Pittenger for help on obtaining psilocybin, Usona Institute for providing
psilocybin, J. Taylor for use of open-field activity boxes, H. Atilgan and H. Ortega for assistance on setting up
video recording, and A. Halberstadt for advice on scoring head-twitch responses. This work was supported by
the Yale Center for Psychedelic Science, NIH/NINDS training grant T32NS041228 (C.L.), and NIH/NIGMS
Medical Scientist Training grant T32GM007205 (N.K.S.). We thank the Yale Center for Advanced Light
Microscopy Facility for their assistance with confocal imaging, supported in part via NIH grant S10OD023598.
Author contributions
L.X.S. and A.C.K. designed the research. L.X.S. performed the two-photon and confocal imaging experiments,
and analyzed the two-photon imaging data. N.K.S. blinded and I.G. analyzed the confocal imaging data. C.L.
performed and analyzed the behavioral experiments. I.G. and K.D. assisted with analyzing the behavioral data.
N.K.S. assisted with the statistical analyses. L.X.S. and A.C.K. wrote the paper, with input from all other
authors.
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Competing interests
A.C.K. received psilocybin from the investigational drug supply program at Usona Institute. The authors
declare no other competing interests.
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420, 812-816 (2002).
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431629doi: bioRxiv preprint
Supplementary Figures
Supplementary Figure 1: Psilocybin increases spine protrusion length in Cg1/M2. (a) Effects of psilocybin or
saline treatment on spine density in Thy1GFP mice, plotted as fold-change from baseline value on Day -3. (b, c)
Similar to (a), plotted separately for females and males. (j – l) Similar to (g – i) for spine head width. Mean ± SEM.
Sample sizes and statistical analyses are provided in Supplementary Table 1.
Supplementary Figure 2. Effects of psilocybin on basal dendrites in PrL/IL and M1. (a) Images of basal
dendritic segments in PrL/IL from coronal brain sections from a Thy1GFP mouse. (b) Effects of psilocybin and saline
on spine density for basal dendrites in PrL/IL. Open circles, individual dendritic segments. (c) Similar to (b) for
spine protrusion length. (d) Similar to (b) for spine head width. (e – h) Similar to (a – d) for M1. Mean, SEM.
Sample sizes and details of the ANOVA models are provided in Supplementary Table 1.
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431629doi: bioRxiv preprint
