Disruption of the non-canonical wnt gene PRICKLE2 leads to cerebellar Purkinje cell abnormalities while cerebellar-mediated behaviors remain intact.

doi:10.21203/rs.3.rs-3112261/v1

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Disruption of the non-canonical wnt gene PRICKLE2 leads to cerebellar Purkinje cell abnormalities while cerebellar-mediated behaviors remain intact.

https://doi.org/10.21203/rs.3.rs-3112261/v1

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published 02 Jan, 2024

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Autism spectrum disorders (ASD) involve brain wide abnormalities that contribute to a constellation of symptoms including behavioral inflexibility, cognitive dysfunction, learning impairments, altered social interactions, and perceptive time difficulties. Although a single genetic variation does not cause ASD, genetic variations such as one involving a non-canonical Wnt signaling gene, Prickle2, has been found in individuals with ASD. Previous work looking into phenotypes of Prickle2 knock-out (Prickle2^−/−) and heterozygous mice (Prickle2^−/+) suggest patterns of behavior similar to individuals with ASD including altered social interaction and behavioral inflexibility. Growing evidence implicates the cerebellum in ASD. As Prickle2 is expressed in the cerebellum, this animal model presents a unique opportunity to investigate the cerebellar contribution to autism-like phenotypes. Here, we explore cerebellar structural and physiological abnormalities in animals with Prickle2 disruption using immunohistochemistry, whole-cell patch clamp electrophysiology, and several cerebellar-associated motor and timing tasks, including interval timing and eyeblink conditioning. Histologically, Prickle2^−/− mice have significantly more empty spaces or gaps between Purkinje cells in the posterior lobules and a decreased propensity for Purkinje Cells to fire action potentials. These structural cerebellar abnormalities did not impair cerebellar-associated behaviors as eyeblink conditioning and interval timing remained intact. Therefore, although Prickle^−/− mice show classic phenotypes of ASD, they do not recapitulate the involvement of the adult cerebellum and may not represent the pathophysiological heterogeneity of the disorder.

cerebellum

autism spectrum disorders

cognition

Purkinje cells

wnt signalling

Autism Spectrum Disorders (ASD) involve neurodevelopmental impairments that are characterized by atypical social interactions, disrupted verbal and non-verbal communication, highly restricted interests, and repetitive behaviors (Geschwind & Levitt, 2007). Although these are core symptoms and identifying markers of ASD, they vary widely in their severity and presentation between affected individuals (Bryson & Smith, 1998).

ASD is associated with a large degree of phenotypic heterogeneity across patients making it difficult to implicate specific brain regions or circuits in disease pathogenesis. Neuroanatomically, patients with ASD have abnormalities in the anterior cingulate cortex, dorsolateral prefrontal cortex, medial frontal cortex, temporal lobe, parietal lobe, amygdala, and hippocampus among other regions (Amaral, Schumann, & Nordahl, 2008; Donovan & Basson, 2017; Palmen, 2004). These abnormalities include microstructural differences, altered connectivity between brain regions, and hyper- and hypoplasia of the developing brain (Donovan & Basson, 2017). Interestingly, the cerebellum is consistently abnormal in ASD and often includes a reduced density and size of Purkinje cells (PCs) throughout the cerebellum in postmortem analyses (Bailey et al., 1998; Fatemi et al., 2012; Oristaglio et al., 2013).

Along with studying ASD in the human brain, animal models have been pivotal to expanding our understanding of the cerebellum in ASD. Importantly, these models mimic human findings of Purkinje cell deficiencies and ASD-like behavior (Mapelli et al., 2022). The Lurcher mouse model of Purkinje cell loss involves a gain of function mutation in the δ2 glutamate receptor gene. Lurcher mice display behaviors similar to humans with ASD including reduced behavioral flexibility, increased activity, and repetitive behaviors (Dickson et al., 2010; Martin et al., 2010). Additionally, a mouse deficient for tuberous sclerosis-I (TSC1) protein in Purkinje cells displays social deficits similar to human patients with ASD (Stoodley et al., 2017; Tsai et al., 2012). These results highlight the importance of the cerebellum and, specifically, the Purkinje cells in ASD.

Alongside the numerous brain regions contributing to ASD symptomology, many candidate genes have been identified, complicating our understanding of the etiology of the disorder (Geschwind, 2011; Muhle et al., 2004). A gene discovered in ASD, Prickle2, was first described in studies looking at patients with interstitial microdeletions of chromosome 3p14. All but one of the patients studied presented either a complete or partial deletion of Prickle2 and all had marked intellectual disability and autism phenotypes (Okumura et al., 2014; Schwaibold et al., 2013). Additionally, the Prickle2 gene was characterized in a knockout mouse model and indicated as a potential model of ASD based on autism-like phenotypes (Sowers et al., 2013). Several characteristic autism-like behaviors were observed, including decreased behavioral flexibility, slower reversal learning, reduced social interest, and reduced dendritic arborization in the hippocampus contributing to impaired performance on hippocampus-dependent learning tasks such as contextual fear conditioning and Barnes maze spatial memory learning. The role of the cerebellum in the pathophysiology of the ASD-like behavior in the Prickle2 knock out mouse (Prickle2 ^−/−) was not considered.

PRICKLE2 is a planar cell polarity (PCP) protein in the non-canonical Wnt signalling pathway (Sowers et al., 2013; Veeman et al., 2003). Murine PRICKLE2 protein is expressed in post-mitotic neurons and has been implicated in neuron maturation and neurite outgrowth via the Dishevelled dependent pathway (Fujimura et al., 2009; Mrkusich et al., 2011; Tissir & Goffinet, 2006). Studies have shown that silencing or downregulating the Prickle2 gene decreases neurite outgrowth in mouse neuroblastoma Neuro2a cell line preparations (Okuda, Miyata, Mori, & Tohyama, 2007).

Prickle2 is present within the mouse cerebellum at embryonic day 17.5, but its involvement in cerebellar pathology relevant to ASD remains to be addressed (Tissir & Goffinet, 2006). Here, we investigated the effect of Prickle2 disruption on the structure and functional roles of the cerebellum, including timing and motor-mediated tasks, that are implicated in ASD.

Prickle2 mutant mice

Gene targeting in TT2 embryonic stem cells was used to generate the Prickle2 mutant mice (Acc. No. CDB0435K) (Yagi et al., 1993) as described (http://www.cdb.riken.go.jp/arg/protocol.html). The Prickle2 mutant mouse line was backcrossed onto the C57bl6/J greater than 10 generations (Sowers et al., 2013). In total, 39 (28 male, 11 female) Prickle2^+/+ and Prickle2^−/− littermates aged 3–5 months with a body weight of at least 20 g were included in this study. Sexes were combined due to no significant sex effects. These experiments were approved by the University of Iowa Institutional Animal Care and Use Committee and adhered to the NIH Guide for the Care and Use of Laboratory Animals. Homozygous and wild-type genotypes were confirmed using PCR (For WT, Primer1: 5’-GAC CTC ATC TAC TTT TAC CAA-3’; Primer2: 5’-TAC TAC CAC CCA CTT TAT TCT-3’. For KO, Primer3: 5’-GGC TCT TTA CTA TTG CTT TAT-3’; Primer2: 5’-TAC TAC CAC CCA CTT TAT TCT-3’).

Immunohistochemistry

Mice (6 Prickle2^+/+ and 5 Prickle2^−/−) were intracardially perfused with PBS followed by 4% paraformaldehyde. The brain was removed and post-fixed in paraformaldehyde overnight. The brain was then stored in 30% sucrose for one day to allow for cryopreservation, indicated by the brain sinking to the bottom of the vial. Brains were sectioned in 40 µm increments on a cryostat (Leica) and stored in cryo-protectant (50% PBS, 20% glycerol, 30% ethylene glycol) at -20 ºC. Slices were mounted and dried overnight before beginning immunostaining procedures. Primary antibodies Calbindin, a reliable marker of cerebellar Purkinje cells (anti-rabbit; Thermofisher; Millipore), GFAP (anti-rat; Thermofisher; Location; 1:500) a marker of Bergmann glia, and DAPI (1:1000) for all neurons, were incubated on the tissue overnight at 4 ºC. Sections were visualized with Alexa Fluor fluorescent secondary antibodies (goat anti-rabbit IgG Alexa 488, goat anti-rat IgG Alexa 568, DAPI 504) and matched with the host primary by incubating for 2 hours. Images were captured using a Zeiss ApoTome.2 (Axio Imager M2). GFAP and DAPI was used for visualization of the Purkinje cells and cerebellar anatomy and were not quantified in these experiments.

Purkinje cell counting

Purkinje cell counts were obtained using Stereo Investigator software (MBF Bioscience, Williston, VT) using the optical fractionator (Olesen et al., 2017). We restricted our analysis to the vermis due to known vermal hypoplasia in ASD (Carper, 2000; Courchesne et al., 1988; Kaufmann et al., 2003). Sagittal cerebellar sections spanning 8 slices to the left and right of the midline (16 total at 40 µm; 640 µm total width of the midline cerebellum) were divided into the cerebellar lobules. Contours were drawn around the Purkinje cell layer of each lobule and randomized locations within a generated grid within the contours were selected by the optical fractionator algorithm. Only cells within the counting frame were included.

Purkinje cell gapping

In the same histological preparations for cell counting, lines were drawn within the Stereo Investigator software to measure the pixel length of each lobule and to measure the perimeter of the lobule. Gaps between calbindin-positive Purkinje cells were then measured and averaged for each lobule. Pixels were then converted to micrometers.

Patch clamp electrophysiology

300-µm parasagittal cerebellum vermis brain slices from adult male animals (6 animals; 3 Prickle2^+/+, 3 Prickle2^−/−) were prepared following previously established protocols (Piochon et al., 2016). Briefly, the cerebellum was dissected from 8- to 12-week-old mice in ice-cold slicing buffer (225 mM sucrose, 26 mM NaHCO3, 1.2 mM KH2PO4, 1.9 mM KCl, 10 mM d-glucose, 1.1 mM CaCl2, and 2 mM MgSO4) bubbled with 95% O2 and 5% CO2. Prepared slices were transferred to oxygenated artificial cerebrospinal fluid (aCSF) (127 mM NaCl, 26 mM NaHCO3, 1.2 mM KH2PO4, 1.9 mM KCl, 10 mM d-glucose, 2.2 mM CaCl2, and 2 mM MgSO4) at 32°C for 1 hour or more, then transferred to a submersion recording chamber perfused with aCSF at 32–34°C a rate of approximately 3 ml/min. Whole-cell recordings of Purkinje neurons were performed using 2.5- to 4-MΩ pipettes. Current-clamp recordings were obtained using a MultiClamp 700B Microelectrode Amplifier (Molecular Devices) and analyzed offline by MATLAB (Mathworks). Experiments were discarded if access resistance varied by more than 20%. Tonic current was injected to hold the membrane voltage stable at -65 mV, and depolarizing current was injected in 50 pA increments. The afterhyperpolarization (AHP) of an action potential (AP) was calculated by finding the minimum of the voltage trace that started at the time of the AP peak and ended at the time of the peak of the following AP. The lowest amplitude current injection that resulted in APs was used, and the average of all AHPs in that sweep is reported here. Data are from 1–3 cells per animal and are plotted as averaged recordings from each cell (≥ 3 recordings per cell).

Motor function

Mice were tested for basic motor impairments, first using distance traveled in an open field, then observing ability to maintain speed with an accelerating rotarod, and finally using time to transverse a balance beam.

Open field

Mice (9 Prickle2^+/+, 12 Prickle2^−/−) were placed in a 43.2 cm x 43.2 cm x 30.5 cm open field arena (Med Associates) for 10 min to assess distance traveled in the arena. Behavior was monitored using real time video imaging via a camera placed above the arena that was synced with CinePlex Studio (Plexon, Dallas, TX) allowing center of mass tracking. The open field test allows for the observation of the animals as they explore unfamiliar terrain and perform general locomotor activity. The distance traveled was calculated using MATLAB (Mathworks).

Rotarod

Mice (9 Prickle2^+/+, 12 Prickle2^−/−) were tested on an accelerating rotarod apparatus (Med Associates, St Albans, VT) consisting of a motor powered rotating rod that accelerated in speed of rotation over time to assess motor learning. For these trials the rotarod was set to accelerate from 4 to 40 rpm over the course of five minutes after which 40 rpm was maintained until the end of the trial. Motor testing was analyzed based on the length of time the animal could maintain running on the rod as it accelerated. Rodents were tested three times per day (at least 15 minutes between trials) for three days by placing them on the rod and recording latency to fall or failure to maintain running for two consecutive rotations should the mouse not independently fall off. The trials were stopped at 500 seconds if the animals did not fall.

Balance Beam

To test motor impairments in the Prickle2 mice, time to transvers a custom-made narrow square beam (84 cm x 1.2 cm x 1.2 cm) was analyzed (9 Prickle2^+/+, 12 Prickle2^−/−). The setup of the balance beam apparatus included a dark enclosure at one end of the beam and an open box at the other end that mice were placed into at the start of the trial. As mice prefer darkness over light and open spaces, they are motivated to transverse the beam to reach the dark enclosure on the opposite end. Time to traverse the beam was recorded over three days with three trials per day.

Delay eyeblink conditioning

Mice (12 animals; 5 Prickle2^+/+, 7 Prickle2^-/-) were trained on delay-275 millisecond eyeblink conditioning. First, animals underwent surgery under isoflurane anesthesia for implantation of electromyographic (EMG) nickel wires and stimulating nickel wires, as described previously (Freeman & Nicholson, 1999). The 2 EMG wires were threaded through the eyelid muscle (orbicularis oculi) and connected via gold pins to a plastic connector. The Teflon coating was stripped near the eyelid to allow for electrical contact with the eyelid to measure eyeblink position. The 2 shock wires were implanted subdermally, immediately caudal to the left eye and then connected to the same plastic connector as the EMG wires. The full assembly was then grounded to a stainless-steel skull screw and cemented with dental acrylic. Mice were connected to a lightweight cable that allowed for free movement inside of the conditioning chamber (BRS/LVE, Inc.).

Eyeblink conditioning was performed by repeatedly pairing a conditioned stimulus (CS- blue LED) with an aversive periorbital shock for the unconditioned stimulus (US; Fig. 4A). Conditioned responses (CRs) were recorded as EMG signals from the nickel wires. A conditioning session was comprised of 100 trials, with a probe trial occurring after every 10 trials. Each trial was separated by a randomized intertrial interval between 15 and 40 seconds. Responses that occurred 100 ms after the CS were considered startle responses and were excluded. Once mice reached criterion of 80% CRs in a single session, they started extinction training where only the CS was presented without the US shock to reduce the association between the CS and US over time. Data were collected using MATLAB scripts for extracting EMG traces, time-locking to events, and quantifying CR properties of latency, amplitude, and timing.

Interval timing task

Mice motivated by 85–90% food restriction were tested on a 12 second fixed interval timing task (9 Prickle2^+/+, 12 Prickle2^−/−; Fig. 5A). Training was performed in an operant chamber (MedAssociates) equipped with a nose poke port containing a yellow LED light (ENV-313W), a pellet dispenser (ENV-203-20), and a house light (ENV-315W). The entire configuration was housed in sound-attenuating chambers (MedAssociates). Nose pokes and the acquisition of rewards were recorded using infra-red beam breaks as the mouse accessed the reward nose poke port. First, animals learned that poking their nose into the nose poke port released their food pellets (20-mg rodent purified pellets, F0071, BioServe). Next, the mice were trained in a 12 second fixed-interval timing task. Rewards were delivered for nose pokes made after 12 seconds had elapsed from the time the nose poke port was illuminated to indicate the trial start. The mice were not punished for responding early but lack of responding for 18 seconds resulted in trial termination and the illuminated nose poke turned off with no reward administered. For rewarded nose pokes, the house light illuminated and remained lit until the animal collected the reward. Between each trial was a 24 ± 6 second pseudorandom interval after which the nose poke hole re-illuminated to indicate the start of a new trial. These trials were repeated for 60 minutes.

To assess interval timing performance we analyzed response efficiency, response start-times, number of responses and number of rewards. Response efficiency, indicating how well an animal estimated the interval, was analyzed as the number of well-timed responses between 11 and 12 s divided by the overall number of responses during the 12 s trial. Efficiency scores closer to 1 reflect a greater number of responses occurring near the to-be-timed interval, indicating more temporally guided performance (Parker, Chen, et al., 2015; Parker, Ruggiero, et al., 2015). Single-trial start times were also analyzed as mice begin by responding at a constant, low rate on individual trials and increases responses in anticipation of reward. Time-response histograms were normalized to total responses to accurately demonstrate timing regardless of response rate.

Statistics

Statistical calculations were performed using GraphPad Prism 9. Data are visualized as mean ± SEM. Data were tested for normality using a Shapiro-Wilk test. When data were not normally distributed, a non-parametric statistical test was used. All histological comparisons were performed within-lobule due to size differences between each lobule, and an unpaired t-test was used for analysis. In the patch clamping data set, the frequency values were analyzed via a two- repeated measures ANOVA; baseline potential, fast afterhyperpolarization, resistance, and capacitance were analyzed with a two-tailed t-test. Behavioral testing was compared using a one-way ANOVA for data sets that occurred in one day and repeated-measures 2-way ANOVA for data sets that spanned multiple days. An alpha level of 0.05 was used for all assessments.

Immunohistochemistry

Immunohistochemical analysis of vermal cerebellar sections for the expression of calbindin indicated no significant differences in the overall number of Purkinje cells between the Prickle2^−/− and Prickle2^+/+ mice (Fig. 1A;1B). However, although posterior lobules (VI-X) had the same number of gapping or space between positively labeled Purkinje cells (Fig. 1C), there was a significant difference in the length of the gaps (Fig. 1D, multiple t-tests per lobule; IV,V p = 0.006; VI p = 0.037; VII p = 0.0004; VIII p = 0.03; IX p = 0.02; X p = 0.012). There were no differences for the total length of the perimeter within each lobule (Fig. 1E).

Patch clamp electrophysiology

Given the prior characterization of Purkinje cell abnormalities in ASD and our finding of increased length of gaps between Purkinje cells in Prickle2^−/− animals, we used whole-cell patch clamp electrophysiology to define their function (Fig. 2A). Purkinje cells recorded from acute sagittal cerebellar vermal slices revealed typical high-frequency trains of action potentials in both Prickle2^−/− and Prickle2^+/+ animals when injected with current, and output frequency was statistically significant (2-way ANOVA, current-by-genotype, p = 0.0067; main effect for current, p < 0.0001; main effect for genotype, p = 0.0019) (Fig. 2B). The baseline membrane potential was unchanged in the Prickle2^−/− mice (Fig. 2C). Interestingly, the peak of the fast after-hyperpolarization was significantly depolarized in Prickle2^−/−, thus the Purkinje cells have less hyperpolarization following an action potential (Fig. 2D, unpaired two tailed t-test, p = 0.0169). The membrane resistance, baseline potential, and capacitance of the Purkinje cells in the Prickle2^−/− vermal cerebellar sections were not significantly different (Fig. 2E-F). Thus, Purkinje cells of Prickle2^−/− mice were less excitable and were unable to maintain firing frequencies that were comparable to Prickle2^+/+ animals when driven by current injection.

Motor Function

Performance of Prickle2 mice was assessed on the open field test, the balance beam test, and the rotarod test revealing no statistically significant differences between genotypes (Fig. 3A; 3B; 3C). Despite the observed Purkinje cell abnormalities, there were no gross motor impairments.

Eyeblink Conditioning

Prickle2 ^−/− and Prickle2^+/+ animals were both able to acquire delay eyeblink conditioning (2-way ANOVA, time-by-genotype, p = 0.1240; Fig. 4A) and extinguish the learned responses with no differences between the genotype (2-way ANOVA, time-by-genotype, p = 0.1342; Fig. 4B). Additionally, there were no significant differences in the CR amplitude, CR duration, CR latency, or percent of startle responses (Fig. 4C-F).

Interval Timing

The efficiency of timing estimation on the 12-second fixed interval timing task (Fig. 5A-B) was not statistically different between Prickle2^+/+ and Prickle2^−/− mice (Fig. 5C). The animals did not differ on the total number of responses (Fig. 5D), the average time they started responding over the training days (Fig. 5E), or the number or rewards obtained (Fig. 5F).

A full or partial deletion of PRICKLE2, a planar cell polarity (PCP) protein in the non-canonical Wnt signalling pathway, is associated with intellectual disability and autism phenotypes (Okumura et al., 2014; Schwaibold et al., 2013). Although Prickle2 is present within the mouse cerebellum at embryonic day 17.5, and continuous research heavily implicates the cerebellum in ASD pathology, our results suggest that brain wide disruption of Prickle2 does not affect cerebellar-mediated tasks that are associated with ASD, despite our observations of abnormal gapping between Purkinje cells (Fatemi et al., 2012; Tissir & Goffinet, 2006).

Purkinje cells are normally arranged in a layer between the granular and molecular layers of the cerebellum. Purkinje cells are the sole output of the computations that take place in the cerebellar cortex, relaying activity to the deep nuclei which project out of the cerebellum to downstream brain structures like the thalamus (Voogd, 1998). Although the overall count of Purkinje cells is not significantly decreased in the Prickle2^−/− mice, our results reveal a notable gapping between Purkinje cells in the posterior cerebellar vermis. The posterior cerebellar lobules of the vermis have been shown to be involved in emotional regulation (Pierce et al., 2022; Schmahmann, 2019). Additionally, some Purkinje cell bodies appear ectopic and stacked-on top of each other or extending into the molecular layer of the cerebellum (see Fig. 1A). Typically, Purkinje cell bodies reside within the Purkinje cell layer.

Given that PRICKLE2 has been implicated in neuron maturation and neurite outgrowth via the Dishevelled dependent pathway, our results showing potential misplacement of Purkinje cells outside the appropriate Purkinje cell layer may be consistent (Fujimura et al., 2009; Mrkusich et al., 2011; Tissir & Goffinet, 2006). The polarity of cells is established with the involvement of Dishevelled non-canonical Wnt signaling, where PRICKLE1 and PRICKLE2 proteins are thought to be involved in the proximal side of a cell during polarity processes (Carreira-Barbosa et al., 2003). However, whether the specific role that PRICKLE2 plays in planar cell polarity could have driven the Purkinje cell gapping requires future studies (Veeman et al., 2003). There is recent evidence from vascular endothelial cells that describes non-canonical Wnt signaling’s role in cellular mechanocoupling (Carvalho et al., 2019). Although Purkinje cells are not mechanically linked together, they do have a vital relationship with neighboring cells through ephaptic coupling that when disrupted, could alter migration patterns during development (Han et al., 2018). Although our data do not support a significant loss of Purkinje cells in the posterior cerebellar vermis, alterations in migration patterns could underly the gapping between Purkinje cells in the Purkinje cell layer. These findings are not consistent with reports of vermal Purkinje cell loss in patients with ASD (Courchesne et al., 1988; Fatemi et al., 2012).

Purkinje cells of Prickle^−/ were less excitable than Prickle2^+/+ mice as evidenced by the I-F curve (Fig. 2B). That is, they generated fewer action potentials for a given excitatory drive. With no stark behavioral changes related to the cerebellum, the differences we observed in the electrophysiology of Purkinje cells could be attenuated downstream to account for normal cerebellar-mediated behavior. Testing this would require looking at downstream areas that receive cerebellar input such as the thalamus to determine whether there is still proper functioning despite the decreased Purkinje cell firing frequency. It is well known that sustained Purkinje cell firing is certainly necessary for proper downstream signaling and learning (Freeman, 2015; Ito, 2002). One potential aspect of the Purkinje cell that could be impaired in Prickle2^−/− mice comes from recent work that shows Purkinje cell afterhyperpolarizations are mediated by BK-type potassium channels, which when downregulated decreases action potential frequency (Dell’Orco et al., 2017; Niday & Bean, 2021). This would fit with our current evidence, given that the fast afterhyperpolarization was more depolarized and produced lower firing frequencies. However, testing BK channels directly would be required to confirm whether they are driving the effect.

It is not known how non-canonical Wnt signaling could play a role in the dynamics of action potentials in Purkinje cells. If BK channels are impacted, there is some evidence that in HEK293 cells, alcohol was shown to regulate BK channel expression through Wnt signaling (Velázquez-Marrero et al., 2016). Whether such a mechanism also operates through prickle2-involved non-canonical Wnt signaling would require further testing. Wnt signaling plays a role in ion channel dynamics which could account for differences in action potentials. Several potassium and calcium channels have been shown to effect Wnt signaling. If Wnt signaling was disrupted, it is possible that ion channels that play a role in metabolic pathways could respond to that feedback and operate differently than normal physiological states (Muccioli et al., 2021). Future Purkinje cell electrophysiological experiments that probe ion channel function through pharmacology or calcium imaging, are needed to define the role of calcium channel activity and potential dysfunction in Prickle2^−/− mice. Additionally, Purkinje cells within each lobule of the cerebellum should be further characterized to define the electrophysiological properties of those that are ectopic, stacked, and those that migrated to the normal position in the Purkinje cell layer. Further subdividing the Purkinje cells in Prickle2^−/− mice could provide further insight into the functional differences and implications of gapping in the Purkinje cell layer.

There are several limitations to this study. Our focus on the cerebellar vermis may not accurately reflect the structural abnormalities reported in humans with ASD and many animal models given recent evidence localizing cognitive function to the lateral cerebellar hemispheres (Hampson & Blatt, 2015; Shipman & Green, 2019; Stoodley et al., 2017). Post-mortem analysis of cerebellar tissue in patients with ASD commonly reveals a loss of Purkinje cells, largely localized to the lateral hemispheres, as well as ectopic Purkinje cell localization outside of the Purkinje cell layer (Bauman & Kemper, 1985; Hampson & Blatt, 2015; Palmen, 2004; Whitney et al., 2008). Additionally, Purkinje cells at the base of the primary fissure of hemispheric Lobule VI (HVI) are known to be critical for proper eyeblink conditioning (Attwell et al., 2001; Heiney et al., 2014; Steinmetz & Freeman, 2014). Given that we observed no differences in eyeblink conditioning, we would hypothesize the Purkinje cells are normal in this region but should be explicitly investigated. Further, aldolase-C/Zebrin II striping of the cerebellum could provide further specificity to the lobular organization of Purkinje cell migration and affected function in the Prickle2^-/- mice. (Brochu et al., 1990).

The cerebellum is necessary for learning and error detection (Popa & Ebner, 2019). Therefore, more robust differences may be identified by analyzing of nuanced aspects of a cerebellar-dependent learning. For eyeblink conditioning, longer interstimulus intervals could be used to make the task more challenging or trace conditioning could be included. Trace eyeblink conditioning differs from delay conditioning where there is co-termination of the CS and US by introducing a separation between the CS and US. This type of learning has been shown to involve longer term memory and downstream brain circuits that receive input from the cerebellum like the thalamus and ultimately the cerebral cortex (Weible et al., 2000). This may be essential as abnormal function of the cerebrocerebellar pathway is implicated in a myriad of neuropsychiatric illnesses like schizophrenia. During interval timing, previous work from our lab has reported inconsistent results from inactivation of the lateral cerebellar nucleus with muscimol (Heslin et al., 2022; Parker et al., 2017). Given the role for the cerebellum in learning, studies investigating cerebellar involvement in Prickle2^−/− mice on the interval timing task may still be warranted at developmental timepoints, or with altered temporal durations, cues, or expected responses timing.

Overall, our results suggest that although Prickle2 and associated PRICKLE2 protein expression in other brain regions is critical for ASD-relevant behaviors, it may not be critical for ASD-relevant behaviors that rely on the cerebellum in adult animals (Sowers et al., 2013). Given the heterogeneity of ASD in animal models of- and humans with- ASD, cerebellar abnormalities and impairment on cerebellar-mediated tasks may be expected to vary in severity and involvement.

Experiment	+/+ mean	Std. dev.	-/- mean	Std. dev.	Statistical test	p-value
Gap size, lobule IV, V (Fig. 1D)	196.22	39.34	402.61	139.81	t-test	0.0068
Gap size, lobule VI	250.09	79.913	793.23	545.01	t-test	0.038
Gap size, lobule VII	246.03	34.63	544.25	129.16	t-test	0.00039
Gap size, lobule VIII	317.46	172.82	598.78	189.49	t-test	0.030
Gap size, lobule IX	247.33	69.57	529.59	245.94	t-test	0.024
Gap size, lobule X	178.42	14.56	273.73	73.74	t-test	0.012
Patch clamping, fast afterhyperpolarization (Fig. 2D)	-56.12	3.44	-51.58	2.74	t-test	0.025

Ethical Approval

All procedures for the handling and experimental use of animals were conducted in accordance with the University of Iowa’s Institutional Animal Care and Use Committee (approved protocol # 7111209).

Competing interests

There are no competing interests associated with this work.

Authors' contributions

Any edits to the author contributions statement? PA, JH, JF, JW, LS, and KP contributed to the conception and design of the present work. PA designed, collected, and analyzed the eyeblink conditioning project with JF. PA, JH, and JW designed, collected, and analyzed the in vitro work recordings. AN and KP collected and analyzed the behavioral data. PA, JH, and KP completed all statistical analyses, and generated figures. AN, PA, JH, and KP contributed to the production of the manuscript. All authors contributed to the article and approved the submitted version.

Funding

None

Availability of data and materials

Data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Disruption of the non-canonical wnt gene PRICKLE2 leads to cerebellar Purkinje cell abnormalities while cerebellar-mediated behaviors remain intact.

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Abstract

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Introduction

Methods

Prickle2 mutant mice

Immunohistochemistry

Purkinje cell counting

Purkinje cell gapping

Patch clamp electrophysiology

Motor function

Open field

Rotarod

Balance Beam

Delay eyeblink conditioning

Interval timing task

Statistics

Results

Immunohistochemistry

Patch clamp electrophysiology

Motor Function

Interval Timing

Discussion

Declarations

References

Additional Declarations

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