The voltage-gated Ca2+ channel subunit α2δ-4 regulates locomotor behavior and sensorimotor gating in mice.

Voltage-gated Ca2+ channels are critical for the development and mature function of the nervous system. Variants in the CACNA2D4 gene encoding the α2δ-4 auxiliary subunit of these channels are associated with neuropsychiatric and neurodevelopmental disorders. α2δ-4 is prominently expressed in the retina and is crucial for vision, but extra-retinal functions of α2δ-4 have not been investigated. Here, we sought to fill this gap by analyzing the behavioral phenotypes of α2δ-4 knockout (KO) mice. α2δ-4 KO mice (both males and females) exhibited significant impairments in prepulse inhibition that were unlikely to result from the modestly elevated auditory brainstem response thresholds. Whereas α2δ-4 KO mice of both sexes were hyperactive in various assays, only females showed impaired motor coordination in the rotarod assay. α2δ-4 KO mice exhibited anxiolytic and anti-depressive behaviors in the elevated plus maze and tail suspension tests, respectively. Our results reveal an unexpected role for α2δ-4 in sensorimotor gating and motor function and identify α2δ-4 KO mice as a novel model for studying the pathophysiology associated with CACNA2D4 variants.

Introduction

Voltage-gated Ca2+ channels mediate Ca2+ signals that initiate a vast array of signaling events including gene transcription, protein phosphorylation, and neurotransmitter release. The main properties of these channels are determined by a pore-forming α1 subunit, while auxiliary β and α2δ subunits regulate the trafficking and some functional aspects of these channels [1]. These subunits are encoded by four genes each [2], with additional functional diversity conferred by extensive alternative splicing [3]. The physiological importance of Cav channels is reflected in the numerous diseases that are linked to mutations in the genes encoding the Cav subunits which include migraine, ataxia, and disorders of vision and hearing [4, 5].

In recent years, variants in Cav encoding genes have been consistently identified in genome-wide association studies of neuropsychiatric disorders. One of the most prominent of such studies analyzed single-nucleotide polymorphisms (SNPs) in ~60,000 individuals and uncovered CACNA1C, the gene encoding Cav1.2, as a major risk gene for schizophrenia, bipolar disorder, major depressive disorder, autism spectrum disorder, and attention deficit hyperactivity disorder (ADHD) [6]. In this study, pathway analysis further revealed an association of other Cav-encoding genes with these disorders, including CACNA2D4 that encodes the α2δ-4 subunit. This result was rather unexpected given that α2δ-4 was thought to be expressed primarily in the retina, where it associates with the Cav1.4 channel and regulates the structure and function of photoreceptor synapses [7-9].

α2δ is an extracellular protein that regulates the cell-surface trafficking of Cav channels [10], but may have additional roles. For example, α2δ-1 binding to thromobospondins promotes synapse formation in a manner that is inhibited by the analgesic and anti-convulsant drug, gabapentin [11]. In cultures of hippocampal neurons, α2δ-1, α2δ-2, and α2δ-3 play essential and redundant roles in regulating the formation and organization of glutamatergic synapses [12]. At the Drosophila neuromuscular junction, α2δ-3 is required for proper synapse morphogenesis—a process that does not involve its association with the Cav2.1 channel [13]. In the retina, the formation of photoreceptor synapses involves the role of α2δ-4 as a Cav1.4 subunit and as a mediator of trans-synaptic interactions of the cell adhesion molecule, ELFN-1, with postsynaptic glutamate receptors [9].

Despite the association of α2δ-4 with neuropsychiatric diseases, whether α2δ-4 contributes to behaviors linked to these disorders is unknown. To address this question, we examined the behavioral phenotypes of α2δ-4 knockout (KO) mice [8].

Materials and methods

Animals

All procedures using animals were approved by the University of Iowa Institutional Animal Care and Use Committee (IACUC protocol #0111262 and #1071502). The α2δ-4 KO mouse line was bred on a C57BL/6 background for at least 20 generations and characterized previously [8]. Experimental animals were bred from homozygous (-/-) α2δ-4 KO mice and age- and sex- matched wild-type (WT) C57BL/6 mice were used as controls. The same cohorts of males (15–25 week old, n = 10 WT, n = 11 KO) and females (11–22 week old, n = 11 WT, n = 11 KO) were used for all behavioral tasks. A separate group of mice (4 week old, n = 4 WT males, n = 4 KO males, n = 4 WT females, n = 4 KO females) were tested for auditory brainstem responses. Before beginning handling and testing, mice were ear punched for identification. All mice were housed in groups of 2–3 animals per cage for the duration of the handling and testing periods with food and water ad libitum. The room in which the mice were housed was maintained on a consistent light cycle with lights on at 09:00 and lights off at 21:00 and testing took place between 08:00 to 13:00. Males and females were tested in separate cohorts at different time points to prevent pheromones on the testing apparatus from impacting results. Mice were generally acclimatized for 30 min in the room in which the assay was conducted prior to initiating the test. A full week was taken between every test to reduce the impact of stress from previous tests on the next result. The order of testing was designed to minimize the impact of preceding assays by performing those with the least stressful tasks first and in the following order: (1) elevated plus maze, (2) light dark box, (3) open field test, (4) prepulse inhibition, (5) rotarod, (6) tail suspension test, and (7) forced swim test.

Prepulse inhibition

The testing apparatus consisted of a startle response box (SR-LAB from San Diego Instruments). A restraint chamber consisted of a clear plastic tube from which the tremble response of the animal could be measured via an accelerometer underneath the chamber. Animals were placed in the restraint chamber and allowed to acclimate to the chamber for 10 min with a consistent background white noise level of 65 dB which was present for the entire experiment. The 25-min testing period was divided into 3 blocks each consisting of 6 or 60 trials. All trials were presented with a randomly spaced intertrial interval ranging from 7 to 15 seconds. The first block consisted of 6 pulse trials at 120 dB. The second block contained 12 of each of the following trial types: standard pulse at 120 dB, no stimulation, prepulse of +4 dB above background, prepulse of +8 dB, and prepulse of +16 dB. The third block consisted of 6 pulse trials at 120 dB. Startle response amplitudes (in mV) were measured in SR-LAB software and %PPI measured as (startle response for pulse alone—startle response for pulse with pre-pulse) / startle response for pulse alone) X 100.

Auditory brainstem responses

Auditory brainstem responses (ABRs) were performed as described previously [14]. Mice were anesthetized with intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). Recordings were conducted on both ears of all animals on a heating pad using electrodes placed subcutaneously in the vertex and underneath the left or right ear. Clicks were square pulses 100 ms in duration, and tone bursts were 3 ms in length at distinct 8-, 16-, and 32 kHz frequencies. ABRs were measured using BioSigRZ software (Tucker-Davis Technologies), with stimulus levels adjusted in 5-dB increments between 25 and 100 dB SPLs in both ears. Electrical signals were averaged over 512 repetitions and ABR threshold was defined as the lowest sound level at which a reproducible waveform was measured.

Elevated plus maze

The testing apparatus consisted of a plus-shaped maze elevated 40 cm above the floor. Two opposing closed and open arms extended from a central zone. Open arms had no walls whereas closed arms were surrounded by gray walls. The floor of the maze was made of gray plastic material. Illumination intensity in the central square was approximately 500 lux. Mice were moved from the home cage to the central square of the maze, always facing the same closed arm. The animals were allowed to explore the maze for 10 min. In the event of a fall, the animal was placed in the central square facing the same closed arm and recording resumed. Time spent in the open and closed arms was evaluated using video recording and Anymaze software.

Light dark box

The testing apparatus consisted of a chamber divided into a light and dark compartment equipped with infrared beam tracking (Med Associates). The apparatus was divided into 2 chambers with a gap in the wall between them. Mice were tested using a very bright light in the light chamber (27,000 lux). Mice were moved from the home cage to the light side of the apparatus facing away from the dark chamber. The animals were allowed to freely explore and move between the chambers for 30 min and the animals’ movements were documented in sequential 5 min intervals via infra-red tracking. Time spent in either compartment was analyzed by Activity Monitor software.

Open field test

The testing apparatus consisted of an open square chamber with walls of 40 cm height and width. Illumination intensity in the central square was approximately 500 lux. Mice were moved from the home cage to the center of the open chamber. The animals were allowed to freely explore the chamber for 10 minutes. Animal behavior was evaluated using video recording and Anymaze software. Relative time spent in the inner and outer portion of the box were taken as a measure of the animals’ anxiety-like behavior. Total distance traveled over the 10 minutes was taken as a measure of the animals’ basal activity level.

Forced swim test

The testing apparatus consisted of a 2-liter beaker filled with 1200 ml of water at room temperature. Mice were placed in the water and monitored for 6 min, then were dried and placed in a recovery cage with a cage warmer. Time spent immobile was recorded, with immobility defined as lack of motion in the hind legs except necessary movement to balance and keep the head above the water.

Tail suspension test

The testing apparatus consisted of a metal bar suspended 30–40 cm above the table. Tails of the mice were wrapped in adhesive tape within the last 1 cm of the tail. A clear plastic tube was placed around the animal’s tail to prevent climbing up the tail and onto the bar. Time spent immobile was recorded, with immobility defined as lack of attempting to move their limbs as described previously [15].

Rotarod test

The testing apparatus consisted of a rotating spindle 3.0 cm in diameter that will increase in speed over the course of the trial (Rotamex 5). Mice were trained for 2 consecutive days with 3 testing trials per mouse each day separated by at least 30 min. For the testing trial, the speed of rotation was increased by 1.2 rpm every 20 s to a maximum of 40 rpm and the latency to fall was recorded. The 6 testing trials were averaged for each mouse.

Statistics

Statistical analysis was done with GraphPad Prism software 8.0 and RStudio. An alpha level of 0.05 was used for all statistical tests. For datasets without repeated measures, data were first tested for normality by the Shapiro–Wilk test and homogeneity by Levene’s test. For parametric data, ANOVA with post hoc Holm-Sidak’s multiple comparisons test was performed. For non-parametric data, Kruskal Wallis tests were used with post hoc Dunn’s multiple comparisons. For data sets with repeated measures, a repeated measures linear mixed model was used with post hoc estimated marginal means. The main effects were reported if there was no significant interaction, and post hoc analysis was performed on the main effects that had more than two levels. Otherwise, post hoc tests were performed and simple main effects were reported using adjusted p value for multiple comparisons. Data were graphically represented as mean ±standard error of the mean (SEM) for each group. Results were considered significant when p < 0.05 (denoted in all graphs as follows: *p < 0.05; **p < 0.01; ***p < 0.001).

Results and discussion

α2δ-4 KO mice were born at normal Mendelian ratios and did not exhibit any overt behavioral phenotypes other than hyperactivity. The control wild-type (WT) strain corresponded to C57BL/6 strain on which the α2δ-4 KO mice were bred for at least 10 generations. Cohorts of male and female mice were analyzed separately, and there were no differences in body weight of the WT and α2δ-4 KO mice used in this study (Table 1).

Table 1

Body weights (g) of animal subjects in this study.

Cohort:	WT F	WT M	KO F	KO M
Mean	21.636	29.550	22.518	29.282
SEM	0.521	1.03	0.790	0.679

Animals (n = 43, 15–25 weeks) were weighed once and prior to initiating the battery of behavioral tests in this study. There was no significant effect of genotype on body weight (F = 0.418, p = 0.521) but males were significantly larger than females (F = 91.5, p < 0.001) by 2-way ANOVA.

Prepulse inhibition is impaired in α2δ-4 KO mice

Sensorimotor gating is a form of pre-attentive processing that is commonly studied in humans and animals using prepulse inhibition (PPI). In this test, a response to a strong acoustic stimulus is generally diminished when it is preceded by a subthreshold stimulus [16]. Reductions in PPI are thought to reflect impairments in working memory in individuals diagnosed with schizophrenia, bipolar disorder, and post-traumatic stress disorder and in animal models of these conditions [17, 18]. Because of the association of Cav-encoding genes with these disorders [6], we tested whether α2δ-4 KO mice exhibit deficits in PPI. WT and α2δ-4 KO were tested for startle responses to a 120 dB acoustic stimulus that was administered alone or after a prepulse stimulus of 4, 8, or 16 dB, and PPI was expressed as the % change in the response amplitude due to the prepulse (%PPI, Fig 1A and 1B). In this assay, there was a significant main effect of both sex (F = 7.876, p < 0.01) and genotype (F = 10.26, p < 0.01), but no interaction between these variables (F = 0.0028, p = 0.958; Fig 1B). PPI was significantly lower for α2δ-4 KO than for WT mice in the cohort of females (p < 0.05 for both 8 and 16 dB prepulse) and males (p < 0.05 for 8 dB, p < 0.01 for 16 dB prepulse). In some mouse strains, relatively low levels of PPI correlate with low basal startle amplitudes [18]. However, basal startle amplitudes were significantly higher in α2δ-4 KO mice than in WT mice (F = 55.50, p < 0.001; Fig 1C). Some studies have shown that patients with schizophrenia have an impaired habituation to the startle pulse [19], which would manifest as a difference in startle response to the 120 dB-stimulus administered without the prepulse (blocks 1–3, Fig 1A). There was no effect of genotype on this parameter (F = 1.580, p = 0.213). Collectively, these results show that α2δ-4 KO mice exhibit impaired PPI without alterations in habituation.

Fig 1

α2δ-4 KO mice exhibit impaired PPI (A), Schematic of PPI session design.

Block 1 & 3 each consist of 6 test pulses (120 dB) alone trials. In block 2, animals were exposed to a period of no stimulation, the test pulse alone, or test pulses preceded by a PPI prepulse of 4, 8, or 12 dB. Each trial type (total of 12) was presented in a randomized order with varying intertrial interval. Each test pulse was 40 ms and prepulse was 20 ms in duration. (B) %PPI evoked by the indicated prepulse intensities. (C) Startle response amplitudes evoked by test pulses without prepulses. *p < 0.05; **p < 0.01; ***p < 0.001 by linear mixed model.

In the retina and cochlea, α2δ proteins support the activity of Cav1.4 and Cav1.3 channels that mediate glutamate release at the specialized ribbon synapse of photoreceptors and inner hair cells, respectively [20]. To determine whether hearing impairment could contribute to weakened PPI in α2δ-4 KO mice, we measured auditory brain stem responses (ABRs). In this assay, elevated ABR thresholds correlate with hearing deficits. In response to click stimuli, α2δ-4 KO males had significantly higher thresholds than WT males (p < 0.05). For pure tone stimuli from 8 kHz to 32 kHz, there was no overall effect of genotype or sex (F = 1.0140, p = 0.318 & F = 2.3092, p = 0.134), but an interaction between genotype and sex (F = 8.0533, p < 0.01) indicated lower thresholds in α2δ-4 KO females than in WT females (p < 0.01, Fig 2). Importantly, all α2δ-4 KO mice displayed functional hearing above 60 dB, the range used in the PPI assays, which argues against the possibility that the reduced PPI of the αα2δ-4 KO mice resulted from hearing loss.

Fig 2

α2δ-4 KO mice exhibit sex-specific alterations in ABRs.

Thresholds (dB) were plotted for WT and α2δ-4 KO mice in response to click and pure tone stimuli of the indicated frequencies. *p < 0.05; **p < 0.01; ***p < 0.001 by Kruskal-Wallis, Dunn’s test, linear mixed model, and estimated marginal means.

α2δ-4 KO mice exhibit anxiolytic and antidepressant phenotypes

Anxiety and depression are common features of a variety of neuropsychiatric disorders, including those associated with CACNA1C variants [21] and have a high rate of comorbidity with schizophrenia [22, 23], ADHD [24], ASD [25], bipolar disorder [23, 26], and major depressive disorder [23, 27]. Therefore, we tested the performance of α2δ-4 KO mice in behavioral assays designed to assess anxiety (open field test, OFT; elevated plus maze, EPM; and light dark box, LD) and depression (forced swim test, FST; and tail suspension test, TST). In the OFT, the animals are placed in the center of an open chamber and the time spent avoiding the center is used as a metric for anxiety-like behavior (i.e., thigmotaxis). In the EPM, the animals are placed in the center of a raised platform with open and closed arms and the time spent avoiding the open arms is taken as an indicator of anxiety-like behavior. While α2δ-4 KO and WT mice did not differ in thigmotaxis in the OFT (p = 0.99, Fig 3A and 3B), α2δ-4 KO mice spent more time in the open arms of the EPM than WT mice (Open η2 = 0.141, p < 0.01 by Kruskal-Wallis; Closed F = 14.206, p < 0.001 by linear mixed model; Fig 3C and 3D). It is unlikely that visual impairment of the α2δ-4 KO mice influenced their abilities to respond to the aversive stimuli of the OFT and EPM since these mice have normal vision in daylight but not dim light conditions [8]. As a further test, we performed the light dark box assay in which avoidance of a chamber with a bright light stimulus is taken as a measure of anxiety-like behavior. The α2δ-4 KO mice spent more time in the lighted chamber than WT mice (η2 = 0.100, p < 0.05 by Kruskal-Wallis) and female mice spent more time in the light chamber than males (η2 = 0.163, p < 0.01 by Kruskal-Wallis) (Fig 3E and 3F). The light intensity used in the lighted chamber was 27,000 lux, which is well above the visual threshold for α2δ-4 KO mice [8]. Taken together, results from the EPM and LD assays support an anxiolytic phenotype in α2δ-4 KO mice.

Fig 3

α2δ-4 KO mice exhibit diminished anxiety-like behaviors.

For WT and α2δ-4 KO mice, graphs show the % total time spent in the inner and outer regions of the chamber in the open field test (OFT) (A,B), open and closed arms of the elevated plus maze (EPM) (C,D), and light and dark chambers in the light-dark assay (LD) (E,F). *p < 0.05; **p < 0.01; ***p < 0.001 by Kruskal-Wallis and linear mixed model.

In the TST and FST, depressive phenotypes are measured as the duration of immobility following suspension of the animal by its tail, or placement of the animal in a beaker of water, respectively. While there were no differences between genotypes in the FST, α2δ-4 KO mice spent significantly less time immobile than WT mice in the TST (F = 15.04, p < 0.001; Fig 4A–4F). These results indicate a task-specific antidepressant- like phenotype in the α2δ-4 KO mice.

Fig 4

α2δ-4 KO mice exhibit diminished depression-like behavior in the tail suspension test.

For WT and α2δ-4 KO mice, the duration spent immobile in the tail suspension test (TST) (A-C) and forced swim test (FST, (D-F) was plotted against time (in 1-min bins) during the assay. A and D represent results for males and females combined while B,C,E,F show data disaggregated by sex. Smooth line represents exponential fits of the results. *p < 0.05; **p < 0.01; ***p < 0.001 by nonlinear regression.

α2δ-4 KO mice exhibit abnormal motor behavior

Abnormal motor behaviors are a common feature of neurodevelopmental disorders including ASD and ADHD [28-30]. Thereofre, we tested motor function of α2δ-4 KO mice in the rotarod assay. In this assay, the mice are placed on a rotating cylinder that is gradually accelerated and the length of time the animal can stay on the cylinder is taken as a measure of balance, coordination, and motor planning [31]. The latency to fall was shorter for α2δ-4 KO than for WT mice (F = 6.457, p < 0.05; Fig 5A). To further assess motor phenotypes in the α2δ-4 KO mice, we analyzed data in the OFT, EPM, and LD assays for aberrant locomotion. In each case, the total distance traveled by α2δ-4 KO (both males and females) mice was significantly greater than for WT mice (OFT η2 = 0.266, p < 0.001; EPM: F = 16.09, p < 0.001; LD: η2 = 0.490, p < 0.001; Fig 5B–5D). These results show that α2δ-4 KO mice exhibit signs of hyperactivity and impairment in motor coordination.

Fig 5

α2δ-4 KO mice exhibit alterations in motor behavior.

For WT and α2δ-4 KO mice, graphs show the latency to fall in the rotarod assay (A) and total distance traveled in the OFT (B), EPM (C), and LD (D) assays. Rotarod: Genotype F = 6.457, p < 0.05; Sex F = 22.543, p < 0.001; Genotype:Sex F = 1.806, p = 0.1867 by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001 by Kruskal-Wallis and one-way ANOVA.

Our results show that α2δ-4 KO mice exhibit a pattern of affective and motor behaviors that resemble those in neuropsychiatric disorders that are linked to variants in Cav-encoding genes [6]. Because α2δ proteins enhance the cell-surface trafficking of Cav channels [5], the phenotypes of α2δ-4 KO mice could result from loss-of function of Cav channels in key brain regions. For example, α2δ-4 could support the activity of Cav1.2 channels in the medial prefrontal cortex (mPFC)—an area involved in sensorimotor gating [32]. In mice lacking the Cav1.2-interacting protein, densin-180 (densin-KO), Cav1.2 is downregulated in the mPFC [33] and there are deficiencies in PPI and a hyperactivity phenotype [34] similar to α2δ-4 KO mice (Figs 1 and 5B–5D). Moreover, deletion of Cav1.2 in the PFC causes anti-depressant behavior in the TST [35], also similar to α2δ-4 KO mice (Fig 4A–4C). However, PFC-specific deletion of Cav1.2 causes increased anxiety-like behavior [36] whereas α2δ-4 KO mice present with an anxiolytic phenotype in the EPM and LD assays (Fig 3C–3F). Thus, the roles of α2δ-4 in regulating PPI, motor behavior, and anxiety may involve distinct signaling pathways that may or may not be characterized by Cav channels.

In this context, it is somewhat paradoxical that α2δ-4 is nearly undetectable in most brain regions by quantitative PCR of the corresponding tissue lysates [37]. α2δ-4 could escape detection by this method if it were expressed differentially across development or in a small subset of neurons implicated in the behaviors that are altered in α2δ-4 KO mice [37, 38]. Notably, α2δ-4 has been detected at low levels by single cell PCR in the sound-amplifying outer hair cells in the cochlea of immature mice [39]. It is unclear how loss of α2δ-4 could lead to improved hearing (i.e., lower ABR thresholds) in α2δ-4 KO females (Fig 2). Possibly, the absence of α2δ-4 could cause homeostatic alterations in other proteins that improve cochlear sound amplification. By the same token, although it is expressed at generally low levels in the brain, α2δ-4 could undergo pathological upregulation in some disease states. For example, α2δ-4 expression is increased in hippocampus of humans and mice following epileptic seizures [40]. Given that α2δ proteins regulate synapse formation in part through trans-synaptic interactions with proteins other than Cav channels [9, 11, 13, 41], aberrant expression of α2δ-4 could cause defects in neuronal connectivity in individuals harboring pathological variants in CACNA2D4.

Although blind under dim-light conditions, α2δ-4 KO mice are expected to have normal vision under the lighting conditions used in our study [8, 9]. To date, alterations in cognitive and/or affective function in individuals diagnosed with CACNA2D4-related vision impairment have not been reported. However, the etiology of most neuropsychiatric disorders is complex and likely involves hundreds to thousands of risk alleles distributed across the genome [42]. Our findings that α2δ-4 KO mice exhibit defects in PPI, motor coordination, and anxiety/depression-related behaviors validate the importance of CACNA2D4 as one such risk allele and that studies of the extra-retinal functions of α2δ-4 warrant further study.

(PDF)

Click here for additional data file.

16 Feb 2022

Myself and the reviewers found the paper to be of interest and generally well performed.  The reviewers did raise some minor questions and comments that require some clarification.  In particular, please ensure all statistical comparisons are clearly explained (as noted by reviewer #2).

Please submit your revised manuscript by Apr 02 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Kevin P.M. Currie, PhD

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript by Klomp et al. is an interesting investigation into the behavioral ramifications of CACNA2D4 deletion in mice. Overall, the authors report a clear behavioral phenotype of KO mice vs. WT, suggesting a role for α2δ-4 in regulating some aspects of prepulse inhibition, locomotor control, and behavioral responses in tests of anxiety-like and depressive-like behaviors.

The report is well-written and the tests have been performed and analyzed appropriately. The subject of this manuscript appears to be of impactful scientific value and thus appropriate for publication in PLOS ONE following minor revisions. The specific critiques listed below deal largely with deficiencies in the details and clarity of the methods used.

Specific comments:

1. Title: if the α2δ-4 is REQUIRED for locomotor behavior and sensorimotor gating in mice, one would expect that loss of this protein expression would lead to a loss of locomotor activity and sensorimotor gating… What the authors have discovered is that loss of α2δ-4 expression alters locomotor behavior and sensorimotor gating in mice. An adjustment to the title seems appropriate.

2. As a general organizational principle, I suggest putting the behavioral testing methods in the same order as the presented results. This is optional, but it can really help the reader who flips back and forth!

3. Add IACUC protocol # to manuscript.

4. Please indicate the number of generations of back-crossing performed in your study population within the methods section.

5. Likewise, provide the breeding colony details (WT and KO animals are littermates from +/- breeding pairs) within the methods section.

6. The authors should replace “(describe details of the cages)” with the details of the cages!

7. Please indicate the max RPM of the rotarod testing.

8. The testing order and age of animals at each test is unclear. A timeline would be a simple way to illustrate how each animal cohort proceeded through the tests.

9. Likewise, the number of animals in each genotype×sex subgroup tested in each assay is unclear. Are we meant to infer that the animals from Table 1 were used in all the behavioral studies as well as the ABRs? It would be best to include group n values in the figure captions.

10. Table 1: there’s no indication what ages each of these mice are. Is there longitudinal data over the course of the study on weight gain across sex & genotype?

11. Figure 2: please adjust the figure legend to match the order of the data presented (WT F, WT M, KO F, KO M)

12. Were any +/- littermates also tested? Do heterozygotes show any phenotypes in other studies?

13. In the abstract and elsewhere the authors suggest that these results indicate a role for α2δ-4 in regulating cognitive functions. While the rotarod test can evaluate aspects of motor skill learning, this can’t adequately be captured by averaging six trials across two days. Overall, the tests performed in this study do not adequately probe whether α2δ-4 genotype alters any aspects of cognition. Thus, such claims should be removed or put into greater context.

Reviewer #2: Mutations of all genes encoding for calcium channel a2d subunits have been linked to a number of neurological and neuropsychiatric disorders. The a2d-4 isoform is predominantly expressed in retinal photoreceptor cells, however, it’s expression level in the brain is extremely low and potential functions of a2d-4 in the CNS outside the retina have not yet been identified. To shed light on potential roles of a2d-4 in the brain, Klomp et al. behaviorally characterized a2d-4 knockout mice. The experiments show a sex-independent hyperactive phenotype. There is also a tendency towards an anxiolytic and anti-depressive behavior, which is a bit more pronounced in female mice. These findings are novel and clearly support a potential role of a2d-4 in the brain. Overall, the experiments are carefully performed, and the study is largely clear and conclusive. However, there are a few points, both in text and data presentation, that need to be addressed before I can recommend publication in PLOS ONE.

1. In the abstract and introduction I suggest being more careful with the wording of the aim of the study, for example: “Despite the association of a2d-4 with neuropsychiatric disease, how a2d-4 contributes to cognitive and affective functions is unknown. To address this question, …” The present study shows that a2d-4 likely plays a role in cognitive and affective functions, but it does not address the “how”.

2. Overall I do not consider it necessary to show all individual data points in table 1. Rather I suggest including means +/-SEM (not StDev as shown, as the comparisons between the groups are done) in the text together with the results of statistics. I am also confused about the statistics presented in table 1: Which test was performed, a 2-way ANOVA or a general linear model? If a 2-way ANOVA was performed, correctly show the results (F, df, and p values) for genotype, sex, and the interaction. I particularly cannot understand the df (9, 10) of the female comparison, this does not seem to fit the experimental design.

3. Importantly, in the context of the following discussion: “a2d-4 could be expressed in a small subset of neurons implicated in these behaviors, thus escaping detection by quantitative PCR in homogenates of specific brain regions (32).” it is necessary to also refer to a previous review (Ablinger et al., PMID 32607809), where this hypothesis (subpopulation of neurons…) was introduced. Another hint for a potential role in the brain is the observation that hippocampal expression increases ~20-fold during development (see ref 32).

Minor points/editorials:

1. Titel: I suggest to remove „Cav“ from the title, as this is a duplication (voltage-gated) and unnecessarily complicates reading.

2. List of authors: I think the corresponding author is missing an “*”. Should be *** (present address Texas?)

3. Also in the abstract and introduction: Either delete “Cav” or write “Voltage-gated Ca2+ channels (CaV)…”

3. Introduction: numerous diseases that “are” linked to mutations in the genes

4. The following sentence is missing the reference: “One of the most prominent of such studies analyzed single-nucleotide polymorphisms (SNPs) in ~60,000 individuals and uncovered CACNA1C ….”

5. Introduction: “a2d ….. (10), but may have additional roles.” I think in this context the critical and redundant role of presynaptic a2d subunits in synapse formation and differentiation should be mentioned (Schöpf et al., 2021).

6. Methods and throughout: correct is “C57BL/6”

7. Methods, delete the following left-over phrase: “(describe details of the cages)”

8. correct time format: 09:00, 20:00, etc.

9. Fig. 1, add F and p values of main factors to the figure legend

10. In the context of figure 3 please also comment on the difference between male and female mice

11. Fig. 4, FST, the fitted curves of one of the conditions is missing in all graphs.

12. delete ….“a2d-4 KO mice” (33) whereas a2d-4 KO mice….

13. differences between male and female mice (e.g. PPI, ABRs and behavioral tests) are presented in the results but not discussed in more detail. Some thoughts/speculations on potential causes for the differences would be helpful.

Looking forward to reading a revised version of the manuscript.

Gerald Obermair

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Gerald Obermair

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.