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. Author manuscript; available in PMC: 2021 Dec 1.
Published in final edited form as: Mol Psychiatry. 2020 Apr 24;26(6):2286–2298. doi: 10.1038/s41380-020-0730-8

Dopamine D1R-neuron cacna1c deficiency: a new model of extinction therapy-resistant post-traumatic stress

Charlotte C Bavley 1,2,3,#, Zeeba D Kabir 1,3,#, Alexander P Walsh 1,3, Maria Kosovsky 1, Jonathan Hackett 1,3, Herie Sun 1, Edwin Vázquez-Rosa 4,5, Coral J Cintrón-Pérez 4,5, Emiko Miller 4,5, Yeojung Koh 4,5, Andrew A Pieper 3,4,5, Anjali M Rajadhyaksha 1,2,3
PMCID: PMC8214244  NIHMSID: NIHMS1708118  PMID: 32332995

Abstract

Post-traumatic stress disorder (PTSD) is characterized by persistent fear memory of remote traumatic events, mental re-experiencing of the trauma, long-term cognitive deficits, and PTSD-associated hippocampal dysfunction. Extinction-based therapeutic approaches acutely reduce fear. However, many patients eventually relapse to the original conditioned fear response. Thus, understanding the underlying molecular mechanisms of this condition is critical to developing new treatments for patients. Mutations in the neuropsychiatric risk gene CACNA1C, which encodes the Cav1.2 isoform of the L-type calcium channel, have been implicated in both PTSD and highly comorbid neuropsychiatric conditions, such as anxiety and depression. Here, we report that male mice with global heterozygous loss of cacna1c exhibit exacerbated contextual fear that persists at remote time points (up to 180 days after shock), despite successful acute extinction training, reminiscent of PTSD patients. Because dopamine has been implicated in contextual fear memory, and Cav1.2 is a downstream target of dopamine D1-receptor (D1R) signaling, we next generated mice with specific deletion of cacna1c from D1R-expressing neurons (D1-cacna1cKO mice). Notably, D1-cacna1cKO mice also show the same exaggerated remote contextual fear, as well as persistently elevated anxiety-like behavior and impaired spatial memory at remote time points, reminiscent of chronic anxiety in treatment-resistant PTSD. We also show that D1-cacna1cKO mice exhibit elevated death of young hippocampal neurons, and that treatment with the neuroprotective agent P7C3-A20 eradicates persistent remote fear. Augmenting survival of young hippocampal neurons may thus provide an effective therapeutic approach for promoting durable remission of PTSD, particularly in patients with CACNA1C mutations or other genetic aberrations that impair calcium signaling or disrupt the survival of young hippocampal neurons.

Introduction

Post-traumatic stress disorder (PTSD) is a debilitating neuropsychiatric condition induced by exposure to a traumatic experience and characterized by mental re-experiencing of the traumatic event, avoidance of potential memory triggers, hyper-reactivity, anxiety, and cognitive deficits [1]. These symptoms are persistent, despite therapeutic approaches such as prolonged exposure therapy, an extinction-based learning process that acutely reduces fear without eradicating the original fear memory [2]. However, for reasons that are not understood, many patients are unable to achieve long-term retention of this new learning, and eventually relapse to the original conditioned fear response, termed “remote fear memory”. Inescapable foot shock and fear conditioning serve as rodent models of PTSD that recapitulate dysfunction in neuronal fear circuits and changes in hippocampal structure and function [3]. Contextual fear conditioning is a particularly good model of PTSD-associated hippocampal dysfunction [4], which bears high relevance to the human condition as hippocampal activity 24 h following exposure to a trauma is predictive of the persistence of PTSD symptoms in patients 3 months later [5]. Interestingly, while patients with PTSD do not show consistent alterations in fear conditioning acquisition [6, 7], they do display unique deficits in contextual processing associated with reduced hippocampal and prefrontal cortex activity [5, 6]. Thus, extending the contextual fear model beyond acquisition to additionally incorporate the anxiety response to remote contextual memory adds clinical relevance.

Although estimates of heritability of susceptibility to PTSD range from 24 to 46% [8–10], suggesting a strong genetic influence, the underlying genetic and molecular mechanisms remain poorly understood. The gene CACNA1C, which encodes the Cav1.2 L-type calcium channel, has recently been identified as a risk factor for PTSD [11]. Aberrations in CACNA1C are also implicated in various neuropsychiatric disorders frequently comorbid with PTSD [12–14], as well as the behavioral effects of stress in rodent models [13, 14]. Understanding the role of CACNA1C in PTSD symptomology could thus help foster development of new treatments for patients. Cav1.2 is a known downstream mediator of dopaminergic signaling [15], which has been implicated in the pathophysiology of PTSD [11, 16]. Here, we report that global heterozygous loss of cacna1c, or loss of cacna1c in dopamine D1R-expressing cells (D1-cacna1cKO), induces exacerbated contextual fear memory at remote time points, without altering the initial process of fear acquisition. This exacerbated remote memory is specific to contextual and not cued fear memory, and is resistant to extinction training. These mice also display deficits in survival of young hippocampal neurons, and ameliorating this deficit by treating D1-cacna1cKO mice with the neuroprotective agent P7C3-A20 protects from persistent remote contextual fear memory. Thus, Cav1.2 is a critical mediator of long-term trauma memory associated with persistent anxiety through regulation of survival of young hippocampal neurons, suggesting new opportunities for therapeutic treatment of patients suffering from PTSD.

Materials and methods

Animals

Male cacna1c heterozygous (Het) mice [17] and conditional knockout mice in which cacna1c was eliminated in dopamine receptor 1 (D1R)-expressing cells by crossing homozygous cacna1c floxed mice with mice expressing Cre recombinase under the control of the drd1 promoter [18], were >8 weeks old at the start of all behavioral experiments. Knockdown was confirmed by quantitative PCR in multiple fear memory-related brain regions in both mutant lines (Supplementary Fig. 1a, b). Mice were maintained on a 12 h light/dark cycle and housed with free access to food and water. All procedures were conducted in accordance with the Weill Cornell Medicine Institutional Animal Care and Use Committee rules.

Fear conditioning and extinction

Contextual fear conditioning and extinction

Contextual fear conditioning and extinction consisted of five training trials on day 1 followed by a 24-h recall test on day 2, followed by 3 days of extinction training and then remote memory tests on days 30, 60, and 180. On day 1, mice were placed in the conditioning apparatus containing a shock chamber enclosed by a sound-attenuated box (Coulbourn, Whitehall, PA, USA). The conditioning apparatus (context A) was a square apparatus (7″W × 7″D × 12″H) with a rod floor and was cleaned with ethanol with a 0.1% peppermint scent between animals. Mice were given 2 min for habituation, followed by five conditioning trials, each consisting of a 30-s tone presentation (2.9 kHz, 84-dB) that co-terminated with a 1-s foot shock (0.7 mA). Conditioning trials were separated by increasing inter-trial intervals. Mice were returned to their home cage after conditioning. On day 2, mice were placed back into the shock chamber, and after 1 min of habituation, freezing was measured during a 4.5 min contextual recall test. Contextual fear extinction on days 3–5 and remote context memory tests on days 30, 60, and 180 followed this same protocol. Behavior was recorded using a camera mounted above the apparatus, and freezing was measured using FreezeView automated analysis (Coulbourn Instruments, Whitehall, PA).

Cue fear conditioning and extinction

Cue fear conditioning and extinction consisted of five training trials on day 1 in context A, a 24-h cue recall test in a novel context B, 3 days of cue extinction training (context B), a 30-day cue recall test (context B), and a 31-day contextual recall test (context A). Context B was a circular apparatus (11.2″H × 10.6″ diameter) with white walls and a blue plastic floor and was cleaned with ethanol with a 0.1% lemon scent between animals. On day 1, animals were conditioned as described for contextual fear conditioning. On day 2, mice received a 24-h cue recall test in a novel circular chamber with green flooring (context B). Following a 2-min habituation period, five 30-s tones were presented and freezing during each of the tones was measured. On days 3–5, mice underwent cued fear extinction following the same protocol as day 2. On day 30, mice received an additional cue recall test, and on the following day (day 31) were placed back into context A for a context recall test. Behavior was recorded using a camera mounted above the apparatus, and freezing was measured using FreezeView automated analysis (Coulbourn Instruments, Whitehall, PA). As all behavioral analyses were performed by objective computerized methods, investigators were not blinded to genotype. For all experiments, outliers were determined using Grubb’s outlier test and removed from the analyses.

Basal locomotion

Horizontal distance traveled (cm) was recorded in a test chamber (27.3 × 27.3 cm) using computer-assisted activity monitoring software (Med Associates) for 1 h.

Elevated plus maze

The elevated plus maze was performed as described previously [17]. The plus maze contained four arms (30.5 cm length), two of which were enclosed by walls (15.2 cm height). Mice were allowed to freely explore the maze for 5 min while their behavior was recorded using a camera secured above the apparatus. Time spent in the open arms was recorded using AnyMaze software (Stoelting).

Morris water maze

A modified Morris water maze (MWM) was used as described previously [19]. A black basin (diameter 38″, depth of 14.5″) was filled with an opaque mixture of water and nontoxic white paint (24–26 °C). Extramaze spatial cues were arranged surrounding the apparatus. The maze was virtually divided into three zones, with one zone containing a submerged platform, designated the “goal” zone for each animal. After a 1 min habituation, 4 days of six training trials were performed. During each trial, the mouse was placed into the water at different starting locations with respect to the hidden platform submerged in the “goal” zone. Latency to find the platform was recorded. Probe trials were conducted 24 h and 30 days following the final training trial. During each probe trial, the hidden platform was removed from the “goal” zone and the percentage of time spent in the “goal” zone was measured using a camera secured above the maze and AnyMaze software (Stoelting).

BrdU staining

Bromodeoxyuridine (BrdU) (Sigma-Aldrich) was administered (50 mg/kg, i.p) daily for 5 days. 24 h following the final injection, mice were euthanized by transcardial perfusion with 4% paraformaldehyde at pH 7.4 and brains were processed for immunohistochemical detection of BrdU. Dissected brains were sectioned into 40-μm-thick free-floating sections and incubated for 2 h in 50% formamide/2x saline-sodium citrate (SSC) at 65 °C. Sections were washed for 5 min in 2x SSC and incubated in 2 m HCL for 30 min at 37 °C. Sections were stained with mouse monoclonal anti-BrdU (1:100, Roche). BrdU+ cells in every 5th section were counted and normalized for dentate gyrus volume using Nikon Metamorph and NIH ImageJ software to determine the number of BrdU+ cells in the dentate gyrus.

P7C3-A20 treatments

For P7C3-A20 experiments, mice underwent fear conditioning on day 1, a context test on day 2, and a cue test on day 3, as described above. One hour following the cue test on day 3, mice were injected with a single bolus of BrdU (150 mg/kg, i.p.). Starting on day 3, mice received an injection of either P7C3-A20 (10 mg/kg, i.p.) or vehicle (5% DMSO, 20% cremophor in 5% dextrose) daily for 30 days. Following 30 days of treatment, mice underwent a context test (day 32) and a cue test (day 33). Next, mice were transcardially perfused, and their dissected brains were processed for staining of BrdU as described above.

Fluoxetine treatments

Mice were injected with fluoxetine (20 mg/kg, i.p.) or saline once a day for 30 days. Following fluoxetine treatment, brains were either extracted for quantification of brain-derived neurotrophic factor (BDNF) (see BDNF ELISA below) or for quantification of hippocampal neurogenesis (see BrdU staining above).

BDNF ELISA

The BDNF Emax ImmunoAssay (ELISA) system (Promega) was used to measure mature BDNF protein levels, with recombinant mature BDNF as a standard. Samples and standards were run in duplicate. Protein was extracted and quantified following the manufacturer’s protocol. Tissue samples were homogenized in lysis buffer (150 mM NaCl, 1% Triton X-100, 25 mM HEPES, 2 mM NaF) containing phosphatase and protease inhibitors followed by incubation with constant rotation at 4 °C for 1 h. Homogenized tissue was centrifuged at maximum speed and the supernatant containing total protein was collected and quantified using the BCA protein assay kit (Thermo Fisher Scientific). Each sample was diluted 1:1 with block and sample buffer (BSB) and pipetted into a 96-well plate previously coated with BDNF antibody in carbonate buffer (25 mM Na2CO3 and 25 mM Na2HCO3, pH 9.7, incubated at 4 °C), followed by blocking with BSB. A second coating of primary anti-human BDNF antibody was added followed by the addition of horseradish peroxidase-conjugated secondary antibody. The colorimetric reaction was initiated by tetramethylbenzidine. The reaction was stopped 10 min later by the addition of 1N HCl, and absorbance was read at 450 nm on a plate reader (iMark Absorbance Microplate Reader, Bio-Rad Laboratories).

Statistical analysis

Statistical analyses were conducted using Graphpad Prism 7.0. For analysis, a t-test, a two-way ANOVA, or a two-way repeated measures (RM) ANOVA was utilized. For ANOVAs with significant interactions, Bonferroni corrected post-hoc tests were conducted. Equality of variances was tested with an F-test (unpaired t-tests) and the Brown–Forsythe and Bartlett tests (ANOVAs). Sample sizes were based on previous work in the lab and are similar to other published work in the field. Sample sizes for individual experiments are indicated in figure legends. Outliers were determined using Grubbs’ Test and were excluded from analyses. As all behavioral analyses were conducted using objective computerized software, investigators were not blinded to subject genotype, but BrdU counts were performed by a blinded investigator. Statistical significance was determined by a p value < 0.05. All data are displayed in figures as mean ± standard error of the mean (SEM).

Results

Cacna1c heterozygous mice display persistent context-associated remote fear memory

People who develop PTSD exhibit normal fear conditioning in response to the trauma [6, 7], but often show long-term contextual processing deficits associated with reduced hippocampal function [5, 6]. We therefore sought to measure remote fear in mice by subjecting both wild-type (WT) and cacna1c Het mice to contextual- and cue-associated fear conditioning, as outlined in Fig. 1a. During training on day 1, mice were subjected to 5 tone-shock pairings in context A. Het and WT mice demonstrated a similar level of sensitivity to electric shock, as there was no difference in freezing between WT and Het mice during the entire test period (Supplementary Fig. 2a) or during tone presentation only (the cue) (Supplementary Fig. 2b) on day 1. On day 2, mice were placed back into context A to test for recall of context-associated fear memory. Here, Het mice demonstrated similar freezing as WT (Fig. 1b). To test the role of cacna1c in remote fear, the same mice were then retested 30, 120, and 180 days later. WT mice exhibited decreased freezing to context at all subsequent time points compared with day 2, indicative of normal attenuation of remote fear memory in mice over time. By contrast, Het mice continued to demonstrate persistently high freezing at all subsequent time points (Fig. 1b; significant interaction (Genotype × day), F(3,72) = 2.76, p = 0.048, two-way RM-ANOVA). This difference in freezing is likely not due to changes in locomotor activity, as WT and Het mice have no differences in basal locomotion [20]. Mice were also tested for cue-associated fear memory at corresponding time points by being placed into a novel context B on days 3, 121, and 181 and exposed to the shock-paired cue tone. Here, WT and Het mice displayed similar levels of freezing (Fig. 1c), indicating that loss of cacna1c specifically causes persistent remote contextual fear.

Fig. 1. Cacna1c heterozygous mice display exaggerated remote contextual fear memory.

Fig. 1

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a Behavioral protocol to measure context- and cue-associated fear memory. b During the long-term context memory test on day 2, WT and Het mice display similar levels of freezing to the shock-paired context, however, during the remote context memory tests on day 30 (***p < 0.001, Bonferroni post-hoc day 2 vs. day 30), day 120 (****p < 0.0001, Bonferroni post-hoc day 2 vs. day 120), and day 180 (****p < 0.0001, Bonferroni post-hoc day 2 vs. day 180), WT but not Het mice demonstrate a decrease in % freezing to context compared with day 2 (WT, n = 14; Het, n = 12). c During the long-term test on day 3 and remote cue memory tests on day 31, day 121, and day 181, WT and Het mice display similar levels of freezing in response to the shock-paired tone in a novel context (WT, n = 13; Het, n = 12). d Behavioral protocol used to measure remote context-associated fear memory following extinction. e During the context memory test on day 2, WT and Het mice display similar levels of freezing to the shock-paired context (WT, n = 7; Het, n = 10), and over the course of contextual fear extinction training, WT mice show a significant decrease in % freezing to context on day 3 (****p < 0.0001, Bonferroni post-hoc day 2 vs. day 3), day 4 (****p < 0.0001, Bonferroni post-hoc day 2 vs. day 4) and day 5 (****p < 0.0001, Bonferroni post-hoc day 2 vs. day 5) and Het mice show a decrease in % freezing to context on day 4 (^^^^p < 0.0001, Bonferroni post-hoc day 2 vs. day 4) and day 5 (^^^^p < 0.0001, Bonferroni post-hoc day 2 vs. day 5) (WT, n = 7; Het, n = 10). f Following extinction training, Het mice show enhanced freezing compared with WT mice during a day 30 remote contextual fear memory test (*p < 0.05, independent samples t-test; WT, n = 7; Het, n = 10). Data are displayed as mean ± SEM.

We next wondered whether Het mice might respond to extinction training as extinction-based therapies to inhibit learned fear are currently employed to treat patients with PTSD [21]. To determine whether the increased freezing displayed by Het mice during the remote contextual fear memory test could be alleviated by acute extinction of the context-associated fear memory, WT and Het mice were subjected to contextual fear conditioning followed by 3 days of contextual fear extinction and a remote context memory test on day 30 (outlined in Fig. 1d). As observed previously (Fig. 1b), there was no difference in freezing between WT and Het mice during the context memory test on day 2 (Fig. 1e). During acute context or cue extinction on days 3 through 5, both WT and Het mice showed acute loss of cue-associated fear memory (Supplementary Fig. 2c; main effect of day, F(3,48) = 12.19, p < 0.0001) and contextually associated fear memory, indicated by a significantly lower percent freezing to context on the final day of extinction compared with the day 2 context test (Fig. 1e; significant interaction (genotype × day), F(3,45) = 4.02, p = 0.01). Despite successful extinction of contextual fear in this acute setting in both genotypes, Het mice again displayed significantly higher freezing compared with WT mice during the remote context memory test on day 30 (Fig. 1f; t(15) = 2.68, p = 0.02). Thus, constitutive cacna1c Het mice show enhanced context-associated remote fear memory even after successful acute extinction of the contextual fear memory. This provides a new preclinical model of treatment-resistant PTSD, in which patients do not experience a sustained long-term response to extinction-based therapy.

Loss of cacna1c in D1R-expressing cells results in persistent context-associated remote fear memory

Next, we wondered what might be the mechanistic role of cacna1c in mediating remote contextual fear. Previous studies have suggested a role for D1Rs in both prolonged remote fear memory [16] and vulnerability to PTSD [22], and Cav1.2 is a downstream target of D1R signaling [15]. We therefore generated and tested D1-cacna1cKO mice, along with WT littermate control mice (D1-cacna1cWT), in contextual fear conditioning as outlined in Fig. 2a. On day 1, D-cacna1cWT and D1-cacna1cKO mice displayed similar levels of freezing to the context (Supplementary Fig. 3a), indicating no difference in shock sensitivity between the groups. Both groups of mice also displayed similar levels of freezing during the context memory test on day 2, as well as successful contextual extinction on days 3–5 (Fig. 2b; main effect of extinction, F(3,36) = 20.79, p < 0.0001, two-way RM-ANOVA). However, during the remote context memory test on day 30, D1-cacna1cKO mice displayed significantly higher freezing than D1-cacna1cWT mice (Fig. 2c; t(12) = 3.055, p = 0.01, independent samples t-test), replicating the phenotype of Het mice. This difference was not due to changes in overall locomotor activity, as there was no difference in locomotion between D1-cacna1cWT and D1-cacna1cKO mice (Supplementary Fig. 3c). Because chronic anxiety is frequently comorbid with PTSD, we additionally tested anxiety-like behavior at remote time points in a separate cohort of animals that had been exposed to fear conditioning. Paralleling exacerbated remote contextual fear, D1-cacna1cKO mice also showed prolonged anxiety-like behavior as indicated by decreased time in the open arm of the elevated plus maze 30 days after fear conditioning that persisted for 60 days (Fig. 2d; significant interaction (genotype × day), F(3,39) = 5.84, p = 0.002). This was also accompanied by enhanced contextual fear at these remote time points in the absence of extinction training (Supplementary Fig. 4a, b; day 30: t(14) = 2.22, p = 0.04; day 60: t(14) = 2.31, p = 0.04). Importantly, no differences in baseline anxiety or fear conditioning-induced anxiety on day 2 were observed between genotypes (Fig. 2d).

Fig. 2. Mice with loss of cacna1c in D1R-expressing cells exhibit exaggerated remote contextual fear memory.

Fig. 2

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a Behavioral protocol used to measure context-associated fear memory and extinction. b During the long-term context memory test (day 2) and over the course of contextual extinction (days 3–5), D1-cacna1cWT and D1-cacna1cKO mice demonstrate similar freezing to the shock-paired context (D1-cacna1cWT, n = 6; D1-cacna1cKO, n = 8). c During the remote context memory test on day 30, D1-cacna1cKO mice display enhanced freezing compared with D1-cacna1cWT mice (**p = 0.01; D1-cacna1cWT, n = 6; D1-cacna1cKO, n = 8). d Twenty-four hours following fear conditioning (FC), both D1-cacna1cWT (***p < 001, Bonferroni post-hoc pre-FC vs. day 2) and D1-cacna1cKO mice (^^^^p < 0.0001, Bonferroni post-hoc pre-FC vs. day 2) demonstrate a decrease in time spent in the open arms of the elevated plus maze compared with time spent prior to fear conditioning. At remote time points (day 30 and day 60), D1-cacna1cKO mice continue to spend less time in the open arms (day 30, ^^^p < 0.001, Bonferroni pre-FC vs. day 30; day 60, ^^^^p < 0.0001, Bonferroni post-hoc pre-FC vs. day 60), while D1-cacna1cWT mice do not. (D1-cacna1cWT, n = 8; D1-cacna1cKO, n = 7). e Behavioral protocol used to measure cue-associated fear memories and extinction. f During the long-term cue memory test on day 2 in a new context B, and over the course of cue extinction training in this context B, D1-cacna1cWT and D1-cacna1cKO display similar freezing to the shock-paired cue (D1-cacna1cWT, n = 9; D1-cacna1cKO, n = 7). g During the remote cue memory test on day 30, following cue extinction training, D1-cacna1cWT and D1-cacna1cKO mice display similar levels of freezing to the shock-paired cue (D1-cacna1cWT, n = 9; D1-cacna1cKO, n = 7). h During the remote context memory test on day 31 in context A, D1-cacna1cKO mice demonstrate enhanced freezing compared with D1-cacna1cWT mice (*p = 0.02; D1-cacna1cWT, n = 9; D1-cacna1cKO, n = 6). Data are displayed as mean ± SEM.

To determine whether enhanced freezing during the remote fear memory test in D1-cacna1cKO mice was specific to context-associated fear memories, as we had observed in Het mice (Fig. 1c), a separate cohort of D1-cacna1cKO and D1-cacna1cWT littermates were subjected to cued fear conditioning and extinction, as outlined in Fig. 2e. Mice were conditioned in context A on day 1 and tested in context B on day 2. As observed in Het mice, no differences in freezing during presentation of the shock-paired tone were observed between D1-cacna1cKO and D1-cacna1cWT mice during conditioning on day 1 (Supplementary Fig. 3b). These mice also performed similarly on the day 2 cue memory test (Fig. 2f), and displayed equivalent cue extinction training on days 3–5 (Fig. 2f; main effect of day, F(3,36) = 20.79, p < 0.0001, two-way RM-ANOVA). Furthermore, during the remote cue memory test on day 30, D1-cacna1cKO mice showed similar levels of freezing as D1-cacna1cWT mice during tone presentation (Fig. 2g). However, when placed back into context A 24 h later (day 31), as expected D1-cacna1cKO mice displayed higher freezing to the context compared with D1-cacna1cWT mice (Fig. 2h; t(13) = 2.60, p = 0.02, independent samples t-test), demonstrating remote contextual fear. Thus, as was observed with Het mice, loss of cacna1c in dopamine D1R-neurons increases remote contextual fear memory without affecting cued fear memory.

Enhanced remote contextual fear memory in D1-cacna1c-deficient mice is accompanied by impaired remote spatial memory

The hippocampus plays a critical role in both context-associated fear and spatial memory, and deficient hippocampal activity during spatial memory tasks correlates with PTSD severity in patients [23]. We therefore wondered whether loss of cacna1c might also impact other forms of hippocampal-dependent memory. To test this, we evaluated mice in the MWM, a hippocampal-dependent task of spatial memory [24]. Mice were trained for 4 days to locate a submerged platform based on spatial cues suspended around the tank, and then tested 1 and 30 days later to measure both recent and remote spatial memory, respectively. During training, both WT and Het mice learned the location of the submerged platform equally well (Fig. 3a; main effect of day, F(3,57) = 36.51, p < 0.0001, two-way RM-ANOVA). During the memory test on day 2, both WT and Het mice also spent significantly more time in the goal zone compared with the other zones (Fig. 3b; significant interaction (genotype × zone), F(2,57) = 5.27, p = 0.008, two-way ANOVA). Similarly, during the remote memory test on day 30, both WT and Het mice continued to spend more time in the goal zone compared with the other zones (Fig. 3c; main effect of zone, F(2,57) = 29.35, p < 0.0001, two-way ANOVA). Thus, Het mice did not show alterations in hippocampal-dependent spatial memory.

Fig. 3. Exaggerated remote contextual fear memory is not due to enhanced spatial memory.

Fig. 3

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a During training on the Morris water maze (MWM), WT and Het mice both learn the location of a submerged platform with similar latency (WT, n = 11, Het, n = 11). b During the 24 h long-term probe test, both WT and Het mice spend significantly more time in the goal zone compared with either zone 1 (****p < 0.0001, Bonferroni post-hoc) or zone 2 (****p < 0.0001, Bonferroni post-hoc). c During the remote memory test on day 30, both WT and Het mice continued to spend more time in the goal zone compared with zone 1 or zone 2 (WT, n = 11, Het, n = 11). d During training on the MWM, D1-cacna1cWT and D1-cacna1cKO mice both learn the location of a submerged platform, with a marginally higher latency observed in D1-cacna1cKO mice compared with D1-cacna1cWT mice (D1-cacna1cWT, n = 7; D1-cacna1cKO, n = 9). e During the 24 h long-term probe test in the MWM, both D1-cacna1cWT and D1-cacna1cKO mice spend significantly more time in the goal zone compared with zone 1 (*p = 0.02, ***p = 0.0002, Bonferroni post-hoc) or zone 2 (****p < 0.0001, Bonferroni post-hoc; D1-cacna1cWT, n = 7; D1-cacna1cKO, n = 9). f During the remote memory probe test on day 30, D1-cacna1cWT mice continue to spend more time in the goal zone relative to zone 1 (*p = 0.02) and zone 2 (#p = 0.12), while D1-cacna1cKO mice spend similar amounts of time in each of the zones (D1-cacna1cWT, n = 7; D1-cacna1cKO, n = 9). Data are displayed as mean ± SEM.

In contrast D1-cacna1cKO mice exhibited impaired remote spatial memory in the MWM. During training, both D1-cacna1cWT and D1-cacna1cKO mice learned the location of the hidden submerged platform (Fig. 3d; main effect of day, F(3,42) = 28.86, p < 0.0001, two-way RMANOVA). During the 24-h memory test, both genotypes also spent significantly more time in the goal zone relative to zone 1 or zone 2 (Fig. 3e; main effect of zone (genotype × zone), F(2,28) = 39.18, p < 0.0001, two-way ANOVA). Interestingly, during the remote spatial memory test on day 30, in which D1-cacna1cWT mice continued to spend more time in the goal zone compared with the other two zones, D1-cacna1cKO mice spent similar amounts of time in each zone (Fig. 3f; significant interaction (genotype × zone), F(2,42) = 4.56, p = 0.016, two-way ANOVA). Thus, D1-cacna1cKO mice, in contrast to their augmented remote contextual fear memory, exhibit impaired remote spatial memory. This is reminiscent of chronic deficits in spatial memory often experienced by patients with PTSD [23].

Loss of cacna1c in D1R-expressing cells reduces survival of young hippocampal neurons

We next turned our attention to investigating the mechanism by which loss of cacna1c in D1R-neurons leads to aberrantly persistent remote contextual fear. Previous studies have shown that contextual fear memory is reduced by enhancing adult hippocampal neurogenesis [25, 26], which is the net product of birth and survival of young hippocampal neurons in the adult brain. D1R-neurons have been linked to this process [27], and we and others have also shown that loss of cacna1c in glutamatergic cells of the forebrain results in lower survival of young hippocampal neurons [28–30]. We thus wondered whether specific loss of cacna1c in D1R-expressing cells would affect this process. In order to broadly examine adult hippocampal neurogenesis, D1-cacna1cKO mice were injected daily with the thymidine analog BrdU (50 mg/kg, i.p.) for 5 days. Mice were perfused 24 h following the final injection, and immunohistochemistry for BrdU was performed in the dentate gyrus. As compared with D1-cacna1cWT mice, D1-cacna1cKO mice showed significantly fewer BrdU+ cells (Fig. 4a, b; t(13) = 3.38, p = 0.005, independent samples t-test), indicating a deficit in adult hippocampal neurogenesis. We next sought to determine whether counteracting this deficit by enhancing survival of young hippocampal neurons in D1-cacna1cKO mice might protect them from aberrantly enhanced remote contextual fear memory after shock. Selective serotonin reuptake inhibitors (SSRIs) have been shown to increase hippocampal neurogenesis [31–33] and are also used to treat patients with PTSD [34, 35]. However, the SSRI medication, fluoxetine is unable to enhance hippocampal neurogenesis in forebrain cacna1c-deficient mice (Supplementary Fig. 5a; significant interaction (genotype × fluoxetine), F(1,20) = 5.98, p = 0.02). This is presumed to be because SSRI-mediated neurogenesis is dependent on expression of BDNF, and cacna1c-deficient mice are aberrantly low in BDNF expression, which is not remedied by treatment with P7C3-A20 [28]. Consistent with this, we find that fluoxetine is unable to increase BDNF levels in cacna1c-deficient mice to levels seen in WT mice (Supplementary Fig. 5b; significant interaction (genotype × fluoxetine), F(1, 23) = 92.59, p < 0.0001).

Fig. 4. Loss of cacna1c in D1R-expressing cells reduces the net magnitude of adult hippocampal neurogenesis.

Fig. 4

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a Representative images of BrdU staining. b D1-cacna1cKO mice have a lower number of BrdU+ cells/mm3 in the dentate gyrus compared with D1-cacna1cWT mice (**p < 0.01, independent samples t-test). c Behavioral protocol used to measure context- and cue-associated fear memories and experimental timeline for P7C3-A20 treatment. d During a long-term context memory test on day 2, D1-cacna1cWT and D1-cacna1cKO mice, who would go on to receive either vehicle (pre-vehicle) or P7C3-A20 (pre-P7C3-A2) injections display similar levels of freezing to the shock-paired context (D1-cacna1cWT- vehicle, n = 7; D1-cacna1cKO- vehicle, n = 6; D1-cacna1cWT- A20, n = 6; D1-cacna1cWT- A20, n = 9). e During a long-term cue memory test on day 3, pre-vehicle and pre-P7C3-A20 D1-cacna1cWT and D1-cacna1cKO mice display similar levels of freezing to the shock-paired tone (D1-cacna1cWT- vehicle, n = 6; D1-cacna1cKO- vehicle, n = 5; D1-cacna1cWT- A20, n = 6; D1-cacna1cWT- A20, n = 9). f During the remote context memory test on day 32, vehicle-treated D1-cacna1cKO mice demonstrate significantly higher freezing to the shock-paired context as compared with vehicle-treated D1-cacna1cWT mice, while P7C3-A20 treated D1-cacna1cKO mice do not (**p < 0.01, Bonferroni post-hoc D1-cacna1cWT vehicle vs. D1-cacna1cKO vehicle). g During a remote cue test on day 33, vehicle-treated and P7C3-A20-treated D1-cacna1cWT and D1-cacna1cKO mice display similar levels of freezing to the shock-paired tone. Data are displayed as mean + SEM.

Given the inefficacy of fluoxetine, we thus employed an alternative method of increasing the net magnitude of hippocampal neurogenesis in D1-cacna1cKO mice. Hippocampal neurogenesis is the balance of proliferation and survival of young hippocampal neurons, and augmentation of either process has been shown to augment the net magnitude of hippocampal neurogenesis. We utilized the neuroprotective agent, P7C3-A20, a neuroprotective agent known to enhance survival of young hippocampal neurons in rodents and primates [36–41], including mice lacking cacna1c in the brain [28, 29]. Following fear conditioning on day 1, a context test on day 2, and a cue test on day 3, D1-cacna1cWT and D1-cacna1cKO mice were injected daily for 30 days with P7C3-A20 (10 mg/kg, i.p.). Mice were treated with P7C3-A20 for 30 days, as the birth, maturation, and functional incorporation of newly born hippocampal neurons in rodents take ~ 4 weeks [40].

During conditioning on day 1 (Supplementary Fig. 6a, b), and during the context test on day 2 (Fig. 4d) and the cue test on day 3 (Fig. 4e), D1-cacna1cWT and D1-cacna1cKO mice that would later receive vehicle (pre-vehicle) or P7C3-A20 (pre-P7C3-A20) showed similar levels of freezing to context and tone. Following 30 days of treatment, mice were again tested in a context test (day 32) and cue test (day 33) (Fig. 4c) to examine remote fear memory. During the context test on day 32, D1-cacna1cKO mice treated with vehicle displayed significantly higher freezing to the context compared with D1-cacna1cWT mice, as expected from above results. Notably, however, D1-cacna1cKO mice treated with P7C3-A20 exhibited remote context fear memory on day 32 equal in magnitude to that seen in D1-cacna1cWT mice (Fig. 4f; interaction (genotype × P7C3-A20), F(1,24) = 3.47, p = 0.075; main effect of genotype, F(1, 24) = 8.79, p = 0.007). This protective effect was associated with an ~2-fold increase in surviving young hippocampal neurons in an additional cohort of D1-cacna1cKO mice 30 days after a single bolus injection of 150 mg/kg BrdU (Supplementary Fig. 7a, b; t(8) = 4.69, p = 0.002). Thus, the aberrantly persistent remote contextual fear memory induced by loss of cacna1c in D1R-expressing cells can be rescued by enhancing survival of young hippocampal neurons. As observed previously, D1-cacna1cWT and D1-cacna1cKO mice displayed similar levels of freezing to tone during a remote cue test on day 33 (Fig. 4e), with P7C3-A20 having no effect (Fig. 4g).

Discussion

We have shown here that both Cav1.2 Het mice and mice specifically lacking Cav1.2 within dopamine D1 receptor-expressing cells (D1-cacna1cKO mice) demonstrate contextual fear conditioning and extinction similarly to WT mice, but that this fear memory becomes exacerbated at remote time points, even in the face of successful extinction. This phenotype is consistent with prolonged and persistent behavioral changes observed in PTSD patients following a traumatic experience, even in many patients who have engaged in extinction therapy.

We also find that Cav1.2 is specifically important for remote contextual fear memory rather than cue fear memory. This is consistent with the hypothesis that PTSD is mediated by alterations in contextual processing [6, 42], and the fact that PTSD patients show alterations of the hippocampus [4–6], a brain region required for successful contextual, but not cue, fear conditioning [43]. Interestingly, this enhanced hippocampal-dependent remote fear memory was accompanied by impaired remote spatial memory in the MWM, demonstrating that the exaggerated remote memory was not driven by a global enhancement in hippocampal-dependent memory. This is consistent with findings in patients with PTSD, in which reduced hippocampal activity during spatial memory tasks is correlated to PTSD symptom severity [23].

Interestingly, loss of Cav1.2 did not appear to alter acute extinction behavior, and successful extinction training did not reduce the exaggerated fear response observed at remote time points. This is consistent with findings in PTSD patients using extinction-based cognitive therapies like eye movement desensitization and reprocessing. These patients often show improvement during these extinction-based therapies, yet their relapse rates remain high [44], suggesting that while PTSD patients are able to form proper extinction memories, this intervention is not sufficient to eliminate long-term PTSD pathology. Though the standard extinction-based therapeutic approach that we applied here cannot sustain reduction of fear in Hets and cKO strains, we do not know whether additional extinction training days might achieve this effect. This will be addressed in future studies, with the caveat that this may be of limited clinical relevance as there is currently no evidence that extending the duration of extinction therapy increases treatment efficacy in human patients with extinction therapy-resistant PTSD.

We also demonstrate that loss of Cav1.2 channels specifically within dopamine D1-expressing cells leads to persistent enhanced remote contextual fear memory. This finding supports data in humans that genes associated with dopaminergic neurogenesis are associated with PTSD risk [11], as well as rodent literature showing that dopamine D1 receptor knockout mice exhibit enhanced remote contextual fear memory and prolonged anxiety-like behavior following fear conditioning [16]. Our data presented here suggest that this role of D1 receptors in remote fear and persistent anxiety-like behavior is mediated through Cav1.2 channels. This is further supported by previous work showing that Cav1.2 is regulated downstream of D1 receptors [15].

Lastly, we find that loss of Cav1.2 within D1-expressing cells impairs the survival of young hippocampal neurons, and that enhancing the survival of these neurons reduces persistent remote fear memory. This suggests that a hippocampal neurogenesis deficit, in the form of decreased survival of young hippocampal neurons, in D1-cacna1cKO mice might be contributing to enhanced remote fear memory. We previously found that loss of Cav1.2 in glutamatergic neurons also resulted in a deficit in neurogenesis caused by a reduction in survival of newborn neurons, rather than a reduction in the proliferation of progenitor cells [28]. In addition to functioning downstream of dopamine D1 receptors, cacna1c also mediates the effects of beta-adrenergic receptor activation [45]. As stimulation of beta-adrenergic receptors enhances neurogenesis in the adult hippocampus [46], future studies will examine the potential role of cacna1c and beta-adrenergic receptors in the exaggerated persistence of fear memory observed in dopamine D1-cacna1cKO mice.

It has already been established that dopamine D1 receptors within the dentate gyrus are critical for stimulation of neurogenesis [27, 47, 48], and our data show that this effect could be mediated by cacna1c. Indeed, we definitively demonstrate that specific loss of cacna1c within D1R-neurons results in reduced hippocampal neurogenesis. One possible mechanism by which cacna1c might impact neurogenesis is through transcriptional regulation of BDNF. BDNF expression is required for survival of young hippocampal neurons [49], and loss of cacna1c reduces BDNF expression in the hippocampus [28]. Future studies that extend our findings to more deeply identify the underlying molecular mechanisms would be expected to provide additional novel therapeutic targets for patients.

Collectively, our data show exacerbated remote fear in both Cav1.2 Het mice and D1-cacna1cKO mice, even in the face of successful acute extinction. This phenotype is reminiscent of the large number of treatment-resistant PTSD patients who suffer from long-term symptoms even after extinction-based cognitive therapy [50]. Indeed, while many PTSD patients are able to effectively form acute extinction memories, this intervention often fails to permanently mask remote fear. The role of Cav1.2 delineated here in remote contextual fear memory, a hippocampal-mediated process, is consistent with reported alterations in hippocampal functioning and contextual processing in PTSD patients [4–6, 42, 43]. Furthermore, persistently enhanced remote contextual fear following selective loss of Cav1.2 within D1R-neurons is compatible with human genetic data indicating that aberrant dopaminergic-mediated neurogenesis is associated with increased risk of PTSD [11]. We have previously reported that Cav1.2 is regulated downstream of D1Rs [15], and here we additionally report that loss of Cav1.2 within D1R-neurons impairs adult hippocampal neurogenesis. Although early reports of human adult neurogenesis [51–56] have been challenged [57], recent work from Tobin et al. [58] has established that adult hippocampal neurogenesis persists in the human brain [51]. Taken together, our results suggest that impaired Cav1.2 signaling in D1R-neurons, leading to reduced survival of young hippocampal neurons, may cause persistent remote contextual fear memory after traumatic stress. Thus, increasing the survival of young hippocampal neurons through neuroprotective treatment may provide a new therapeutic approach for PTSD patients harboring mutations in cacna1c or other impairments in calcium signaling or hippocampal neurogenesis.

Supplementary Material

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Acknowledgements

This work was supported by grants to AMR by the National Institute of Drug Abuse, The Hartwell Foundation and the Paul Fund, and to AAP by the Brockman Foundation, the Elizabeth Ring Mather & William Gwinn Mather Fund, the S. Livingston Samuel Mather Trust, the G.R. Lincoln Family Foundation, and Gordon & Evie Safran. CCB was supported by a T32 grant from NIDA, a TL1 grant from the National Center for Advancing Translation Sciences/NIH, and the Frank & Blanche Mowrer Memorial Fellowship. EV-R was also supported by the Training Program in Free Radical and Radiation Biology from the University of Iowa (T32 CA078586). Some of this material is the result of work supported with resources and the use of facilities at the Louis Stokes VA Medical Center in Cleveland.

Footnotes

Supplementary information The online version of this article (https://doi.org/10.1038/s41380-020-0730-8) contains supplementary material, which is available to authorized users.

Data availability

The datasets generated during this study are available from the corresponding authors upon reasonable request.

Conflict of interest AAP is a consultant for Proneurotech, Inc. All other authors declare no conflicts of interest.

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