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. 2013 Aug 19;154(11):4316–4327. doi: 10.1210/en.2013-1257

Restoration of Dopamine Signaling to the Dorsal Striatum Is Sufficient for Aspects of Active Maternal Behavior in Female Mice

Charles W Henschen 1, Richard D Palmiter 1, Martin Darvas 1,✉
PMCID: PMC5398593  PMID: 23959937

Abstract

Striatal dopamine (DA) is important for motivated behaviors, including maternal behavior. Recent evidence linking the dorsal striatum with goal-directed behavior suggests that DA signaling in the dorsal striatum, not just the nucleus accumbens, could be involved in maternal behavior. To investigate this question, we tested the maternal behavior of mice with DA genetically restricted to the dorsal striatum. These mice had a mild deficit in pup retrieval but had normal licking/grooming and nursing behavior; consequently, pups were weaned successfully. We also tested a separate group of mice with severely depleted DA in all striatal areas. They had severe deficits in pup retrieval and licking/grooming behavior, whereas nursing behavior was left intact; again, pups survived to weaning at normal rates. We conclude that DA signaling in the striatum is a part of the circuitry mediating maternal behavior and is specifically relevant for active, but not passive, maternal behaviors. In addition, DA in the dorsal striatum is sufficient to allow for active maternal behavior.

The care of their young by mammalian mothers encompasses a variety of behavioral adaptations, including nursing, licking, and grooming of pups, as well as retrieving them to a nest. In recent years, dopamine (DA) has been established as an important player in the circuitry mediating rodent maternal behavior. Most research has focused on the mesolimbic DA projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) in the ventral striatum. DA is released in the NAc during maternal behavior (1, 2), and increased DA release is associated with stronger maternal responses (3). The medial preoptic area (MPOA) is important for the motivational aspects of maternal behavior (4–6) and has been shown to stimulate the VTA (7, 8). MPOA lesions reduce NAc activity during maternal behavior (9). Furthermore, inhibition of DA D1 receptors (D1Rs) in both the NAc (10, 11) and the MPOA (12) disrupts maternal behavior, whereas stimulation of D1Rs in the NAc facilitates maternal behavior (13, 14). Activation of D1Rs in the NAc is thought to facilitate maternal behavior by promoting γ-aminobutyric acid-ergic inhibition of the ventral pallidum (15, 16).

DA seems to be especially important for active, goal-directed maternal behaviors, such as pup retrieval, licking, and grooming, and less important for passive behaviors, such as nursing (12, 17–19). Recent research has implicated the dorsal striatum, which receives dopaminergic projections from the substantia nigra pars compacta (SNc), in goal-directed behavior (20–23), suggesting that this circuit may influence maternal behavior as well. There have been few studies exploring the role of dorsal striatal DA on maternal behavior. Hansen et al (24) observed no effect of 6-hydroxydopamine (6-OHDA) lesions of the dorsal striatum on maternal behavior, but another experiment showed a dose-dependent reduction of licking behavior after infusion of a nonselective DA receptor antagonist into the dorsal striatum (10).

To clarify the role of striatal DA in maternal behavior, we tested 2 genetic models in their abilities to rear pups successfully and to perform maternal behavior when challenged. In one model, DA levels in the entire striatum are approximately 5% of normal levels. These mice were generated by inactivating the tyrosine hydroxylase (Th) gene in cells expressing the DA transporter (DAT), which reduces striatal DA while sparing DA synthesis in neurons that do not express the DAT. The other genetic model has inactive Th alleles in DA neurons that can be reactivated by expression of Cre recombinase. These DA-deficient (DD) mice have severe deficits in motivation, feeding, and locomotion and require daily injections of L-3,4-dihydroxyphenylalanine (L-Dopa) for survival (25). Injection of canine adenovirus 2 expressing Cre recombinase (CAV2-Cre) reactivates Th alleles in neurons projecting to the site of injection, thereby restoring dopaminergic signaling to the injected area. Our lab has previously shown that restoration of DA to the dorsal striatum in DD mice rescues feeding behavior and locomotor activity (25), as well as several learning and memory tasks (26–29). By comparing the maternal abilities of mice with severely limited striatal DA and mice with DA restored only to the dorsal striatum, we elucidated the relationship between striatal DA and maternal behavior.

Materials and Methods

Animals

All experiments were conducted in accordance with National Institutes of Health guidelines and approved by the University of Washington Institutional Animal Care and Use Committee. Hypodopaminergic (TH:DAT knockout or TH:DAT KO) mice were generated by breeding mice with 2 floxed Th alleles (Thlox/lox) with mice carrying one null Th allele (ThΔ/+) and an allele with Cre recombinase expressed under the control of the Slc6a3 gene encoding DAT (30); controls for TH:DAT KO mice carried 1 wild-type Th allele and the Cre recombinase allele (Thlox/+; Slc6a3Cre/+). These mice were maintained on a C57Bl/6 background. Flox-stop DD mice (Thfs/fs, DbhTh/+; referred to here as DD mice) were generated as described (25) and maintained on a mixed C57Bl/6 × 129/SvEv genetic background. Controls for DD mice had at least 1 wild-type Th and Dbh allele; consequently, they have normal levels of DA and norepinephrine, respectively. Beginning at approximately 14 days of age, DD mice received daily ip injections of L-Dopa (50 mg/kg; Sigma-Aldrich) dissolved in saline solution containing 2.5 mg/mL ascorbic acid. Mice were housed under a 12-hour light, 12-hour dark cycle (lights on at 7 am) in a temperature-controlled environment with food and water provided ad libitum. CAV2-Cre was generated and prepared as described (31). The viral preparation had a titer of 2 × 1012 particles/mL. Bilateral injections of CAV2-Cre (0.5 μL) into the posterior dorsal region of the striatum (at bregma, ±1.75 mm lateral to midline and 3 mm ventral from the skull surface) were performed on 6- to 8-week-old female DD mice, as well as control mice with 1 normal Th allele (referred to as sham controls), under isoflurane anesthesia. We injected a total of 9 sham control and 14 DD mice. L-Dopa treatment ceased 2 weeks after viral injection of DD mice, and mice that maintained their body weight for 2–3 weeks without L-Dopa were considered virally rescued (virally rescued DD [vrDD]) and included in the experiment. All 9 injected sham control mice were included and 11 of the 14 injected DD mice were included. The 3 injected DD mice that were excluded presumably had insufficient reactivation of Th, as evidenced by their inability to survive without continuation of L-Dopa treatment.

Maternal behavior assays

At 12–18 weeks of age, female mice were separated from their littermates, and each one was housed with a C57Bl/6 male. During the last week of pregnancy, the male was removed. The behavior assay was adapted from a procedure by Miller and Lonstein (12). Two behavior tests for each litter were conducted: one between 5 and 7 days (average of 6 d) and a second between 9 and 12 days (average of 10 d) after parturition. On test days, pups were removed from their dams in the home cage at 12:30 pm (±30 min) and placed in a clean filter-top cage with a heating pad under one half of the cage. Dams remained in their home cages with 5 pieces of chow distributed on the floor of the cage. After a 3-hour separation, maternal behavior tests were conducted. First, the top of the home cage was removed. An opaque covering was placed over the side of the cage containing the nest to keep the nest area relatively dim. The dam was allowed to habituate to these conditions for 5 minutes with no pups present. Then, with the dam on the side of the cage containing the nest, 4 pups were distributed on the opposite side of the cage. Any remaining pups in the litter were left in the extra cage atop the heating pad. The actions of the dam were recorded for the next 30 minutes. The latencies to retrieve each pup, begin licking/grooming, and begin nursing were recorded, as well as the instance and duration of licking/grooming and nursing, and nonmaternal behaviors such as nest rearranging, self-grooming, and eating. Trials were also screened for digging and treading behaviors, which are indicative of high maternal stress (32) and have been shown to impact on maternal behavior (33, 34). Pups that crawled back to the nest on their own were not included in subsequent analyses. Nursing and licking/grooming were counted whether they took place in the nest or not. At the end of the test, any remaining pups were reintroduced into the home cage. Data were confirmed later by video analysis. Activity levels were measured by EthoVision software (Noldus). Between 18 and 21 days after parturition, the pups were killed, and another C57Bl/6 male was introduced into the female's cage. The same procedures were followed for her second litter, which was also killed 18–21 days after parturition. Only dams with litters of 4 or more pups were used for behavioral analysis.

Immunohistochemistry and DA measurement

At 29–37 weeks of age, all dams were killed and subjected to either of the 2 following procedures. After transcardial perfusion (4% paraformaldehyde) of deeply anesthetized mice, brains were removed, cryoprotected in 30% sucrose, frozen in isopentane, and sectioned on a cryostat. Staining for TH and DAT was conducted on 30-μm sections using the following primary antibodies: rabbit anti-TH (1:2000; Millipore Bioscience Research Reagents) and rat anti-DAT (1:1000; Millipore Bioscience Research Reagents). Immunofluorescence was detected using the following IgG secondary antibodies: Alexa Fluor 594 (TH, 1:200; Jackson ImmunoResearch) and Dy-Light 488 (DAT, 1:400; Jackson ImmunoResearch). Antibody information is detailed in Supplemental Table 1, published on The Endocrine Society's Journals Online web site at http://endo.endojournals.org. Alternatively, for measurement of DA content, tissue punches (1 mm diameter; 2 mm thick) were collected after cervical dislocation, immediately placed in liquid nitrogen, and stored at −80°C until analysis. HPLC coupled with electrochemical detection was used to measure DA content by the Neurochemistry Core Laboratory at the Center for Molecular Neuroscience Research of Vanderbilt University (Nashville, Tennessee).

Statistical tests

All collected data were analyzed using GraphPad Prism software. Student's t test was used for HPLC analysis of DA levels. Litter size, pup survival, and locomotion were analyzed by 2-way ANOVA with genotype and litter number as variables. Log-rank (Mantel-Cox) curve analysis was used for pup-retrieval data. Licking/grooming, nursing, and nonmaternal behaviors were analyzed by 2-way ANOVA with genotype and pup age as variables. If significant main effects of group factors were confirmed, Bonferroni's multiple comparison post hoc tests were used. All data points are reported as mean ± SEM. Significance is reported in the text and figure legends. Differences were considered significant if P < .05.

Results

Striatal hypodopaminergic TH:DAT KO mice

We tested 2 mouse models of DA deficiency for their effects on maternal behavior. In the TH:DAT KO model, the striatum is deficient in DA, whereas other brain regions have relatively normal DA signaling. Figure 1A shows the alleles that were used to generate TH:DAT KO mice (ThΔ/lox; Slc6a3Cre/+) and their controls (Th+/lox; Slc6a3Cre/+). Analysis of DA levels by HPLC in dams that were used in the behavior study revealed that TH:DAT KO mice had approximately 5% normal DA levels in both the dorsal and ventral striatum (Student's t test, P < .01) and approximately 20% in the hypothalamus (Student's t test, P < .05) compared with controls (Figure 1B). In other brain regions, DA levels were normal (data not shown). Figure 1, C and D, illustrates the DA levels in TH:DAT KO mice compared with wild-type mice.

Figure 1.

Figure 1.

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Two mouse models of DA deficiency. (A) Schematic representation illustrating the TH:DAT KO model, in which Cre recombinase disrupts the Th allele in neurons expressing the Slc6a3 allele (which codes for DAT). (B) DA levels in TH:DAT KO and control mice (each N = 5). DA levels in TH:DAT KO are diminished to approximately 5% of control levels in both dorsal and ventral striatum, and to approximately 20% of control levels in the MPOA. (C) Illustration of a TH:DAT KO mouse brain, showing depleted DA levels in the striatum and diminished DA levels in the MPOA. (D) Illustration of a control mouse brain, showing full DA levels in the striatum and hypothalamus. (E) Schematic representation illustrating the DD model, in which Th is rendered nonfunctional by a stop cassette flanked by loxP sites. A functional Th gene is inserted into one Dbh allele in order to allow for normal norepinephrine expression. (F) Viral rescue of DD mice. TH expression is restored to the dorsal striatum in DD mice by injection of CAV2-Cre into this region and retrograde transport to the midbrain. (G) DA levels by vrDD (N = 5) and sham control mice (N = 7). DA levels in vrDD mice are restored to approximately 25% of control levels in the dorsal striatum, which is above the threshold necessary for performance of most DA-dependent behaviors (27). DA levels in are restored to only approximately 9% of control levels in the ventral striatum and approximately 1% of control levels in the MPOA. (H) Illustration of a vrDD mouse brain, showing functionally restored DA levels in the dorsal striatum and small amounts of DA in other regions. *, P < .05; **, P < .01.

Partial restoration of DA signaling to the dorsal striatum of DD mice

In the second model of DA deficiency, we selectively restored DA signaling to the dorsal striatum in DD mice by injecting CAV2-Cre bilaterally into the dorsal striatum of mice with conditional flox-stop Th alleles (Figure 1, E and F). The virus was retrogradely transported to the SNc, where it activated Th expression in dopaminergic neurons (25). Mice with successful viral rescue (vrDD) that were able to maintain body weight without further L-Dopa administration were included in these experiments. The sham controls were injected with CAV-2 Cre in the dorsal striatum, but they already had a functional Th allele, so the injections had no effect. Analysis of striatal tissue of dams used in the behavior study revealed that DA was restored to approximately 25% of control levels in the dorsal striatum and approximately 9% of control levels in the ventral striatum, which in both cases was significantly lower than in control animals (Student's t test, P < .01) (Figure 1G); DA in the MPOA was less than 1% when compared with control levels (Student's t test, P < .01) (Figure 1G). Figure 1H depicts DA distribution in the brain of vrDD mice. Immunohistochemical analysis of sham control and vrDD mice confirmed that TH was selectively restored in the dorsal striatum (Figure 2, A–D) and SNc (Figure 2, E–H) of vrDD mice, with almost no immunostaining in the ventral striatum (Figure 2, A–D) or VTA (Figure 2, I–L) in vrDD mice.

Figure 2.

Figure 2.

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Restoration of dopaminergic signaling in midbrain and striatum in vrDD mice, as illustrated by immunostaining of TH (red) and DAT (green) in representative coronal sections from brains of vrDD and sham control mice. DD mice have no TH staining in either region (25). (A–C) TH staining in the striatum (A), SNc (B), and VTA (C) of control mice. (D–F) DAT staining in the striatum (D), SNc (E), and VTA (F) of control mice. (G–I) TH staining in the striatum (G), SNc (H), and VTA (I) of vrDD mice. (J–L) DAT staining in the striatum (J), SNc (K), and VTA (L) of vrDD mice. TH expression in vrDD mice was partially restored in the dorsal striatum, with little or no TH expression in the ventral striatum. Several TH-positive DA neurons were found in SNc sections of vrDD mice. Very few TH-positive cells were visible in the VTA of vrDD mice. Arrowheads show TH-positive stained cells and fibers in sections of vrDD mice.

Breeding success and pup survival were unaffected by striatal DA depletion

The first step in our analysis was to determine whether female mice from each genetic model were fertile and able to care for their young adequately enough for pups to reach weaning. The number of animals per group, pregnant dams, age of dams at first and second birth, total number of litters produced, and the number of litters analyzed for maternal behavior during recorded trials are summarized in Table 1. All TH:DAT KO (N = 8) and control (N = 9) mice that were placed with a male became pregnant. Dam ages were not significantly different between TH:DAT KO dams and controls for either litter. One TH:DAT KO dam and one control dam had too few pups (<4) in both litters and were not included in behavior tests. One TH:DAT KO dam was euthanized at her second parturition due to dystocia; 2 control dams could not become pregnant a second time, one of which had a small (2 pups) first litter. Two-way ANOVA of the number of pups born per litter by TH:DAT KO and control mice (Figure 3A) revealed no significant differences for genotype (F(1,28) = 0.51; P > .05), litter number (F(1,28) = 1.22; P > .05), or genotype per litter number interactions (F(1,28) = 0.04; P > .05). No differences in pup survival to weaning (Figure 3B) were detected between TH:DAT KO and controls as indicated by 2-way ANOVA, which yielded no significant effects for genotype (F(1,28) = 0.42; P > .05), litter number (F(1,28) = 0.88; P > .05), and genotype per litter number interactions (F(1,28) = 0.02).

Table 1.

Summary of Dams and Litters Tested

TH: DAT KO
 vrDD
Control TH: DAT KO Sham vrDD
Total number of animals 9 8 9 11
Number of pregnant dams 7 7 9 11
Average age at birth of 1st litter (weeks) 18.2 18.1 18.2 22.2
Average age at birth of 2nd litter (weeks) 26.6 26.2 28.3 29.5
Total number of produced litters 16 16 18 21
Number of 1st litters analyzed 7 7 9 10
Number of 2nd litters analyzed 6 6 9 7
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Figure 3.

Figure 3.

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Pup survival rate is not affected by striatal DA depletion or restriction, whereas litter size of the second litter only is diminished in vrDD dams. (A) Number of mice in each litter at parturition in TH:DAT KO dams (N = 8) and controls (N = 9). (B) Pup survival rate in each litter, as expressed by the percentage of pups that survived to weaning age, in TH:DAT KO dams (N = 8) and controls (N = 9). (C) Number of mice in each litter at parturition in vrDD dams (N = 11) and sham controls (N = 9). (D) Pup survival rate in each litter, as expressed by the percentage of pups that survived to weaning age, in vrDD dams (N = 11) and sham controls (N = 9). Data are expressed as mean ± SEM. *, P < .05; **, P < .01. n.s., not significant.

All sham and vrDD mice that were placed with a male became pregnant. Dam ages were not significantly different between vrDD dams and sham controls for either litter. One vrDD mouse had small litters (<4 pups) on both first and second parturition and was excluded from behavior experiments. Two additional vrDD mice had too few pups in their second litters and were not included in the recordings for that litter; one vrDD dam could not become pregnant a second time. In vrDD dams, 2-way ANOVA of the number of pups born per litter (Figure 3C) indicated a significant effect of genotype (F(1,33) = 8.93; P < .01) but not litter number (F(1,33) = 1.64; P > .05) or litter number per genotype interaction (F(1,33) = 0.99; P > .05). Post hoc pair-wise comparisons confirmed a significant reduction of litter size only for the second litter of vrDD mice (Bonferroni; P < .05). However, 2-way ANOVA of pup survival in vrDD mice (Figure 3D) showed no significant effects of genotype (F(1,33) = 1.06; P > .05), litter number (F(1,33) = 0.88; P > .05), or genotype per litter number interaction (F(1,33) = 0.39; P > .05). Therefore, neither severe depletion of striatal DA nor restriction of DA to the dorsal striatum had any effect on pup survival rate, but the size of the second litter was reduced in vrDD mice.

Active maternal behaviors were impaired in TH:DAT KO dams

Having demonstrated that females in both models of DA deficiency were able to rear pups successfully, we examined the mother-pup interactions of TH:DAT KO mice to determine whether striatal DA contributes to maternal behavior. For their first litter, TH:DAT KO mice had dramatically longer pup-retrieval latencies than controls when pups were approximately 6 days old (Log-rank, χ2 [1, N = 56] = 40.79; P < .01) (Figure 4A) and when pups were approximately 10 days old (Log-rank, χ2 [1, N = 48] = 30.09; P < .01) (Figure 4B). For their second litter, TH:DAT KO mice also had much longer pup-retrieval latencies than controls when pups were approximately 6 days old (Log-rank, χ2 [1, N = 46] = 26.23; P < .01) (Figure 4C) and when pups were approximately 10 days old (Log-rank, χ2 [1, N = 41] = 36.55; P < .01) (Figure 4D).

Figure 4.

Figure 4.

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TH:DAT KO mice have impaired active maternal behaviors and delayed latency to begin nursing. Data from the first litter (each N = 7) and the second litter (each N = 6) of TH:DAT KO and control mice (Ctrl) are shown. (A–D) Pup retrieval latencies and percentage of total number of pups retrieved as a function of time in TH:DAT KO dams and controls at approximately 6-day pup age (A and C) and approximately 10-day pup age (B and D) for the first litter (A and B) and the second litter (C and D). Pups that crawled back to the nest on their own are not included. Data points represent retrieval times for individual pups. (E and F) Licking and grooming behavior for the first litter in TH:DAT KO dams and controls, including latency to begin licking and grooming (E) and total licking and grooming time (F). (G and H) Nursing behavior for the first litter in TH:DAT KO dams and controls, including latency to begin nursing (G) and total nursing time (H). (I and J) Licking and grooming behavior for the second litter in TH:DAT KO dams and controls, including latency to begin licking and grooming (I) and total licking and grooming time (J). (K and L) Nursing behavior for the second litter in TH:DAT KO dams and controls, including latency to begin nursing (K) and total nursing time (L). (E–L) Data are expressed as mean ± SEM. *, P < .05; **, P < .01. n.s., not significant.

DAT:TH KO mice were also impaired in licking/grooming and nursing behaviors. For their first litter, 2-way ANOVA revealed significant main effects of genotype for latency to begin licking/grooming pups (F(1,24) = 12.02; P < .01) (Figure 4E), total licking/grooming duration (F(1,24) = 18.81; P < .01) (Figure 4F), and latency to start nursing pups (F(1,24) = 4.33; P < .05) (Figure 4G) but not for total nursing duration (F(1,24) = 3.36; P > .05) (Figure 4H). Similar results were observed for their second litter. Two-way ANOVA revealed significant main effects of genotype for latency to begin licking/grooming pups (F(1,20) = 5.35; P < .05) (Figure 4I), total licking/grooming duration (F(1,20) = 12.68; P < .01) (Figure 4J), and latency to start nursing pups (F(1,20) = 6.61; P < .05) (Figure 4K) but not for total nursing duration (F(1,20) = 2.08; P > .05) (Figure 4H).

Active maternal behaviors were restored in vrDD dams

We also examined whether DA signaling in the striatum alone is sufficient for maternal behavior. We tested vrDD mice and sham controls in the same maternal behavior paradigms as the TH:DAT KO mice. For the first litter, vrDD dams did not take longer to retrieve either their approximately 6-day-old pups (Log-rank, χ2 [1, N = 75] = 0.99; P > .05) (Figure 5A), or their approximately 10-day-old pups (Log-rank, χ2 [1, N = 62] = 0.08; P > .05) (Figure 5B) than sham controls. For the second litter, vrDD dams took longer to retrieve their approximately 6-day-old pups than sham controls (Log-rank, χ2 [1, N = 61] = 5.64; P < .05) (Figure 5C) but did not take longer to retrieve their approximately 10-day-old pups (Log-rank, χ2 [1, N = 57] = 0.33; P > .05) (Figure 5D).

Figure 5.

Figure 5.

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The vrDD mice have mostly restored pup retrieval behavior and intact licking, grooming, and nursing behavior. Shown are data from the first litter of vrDD (N = 10) and sham control (N = 9) mice and their second litter (vrDD N = 7, sham control N = 9). (A–D) Pup retrieval latencies and percentage of total number of pups retrieved as a function of time in vrDD dams and sham controls at approximately 6-day pup age (A and C) and approximately 10-day pup age (B and D) for the first litter (A and B) and the second litter (C and D). Pup retrieval behavior was restored in vrDD dams except for the second litter at the approximately 6-day pup age level. Pups that crawled back to the nest on their own are not included. Data points represent retrieval times for individual pups. (E and F) Licking and grooming behavior for the first litter in vrDD dams and controls, including latency to begin licking and grooming (E) and total licking and grooming time (F). (G and H) Nursing behavior for the first litter in vrDD dams and controls, including latency to begin nursing (G) and total nursing time (H). (I and J) Licking and grooming behavior for the second litter in vrDD dams and controls, including latency to begin licking and grooming (I) and total licking and grooming time (J). (K and L) Nursing behavior for the second litter in vrDD dams and controls, including latency to begin nursing (K) and total nursing time (L). (E–L) Data are expressed as mean ± SEM. *, P < .05; **, P < .01. n.s., not significant.

Licking/grooming and nursing behaviors were restored in vrDD mice. For their first litter, 2-way ANOVA showed no significant differences between vrDD dams and sham controls in latency to begin licking/grooming (F(1,34) = 0.04; P > .05) (Figure 5E), total licking/grooming time (F(1,34) = 0.28; P > .05) (Figure 5F), latency to initiate nursing (F(1,34) = 0.05; P > .05) (Figure 5G), or total nursing time (F(1,34) = 0.69; P > .05) (Figure 5H). Similar results were obtained for their second litter. Two-way ANOVA revealed no significant effects of genotype for latency to begin licking/grooming pups (F(1,27) = 1.56; P > .05) (Figure 5I), total licking/grooming duration (F(1,27) = 1.26; P > .05) (Figure 5J), latency to start nursing pups (F(1,27) = 0.14; P > .05) (Figure 5K), or total nursing duration (F(1,27) = 0.49; P > .05) (Figure 5H). Taken together, these results indicate that vrDD dams have essentially normal maternal behavior.

Locomotor activity and nonmaternal behaviors are not altered in TH:DAT KO mice or vrDD mice

To rule out the possibility that differences in maternal behavior are caused by differences in locomotor activity, we used the videos from the behavior tests to analyze dams' distance traveled during the tests. We could only examine tests involving approximately 6-day-old pups, because the older pups interfered with the video tracking. One video from the vrDD group (first litter) was not included in the analysis, because the dam could not be tracked reliably throughout the test. For the TH:DAT KO group, 2-way ANOVA revealed no significant main effect of genotype (F(1,22) = 0.02; P > .05) (Figure 6A), although there was a significant main effect of litter number (F(1,22) = 6.92; P < .05) (Figure 6A), indicating more activity during tests of the second litter. For the vrDD group, 2-way ANOVA revealed no significant main effect of genotype (F(1,30) = 0.39; P > .05) (Figure 6B) or for litter number (F(1,30) = 3.23; P > .05) (Figure 6B). These findings suggest that deficits in maternal behavior were not caused by hypoactivity.

Figure 6.

Figure 6.

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TH:DAT KO mice and vrDD dams have normal levels of locomotion during maternal behavior tests. (A) Distance moved by TH:DAT KO dams and controls (Ctrl) during tests of the first litter (each N = 7) and the second litter (each N = 6) at the approximately 6-day pup age level. (B) Distance moved by vrDD dams and sham controls during tests of the first litter (each N = 9) and the second litter (sham control N = 9, vrDD N = 7) at the approximately 6-day pup age level. Data are expressed as mean ± SEM. No significant genotype effects were found.

In addition to maternal behaviors, nonmaternal behaviors (nest rearranging, eating, self-grooming, digging/treading, and rearing) were also analyzed. These data are presented in Supplemental Table 2. The only significant differences were in nest rearranging during the second litter in both TH:DAT KO and vrDD dams. In TH:DAT KO dams, 2-way ANOVA indicated significant main effects of genotype (F(1,20) = 5.38; P < .05) (Supplemental Table 1) and pup age (F(1,20) = 6.58; P < .05) (Supplemental Table 1). Bonferroni's post hoc comparisons showed that TH:DAT KO mice performed more nest-rearranging behavior than controls only in tests with approximately 6-day pups (P < .05). Likewise, in vrDD dams, 2-way ANOVA of nest-rearranging behavior revealed a significant main effect of genotype (F(1,27) = 20.02; P < .01) (Supplemental Table 1) and a significant interaction effect (F(1,27) = 6.63; P < .05) (Supplemental Table 1), with post hoc comparisons showing a difference only at the approximately 10-day age level (Bonferroni, P < .01). All other ANOVAs of nonmaternal behaviors showed no differences (all P > .05) (Supplemental Table 1).

Discussion

The TH:DAT KO model described here is ideal for analyzing the effect of hypodopaminergic signaling on maternal behavior, because these mice have severely depleted DA in the striatum (5% of normal) and reduced levels of DA in hypothalamus (20% of normal) but have normal DA levels in other brain regions. They survive and reproduce without pharmacological intervention, but dams were impaired in pup retrieval and licking/grooming behavior. Despite their poor performance in maternal behavior tests, their pups survived to weaning age at normal rates. Although lesioning the DA system in rats has been reported to adversely affect pup survival (35), our findings indicate that normal DA signaling in striatum and hypothalamus is necessary for normal maternal behavior but not for pup survival under controlled laboratory conditions. TH:DAT KO mice had normal activity levels during the tests, so their deficits in maternal behavior stem from problems in motivation rather than locomotion. They also showed minimal differences from controls in other nonmaternal behaviors. Some TH:DAT KO dams spent time eating, whereas no controls ate at all during the tests, but the TH:DAT KO dams that did not eat were also impaired in pup retrieval and licking/grooming behavior. Nest-rearranging behavior was increased in TH:DAT KO dams, but only at 1 time point. Taken together, these data suggest that although TH:DAT KO dams were as active as controls, fewer of their movements were directed toward pups. Because the 3-hour separation before each behavior test was potentially stressful for dams, we cannot definitively rule out the possibility that the deficits observed in TH:DAT KO dams were due to stress hypersensitivity. However, this explanation is unlikely, because these mice did not engage in more stress-related behaviors (treading, self-grooming) than controls.

The DD line of mice is a more complete model of hypodopaminergia (<1% of normal DA levels in all brain areas), but we could not test those mice because of their extreme hypoactivity and complete lack of exploratory locomotion (36). Furthermore, the daily L-Dopa injections necessary to keep them alive would confound the results due to bursts of activity that occur after each injection (37).

The TH:DAT KO model does not allow us to differentiate between striatal subregions where DA signaling may be important for maternal behavior, so we used a viral rescue strategy to examine the role of dorsal striatal DA in maternal behavior. The vrDD mice have partially restored DA levels only in the dorsal striatum, but they display normal locomotor behavior and can survive without L-Dopa intervention. It is important to note that DD mice have no developmental disturbances of the nervous system. Excitatory inputs to their DA neurons are intact, and they have rapid biological responses to pharmacological restoration of DA signaling (38). However, postsynaptic target cells of DA neurons in the striatum become hypersensitive to DA signaling in DD mice (39). After viral restoration of DA signaling in vrDD mice, this hypersensitivity disappears in the striatal area where TH expression is restored (25). Although inactivation of the main types of striatal DA receptors, D1 and D2, does not affect fertility in mutant mice (40, 41), deficits in maternal behavior have been reported for mice lacking D2 receptors (42). No such information is available for mice lacking D1Rs. Because rescue of DA signaling in vrDD mice restores signaling through both D1 and D2 receptors, our study does not allow us to distinguish which striatal DA receptor signaling pathway is critical.

The vrDD mice performed as much licking/grooming behavior as sham controls and retrieved pups almost as efficiently as controls, demonstrating that DA in the dorsal striatum enables active maternal behavior. Although vrDD mice had a deficit in pup retrieval of their second litter when the pups were approximately 6 days old, the deficit disappeared when the same pups were approximately 10 days old, when pups' vocalizations become more complex (43). We observed elevated nest-rearranging behavior in vrDD mice compared with sham controls, but this difference did not appear to affect retrieval, licking/grooming, or nursing, and there were no other differences in nonmaternal behaviors or activity levels between these groups. Although DA signaling in the ventral striatum has repeatedly been shown to modulate active maternal behavior in rats (2, 3, 11, 16), it does not appear to be essential in the mice used in our study. We cannot rule out the possibility that DA in the ventral striatum is also sufficient for maternal behavior; if so, there may be redundant pathways of DA signaling in dorsal and ventral striatum. Because vrDD dams showed some deficit in pup retrieval, it is likely that both striatal subregions or the hypothalamus contribute to normal maternal behavior.

To our knowledge, this is the first time that the dorsal striatum has been clearly implicated in maternal behavior. Although Keer and Stern (10) found a deficit in licking behavior after injection of the nonselective DA receptor antagonist cis-flupenthixol into the dorsal striatum, the effect was stronger when the drug was injected into the NAc, and there was no effect on pup retrieval behavior after a dorsal injection. Hansen et al (24) found no deficits in maternal behavior after a 6-OHDA lesions of the dorsal striatum, but the authors reported that DA levels were still 30% of normal. Previous studies of vrDD mice showed that mice can perform many DA-dependent behaviors with this degree of DA depletion in the dorsal striatum (27–29, 44), so these 6-OHDA lesions may be insufficient to affect maternal behavior.

The DA detected in the ventral striatum of the vrDD mice probably represents slight misplacement of punches during sample collection, because TH staining in the ventral striatum was not observed, and we have been unable to measure functional DA signaling in the ventral striatum after dorsal viral injections (25, 44). Furthermore, DA levels in the ventral striatum of vrDD mice were comparable with those in TH:DAT KO mice. Hence, even if there were a small amount of DA in the ventral striatum of vrDD mice, this would not be sufficient to support maternal behavior. There is evidence that the SNc receives projections from both the MPOA and the ventral bed nucleus of the stria terminalis (45), another nucleus implicated in the onset of maternal behavior (6, 9, 46, 47). Although it is not yet known whether the MPOA and ventral bed nucleus of the stria terminalis stimulate the firing of DA neurons in the SNc as they do in the VTA, this is one potential mechanism for nigro-striatal involvement in maternal behavior.

There is an important distinction between the present vrDD strategy and previous studies of DA's role in maternal behavior. Lesions and pharmacological interventions are subtractive, removing an aspect of DA signaling from an intact neural network to test whether the circuit in question is necessary for maternal behavior (4, 9, 16, 24, 35, 48). Our strategy is restorative, rescuing a particular pathway of DA signaling in mice otherwise lacking DA in all brain areas; thus, emphasizing the impact of DA signaling in the dorsal striatum to the circuitry mediating active maternal behavior. Likewise, the TH:DAT KO model differs from previous studies, because it involves the cell-specific inactivation of DA synthesis rather than the killing of DA neurons or blockade of receptors. Lesions that kill DA neurons could alter other aspects of neurotransmission in addition to DA signaling (49, 50); pharmacological intervention is transitory and may be nonspecific.

Although not a focus of these studies, it is noteworthy that both vrDD and TH:DAT KO females became pregnant and gave birth to normal size litters, indicating that limited DA signaling in the striatum is sufficient for normal endocrine control of oocyte maturation, ovulation, mating behavior, and parturition for at least 2 cycles. However, dopaminergic neurons in the arcuate hypothalamus regulate prolactin production; vrDD mice lacking DA production by these neurons massively overproduce prolactin by 9 months of age, which may have reproductive consequences (51). It is also noteworthy that vrDD males are fertile and sire normal-size litters. Thus, DA signaling in the dorsal striatum is sufficient for sexual behavior in both sexes.

Previous findings suggest that disruption of the mesolimbic DA pathway and its input from the MPOA impairs active but not passive maternal behavior (10, 12, 16, 24, 48). Passive behaviors like nursing are performed after the mammal has achieved the goal of the appetitive behavior. Such behaviors are thought to be more reflexive than voluntary, mediated by hypothalamic interactions with the brainstem rather than motivational systems in the telencephalon (6, 17). Active behaviors are performed to help the animal achieve a particular goal, and these behaviors depend on the striatal DA system (6, 17, 52). Hence, it is surprising that passive nursing behavior was reduced in TH:DAT KO mice. Although TH:DAT KO dams spent had longer latencies to begin nursing than controls, this delay is largely a function of their longer pup-retrieval latencies, so their lack of striatal DA may not directly impair nursing behavior. Similar patterns have been observed in other realms of behavior: DD mice will not seek food but still have the capacity to consume it when it is delivered to their mouths (37, 44), and they have a normal preference for a sucrose solution (53).

Access to pups can serve as a reward (4) and can be preferred over access to drugs of abuse (54). Therefore, it is likely that motivation for active maternal behaviors is mediated in part by the same circuitry that mediates reward-oriented, goal-directed behaviors. Release of striatal DA energizes animals to perform these behaviors (55–57) and allocates energy resources toward goal-directed behavior (58), potentially enabling dams to respond maternally to sensory cues of nearby pups. For vrDD mice in the present study, we chose an injection site in the caudal dorsal striatum that has been implicated in goal-directed behavior (23, 59). Although we show that DA in this region is sufficient to motivate maternal behavior at levels close to normal, other striatal subregions may still contribute. In fact, there is mounting evidence for a more nuanced system of striatal organization than the traditional dorsal-ventral division, with multiple subregions playing different roles and some functional overlap between the dorsal striatum and the NAc (22, 27, 28, 60, 61). Future studies examining other striatal subregions and their interactions with the MPOA and other key circuits will help reveal a more accurate picture of the role of striatal DA in maternal behavior. Furthermore, there is evidence that maternal care can influence adult phenotypes related to anxiety and stress (62), as well as hippocampal development (63), so it would be interesting to conduct future experiments to examine the behavior of adult mice reared by hypodopaminergic dams. Due to the breeding setup in the present study, pups were not homozygous for any of the mutations present in the dams and hence presumably normal in their DA levels. Maternal care by wild-type animals for hypodopaminergic pups would be another interesting future topic for investigation.

Acknowledgments

We thank Jeffrey Gibbs for maintaining the DD mouse colony and helping with some of the behavior assays and Dr Miguel Chillon (Vector Production Unit of Centre de Biotecnologia Animal i Teràpia Gènica at Universitat Autonoma Barcelona, Barcelona, Spain) for initially providing us with the CAV2-Cre virus.

This work was supported in part by the Howard Hughes Medical Institute and the Pacific Northwest Udall Center Grant NS062684.

Disclosure Summary: The authors have nothing to disclose.

Funding Statement

This work was supported in part by the Howard Hughes Medical Institute and the Pacific Northwest Udall Center Grant NS062684.

Footnotes

Abbreviations:
CAV2-Cre
canine adenovirus 2 expressing Cre recombinase
DA
dopamine
DAT
DA transporter
DD
DA deficient
D1R
D1 receptor
L-Dopa
L-3,4-dihydroxyphenylalanine
KO
knockout
MPOA
medial preoptic area
NAc
nucleus accumbens
6-OHDA
6-hydroxydopamine
SNc
substantia nigra pars compacta
Th
tyrosine hydroxylase
vrDD
virally rescued DD
VTA
ventral tegmental area.

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