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. Author manuscript; available in PMC: 2019 May 1.
Published in final edited form as: J Neurochem. 2018 Feb 8;145(4):287–298. doi: 10.1111/jnc.14307

Alterations in mGlu5 receptor expression and function in the striatum in a rat depression model

Li-Min Mao 1, John Q Wang 1,2,*
PMCID: PMC5980680  NIHMSID: NIHMS934720  PMID: 29337350

Abstract

Major depressive disorder is a common form of mental illness. Many brain regions are implicated in the pathophysiology and symptomatology of depression. Among key brain areas is the striatum that controls reward and mood and is involved in the development of core depression-like behavior in animal models of depression. While molecular mechanisms in this region underlying depression-related behavior are poorly understood, the glutamatergic input to the striatum is believed to play a role. In this study, we investigated changes in metabotropic glutamate (mGlu) receptor expression and signaling in the striatum of adult rats in response to prolonged (10–12 weeks) social isolation, a pre-validated animal paradigm modeling depression in adulthood. We found that mGlu5 receptor protein levels in the striatum were increased in rats that showed typical depression- and anxiety-like behavior after chronic social isolation. This increase in mGlu5 receptor expression was seen in both subdivisions of the striatum, the nucleus accumbens and caudate putamen. At subcellular and subsynaptic levels, mGlu5 receptor expression was elevated in surface membranes at synaptic sites. In striatal neurons, the mGlu5-associated phosphoinositide signaling pathway was augmented in its efficacy after prolonged social isolation. These data indicate that the mGlu5 receptor is a sensitive substrate of depression. Adulthood social isolation leads to the upregulation of mGlu5 receptor expression and function in striatal neurons.

Keywords: Caudate putamen, nucleus accumbens, mGlu1, metabotropic glutamate receptor, anhedonia, antidepressant, social isolation

Graphical abstract

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This study was carried out to monitor changes in group I metabotropic glutamate (mGlu) receptors in the striatum in a rat depression model. Striatal mGlu5 receptor proteins were elevated in adult rats that showed typical depression- and anxiety-like behavior afterchronic social isolation. mGlu5 receptors were elevated in surface membranes at synaptic sites. The mGlu5-associated inositol-1,4,5-triphosphate (IP3) signaling was augmented in socially isolated rats. Thus, adulthood social isolation leads to the upregulation of mGlu5 receptors in striatal neurons.

Group I metabotropic glutamate (mGlu) receptors (mGlu1/5) are coupled to Gαq proteins (Niswender and Conn, 2010). Stimulation of these receptors activates phospholipase Cβ1, which hydrolyzes phosphoinositide into inositol-1,4,5-triphosphate (IP3) and diacylglycerol. The former releases Ca2+ from internal stores, while the latter activates protein kinase C. Group I mGlu receptors are broadly expressed in the brain and are typically distributed postsynaptically (Niswender and Conn, 2010). In the striatum, including the nucleus accumbens (NAc) and caudate putamen (CPu), mGlu1 and especially mGlu5 receptors are enriched in medium spiny projection neurons (Testa et al., 1994; Tallaksen-Greene et al., 1998), implying roles of these receptors in normal striatal neuronal activities and in the pathogenesis of various psychiatric disorders (Niswender and Conn, 2010).

Major depressive disorder is a common form of mental illness and is classified into multiple subtypes based on distinct subsets of syndromes. Various brain regions are implicated in depression, and a recent focus is the NAc (Nestler and Carlezon, 2006). As a central site within the limbic circuits that control the rewarding effects of natural rewards (such as food and sex) and drugs of abuse, the NAc plays a role in anhedonia (decreased ability to experience pleasure) and reduced motivation, two core symptoms in most individuals with depression. Indeed, early studies in various animal models support the role of the NAc in unique behavioral phenotypes that are likely relevant to depression (Zacharko and Anisman, 1991; Nestler and Carlezon, 2006). One example is that cAMP response element binding protein activity in the NAc is associated with depression- and/or anxiety-like behavior and antidepressant-like activity (Pliakas et al., 2001; Barrot et al., 2002; Newton et al., 2002; Carlezon et al., 2005; Dinieri et al., 2009; Wallace et al., 2009; Green et al., 2010). Apparently, the NAc serves as one of key substrates critical for the pathophysiology and symptomatology of depression, although underlying molecular mechanisms within this region are poorly understood.

Accumulating evidence implies a role of glutamatergic transmission in major depression and antidepressant activity (Paul and Skolnick, 2003; Bleakman et al., 2007; Vose and Stanton, 2017). Among all glutamate receptor subtypes investigated, group I mGlu receptors are of particular interest (Pilc et al., 2008). The mGlu5 antagonists consistently produced antidepressant-like effects in several models of depression (Tatarczynska et al., 2001; Pilc et al., 2002; Palucha et al., 2005; Li et al., 2006; Belozertseva et al., 2007). In support of pharmacological data, mGlu5 knockout mice displayed an antidepressant-like behavioral phenotype, a significant decrease in the immobility (Li et al., 2006). These data support the mGlu5 receptor as an important regulator of depression. How mGlu5 receptors are linked to depression is unclear at present due to limited studies. It is generally thought that the mGlu5 receptor constitutes a critical element within a network of synaptic proteins that together undergo adaptive changes in response to chronic stress. By working in concert with other glutamate receptors, e.g., ionotropic N-methyl-D-aspartate (NMDA) receptors and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, mGlu5 receptors contribute to the remodeling of excitatory synaptic transmission related to the pathogenesis of depression. In fact, mGlu5 receptors are known to potentiate NMDA receptors (Huang and van den Pol, 2007; Rosenbrock et al., 2010) and the NMDA receptor antagonists, like mGlu5 antagonists, produced antidepressant effects (Tokita et al., 2012; Dutta et al., 2015; Aleksandrova et al., 2017; Jaso et al., 2017).

To date, neurochemical studies monitoring changes in mGlu1/5 expression and function in depressed animal brains are limited, especially in the striatum (aan het Rot et al., 2009). Thus, we initiated this study to investigate alterations in mGlu1/5 expression and function in the NAc and CPu in an animal paradigm of depression, a prolonged social isolation in adult rats. This depression model in adulthood is unique because it characteristically induced anhedonia/depression-like behavior in species (rodents) that have important social interactions (Wallace et al., 2009) and is particularly useful for modeling depression in adulthood animals for a purpose to explore long-lasting neuroplasticity associated with enduring depression-related behavior (Krishnan and Nestler, 2011).

Materials and Methods

Animals

Wistar male rats (RRID:RGD_2312511; catalog #: 2312511; Charles River, New York, NY) were used. The study was not pre-registered. These animals arrived at 7–8 weeks of age (200–225 g) and acclimated to the facility for at least 5 days before the experiment. They were housed in a controlled environment at a constant temperature of 23°C and humidity of 50 ± 10% with food and water available ad libitum. The animal room was on a 12-h/12-h light/dark cycle (lights on between 7 a.m. and 7 p.m.). Animals were used in accordance with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee (University of Missouri-Kansas City, reference #: 1006-3) has approved the animal protocol. The Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines have been followed.

Social isolation

Prolonged adulthood social isolation was conducted utilizing a recently validated chronic stress model, which has demonstrated to produce a discrete set of depression- and anxiety-like symptoms (Wallace et al., 2009). Briefly, isolated rats were housed in home cages individually (one per cage) for 10–12 weeks (Fig. 1A). Other rats were housed two animals per cage for the same period of time and served as controls. After 10–12 weeks of social isolation, rats were used for behavioral assessments. Rats were then anesthetized by an intraperitoneal injection of sodium pentobarbital (65 mg/kg) and sacrificed for neurochemical assays to monitor changes in subcellular and subsynaptic expression of mGlu1/5 receptors or in mGlu5-mediated IP3 production. Sodium pentobarbital was chosen to ensure deep anesthesia prior to decapitation. Animals were randomly divided into different groups using a computer-generated randomization table (GraphPad software/QuickCalcs, La Jolla, CA). Sample size was determined by the sample size calculation with alpha = 0.05 and beta = 0.2 (80% power). There were no sample size differences between the beginning and end of the experiments. The criteria for inclusion/exclusion were based on the health state of animals. The animals that were healthy and showed no sign of illness as evaluated by the body weight and visual observations were used in the analysis.

Figure 1. Chronic social isolation in adult rats induces depression- and anxiety-like behavior.

Figure 1

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(A) Timeframe for behavioral and neurochemical assessments. Following 10–12 weeks of prolonged social isolation, rats underwent behavioral tests, such as sucrose intake and elevated plus maze, prior to striatal tissue collection for neurochemical assays. (B) Effects of social isolation (SI) on sucrose intake. (C) Effects of social isolation on sucrose preference. (D) Effects of social isolation on the time spent in open arms. (E) Effects of social isolation on open arm entries. Note that social isolation significantly reduced sucrose drinking (B) and sucrose preference (C) as measured during a period of 24 h. Social isolation also reduced the time spent in open arm exploration (D) and the number of entries to open arms (E) during the elevated plus maze test over 5 min. Data are presented as means ± standard error of the mean (n = 6 − 14 per group) with 'n' equal to the number of animals. *P < 0.05 versus double-housed control rats (Student's t-test). P values = 0.025 (B), 0.028 (C), 0.020 (D), and 0.022 (E).

Sucrose preference

Preference of sucrose liquid intake was measured as an operational index of anhedonia (reduced responsiveness to a pleasurable stimulus). A modified two-bottle-choice paradigm was utilized as described previously (Wallace et al., 2009). Briefly, rats were initially habituated to two bottles of water for 5 days. Rats were then allowed unlimited access to two bottles, one containing tap water and another one containing 1% (w/v) sucrose, for 24 h. The placement of the bottles was switched after about 12 h to control the left-versus-right side preference of drinking behavior. Water and sucrose solutions consumed were measured and expressed as a preference for sucrose, which was calculated as the percentage of the volume of sucrose consumed (ml per 24 h) divided by the total fluid (sucrose + water) intake (ml per 24 h).

Elevated plus maze

The elevated plus maze was used to assess anxiety-like behavior (Walf and Frye, 2007). Two enclosed and two open arms are arranged in a plus shape (61 cm from the floor, 10 × 50 cm arms). Rats were placed onto the central area of the plus maze facing an open arm. Behavior was recorded and scored in a blind manner. Anxiety-like behavior was analyzed as the time spent in the open arms (s) and the ratio of open arm entries to the total number of entries (expressed as a percentage) during a 5-min freely exploring period.

Surface cross-linking assays

Surface cross-linking assays to detect surface versus intracellular mGlu1/5 receptor expression in striatal neurons of adult rat brains were performed as described previously (Boudreau and Wolf, 2005; Mao et al., 2009; Herrold et al., 2013). Briefly, rats were anesthetized and decapitated. Rat brains were removed and cut into coronal sections (300–400 µm) with a vibratome (Leica VT1200S, Buffalo Grove, IL). The NAc and CPu were rapidly dissected and added into Eppendorf tubes filled with ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM) 10 glucose, 124 NaCl, 3 KCl, 1.25 KH2PO4, 26 NaHCO3, 2 MgSO4, and 2 CaCl2, bubbled with 95% O2–5% CO2, pH 7.4. A bis(sulfosuccinimidyl)suberate (BS3) agent (Pierce, Rockford, IL) was added to 2 mM and incubated for 1 h with gentle agitation at 4°C. The cross-linking reaction was terminated by quenching with 20 mM of glycine (10 min, 4°C). The sections were then washed four times in cold phosphate buffered saline solutions (5 min each) and sonicated in ice cold isotonic sucrose homogenization buffer containing 0.32 M sucrose, 10 mM HEPES, pH 7.4, 2 mM EDTA, 1% sodium dodecyl sulfate (SDS), and a protease/phosphatase inhibitor cocktail (ThermoFisher Scientific, Rochester, NY). Protein concentrations were measured with a Pierce bicinchoninic acid assay kit (ThermoFisher) and saved in −80°C before used in immunoblot analysis.

Fractionation of synaptic and extrasynaptic proteins

Enriching proteins from synaptic and extrasynaptic membrane pools was performed according to a subsynaptic fractionation procedure that we used previously (Mao et al., 2013). Briefly, rats were anesthetized and decapitated. The striatum from removed brains was homogenized in isotonic sucrose homogenization buffer containing 0.32 M sucrose, 10 mM HEPES, pH 7.4, 2 mM EDTA, and a protease/phosphatase inhibitor cocktail (ThermoFisher). The homogenate was centrifuged at 760 g (10 min, 4°C). The pellet was resuspended, re-homogenized, and centrifuged at 760 g. The combined supernatant from the first and second centrifugation at 760 g was then centrifuged at 10,000 g (15 min, 4°C) to generate the supernatant containing cytosol proteins and the pellet 2 (P2) containing crude synaptosomal plasma membranes. P2 was washed and centrifuged to produce the final P2 pellet. This P2 was resuspended in the sucrose homogenization buffer containing Triton X-100 (0.5%, v/v). The suspension was incubated with gentle rotation for 20 min (4°C). After centrifugation for 20 min (32,000 g), the pellet enriched with Triton X-100-insoluable synaptic membranes and the supernatant enriched with Triton X-100-soluable extrasynaptic membranes were obtained. The extrasynaptic fraction was concentrated if needed by acetone precipitation (−20°C overnight and then centrifugation at 3,000 g). The concentrated extrasynaptic pellet and the synaptic fraction were solubilized in the sucrose-Triton buffer containing 1% SDS and a protease/phosphatase inhibitor cocktail (ThermoFisher). Protein concentrations were determined and samples were stored at −80°C until use.

Western blot analysis

Western blots were performed as described previously (Jin et al., 2013). Briefly, protein samples without heat were loaded with lithium dodecyl sulfate loading buffer and separated on 4–12% Tris-glycine gels (Invitrogen, Carlsbad, CA). They were then transferred to polyvinylidene fluoride membranes. Membranes were incubated with a primary antibody overnight at 4°C. This was followed by 1-h incubation of a horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (1:2,000–5,000) at room temperature. Immunoblots were developed with the enhanced chemiluminescence reagent (GE Healthcare Life Sciences, Piscataway, NJ). MagicMark XP Western protein standards (Invitrogen) were used for protein size determination. The density of immunoblots was measured using the NIH ImageJ (Bethesda, MD). Samples were normalized to total protein loading in Ponceau S stained membranes (Gustin et al., 2010; Eagleson et al., 2013; Bermejo et al., 2014). For mGlu1a and mGlu5 receptor immunoblots, no reducing agents, such as dithiothreitol, and antioxidants were used.

Striatal slice preparation

Rat striatal slices were prepared as described previously (Jin et al., 2013). Briefly, after 10–12 weeks of social isolation, rats were anesthetized and decapitated. Brains were removed and cut using a Leica VT1200S vibratome. Slices were preincubated at 30°C in ACSF for recovery. After the solution was replaced for an additional preincubation (10–20 min), drugs were added and incubated at 30°C.

IP3 Assays

Cellular IP3 levels were measured in rat striatal slices using a rat IP3 ELISA Kit from CUSABIO (Wuhan, China) according to the manufacturer's instructions.

Antibodies and pharmacological agents

Primary antibodies used in this study include a mouse antibody against mGlu1a (RRID:AB_396369; BD Biosciences, San Jose, CA), postsynaptic density protein 95 (PSD-95) (RRID:AB_2292909; UC Davis/NIH NeuroMab Facility, Davis, CA), or α-actinin (RRID:AB94325; MilliporeSigma, Billerica, MA), or a rabbit antibody against mGlu5 (RRID:AB_2295173; MilliporeSigma), calnexin (RRID:AB_2243890; Santa Cruz Biotechnology, Dallas, TX), or actin (RRID:AB_476693; MilliporeSigma). Validation data for antibodies are available from the companies. Pharmacological agents include 3-methyl-aminothiophene dicarboxylic acid (3-MATIDA, Tocris Bioscience, Minneapolis, MN), 3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine hydrochloride (MTEP, Tocris), and (RS)-3,5-dihydroxyphenylglycine (DHPG, Tocris). All drugs were freshly prepared at the day of experiments.

Statistics

Data were evaluated statistically using GraphPad Prism 6 (RRID:SCR_002798; GraphPad software, La Jolla, CA), following tests for the normality of data. No test for outliers was conducted on the data obtained in the study. No rats were excluded from the analysis. We used a one-way analysis of variance (ANOVA) followed by a Bonferroni (Dunn) comparison of groups using least squares-adjusted means or a two-tailed unpaired Student's t-test for two-group comparison. A value of P < 0.05 was considered as a statistically significant level. Exact P values are provided in the figure legend.

Results

Depression- and anxiety-like behavior in socially isolated animals

Prolonged social isolation in adulthood has been demonstrated to produce depression-like behavioral phenotypes (Wallace et al., 2009). Anhedonia, a core symptom of depression and a phenotype that can be measured objectively in rodents, was discovered as a decrease in natural reward-related behavior (deficits in sucrose preference) in socially isolated rats (Wallace et al., 2009). Thus, for the first step of this study, we attempted to validate our depression model by measuring the occurrence of anhedonic behavior. Using two-bottle tests, we found that chronic social isolation (10–12 weeks) produced a significant decrease in sucrose intake in isolated rats compared with control (double-housed) rats (Fig. 1B). Similarly, sucrose preference was reduced in socially isolated rats relative to control rats (Fig. 1C). These results support our prolonged social isolation in adulthood as an animal model with depression-like phenotypes. Since symptoms of depression and anxiety often occur together, we assessed anxiety-like behavior in socially isolated rats. In the elevated plus maze test, we found that prolonged social isolation significantly reduced the time spent in open arm exploration (Fig. 1D) and the number of open arm entries (Fig. 1E). This parallel occurrence of anxiety-like behavior in isolated rats further supports the validity of social isolation as a depression model.

Surface versus intracellular expression of mGlu1/5 receptors

We used a BS3 cross-linking method to assay the surface versus intracellular expression of mGlu1/5 receptors under normal conditions. BS3 is a membrane-impermeable reagent which selectively cross-links surface membrane-bound receptors to form high molecular weight aggregates as opposed to unlinked intracellular receptors with a normal molecular weight. These BS3-linked surface and BS3-unlinked intracellular receptors can be readily separated by gel electrophoresis due to their different molecular weights. As shown in Fig. 2A and 2B, predominant mGlu1a and mGlu5 dimers (250–280 kDa), a major functional form of mGlu1/5 receptors in neurons in vivo (Romano et al., 2001; Shaffer et al., 2010), were displayed in normal NAc tissue from control rats without BS3 treatment. A small amount of the receptors was also displayed in a monomer form (130–150 kDa) in control samples. In the BS3-treated NAc tissue, high molecular bands of mGlu1a and mGlu5 were seen, which reflect BS3-linked surface receptor aggregates. A BS3-unlinked dimer band of either the receptor was shown as intracellular receptors. The selectivity of BS3 was confirmed by the lack of the BS3 cross-linking effect on α-actinin, an intracellular protein (Fig. 2C). Subsequent quantification analysis reveals a significantly less amount of intracellular mGlu1a and mGlu5 dimers in BS3-treated tissue compared with control tissue from the NAc (Fig. 2D). Similar results were seen in the CPu (Fig. 2E). Thus, under normal conditions, mGlu1a and mGlu5 receptors in NAc and CPu neurons are expressed in a higher proportion in surface than intracellular membranes.

Figure 2. Normal surface and intracellular expression of metabotropic glutamate (mGlu) receptors (mGlu1a and mGlu5) in the rat striatum.

Figure 2

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(A and B) Representative immunoblots showing surface and intracellular expression of mGlu1a (A) and mGlu5 (B) receptors in the nucleus accumbens (NAc). Rat NAc tissue was treated with bis(sulfosuccinimidyl)suberate (BS3) or vehicle control (Con), followed by immunoblot analysis (2.5 µg per lane). (C) A representative immunoblot showing expression of α-actinin in control and BS3-treated NAc tissue. (D and E) The percentage of intracellular mGlu1a and mGlu5 dimers in the NAc (D) and caudate putamen (CPu) (E). Total and intracellular receptors in the dimer form were measured from control tissue and BS3-treated tissue, respectively. Data are presented as means ± standard error of the mean (n = 3 per group) with 'n' equal to the number of animals.

Effects of social isolation on mGlu1/5 receptor expression in the NAc

We next wanted to investigate the impact of social isolation on mGlu1/5 receptor expression in striatal neurons. Social isolation had an insignificant impact on mGlu1 expression in NAc neurons. As shown in Fig. 3A, no significant difference in the surface pool of mGlu1a receptors was observed between socially isolated and double-housed control rats, although a tendency of increase was observed in isolated rats. An amount of mGlu1a receptors in the intracellular pool of isolated rats showed little change as compared with control animals. As a result, total mGlu1a receptor levels (surface + intracellular) and the surface to intracellular ratio did not significantly differ between two groups of rats (Fig. 3A and 3B). In contrast to mGlu1a receptors, mGlu5 expression in the NAc was altered to a significant level by social isolation. The surface level of mGlu5 receptors in isolated rats was elevated as compared to control rats (Fig. 3C). A small but significant increase in intracellular mGlu5 expression was also seen in isolated animals. Total mGlu5 levels (surface + intracellular) were elevated as a result of overall increases in both compartments. Moreover, since mGlu5 expression was increased in the surface pool more than the intracellular pool, the surface to intracellular ratio was enhanced in isolated animals (Fig. 3D). Social isolation did not alter expression of a control protein, α-actinin, in NAc neurons (Fig. 3E). These results indicate that social isolation enhances mGlu5 receptor expression in the NAc and enhanced mGlu5 expression primarily occurs in the surface compartment of NAc neurons.

Figure 3. Effects of social isolation on surface and intracellular expression of metabotropic glutamate (mGlu) receptors (mGlu1a and mGlu5) in the rat nucleus accumbens.

Figure 3

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(A and B) Effects of social isolation (SI) on surface and intracellular (Intra) expression of mGlu1a receptors and on the surface to intracellular (S:I) ratio of mGlu1a receptors. (C and D) Effects of social isolation on surface and intracellular expression of mGlu5 receptors and on the S:I ratio of mGlu5 receptors. Note that the socially isolated rats showed a marked increase in surface expression of mGlu5 receptors compared with double-housed control (Con) animals. (E) Effects of social isolation on expression of α-actinin. Representative immunoblots are shown left to the quantified data. Data are presented as means ± standard error of the mean (n = 8 per group) with 'n' equal to the number of animals. *P < 0.05 versus double-housed control animals (Student's t-test). P values for mGlu5 = 0.003 (Surface), 0.038 (Intra), 0.003 (Surface + Intra), and 0.043(S:I).

Effects of social isolation on mGlu1/5 receptor expression in the CPu

The results observed in the CPu generally resemble those seen in the NAc. First, mGlu1a expression in this region remained stable following social isolation. In both surface and intracellular pools, the mGlu1a level showed a minimal change in isolated rats compared to control rats (Fig. 4A). Total mGlu1a levels (Fig. 4A) and the surface to intracellular ratio (Fig. 4B) were thus not different between the two groups. Second, mGlu5 expression in the CPu stayed sensitive to social isolation. The mGlu5 level in the surface fraction of isolated rats was substantially higher than that of control rats (Fig. 4C). Intracellular mGlu5 levels also showed an increase tendency (although P > 0.05). The surface to intracellular ratio was greater in isolated rats due to a greater increase in mGlu5 expression in the surface than intracellular fraction (Fig. 4D). Finally, α-actinin expression remained unchanged in isolated animals relative to control animals (Fig. 4E).

Figure 4. Effects of social isolation on surface and intracellular expression of metabotropic glutamate (mGlu) receptors (mGlu1a and mGlu5) in the rat caudate putamen.

Figure 4

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(A and B) Effects of social isolation (SI) on surface and intracellular (Intra) expression of mGlu1a receptors and on the surface to intracellular (S:I) ratio of mGlu1a receptors. (C and D) Effects of social isolation on surface and intracellular expression of mGlu5 receptors and on the S:I ratio of mGlu5 receptors. Note that the socially isolated rats showed an increase in surface expression of mGlu5 receptors compared with double-housed control (Con) animals. (E) Effects of social isolation on expression of α-actinin. Representative immunoblots are shown left to the quantified data. Data are presented as means ± standard error of the mean (n = 8 per group) with 'n' equal to the number of animals. *P < 0.05 versus double-housed control animals (Student's t-test). P values for mGlu5 = 0.001 (Surface), 0.168 (Intra), 0.005 (Surface + Intra), and 0.034(S:I).

Effects of social isolation on synaptic versus extrasynaptic expression of mGlu1/5 receptors

To determine whether the enhanced surface expression of mGlu5 receptors occurs in a region including the synaptic site, we monitored changes in the amount of mGlu5 proteins enriched from synaptic versus extrasynaptic pools of striatal neurons. PSD-95, a known marker at synaptic sites, was abundant and absent in synaptic and extrasynaptic membranes, respectively, in our fractionation samples from the normal rat striatum (Fig. 5A). In contrast, calnexin, a Ca2+-binding protein present in extrasynaptic membranes (Davies et al., 2007; Ferrario et al., 2011), was seen in extrasynaptic but not synaptic fractions (Fig. 5A). These results demonstrate that the fractionation procedure we used was sufficient in enriching proteins from distinct subsynaptic compartments. Using synaptic and extrasynaptic samples, we then quantified the relative abundance of mGlu1a and mGlu5 proteins in these pools in the normal adult rat striatum. mGlu1a proteins were present at a higher level in synaptic than extrasynaptic fractions (Fig. 5B). Similarly, mGlu5 was more abundant in synaptic than extrasynaptic locations. Thus, mGlu1a and mGlu5 receptors are normally concentrated at synaptic sites, while a small but significant amount of both receptors is present at extrasynaptic sites. In socially isolated rats, no significant change in mGlu1a expression was seen at both synaptic and extrasynaptic sites as compared to control rats (Fig. 5C). Remarkably, mGlu5 levels at synaptic sites were markedly elevated in isolated rats relative to control rats (Fig. 5D). A smaller increase in mGlu5 expression was also seen at extrasynaptic sites (P < 0.05). These data indicate that social isolation increases the redistribution of mGlu5 receptors in synaptic and, to a smaller extent, extrasynaptic locations.

Figure 5. Effects of social isolation on synaptic and extrasynaptic expression of metabotropic glutamate (mGlu) receptors (mGlu1a and mGlu5) in the rat striatum.

Figure 5

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(A) Representative immunoblots showing expression of a synaptic marker postsynaptic density protein 95 (PSD-95) and an extrasynaptic marker calnexin in synaptic (Syn) and extrasynaptic (Extrasyn) samples. (B) Distribution of mGlu1a and mGlu5 receptors in synaptic and extrasynaptic compartments. (C and D) Effects of social isolation (SI) on synaptic versus extrasynaptic expression of mGlu1a (C) and mGlu5 (D) receptors. Note that mGlu5 expression was elevated in synaptic and extrasynaptic locations. Representative immunoblots are shown left to the quantified data (B–D). Data are presented as means ± standard error of the mean (n = 6 per group) with 'n' equal to the number of animals. *P < 0.05 versus double-housed control animals (Student's t-test). P values for mGlu5 = 0.009 (Syn) and 0.014 (Extrasyn).

Effects of social isolation on mGlu5-IP3 signaling

Given that surface and synaptic expression of mGlu5 receptors was upregulated in socially isolated rats, we next wanted to examine whether these biochemical changes lead to modifications of mGlu5 function. mGlu5 receptors activate the Gαq-coupled pathway to produce IP3 molecules (Niswender and Conn, 2010). We thus measured the cytosolic IP3 level as a readout of classical function of mGlu5 receptors. To this end, rats were sacrificed after 10–12 weeks of social isolation and striatal slices were prepared for IP3 assays. An mGlu1/5 agonist DHPG was added to slices to activate mGlu5 receptors in the presence of an mGlu1 antagonist 3-MATIDA (10 µM). As shown in Fig. 6A, adding DHPG (75 µM, 20 s) to striatal slices of normal rats induced a rapid increase in IP3 levels. This increase was confirmed to be mediated by mGlu5 receptors by the observation that pretreatment with an mGlu5 antagonist MTEP (10 µM, 30 min prior to DHPG) completely blocked the IP3 formation induced by DHPG. After 10–12 weeks of social isolation, an increase in IP3 levels was induced by DHPG in striatal slices of isolated rats (Fig. 6B). Noticeably, the degree of this increase was greater than that seen in control rats. Thus, social isolation augments mGlu5 function in striatal neurons as measured by the agonist-stimulated IP3 formation.

Figure 6. Effects of social isolation on the metabotropic glutamate (mGlu) receptor (mGlu5)-mediated inositol-1,4,5-triphosphate (IP3) production in rat striatal neurons.

Figure 6

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(A) Effects of the mGlu5 antagonist MTEP on the DHPG-stimulated IP3 formation. Data were analyzed using analysis of variance (ANOVA): F(3,20) = 7.274, n = 24, P = 0.002. (B) Effects of social isolation (SI) on the DHPG-stimulated IP3 formation. Data were analyzed using ANOVA: F(3,26) = 16.77, n = 30, P < 0.0001. Note that the DHPG-stimulated IP3 formation was augmented in socially isolated rats compared to control rats. Experiments were conducted in rat striatal slices in the presence of an mGlu1 antagonist 3-MATIDA (10 µM). DHPG (75 µM) was applied for 20 s. MTEP was applied at 10 µM 30 min before and during 20-s incubation of DHPG (A). Data are presented as means ± standard error of the mean (n = 6–9 per group) with 'n' equal to the number of striatal slices (A) or animals (B). *P < 0.05 versus vehicle (B) or vehicle + vehicle (A). + P < 0.05 versus vehicle + DHPG (A).

Discussion

This study investigated responses of mGlu1/5 receptors in their subcellular and subsynaptic expression to prolonged social isolation in adult rats. We focused on a reward-related brain area, the striatum, which is enriched with glutamatergic afferents and glutamate receptors and is actively involved in mood control. We found that mGlu5 receptors were sensitive to social isolation. Levels of this receptor in the surface compartment of NAc neurons were markedly increased in isolated rats compared to double-housed control animals. The increase was not confined to the NAc as similar increases occurred in the CPu. At synaptic and, to a lesser extent, extrasynaptic sites, mGlu5 receptor expression was elevated in isolated rats. In contrast to mGlu5 receptors, mGlu1a receptors were less sensitive to social isolation. Functionally, the mGlu5-mediated IP3 signaling was enhanced in the striatum of isolated rats. These data indicate that mGlu5 receptors in expression and function undergo a positive adaptive change in striatal neurons in response to prolonged social isolation.

mGlu5 receptors are localized postsynaptically in two subpopulations of striatal medium spiny neurons (MSN), i.e., striatonigral neurons bearing D1 receptors (direct pathway) and striatopallidal neurons bearing D2 receptors (indirect pathway) (Tallaksen-Greene et al., 1998). Group I mGlu receptors are known to induce synaptic plasticity at glutamatergic synapses throughout the striatum. One of the best-characterized forms of synaptic plasticity is long-term depression (LTD) (reviewed in Luscher and Huber, 2010). Remarkably, the mGlu1/5 agonist DHPG-induced LTD in MSNs of the direct and indirect pathways differentially relies on dopamine signals. In particular, DHPG-LTD in the indirect pathway requires D2 receptor activation (Kreitzer and Malenka, 2005; 2007). In this study, hyperactive mGlu5 receptors were discovered in the striatum of socially isolated rats. This adaptive change may have a significant impact on mGlu5-LTD. It is likely that upregulated mGlu5 receptors serve as an element critical for the remodeling of synaptic plasticity such as mGlu5-LTD during the course of social isolation. Future studies will have to explore changes in mGlu5-LTD at excitatory synapses onto distinct subsets of MSNs, especially D2-bearing striatopallidal neurons, and elucidate roles of the mGlu5-LTD plasticity in depressive behavior.

Several animal models of ‘active’ stress, including foot shock, restraint stress, social defeat and learned helplessness, produce depression- and anxiety-like behavior and have been extensively utilized in relevant studies. As compared to active stress, ‘passive’ models of stress, such as social isolation, have been less studied, even though social isolation and loneliness seem to have particular relevance in mimicking certain subtypes of human depression and anxiety (Hall, 1998; Heinrich and Gullone, 2006; Grippo et al., 2007). Early studies focused on isolation rearing during the early life stages, either in neonatal pups or adolescents, with measuring life-long behavioral abnormalities in adulthood (Hall, 1998). Recent studies used a very different model in which rodents undergo prolonged exposure (weeks to months) to social isolation in adulthood (Wallace et al., 2009). This model produced a characteristic anhedonic feature. It also induced anxiety-like behavior. Since it responded uniquely to chronic, but not acute, antidepressant administration (Wallace et al., 2009), it closely models a human depression condition that antidepressants usually show the effect in depressed humans after many weeks of treatment. Moreover, compared to other stressors, social isolation has its strength since it relies on social interactions and has a good construct validity (Krishnan and Nestler, 2011). In this study, we also observed anhedonia (deficits in sucrose preference) and anxiety-like behavior following chronic social isolation in adult rats. This confirms the utility of social isolation in modeling depression in adulthood. Given the advantage of this model in exploring experience-dependent neuroplasticity (Nestler and Hyman, 2010; Krishnan and Nestler, 2011), it is rational and tempting to use this model to examine plastic changes in mGlu1/5 receptors in relation to depression.

The Flinders Sensitive Line (FSL) rats have been proposed to be a model of retarded depression (Overstreet, 2002). No significant difference in mGlu5 densities in the NAc as detected by autoradiography with a radiolabeled mGlu5 antagonist (ABP688) was found between FSL and SD control rats (Kovacevic et al., 2012). The mGlu5 density in the median part of the CPu was slightly lower in FSL than SD rats. Reduced densities of mGlu5 but not mGlu1 receptors were also observed in the prefrontal cortex, cingulate cortex, thalamus, and hippocampus in individuals with major depressive disorder compared to psychiatrically healthy subjects, although long-term effects of antidepressant treatment on mGlu5 receptor expression cannot be fully excluded (Deschwanden et al., 2011). In a chronic mild stress model, mGlu5 proteins were increased in the CA1 and decreased in the CA3 of the rat hippocampus (Wieronska et al., 2001). Similarly, in the hippocampus of congenitally learned helpless rats, mGlu5 receptors were enhanced in their protein levels and signaling efficacy (Pignatelli et al., 2013). However, in the NAc, active stress (social defeat and electrical food shock) caused a reduction of mGlu5 receptors in susceptible mice as compared to resilient or control mice (Shin et al., 2015). No report has described the response of mGlu1/5 receptors in the striatum to a passive stress model (such as social isolation), to our knowledge, until this study. We found that mGlu5, although not mGlu1, receptor expression was increased in the NAc and CPu of socially isolated rats. It appears that mGlu5 receptor expression is regulated differently, depending on brain regions and models of stress. This may obligate the receptor to play a distinct role in different types, stages, and severity of symptoms derived from various models of depression.

In addition to the monoaminergic system, the glutamatergic transmission is implicated in the pathophysiology, symptomatology, and even etiology of depression (Paul and Skolnick, 2003; Bleakman et al., 2007; Tokita et al., 2012; Vose and Stanton, 2017). To date, pharmacological studies have documented that NMDA receptor antagonists and positive modulators of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors possess the antidepressant property (Paul and Skolnick, 2003; Tokita et al., 2012; Dutta et al., 2015; Aleksandrova et al., 2017; Jaso et al., 2017). Regarding mGlu5 receptors, a number of studies show that mGlu5 antagonists (MPEP and MTEP) produced antidepressant-like effects in a variety of preclinical depression models (Tatarczynska et al., 2001; Pilc et al., 2002; Wieronska et al., 2002; Palucha et al., 2005; Li et al., 2006; Molina-Hernandez et al., 2006; Belozertseva et al., 2007; Pomierny-Chamiolo et al., 2010; Liu et al., 2012). Similarly, mGlu5 knockout mice have an antidepressant-like nature (Li et al., 2006). The data support that the mGlu5 antagonism exerts an antidepressant effect, although how mGlu5 antagonists achieve this effect is unclear. Based on results from the current work, it is likely that chronic social isolation causes long-lasting adaptation of mGlu5 receptors in striatal neurons, i.e., an increase in mGlu5 receptor expression. This leads to hyperactivity of mGlu5 receptor signaling, which thereby contributes to depression-related behavior. Given the central role of the striatum in processing reward responses, mGlu5 receptors in the striatum may regulate at least in part anhedonic symptoms. It should be noted that a recent study found that NAc mGlu5 receptors promote resilience but not susceptibility to depression since mGlu5 knockout mice displayed more depression-like behaviors than control mice in active stress models of depression (food shock and social defeat) (Shin et al., 2015). This underscores the complexity of roles of NAc mGlu5 receptors in distinct symptoms in response to different stressors. Future pharmacological studies with mGlu5 selective agents can be conducted to analyze the accurate role of striatal mGlu5 receptors in processing the depressive properties of socially isolated rats.

In addition to unipolar depression, mGlu5 receptors may be implicated in bipolar depression. The classic mood stabilizer lithium that is used to treat bipolar disorder reduced surface mGlu5 receptor expression and mGlu5-Ca2+ signaling in hippocampal neurons (Sourial-Bassillious et al., 2009), raising the possibility that the therapeutic effect of lithium may be in part related to its antagonist properties at mGlu5 receptors. Consistent with this, the mGlu5 negative allosteric modulator DSR-98776 exhibited potent antidepressant and antimanic activity (Kato et al., 2015). Thus, there exists increasing interest in exploring mGlu receptors in their roles in the neuropathology of bipolar depression and as an emerging target for pharmacotherapy (Blacker et al., 2017).

Compared to mGlu5 receptors, data on mGlu1 receptors in their roles in depression and antidepressant activity are limited (Lesage and Stechler, 2010). A systemic injection of the mGluR1 antagonist JNJ-16567083 produced antidepressant-like effects in two behavioral despair tests (forced swim test and tail suspension test) in rats and mice (Belozertseva et al., 2007; Molina-Hernandez et al., 2008). However, while JNJ-16567083 reduced immobility in the tail suspension test (Belozertseva et al., 2007); this effect was not picked up with structurally related compounds (Lesage and Stechler, 2010). In this study, we have not observed a significant change in mGlu1 receptor expression in the striatum in our depression model. Future work will clarify the model- and brain region-specific changes in a specific event of various mGlu1 activities (posttranslational modification, dimerization, trafficking, endocytosis, additional signaling pathways, etc.) in response to depression and roles of these changes in constructing long-lasting mGlu1 receptor plasticity related to distinct subsets of depressive symptoms.

Acknowledgments

This work was supported by NIH grants R01DA10355 (JQW) and R01MH61469 (JQW).

Abbreviations

ACSF

artificial cerebrospinal fluid

ANOVA

analysis of variance

BS3

bis(sulfosuccinimidyl)suberate

CPu

caudate putamen

DHPG

(RS)-3,5-dihydroxyphenylglycine

FSL

Flinders Sensitive Line

IP3

inositol-1,4,5-triphosphate

LTD

long-term depression

3-MATIDA

3-methyl-aminothiophene dicarboxylic acid

mGlu

metabotropic glutamate

MSN

medium spiny neurons

NAc

nucleus accumbens

NMDA

N-methyl-D-aspartate

PSD-95

postsynaptic density protein 95

SD

Sprague-Dawley

SDS

sodium dodecyl sulfate

SI

social isolation

MTEP

3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine hydrochloride

RRID

research resource identifier

Footnotes

Conflict of interest: The authors declare that there are no potential conflicts of interest.

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