Ionotropic and metabotropic glutamate receptors regulate protein translation in co-cultured nucleus accumbens and prefrontal cortex neurons

Abstract

The regulation of protein translation by glutamate receptors and its role in plasticity have been extensively studied in the hippocampus. In contrast, very little is known about glutamatergic regulation of translation in nucleus accumbens (NAc) medium spiny neurons (MSN), despite their critical role in addiction-related plasticity and recent evidence that protein translation contributes to this plasticity. We used a co-culture system, containing NAc MSNs and prefrontal cortex (PFC) neurons, and fluorescent non-canonical amino acid tagging (FUNCAT) to visualize newly synthesized proteins in neuronal processes of NAc MSNs and PFC pyramidal neurons. First, we verified that the FUNCAT signal reflects new protein translation. Next, we examined the regulation of translation by group I metabotropic glutamate receptors (mGluRs) and ionotropic glutamate receptors by incubating co-cultures with agonists or antagonists during the 2-h period of non-canonical amino acid labeling. In NAc MSNs, basal translation was modestly reduced by blocking Ca²⁺-permeable AMPARs whereas blocking all AMPARs or suppressing constitutive mGluR5 signaling enhanced translation. Activating group I mGluRs with dihydroxyphenylglycine increased translation in an mGluR1-dependent manner in NAc MSNs and PFC pyramidal neurons. Disinhibiting excitatory transmission with bicuculline also increased translation. In MSNs, this was reversed by antagonists of mGluR1, mGluR5, AMPARs or NMDARs. In PFC neurons, AMPAR or NMDAR antagonists blocked bicuculline-stimulated translation. Our study, the first to examine glutamatergic regulation of translation in MSNs, demonstrates regulatory mechanisms specific to MSNs that depend on the level of neuronal activation. This sets the stage for understanding how translation may be altered in addiction.

1. Introduction

The ability to encode an experience and produce long-lasting changes in behavior requires synaptic modifications dependent on the synthesis of new proteins (Sutton and Schuman, 2006; Zukin et al., 2009). It is well established that dendritic protein translation is regulated by excitatory synaptic transmission and that this is vital for plasticity at excitatory synapses; furthermore, aberrant translation profoundly influences neuronal function and is a key feature of certain brain disorders (Buffington et al., 2014; Liu-Yesucevitz et al., 2011; Steward and Schuman, 2003; Sutton and Schuman, 2005; Swanger and Bassell, 2013).

Protein translation has been extensively studied in hippocampus and cortex, especially in relation to autism-spectrum disorders (Aakalu et al., 2001; Bassell and Warren, 2008; Bhakar et al., 2012; Huber et al., 2000; Huber et al., 2001; Osterweil et al., 2010; Sidorov et al., 2013; Sutton et al., 2006; Waung and Huber, 2009). Recent evidence suggests that alterations in protein translation in reward-related brain regions contribute to cellular and behavioral plasticity in animal models of drug addiction (Huang et al., 2016; Placzek et al., 2016a; Placzek et al., 2016b; Scheyer et al., 2014; Werner et al., 2018). The nucleus accumbens (NAc) is a critical component of the brain’s reward system, serving as a gateway where cortical, limbic, and motor circuits interface to interpret sensory and motivational stimuli and generate adaptive motivated behaviors; GABAergic medium spiny neurons (MSNs) are the principal neurons in the NAc, comprising 90-95% of cells in this region (Sesack and Grace, 2010). Signaling molecules regulating translation have been studied in the NAc (e.g., mTOR; (Dayas et al., 2012; Neasta et al., 2014)) but little is known about glutamatergic regulation of translation in these GABAergic principal neurons, aside from a recent study focusing on effects of cocaine exposure (Stefanik et al., 2018), and it is possible that glutamatergic regulation in GABAergic MSNs differs from what has been found in phenotypically distinct glutamatergic principal neurons in hippocampus and cortex. It is important to understand the regulation of translation in NAc MSNs, not only because of their importance for addiction and other brain disorders (Plotkin and Surmeier, 2015; Surmeier et al., 2014; Wolf, 2016), but also because of growing evidence that some forms of plasticity in MSNs depend upon protein translation, both under normal conditions (Yin et al., 2006) and in animal models of disease (Santini et al., 2013; Scheyer et al., 2014; Smith et al., 2014).

As a first step in addressing this gap in knowledge, we characterized the regulation of protein translation in cultured MSNs, which are amenable to direct measurement of translation. We utilized a co-culture system consisting of NAc MSNs from postnatal day 1 (P1) rats and prefrontal cortex (PFC) neurons obtained from P1 mice expressing enhanced cyan fluorescent protein (ECFP). The PFC neurons establish excitatory synapses onto the MSNs, which would be absent in cultures composed exclusively of NAc neurons, but can be distinguished from NAc neurons based on cyan fluorescence (Reimers et al., 2014; Sun et al., 2008; Sun and Wolf, 2009).

To assess protein translation, we tagged newly synthesized proteins by incorporating the non-canonical amino acid azidohomoalanine (AHA) and visualized them using click chemistry and a fluorescent tag. This method, fluorescent noncanonical amino acid tagging (FUNCAT), has been used previously in other culture systems (Cohen et al., 2013; Dieterich et al., 2010; Fallini et al., 2016; Hsu et al., 2015; Liu and Cline, 2016; tom Dieck et al., 2015; Tom Dieck et al., 2012; Younts et al., 2016). We focused on regulation of translation by ionotropic and group I metabotropic glutamate receptors (mGluR). Our results show that: 1) the regulation of protein translation in NAc MSNs occurs through both overlapping and distinct mechanisms from those operating in PFC cells, and 2) these mechanisms can change depending on the activity level of the cell. These results highlight the heterogeneity of translational regulation across brain regions and different activity states, and provide foundational information for future studies of the relationship between protein translation and NAc function.

2. Methods

2.1. Animals

All animal use procedures were approved by the Rosalind Franklin University of Medicine and Science Institutional Animal Care and Use Committee and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Pregnant Sprague Dawley rats (Harlan, Indianapolis, IN) were obtained at 18-20 days of gestation and housed individually in breeding cages. One-day-old (P1) offspring were decapitated and used to obtain NAc neurons. PFC neurons were obtained from P1 offspring of homozygous enhanced cyan fluorescent protein (ECFP)-expressing mice (strain: B6.129(ICR)-Tg(ACTB-ECFP)1Nagy/J; Jackson Laboratory, Bar Harbor, ME). This homozygous ECFP transgenic mouse strain was maintained in house by mating ECFP male and female mice. All offspring express ECFP.

2.2. Postnatal nucleus accumbens/prefrontal cortex co-cultures

This NAc/PFC co-culture system has previously been described in detail (Sun et al., 2008; Sun and Wolf, 2009). Briefly, the medial PFC of ECFP-expressing P1 mice was dissected and dissociated with papain (20-25U/mL; Worthington Biochemical, Lakewood, NJ) at 37°C for 30 minutes. The cells were then plated at a density of 30,000 cells/well onto coverslips coated with poly-D-lysine (0.1mg/mL; Sigma, St. Loius, MO). One to three days later, the NAc from P1 rats was also dissected and dissociated with papain. These NAc cells were plated at a density of 30,000 cells/well with the PFC cells described previously. The NAc/PFC co-cultures were grown in Neurobasal media (Invitrogen, Carlesbad, CA), supplemented with 2mM GlutaMAX, 0.5% Gentamicin, and 2% B27 (Invitrogen). Half of the media was replaced every 4 days. Cultures were used for experiments between 14 and 21 days in vitro.

2.3. FUNCAT

Direct detection of de novo protein synthesis was achieved using a strategy adapted from previous studies (Dieterich et al., 2010; Hinz et al., 2013; Tom Dieck et al., 2012). Cells were grown for 14-21 days in supplemented Neurobasal media (see previous section) until the day of the experiment when media was replaced with supplemented methionine-free DMEM (Invitrogen) for 30 minutes. This methionine (Met) starvation enhances tagging by the non-canonical amino acid L-azidohomoalanine (AHA, Life Technologies), which incorporates at methionine codons. After Met starvation, AHA (1 mM, pH 7.4) was added to the wells (with or without test drugs) for 2 hours. In addition, every experiment included a control group (4-6 wells) in which, after Met starvation, DMEM supplemented with 1 mM Met was added for 2 hours. This group was used to define the background signal in the absence of AHA incorporation for each experiment (see Experimental design and statistical analysis, below). After AHA or Met incubation, cells (on coverslips) were washed, fixed in 4% PFA for 15 minutes, and stored in PBS until click chemistry reactions. Cells were permeabilized with 0.25% Triton X-100, washed with 3% BSA, and the click chemistry reaction was performed by incubating the coverslips upside-down on droplets of PBS, pH 7.8, containing a Cy-5 conjugated copper-free click chemistry reagent, dibenzocyclooctyne (DBCO, 20nM, Click Chemistry Tools, Scottsdale, AZ), for 30 minutes. Coverslips were then mounted on slides using ProLong Gold Antifade mountant (ThermoFisher, Waltham, MA). In preliminary experiments, we compared Cy5 staining in MSN processes after 30 min versus 2 hours of AHA labeling. The 30 min labeling period did not result in a sufficiently robust and consistent signal for quantification of both decreases and increases in its magnitude in response to experimental manipulations (data not shown).

2.4. Drug treatments

Drugs were incubated for 2 hours along with AHA as described above. Drugs and concentrations are as follows: anisomycin (40 μM, Sigma); colchicine (30 μM, ThermoFisher); 3-((2-Methyl-4 thiazolyl)ethynyl)pyridine (MTEP; 1 μM, Sigma); LY367385 (50 μM, Tocris); JNJ16259685 (JNJ; 100 μM, Tocris); 3,5-dihydroxyphenylglycine (DHPG; 50 μM, Tocris); D-(−)-2-Amino-5-phosphonopentanoic acid (APV; 50 μM, Tocris); 1-Naphthyl acetyl spermine (naspm; 100 μM, Tocris); 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX; 10 μM, Tocris); N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251; 2 μM, Tocris); LY344545 (100 μM, gift of Eli Lilly); and [R-(R*,S*)]-6-(5,6,7,8-Tetrahydro-6-methyl-1,3-dioxolo[4,5-g]isoquinolin-5-yl)furo[3,4-e]-1,3-benzodioxol-8(6H)-one (bicuculline; 20 μM, Tocris).

2.5. Immunocytochemistry

For experiments combining FUNCAT with immunocytochemistry, cells that had previously undergone the AHA-DBCO click chemistry protocol (described above) were washed and then blocked for 1 hour in 3% bovine serum albumin (BSA) in PBS, pH 7.4, at room temperature before being incubated with 1:200 rabbit anti-synaptophysin (080139; ThermoFisher) or 1:1000 mouse anti-S6 ribosomal protein (2317; Cell Signaling Technology) primary antibodies overnight at 4°C. Coverslips were then washed with PBS and incubated in 1:500 Cy3-conjugated rabbit anti-mouse (for synaptophysin; GE Healthcare, Buckinghamshire, UK) or goat anti-rabbit (for S6; GE Healthcare, Buckinghamshire, UK) secondary antibody for 1 hour at room temperature before being mounted on slides using ProLong Gold Antifade mountant. Image analysis is described in the next section.

2.6. Experimental design and statistical analysis

We analyzed the FUNCAT signal in processes of NAc MSNs and nearby PFC pyramidal neurons. All experimental groups from the same culture preparation were processed simultaneously. For each cell type (MSN or PFC pyramidal neuron), three to six cells per well from four to six wells were analyzed for each experimental group. NAc MSNs were distinguished from other neuronal cell types based on morphology and from ECFP-expressing PFC cells based on the absence of fluorescent signal (Sun et al., 2008). Among the PFC neurons, morphology was used to identify pyramidal neurons for analysis. Cells were selected under phase contrast to avoid experimenter bias and images were coded to ensure that analysis was blind to experimental condition. For each image, fluorescence intensity of the AHA labeling was measured using the total area in a fixed 15 μm length of a process. We analyzed processes located 15-30 μm from the soma because processes in this range receive the highest density of synaptic contacts (Sun et al., 2008) and because manipulating glutamate or DA transmission in the cultures leads to trafficking of AMPARs, NMDARs and group I mGluRs that can be detected in this population of neuronal processes, suggesting relevance to the DA-glutamate interactions important for addiction (Chao et al., 2002; Loweth et al., 2014a; Mangiavacchi and Wolf, 2004a, b; Reimers et al., 2014; Sun et al., 2008; Sun and Wolf, 2009; Werner et al., 2017). It is very unlikely that axons were included in our analysis, as they are readily distinguishable from dendrites based on size and branching. For example, a prior study of cultured NAc MSNs demonstrated that axons are much thinner and exhibit far less branching (Shi and Rayport, 1994). The background threshold was set based on an average of background fluorescence in unstained processes in the Met wells that were included as negative controls in every experiment (see section titled FUNCAT). Because the typical signal intensity in Met groups was negligible (approximate corrected value ~0) compared to AHA-treated cells, we have excluded Met groups from the graphs for simplicity of presentation and, to analyze drug effects, we normalized data from experimental groups (AHA + drug) to the mean of control wells treated with AHA only (no test drugs). However, the Met group, the AHA-only group, and AHA + drug groups were included in ANOVAs (F values>22.74, p’s<0.0001). All significant Tukey’s post-hoc statistics reported are based on comparisons to the AHA group unless otherwise noted. Experimental data were analyzed for significance using Prism 6 (Graphpad) software. Statistical significance was defined as p<0.05 and all data are presented as mean +/− SEM.

3. Results

3.1. Validation of FUNCAT in cultured NAc MSNs

We began by adapting the FUNCAT approach to detect and quantify newly translated proteins in NAc MSNs. This approach utilizes a non-canonical amino acid bearing an azide group (azidohomoalanine or AHA), which incorporates into newly synthesized proteins at Met codons (Dieterich et al., 2010; Hinz et al., 2013; tom Dieck et al., 2015). Newly translated proteins can then be visualized by conjugating the azide-bearing AHA to an alkyne affinity tag bound to a flouorophore. This can be accomplished using selective Cu(I)-catalyzed [3+2] azide-alkyne cycloaddition (CuAAC click chemistry) (Dieterich et al., 2010; Dieterich et al., 2007; Dieterich et al., 2006) or in copper-free conditions using fluorescent reporters containing dibenzylcyclooctyne (DBCO) (Bagert et al., 2014; Sletten and Bertozzi, 2009; tom Dieck et al., 2015).

In preliminary experiments, we compared Cy5-conjugated click chemistry tools in both copper-catalyzed and copper-free conditions and found equivalent fluorescence signals using either method (data not shown). Based on these studies, we conducted all subsequent click chemistry experiments using copper-free conditions and DBCO, which has the advantage of being able to detect newly synthesized proteins at low concentrations without a multi-step labeling procedure.

We used this approach to measure translation in co-cultures. Between 14 to 21 days after plating, co-cultures were incubated with 1 mM AHA for 2 hours [concentration and timing were based on prior studies; (Dieterich et al., 2010; Hsu et al., 2015; Tom Dieck et al., 2012)] and then processed for click chemistry to tag AHA with DBCO-Cy5 for visualization. Since AHA is incorporated into newly synthesized proteins at Met codons, some wells from every experiment were incubated with an equivalent concentration of Met (rather than AHA) prior to click chemistry to serve as negative controls for defining baseline levels of fluorescence. Cy5 labeling was assessed using fluorescence microscopy and analyzed with Metamorph software. As detailed in Materials and Methods, we distinguished NAc from PFC neurons based on cyan fluorescence (Sun et al., 2008) (Fig. 1A) and used previously described morphological criteria to identify dendritic processes (Shi and Rayport, 1994; Sun et al., 2008). We acquired images of processes located 15-30 μm away from the soma. Fluorescent labeling was present in the soma after incubation with either Met (not shown) or AHA (Fig. 1A) followed by click chemistry. However, labeling in processes was far more robust in AHA-treated cells (~20-fold greater) than in Met controls, and this labeling was essentially eliminated if anisomycin, an inhibitor of translation, was present during the AHA incubation period (Fig. 2A, B; Tukey’s post hoc test, p<0.0001). Processes of cells that were incubated with AHA but not processed for click chemistry were found to exhibit very low levels of fluorescent labeling similar to that observed in the Met group (data not shown). Interestingly, the AHA signal in processes of PFC pyramidal neurons was higher than in NAc MSNs (Fig. 1B). We speculate that this reflects higher neuronal activity leading to higher protein translation in glutamatergic PFC pyramidal neurons versus GABAergic MSNs.

Figure 1. Detection of newly translated proteins in processes of identified NAc and PFC neurons using FUNCAT.

A, Representative images showing morphology (left), somatic CFP fluorescence, and AHA signal in co-cultured NAc and PFC neurons. Left panel, Processes visualized under phase contrast show that neuronal morphology is not compromised by FUNCAT processing. Middle panel, PFC cells obtained from ECFP mice can be distinguished from NAc cells from rats based on somatic CFP fluorescence. Right panel, AHA signal can be detected in both cell types. B, AHA signal intensity is different between NAc medium spiny neurons and PFC pyramidal neurons, suggesting a higher rate of basal translation in the PFC neurons. *p<0.05

Figure 2. Characterization of AHA-tagged proteins in processes of NAc MSNs.

Co-cultured NAc and PFC neurons were incubated with methionine (Met, 1 mM), the non-canonical amino acid azidohomoalanine (AHA, 1 mM), or 1 mM AHA plus the protein synthesis inhibitor anisomycin (40 μM) (AHA + Aniso) for 2 hours and then tagged using click chemistry with 20 nM DBCO-Cy5 for visualization of AHA. A, Top panels show representative images of NAc MSN processes under phase contrast and lower panels show Cy5 signal detected with fluorescence microscopy. Co-incubation of AHA with anisomycin reduced Cy5 fluorescence in NAc MSN processes nearly to the level detected in the Met control group, confirming that Cy5 fluorescence reflects AHA incorporation into newly translated proteins. We note that Cy5 signal was detected in the soma of all groups (not shown). Scale bar = 50 μm. B, Quantification of Cy5 fluorescence in processes of NAc MSNs for the experimental groups shown in panel A (Met, AHA, and AHA + Aniso). In addition, data are shown for a negative control group treated with AHA but no DBCO-Cy5 (No Click). The Cy5 signal was markedly higher in the AHA group relative to all other groups (**** p<0.0001). Data are presented as arbitrary fluorescent units (au). C, Blocking microtubule-dependent transport of newly synthesized proteins from the soma, using the microtubule polymerization inhibitor colchicine (Colc), did not impact basal protein translation in NAc MSN dendritic segments measured using AHA incorporation (left bars). Colchicine also failed to affect bicuculline (Bicuc)-stimulated protein translation in processes of NAc MSNs (right bars). D, A trend towards a decrease in the AHA signal in NAc MSN dendritic segments was observed with increasing distance from the soma (one-way ANOVA, p=0.12). Numbers correspond to measures made starting 15, 30 or 45 μm from the soma. The number of processes analyzed is indicated within each bar. *p<0.05 indicates main effect of bicuculline on the AHA signal.

While our methods are not sufficient to distinguish local versus somatic protein translation, we performed several studies to provide a preliminary assessment of a possible contribution of local translation. First, we utilized colchicine, which inhibits microtubule polymerization (Fischer and Schmatolla, 1972; Shan et al., 2003), to inhibit the transport of newly synthesized proteins from the soma to the processes. This strategy has been employed in other studies (e.g., (Liu and Cline, 2016; Younts et al., 2016)) to evaluate the contribution of local versus somatic translation. Fig. 2C shows that the AHA signal in processes did not differ significantly in vehicle- versus colchicine-treated cells, either under basal conditions or when translation was stimulated using the GABA_A antagonist bicuculline (we show in subsequent experiments that bicuculline stimulates translation by disinhibiting excitatory transmission). As a second approach, we examined whether the AHA signal falls off as a function of distance from the soma, which would be expected if there was a substantial contribution from somatic protein translation (Fig. 2D). While a trend towards a decrease was observed (one-way ANOVA: F(2,66)=2.22, p=0.12), we have previously observed a very similar distant-dependent decline in synaptic contacts onto MSNs in the same co-culture system (Sun et al., 2008), which could reduce translation driven by synaptic transmission in more distal processes. Third, we processed co-cultures for FUNCAT and then performed immunocytochemistry to detect the ribosomal subunit S6, a marker of translation, in MSN processes (Biever et al., 2015b). We found considerable but not complete overlap (~76% of S6 staining co-localizes with AHA signal; based on analysis of processes from 25 neurons in 5 different wells) between S6 expression and sites of AHA incorporation in processes (Fig. 3), a result similar to that reported in other systems (Aakalu et al., 2001; Biever et al., 2015a; Graber et al., 2013). Finally, it is well known that synaptic transmission regulates dendritic protein translation (Swanger and Bassell, 2013), and therefore sites of AHA incorporation should be in the vicinity of synapses. To test this, we processed additional co-cultures for FUNCAT and then performed immunocytochemistry to detect the synaptic marker synaptophysin. We observed adjacent rather than overlapping staining, consistent with prior results (Aakalu et al., 2001) and with work showing that polyribosomes are predominantly located in the dendritic shaft beneath spines rather than in spines themselves (Ostroff et al., 2002; Steward and Levy, 1982). Fig. 4 shows an example of AHA signals in proximity to synaptophysin staining. These results are not sufficient to establish a relationship between local translation and our measured AHA signal in processes, but they indicate that the possibility of a contribution of local translation to this signal should not be ruled out. Regardless of where translation is occurring, the diffuse nature of the AHA staining observed in processes (e.g., Fig. 2A, lower middle panel) likely reflects movement of newly translated proteins away from their initial site of translation during the 2 hour AHA-labeling period.

Figure 3. AHA incorporation in processes of NAc MSNs co-localizes with a marker of translation, ribosomal subunit S6.

Co-cultured NAc and PFC neurons were incubated for 2 hours with 1 mM AHA, tagged with 20 nM DBCO-Cy5 (red), and then immunostained for the ribosomal subunit S6 (green). Metamorph analysis of fluorescence microscopy images indicated that most of the area of Cy5 staining (~76%) co-localizes with S6 staining (see Results for more information).

Figure 4. AHA incorporation in processes of NAc MSNs is in proximity to synapses.

Co-cultured NAc and PFC neurons were incubated for 2 hours with 1 mM AHA, tagged with 20 nM DBCO-Cy5, and immunostained for the synaptic marker, synaptophysin. We observed predominantly adjacent rather than overlapping staining, consistent with prior studies (see Results).

For the quantitative analysis of AHA labeling depicted in Figs. 2B-D and discussed above, we compared protein translation between experimental groups by measuring AHA fluorescence in a fixed length (100 μm) of each dendritic process. To verify that the same results are obtained when the AHA signal is normalized to area of these dendritic segments, and at the same time address the possibility that FUNCAT processing may affect this area, we analyzed dendritic area for experimental groups shown in Figs. 2B and C. Our results indicated no significant changes in dendritic area following FUNCAT processing. Thus, relative to untreated control cultures, both Met and AHA groups exhibited a small trend towards increased dendritic area that did not reach statistical significance in either case (one-way ANOVA, F(3,68)=1.87, p=0.14; No treatment vs. Met: 9% increase, Tukey’s post hoc test p=0.82; No treatment vs. AHA: 10% increase, Tukey’s post hoc test p=0.68). Combined treatment with AHA and anisomycin did not impact dendritic area compared to the AHA only group (Tukey’s post hoc test p=0.72). The fact that the same trend towards increased dendritic area was observed in Met and AHA groups indicates that it is likely a response to the extensive processing required for the FUNCAT technique (see Methods), not a response specifically related to AHA incorporation into nascent proteins. Importantly, regardless of whether we measured the AHA signal for a fixed length of process (as shown in Figs. 1B and C) or normalized this signal to the area of each dendritic segment, the analysis indicated the same magnitude of effects for anisomycin (t₁₉=0.740, p=0.47), colchicine (t₁₉=0.811, p=0.43), and bicuculline (t₁₇=0.426, p= 0.68) versus AHA controls. These results indicate that changes in dendritic area do not contribute to observed drug-induced changes in the AHA signal.

Overall, these results establish that AHA is incorporating into nascent polypeptide chains in NAc MSNs and that FUNCAT can therefore be used to assess protein translation in these neurons. We went on to characterize the role of group I mGluRs and ionotropic glutamate receptors in regulating protein translation in MSNs, as well as PFC pyramidal neurons, under conditions of basal synaptic transmission and after activation of excitatory synaptic transmission with the GABA_A receptor antagonist bicuculline.

3.2. Regulation of translation by group I mGluRs under conditions of basal synaptic transmission

In hippocampal neurons, protein translation is activated by group I mGluRs (Waung and Huber, 2009; Weiler and Greenough, 1993). We examined whether these receptors also regulate protein translation in NAc MSNs under basal conditions. For this and the other pharmacological studies described below, we took the approach of incubating co-cultures with antagonists to detect regulation of translation by ongoing transmission at group I mGluRs. This strategy has been used in other brain regions (Osterweil et al., 2010) and is valid in our co-cultures because we have established that there is ongoing glutamate transmission under basal conditions (Loweth et al., 2014a; Reimers et al., 2014; Sun and Wolf, 2009).

We began by treating co-cultures with two highly selective mGluR1 antagonists: LY367385 (LY; 50 μM), a competitive antagonist (Clark et al., 1997), and JNJ16259685 (JNJ; 100 μM), a noncompetitive antagonist (Lavreysen et al., 2004). Each antagonist was present throughout the 2-hour incubation with AHA. We found no effect of either drug on translation in either NAc MSNs (Tukey’s post hoc tests, AHA vs. LY p=0.51; AHA vs. JNJ, p=0.99) or in PFC pyramidal neurons (Tukey’s post hoc tests, AHA vs. LY p=0.82; AHA vs. JNJ p=0.64) studied in the same co-cultures (Fig. 5).

Figure 5. Blocking mGluR1 does not alter protein translation in processes of NAc MSNs or PFC pyramidal neurons.

Co-cultured NAc and PFC neurons were incubated with 1 mM AHA +/− drugs for 2 hours and tagged with 20 nM DBCO-Cy5. The addition of the competitive mGluR1 antagonist LY367385 (LY; 50 μM) or the noncompetitive mGluR1 antagonist JNJ16259685 (JNJ; 100 μM) failed to alter AHA incorporation in processes of NAc MSNs or PFC pyramidal neurons. Number of processes analyzed is indicated within the bars.

Next, we assessed the role of mGluR5 in the regulation of protein synthesis. To our surprise, the mGluR5 noncompetitive antagonist/negative allosteric modulator MTEP (1 μM) significantly increased protein translation in NAc MSNs (Tukey’s post hoc test, p=0.0003; Fig. 6A, left panel), suggesting that mGluR5 activity in the cultures is suppressing translation, an effect opposite to that observed in hippocampus (Waung and Huber, 2009; Weiler and Greenough, 1993). In contrast, MTEP had no effect on translation in PFC pyramidal neurons in the same co-cultures (Tukey’s post hoc test, p=0.93; Fig. 6A, right panel), further demonstrating that the same receptor (mGluR5) can exert different effects on translation depending on the cell type. We also evaluated a competitive mGluR5 antagonist, LY344545 (100 μM) (Doherty et al., 2000), to distinguish between an effect of MTEP on agonist-driven mGluR5 activity versus constitutive ligand-independent mGluR5 activity. The latter would not be affected by a competitive antagonist. We found no effect of the competitive mGluR5 antagonist LY344545 in either cell type (NAc: Tukey’s post hoc test, p=0.99; PFC: Tukey’s post hoc test, p=0.97). These results suggest that MTEP is working by inhibiting constitutive mGluR5 activity in the cultured NAc neurons.

Figure 6. A noncompetitive mGluR5 antagonist, but not a competitive antagonist, increases protein translation in processes of NAc MSNs.

Co-cultured NAc and PFC neurons were incubated with 1 mM AHA +/− drugs for 2 hours and tagged with 20 nM DBCO-Cy5. A, The noncompetitive mGluR5 antagonist MTEP (1 μM) significantly increased translation in processes of NAc MSNs, but not PFC pyramidal neurons. B, The competitive mGluR5 antagonist LY344545 (100 μM) failed to alter protein translation in either cell type. Number of processes analyzed is indicated within the bars. *** p<0.001 vs. AHA

Because the directionality of MTEP’s effect in MSNs was surprising in light of mGluR5 actions in hippocampus (above), we considered the alternate possibility that inhibition of mGluR5 activity augments translation indirectly, by disrupting mGluR5-mediated synaptic depression. In MSNs in the intact brain, group I mGluR-mediated synaptic depression is elicited by postsynaptic mGluR5 activation leading to endocannabinoid formation, stimulation of presynaptic CB1Rs, and depression of glutamate release (Gerdeman et al., 2002; McCutcheon et al., 2011; Robbe et al., 2002). If this mechanism for synaptic depression exists in our cultures, interfering with it, by blocking mGluR5, would disinhibit MSNs, which might indirectly increase translation. To test this, we determined if disrupting synaptic depression at a different point in the pathway – the CB1R – would lead to a similar increase in protein synthesis. However, we found no effect of the CB1R antagonist AM251 (2 μM; 2 hours) on translation in either NAc MSNs (Tukey’s post hoc test, p=0.84) or PFC pyramidal neurons (Tukey’s post hoc test, p=0.84; Fig. 7).

Figure 7. Blocking CB1R signaling does not alter protein translation in processes of cultured NAc MSNs or PFC pyramidal neurons.

Co-cultured NAc and PFC neurons were incubated with 1 mM AHA +/− the CB1R antagonist AM251 (2 μM) for 2 hours and tagged with 20 nM DBCO-Cy5. Incubation with AM251 failed to alter protein translation in either cell population. Number of processes analyzed is indicated within the bars.

We also examined the effect of the non-selective group I mGluR agonist DHPG (50 μM). DHPG was applied during the entire 2 hours of AHA incubation, either alone or in the presence of antagonists of mGluR1 or mGluR5 (50 μM LY367385 and 1 μM MTEP, respectively). In NAc MSNs, DHPG significantly increased translation (Tukey’s post hoc test, p=0.0009), and the mGluR1 antagonist LY367385, which produced no effect on its own (Fig. 5), blocked this effect of DHPG (Tukey’s post hoc test, DHPG vs. DHPG+LY, p=0.0004; Fig. 8, left panel). The mGluR5 noncompetitive antagonist MTEP, which increased translation in NAc MSNs on its own (Fig. 6), augmented the effect of DHPG in MSNs (Tukey’s post hoc test, DHPG vs. DHPG+MTEP, p=0.05; Fig. 8, left panel). In PFC pyramidal neurons, effects of DHPG were variable, such that only a trend towards increased translation was obtained even after analyzing a substantial number of processes (51 processes; Tukey’s post hoc test, DHPG vs. AHA only, p=0.16; Fig. 8, right panel). However, translation was significantly lower in PFC pyramidal neurons incubated with LY + DHPG compared to neurons incubated with DHPG only (Tukey’s post hoc test, p=0.003), suggesting that stimulation of mGluR1 leads to enhanced translation; in contrast, there was no significant difference between PFC pyramidal neurons incubated with MTEP + DHPG compared to neurons incubated with DHPG only (Tukey’s post hoc test, p=0.60; Fig. 8, right panel).

Figure 8. The effect of group I mGluR stimulation on translation is mediated by mGluR1, not mGluR5, in NAc MSNs.

Co-cultured NAc and PFC neurons were incubated with 1 mM AHA +/− drugs for 2 hours and tagged with 20 nM DBCO-Cy5. Left: Incubation with the group I mGluR agonist DHPG (50 μM) significantly enhanced translation in processes of NAc MSNs and this was blocked by co-incubation with the mGluR1 antagonist LY367385 (LY; 50 μM). Co-incubation with DHPG and the mGluR5 antagonist MTEP (1 μM) enhanced protein translation above the level produced by DHPG alone. Right: In PFC pyramidal neurons, the effect of DHPG alone did not reach statistical significance, but translation was significantly lower after incubation with LY + DHPG compared to DHPG alone. Co-incubation with MTEP did not significantly alter the effect of DHPG in PFC pyramidal neurons. Number of processes analyzed is indicated within the bars. *p≤0.05, ***p<0.001, ****p<0.0001 vs. AHA or as indicated

Overall, these results indicate that, in NAc MSNs, there is constitutive mGluR5 activity under basal conditions that is suppressing protein translation, but when group I mGluRs are stimulated with DHPG, protein translation is increased via mGluR1 activation. In PFC pyramidal neurons, blocking group I mGluRs has no effect on basal translation, but DHPG-induced translation is similarly dependent upon mGluR1 activation.

3.3. Regulation of translation by ionotropic glutamate receptors under conditions of basal synaptic transmission

In hippocampal neurons, activation of glutamate receptors can be associated with increased translation (e.g., (Gong et al., 2006)), but spontaneous glutamate release acting at NMDARs can suppress translation (Autry et al., 2011; Kavalali and Monteggia, 2015; Nosyreva et al., 2013; Sutton et al., 2006; Sutton et al., 2007; Sutton et al., 2004). Furthermore, there is evidence in PFC that blocking NMDARs on interneurons may indirectly increase protein translation in pyramidal neurons (Dwyer and Duman, 2013). In the case of hippocampus and superior colliculus, the ability of NMDAR activation to suppress translation depends on Ca²⁺ influx through the NMDAR, Ca²⁺-induced Ca²⁺ release, and phosphorylation of eEF2 (Reese and Kavalali, 2015; Scheetz et al., 2000). In NAc MSNs grown in this co-culture system, high levels of Ca²⁺-permeable AMPA receptors (CP-AMPARs) are expressed on the cell surface (Sun and Wolf, 2009), indicating that both NMDARs and CP-AMPARs are potential sources of synaptic Ca²⁺ entry into MSNs.

We therefore examined whether NMDARs or CP-AMPARs regulate protein translation in MSNs, as well as PFC pyramidal neurons in the same co-cultures, under conditions of basal synaptic transmission. As noted above, we have previously demonstrated ongoing glutamate transmission in our co-cultures under basal conditions (Loweth et al., 2014a; Reimers et al., 2014; Sun and Wolf, 2009), so it is reasonable to use an antagonist strategy to reveal a regulatory effect of ongoing glutamate receptor transmission. We found that the NMDAR antagonist APV (50 μM; 2 hours) did not significantly alter translation in either NAc MSNs (Tukey’s post hoc test, p=0.27) or PFC pyramidal neurons (Tukey’s post hoc test, p=0.16) in the same co-cultures, although a trend towards a reduction was found in both cell types (Fig. 9). This contrasts with what was expected based on the work described in the previous paragraph (see Discussion for possible explanations). However, compared to cells incubated with AHA alone, NAc MSNs treated with the selective CP-AMPAR antagonist naspm (100 μM; 2 h) exhibited a significant reduction in translation (Tukey’s post hoc test, p=0.0008; Fig. 9). Naspm had no effect in PFC pyramidal neurons (Tukey’s post hoc test, p=0.65; Fig. 9), but it should be noted that levels of CP-AMPARs on the surface of PFC neurons in this co-culture system have not been evaluated.

Figure 9. CP-AMPARs, but not NMDARs, regulate protein translation in processes of NAc MSNs under basal conditions.

Co-cultured NAc and PFC neurons were incubated with 1 mM AHA +/− drugs for 2 hours and tagged with 20 nM DBCO-Cy5. Left: Incubation of co-cultures with the CP-AMPAR antagonist naspm (100 μM), but not the NMDAR antagonist APV (50 μM), significantly reduced translation in cultured NAc MSNs. Right: Neither drug significantly affected translation in PFC pyramidal neurons. Number of processes analyzed is indicated within the bars. ***p<0.001 vs. AHA

Using additional co-culture plates, we repeated the naspm study and compared naspm to NBQX, which blocks all AMPARs. In this experiment we replicated the reduction in translation in MSNs after naspm (Tukey’s post hoc test, p=0.02) and the lack of effect of naspm in PFC neurons (Tukey’s post hoc test, p=0.86). In contrast, NBQX (10 μM; 2 hours) robustly increased translation in both cell types (MSNs: Tukey’s post hoc test, p<0.0001; PFC pyramidal neurons: Tukey’s post hoc test, p=0.0004; Fig. 10). To verify that this reflects blockade of glutamate synaptic transmission, we repeated the NBQX experiment in cultures containing NAc neurons but no PFC neurons (i.e., glutamatergic neurons are eliminated). In the NAc-only cultures, NBQX produced only a nonsignificant trend towards increased translation (Tukey’s post hoc test, p=0.18), which we attribute to the presence of ambient glutamate derived from the L-glutamine added to the culture media.

Figure 10. Two hours of AMPAR blockade increases protein translation in processes of both NAc MSNs and PFC pyramidal neurons.

Co-cultured NAc and PFC neurons were incubated with 1 mM AHA +/− drugs for 2 hours and tagged with 20 nM DBCO-Cy5. The AMPAR antagonist NBQX (10 μM) significantly enhanced translation in both NAc MSNs and PFC pyramidal cells. This is in contrast to the selective CP-AMPAR antagonist, naspm (100 μM), which reduced translation in NAc neurons but not PFC neurons. Number of processes analyzed is indicated within the bars. *p<0.05, ***p<0.001 vs. AHA or as indicated

Overall, these results indicate that, under conditions of basal synaptic transmission in NAc MSNs, AMPARs rather than NMDARs regulate translation and, surprisingly, opposite effects are observed after blockade of CP-AMPARs (naspm) versus all AMPARs (NBQX). The increase in translation produced by NBQX was also observed in PFC pyramidal neurons and may represent a homeostatic response to blockade of AMPARs for 2 hours (see Discussion).

3.4. Regulation of translation by glutamate receptors after increasing synaptic transmission using bicuculline

To further investigate the effects of neuronal activity on translation in cultured neurons, we conducted a series of experiments with bicuculline, a competitive antagonist of GABA_A receptors that increases excitatory synaptic transmission by disinhibiting cultured neurons (Turrigiano and Nelson, 2004) and has previously been shown to increase translation in cultured hippocampal neurons (Gong et al., 2006; Hsu et al., 2015; Wells et al., 2001). Consistent with these results, incubation of co-cultures with bicuculline (20 μM; 2 hours) increased translation in both NAc MSNs and PFC pyramidal neurons (MSNs: Tukey’s post hoc test, p values < 0.02; PFC pyramidal neurons: Tukey’s post hoc test, p values < 0.04; Figs. 11 and 12, respectively). To determine the mechanism by which bicuculline is acting to increase translation, we co-incubated the cells with bicuculline plus glutamate receptor antagonists. The effect of bicuculline in NAc MSNs was completely abolished in the presence of LY367385 (Tukey’s post hoc test, Bicuc vs. Bicuc+LY, p<0.0001; Fig. 11A), MTEP (Tukey’s post hoc test, Bicuc vs. Bicuc+MTEP, p=0.0005; Fig. 11B), NBQX (Tukey’s post hoc test, Bicuc vs. Bicuc+NBQX, p=0.0003; Fig. 12A), or APV (Tukey’s post hoc test, Bicuc vs. Bicuc+APV, p=0.0007; Fig. 12B); of these, only NBQX reduced translation to below levels observed under control conditions (Fig. 12B). However naspm (100 μM), which produced a modest reduction in basal translation, did not significantly affect the bicuculline-induced increase in translation in NAc MSNs (Tukey’s post hoc test, Bicuc vs. Bicuc+naspm, p=0.67; Fig. 12C). These results indicate that group I mGluRs, NMDARs and AMPARs (but not CP-AMPARs) all mediate the bicuculline-induced increase in translation in NAc MSNs.

Figure 11. Group I mGluR antagonists block bicuculline-induced increases in protein translation in NAc MSNs.

Co-cultured NAc and PFC neurons were incubated for 2 hours with the competitive GABA_A receptor antagonist bicuculline (Bicuc; 20 μM) in combination with either the mGluR1 antagonist LY367385 (LY; 50 μM; panel A) or the mGluR5 antagonist (MTEP) (1 μM; panel B). AHA (1 mM) was present in all groups. Bicuculline increased translation in processes of both NAc MSNs and PFC pyramidal neurons. This effect was blocked in NAc MSNs by either the mGluR1 antagonist or the mGluR5 antagonist. In PFC neurons, the bicuculline-induced increase in translation was not affected by the mGluR1 antagonist, but a modest contribution of mGluR5 cannot be ruled out since the Bicuc + MTEP group did not differ significantly from either the AHA group or the Bicuc group. *p<0.05, **p<0.005, ***p<0.001 vs. AHA or as indicated

Figure 12. Ionotropic glutamate receptor antagonists NBQX and APV, but not naspm, block bicuculline-induced increases in protein translation in processes of NAc and PFC neurons.

Co-cultured NAc and PFC neurons were incubated for 2 hours with the competitive GABA_A receptor antagonist bicuculline (Bicuc; 20 μM) in combination with an AMPAR or NMDAR antagonist. AHA (1 mM) was present in all groups. A, The AMPAR antagonist NBQX (10 μM) blocked the increase in translation produced by neuronal activation with bicuculline in processes of both NAc MSNs and PFC pyramidal neurons. B, The NMDAR antagonist APV (50 μM) blocked the bicuculline-induced increase in translation in both NAc MSNs and PFC pyramidal neurons. C, The selective CP-AMPAR antagonist naspm (100 μM) had no effect on bicuculline-induced translation in either cell type. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001 vs. AHA or as indicated

In PFC pyramidal neurons, the bicuculline-induced increase in translation was not altered by the mGluR1 antagonist (Fig. 11A). Results with the mGluR5 antagonist MTEP were less clear-cut. The AHA signal in the Bicuc+MTEP group was intermediate between the AHA control group and the Bicuc group but did not differ significantly from either of these groups, although there was a trend towards lower translation in the Bicuc+MTEP group compared to the Bicuc group (Tukey’s post hoc test, p=0.31). These results may suggest a modest mGluR5-dependent activation of translation in PFC pyramidal neurons after bicuculline (Fig. 11B). Both NBQX (Fig. 12A) and APV (Fig. 12B), but not naspm (Fig. 12C), abolished the effect of bicuculline on translation (NBQX: Tukey’s post hoc Bicuc vs. Bicuc+NBQX, p=0.0003; APV: Tukey’s post hoc test, Bicuc vs. Bicuc+APV, p=0.0007). These results indicate that ionotropic glutamate receptors play a dominant role in regulating translation during periods of bicuculline stimulation in PFC pyramidal neurons.

4. Discussion

Protein translation is regulated at multiple levels. For example, many intracellular factors and signaling pathways regulate translation (Buffington et al., 2014), and this intracellular machinery can in turn be influenced by excitatory synaptic transmission (Kavalali and Monteggia, 2015; Sutton et al., 2006). Because of the potential importance of protein translation in cocaine-related synaptic plasticity (see Introduction), many studies have assessed effects of cocaine on intracellular translational machinery within the NAc (for review see: (Dayas et al., 2012; Neasta et al., 2014)) and we have recently shown that alterations in this machinery can be detected months after the last drug exposure in association with persistent cocaine craving (Werner et al., 2018). Here we focused on upstream regulation of translation by excitatory synaptic transmission. We used FUNCAT to characterize regulation of translation in processes of co-cultured NAc MSNs and PFC pyramidal neurons under basal conditions and following neuronal activation via the GABA_A receptor antagonist bicuculline. Our results suggest a complex coordination of glutamate receptor subtypes in regulating translation that depends upon cell type (PFC pyramidal neuron versus NAc MSN) and the level of neuronal activity (Table 1).

Table 1.

Summary of changes in AHA signal in response to alterations in ionotropic or metabotropic glutamate receptor activity

		NAc Medium Spiny Neuron
Receptor		mGluR1		mGluR5		Endocannabinoid	NMDA	AMPA	CP-AMPA	GABAA
Drug		LY367385	JNJ16259685	MTEP	LY344545	AM251	APV	NBQX	NASPM	Bicuculline
Neural State	Basal	–	–	↑	–	–	–	↑	↓	n/d
	Bic Stimulated	↓	n/d	↓	n/d	n/d	↓	↓	–	↑
		PFC Pyramidal Neuron
Receptor		mGluR1		mGluR5		Endocannabinoid	NMDA	AMPA	CP-AMPA	GABAA
Drug		LY367385	JNJ16259685	MTEP	LY344545	AM251	APV	NBQX	NASPM	Bicuculline
Neural State	Basal	–	–	–	–	–	–	↑	–	n/d
	Bic Stimulated	–	n/d	– *	n/d	n/d	↓	↓	–	↑
		NAc Medium Spiny Neuron		PFC Pyramidal Neuron
	Receptor	mGluR1	mGluR5	mGluR1	mGluR5
	Drug	LY367385	MTEP	LY367385	MTEP
Neural State	DHPG Stimulated	↓	–	↓ **	–

Abbreviations and symbols: NAc, nucleus accumbens; PFC, prefrontal cortex; ↑, increase; ↓, decrease; –, no change; n/d, not determined

^*The AHA signal in the Bicuc+MTEP group was intermediate between the AHA control group and the Bicuc group, but did not differ significantly from either of these groups; this may indicate a role for mGluR5 in mediating the Bicuc-induced increase in translation in the PFC pyramidal neurons (see Fig. 11).

^**The DHPG group showed only a trend towards an increase relative to the AHA control group, but the LY+DHPG group was significantly decreased compared to DHPG alone, suggesting that stimulation of mGluR1 may lead to enhanced translation in PFC pyramidal neurons (see Fig. 8).

4.1. Advantages and limitations of the experimental approach

Our ultimate goal is to understand how protein translation is related to the neuronal plasticity that underlies drug addiction. Cultured neurons offer advantages for mechanistic studies due to ease of manipulating synaptic transmission and the ability to selectively measure signals in different cellular compartments, and might be employed to study effects of maternal drug exposure or transgenerational effects of drug exposure by preparing cultures from offspring of drug-exposed animals. However, studies conducted in vivo or using ex vivo tissue offer obvious advantages for assessing how translation changes in the adult brain following drug exposure. For this reason, while the present study was underway, a complementary study was performed that focused on comparing rats that self-administered saline versus cocaine (Stefanik et al., 2018); this study used different labeling methods because we were unable to validate the FUNCAT signal in freshly dissected brain tissue. The present results in cultured MSNs will inform future studies of translational regulation in NAc tissue from rats exposed to cocaine and other drugs of abuse.

Although the present study measured newly translated proteins in neuronal processes, we acknowledge that our methods are not adequate to distinguish the contribution of somatic versus dendritic translation to our signal. Nevertheless, several findings suggest that a portion of the AHA signal measured in neuronal processes may be attributable to dendritic translation. Most notably, blocking microtubule-dependent transport of newly synthesized proteins from the soma with colchicine did not significantly alter translation under either basal or bicuculline-stimulated conditions. Furthermore, the spatial relationship between AHA, ribosomal and synaptic staining is consistent with prior studies of dendritic translation (e.g., (Aakalu et al., 2001)).

Another limitation of the present study is that we did not distinguish between subpopulations of neurons within each region. It would be particularly interesting to know if translation is differentially regulated in MSNs expressing D1 dopamine receptors versus MSNs expressing D2 dopamine receptors. We did not undertake this analysis in the present study because young neurons in culture exhibit significantly less segregation of D1 and D2 receptors relative to mature neurons (see (Sun et al., 2008)), which would limit the applicability of our results to the intact NAc of adult rats.

4.2. Group I mGluRs and regulation of translation

In the present work, we show that group I mGluRs regulate translation differently depending on cell type and the level of neuronal activation. In MSNs studied under basal conditions, the ability of a noncompetitive, but not a competitive, mGluR5 antagonist to enhance translation suggests that constitutive ligand-independent activity of mGluR5 is normally suppressing translation. The same noncompetitive mGluR5 antagonist also increased translation in freshly dissected adult NAc tissue (Stefanik et al., 2018). Constitutive mGluR5 activity has previously been observed under conditions where mGluR5 is uncoupled from long Homer proteins (Ade et al., 2016; Ango et al., 2001; Ronesi et al., 2012). Future studies could examine if this accounts for constitutive mGluR5 activity in cultured NAc MSNs and whether “stronger” mGluR5-long Homer coupling explains absence of this effect in PFC pyramidal neurons.

When co-cultures were treated with DHPG to activate group I mGluRs, we observed a robust and reliable increase in translation in NAc MSNs that was blocked by an mGluR1 antagonist. The response to DHPG in PFC pyramidal neurons was highly variable but also relied on mGluR1. If bicuculline was used to more broadly activate excitatory transmission, the resulting increase in translation in NAc MSNs was blocked by either mGluR1 or mGluR5 antagonists, as well as by ionotropic glutamate receptor antagonists, while increased translation in PFC pyramidal neurons mainly relied upon ionotropic glutamate receptor stimulation, with a possible contribution from mGluR5 (see Section 3.4). These results suggest that bicuculline and DHPG activate distinct signaling cascades and that group I mGluRs may be more important in controlling translation in MSNs compared to PFC pyramidal neurons.

While constitutive mGluR5 activity suppresses translation in NAc MSNs, ligand-driven mGluR5 activity enhances translation in hippocampal neurons (Waung and Huber, 2009) through a beta-arrestin pathway (Eng et al., 2016; Stoppel et al., 2017). Group I mGluR function differs in other ways between the two cell types. In hippocampus, mGluR5-mediated LTD is expressed postsynaptically through a mechanism requiring protein synthesis and endocytosis of AMPARs (Huber et al., 2000; Luscher and Huber, 2010). In contrast, mGluR5-mediated LTD in striatal MSNs is expressed presynaptically through a CB1R-dependent mechanism (Choi and Lovinger, 1997; Gerdeman et al., 2002; Robbe et al., 2002) and is independent of protein translation within the MSN (Jung et al., 2012; Yin et al., 2006). Interestingly, group I mGluR LTD in PFC pyramidal neurons is mechanistically similar to that observed in MSNs (Lafourcade et al., 2007), yet mGluR5 regulation of translation can differ between MSNs and PFC pyramidal neurons (Fig. 6A). It may be relevant that MSNs, in the intact NAc (Loweth et al., 2014b; McCutcheon et al., 2011) and in primary cultures (Loweth et al., 2014a; Pick et al., 2017), also express, under certain conditions, an mGluR1-dependent LTD that depends on removal of CP-AMPARs; this has not been described in PFC neurons. Further complicating the picture, group I mGluRs can regulate protein synthesis through multiple pathways (Gallagher et al., 2004; Hou and Klann, 2004; Ronesi and Huber, 2008; Waung and Huber, 2009).

4.3. Ionotropic glutamate receptors and regulation of translation

Spontaneous (minis) and evoked (action-potential dependent) glutamate transmission involve different vesicle populations and influence distinct postsynaptic compartments and signaling cascades (Kavalali, 2015; Kavalali and Monteggia, 2012; Sutton and Schuman, 2009). In hippocampus, high levels of evoked glutamate transmission are associated with increased translation (Gong et al., 2006), whereas spontaneous NMDAR transmission negatively regulates translation in the hippocampus (Kavalali and Monteggia, 2015; Sutton et al., 2004). Contrary to what would have been expected from this hippocampal work, when we incubated cultures with APV under basal conditions we did not observe significant changes in translation in either MSNs or PFC pyramidal cells, despite the fact that APV should have blocked effects of both spontaneous and action-potential dependent glutamate release at NMDARs. Absence of an APV effect could reflect a lower level of spontaneous glutamate release in our culture system. Interestingly, in freshly dissected NAc tissue from the adult rat, we have observed increased translation after exposure to APV (Stefanik et al., 2018).

Whereas NMDAR blockade did not influence translation in NAc MSNs studied under basal conditions, blocking all AMPARs with NBQX resulted in increased translation while, surprisingly, blocking only CP-AMPARs with naspm reduced translation. Increased translation after blocking all AMPARs has been reported previously in hippocampus (Sutton et al., 2004). In our co-culture system, we have previously shown that long-term blockade of all AMPARs (48 or 72 hours with CNQX) leads to a scaling up of excitatory synaptic transmission that depends upon protein translation, whereas long-term blockade of NMDARs (72 hours with APV) did not produce this effect (Sun and Wolf, 2009). It is possible that the shorter-term blockade of all AMPARs tested here (2 hours) similarly elicits homeostatic effects within the neurons that include an increase in translation. Blockade of NMDARs or CP-AMPARs may not be sufficient to produce such an effect. In the case of NMDARs, there may not be sufficient activation under basal conditions due to Mg²⁺ block. CP-AMPARs are highly expressed on the surface of cultured MSNs (Sun and Wolf, 2009), but the selective CP-AMPAR antagonist naspm not only failed to mimic the increased translation produced by NBQX, but reduced translation in MSNs under basal conditions; this may indicate that signaling through CP-AMPARs is compartmentalized so as to exert different effects on translation compared to other glutamate receptors. Interestingly, CP-AMPARs are important for glutamate-mediated stimulation of protein translation in axons (Hsu et al., 2015).

In PFC pyramidal neurons studied under basal conditions, NBQX elicited an increase in translation, as observed in NAc MSNs. A homeostatic increase in translation after AMPAR blockade may therefore be common to both cell types. No effect of naspm was observed, but this is difficult to interpret because we have not established whether CP-AMPARs are present on the surface of PFC pyramidal neurons in our co-culture system.

Bicuculline, which disinhibits excitatory transmission, increased translation in both MSNs and PFC pyramidal neurons. This was blocked in both cell types by APV or NBQX, but not by the CP-AMPAR antagonist naspm; in MSNs, it was also blocked by group I mGluR antagonists (above). These results suggest that both NMDARs and AMPARs contribute to the enhancement in excitatory transmission that drives increased protein translation after bicuculline, similar to previous findings in hippocampal cultures (Gong et al., 2006). This contrasts with different effects of each glutamate receptor subtype in MSNs or PFC pyramidal neurons under basal conditions. It is possible that increased synaptic activity alters postsynaptic signaling cascades so as to change the cell’s response to activation of specific glutamate receptors.

4.4. Conclusions

Using FUNCAT, we have characterized the regulation of protein translation by ionotropic and metabotropic glutamate receptors in co-cultured NAc MSNs and PFC pyramidal neurons. We observed a complex interplay between glutamate receptor subtypes that depended on the level of neuronal activation and differed to some degree between MSNs and pyramidal neurons (Table 1). It will be important in the future to determine if there is a difference in the mRNA populations that are regulated by each glutamate receptor subtype and whether this differs between distinct populations of cells within a particular region (e.g., MSNs that express D1 receptors versus D2 receptors) or between cells in different regions. However, the present results provide a critical foundation for future work on how protein translation in NAc and PFC neurons is regulated under normal conditions and how it may be altered by drugs of abuse.

Highlights.

• How glutamate transmission regulates protein translation in NAc MSNs is unknown.

• We studied glutamate receptor regulation of translation in NAc/PFC co-cultures.

• AMPARs, NMDARs, mGluR1 and mGluR5 differently regulate translation.

• Regulatory mechanisms depend on cell type and level of neuronal activation.

• A foundation is provided for understanding aberrant translation in drug addiction.