The procedures to elicit place-conditioning were identical to those described previously by our group (e.g., Ary et al., 2013), with separate groups of mice injected with intraperitoneal (i.p.) injections of 3, 10, or 30 mg/kg cocaine (National Institute on Drug Abuse, Bethesda, MD, United States). Animals were habituated to both compartments of the two-compartment apparatus for 15 min during a pre-conditioning testing (PreTest) and then were subjected to daily, 15-min, conditioning sessions, alternating between cocaine and saline, over the course of 8 days. The day following the last conditioning session, animals were allowed free-access to both compartments in a post-conditioning test. The behavior of the mice was recorded throughout the experiment using a digital video-tracking system and ANYMaze software (Stoelting, Co., Wood Dale, IL, United States). The difference in the time spent in the cocaine- versus saline-paired compartment on a post-conditioning test served to index cocaine reward/aversion (Conditioned Place-Preference Score or CPP Score). The data were analyzed using a Genotype (+/+, +/−, −/−) × Sex × Dose (3, 10, and 30 mg/kg cocaine) × Side (paired vs. unpaired) ANOVA, with repeated measures on the Side factor. The data for locomotor activity during the PreTest and cocaine-induced locomotion were published previously in Park et al. (2013). A follow-up study employed identical placeconditioning procedures to assay the effects of intra-NAC AAV-Homer1a infusion upon genotypic differences in novelty-induced locomotion during the PreTest, habituation of locomotor activity (changes in saline-induced locomotion observed during the four saline-conditioning sessions), sensitization of cocaine-induced locomotor hyperactivity (changes in locomotor activity observed during the four cocaine-conditioning sessions; 10 mg/kg) and cocaine-conditioned reward (CPP Score following conditioning).

#### 2.4 Construction of AAV-Homer1a

The rat Homer1a coding sequence was amplified using wholebrain cDNA, and the PCR was product expressed as an N-terminal fusion protein with the hemagglutinin (HA)-tag in a rAAV backbone containing the 1.1 kb CMV enhancer/chickenactin (CBA) promoter, 800 bp human interferon scaffold attachment region inserted 5\_ of the promoter, the woodchuck post-transcriptional regulatory element (WPRE), and the bovine growth hormone polyA flanked by inverted terminal repeats (AAV-Homer1a, AAV-Homer1c). The same AAV-CBA-WPREbGH backbone encoding the enhanced green fluorescent protein (EGFP) was used as control (AAV-GFP). As described previously (Hauck et al., 2003), AAV pseudotyped vectors virions containing a 1:1 ratio of AAV1 and AAV2 capsid proteins with AAV2 intertrigeminal regions were generated. For this, human embryonic kidney 293 cells were transfected with the AAV cisplasmid, the AAV1 (pH21) and AAV2 (pRV1) helper plasmids (pF6), and the adenovirus helper plasmid by standard calcium phosphate transfection methods. Cells were harvested at 48 h after transfection, and the vector purified using heparin affinity columns as described previously (During et al., 2003). As also described previously (Clark et al., 1999), genomic titers were determined using the Prism 7700 sequence detector system (Applied Biosystems, Foster City, CA, United States) with primers designed to WPRE.

### 2.5 Surgical Procedures and AAV Infusion

The NAC is a limbic structure implicated in both the psychomotor-activing and rewarding/reinforcing properties of drugs of abuse (e.g., Morein-Zamir and Robbins, 2015; Scofield et al., 2016). Homer1a is induced within the NAC by various drugs of abuse (Szumlinski et al., 2008a; Marton et al., 2015) and Homer1a−/− mice exhibit impaired cocaine-induced neurochemical sensitization within this region (Park et al., 2013). Thus, under isoflurane anesthesia, we conducted craniotomies to implant bilateral guide cannulae (20-gauge; 10-mm long), 2 mm above the NAC of mice (see Lominac et al., 2005; Cozzoli et al., 2009; Ary et al., 2013), for the purposes of conducting in vivo microdialysis measurement of glutamate and dopamine (see section 2.6 below) and/or infusing AAVs carrying either cDNA for Homer1a (AAV-Homer1a) or a scrambled DNA sequence as a control (AAV-control) to determine an active role for NAC Homer1a expression in regulating neurochemistry and behavior (see Lominac et al., 2005; Cozzoli et al., 2009 for AAV details). A Kopf stereotaxic device held the animal's head level and holes were drilled based on coordinates from bregma: (AP: +1.3 mm; ML: ±1.0 mm; DV: −2.3 mm) (Paxinos and Franklin, 2013). Guide cannulae were fixed to the skull with dental resin, surgical incisions were closed with a tissue adhesive.

For AAV-naïve mice undergoing microdialysis procedures, dummy cannulae (24 gauge; length equivalent to guide cannulae) was then placed inside the guide cannulae to prevent contamination or blockade and animals were allowed a minimum of 5 days recovery prior to neurochemical testing. Our AAVs are considered BSL1 and thus, standard protective personal equipment were employed during AAV infusion. For animals receiving AAV infusions, microinjectors (33-gauge, 12 mm long) where then lowered down the guide cannulae and the AAVs infused at a rate of 0.05 µl/min for 5 min (total volume/side = 0.25 µl). Microinjectors were left in place for an additional 5 min to allow for diffusion away from the microinjector tip prior to removal and occlusion of the guide cannulae. AAV-infused mice were allowed a minimum of 3 weeks recovery to achieve maximal neuronal transduction (Klugmann and Szumlinski, 2008). AAV transduction within the NAC was verified using standard immunohistochemical staining procedures for the hemagglutanin (HA) tag using a mouse anti-HA primary antibody (Covance, Princeton, NJ, United States), and straining visualized using a M.O.M. Detection Kit (Vector Laboratories, Burlingame, CA, United States), as conducted previously (Szumlinski et al., 2004, 2005b, 2017; Lominac et al., 2005; Cozzoli et al., 2009; Ary et al., 2013).

### 2.6 In Vivo Microdialysis

Homer1a−/− mice do not exhibit cocaine-induced dopamine or glutamate sensitization in the NAC (Park et al., 2013). Thus, a series of in vivo microdialysis experiments were conducted to relate this sensitization-resistant phenotype to biochemical indices of neuronal function within this region. For all in

vivo microdialysis experiments, a microdialysis probe (24 gauge; 12 mm in length with ∼1.0 mm active membrane) was lowered unilaterally into one of the guide cannulae and perfused with artificial CSF (146 nM NaCl, 1.2 mM CaCl2, 2.7 mM KCl, 1.0 mM MgCl2, pH = 7.4) at a rate of 2 µl/min. Dialysate collection began after 3 h of probe equilibration and occurred in 20 min intervals into vials containing 10 µl of preservative [10% methanol (v/v), 15% acetonitrile (v/v), 150 mM NaPO4, 4.76 mM citric acid, 3 mM SDS, 50 mM EDTA, pH = 5.6]. Dialysate was stored at −80◦C until analysis by HPLC (see section 2.6 below). Microdialysis probe localization within the NAC was verified using standard cresyl violet staining procedures (for AAV-naïve mice) or upon immunohistochemical localization of AAV transduction (see above), followed by examination of tissue under light microscope. In nearly all subjects, microdialysis probes were localized primarily to the shell subregion or to the interface between the shell and core subregions (see section 3).

#### 2.6.1 DHPG-Stimulated Glutamate Release

Homer proteins are critical regulators of mGlu1/5 function in vitro and in vivo (cf. Ango et al., 2000, 2001; Shiraishi-Yamaguchi and Furuichi, 2007; Worley et al., 2007; Szumlinski et al., 2008a; Fagni, 2012; Constantin, 2016). Thus, we compared the effects of Homer1a versus pan-Homer1 deletion upon an in vivo index of mGlu1/5 function – agonist-stimulated glutamate release (Swanson et al., 2001; Szumlinski et al., 2004, 2017). For this, Homer1a+/+,−/− and pan-Homer1+/+,−/− mice were perfused within increasing concentrations of the mGlu1/5 agonist DHPG [(S)-3,5-dihydroxyphenylglycine; 0, 3, 30, and 300 µM; Tocris Cookson, Ballwin, MO, United States], via the microdialysis probe in 1-h intervals. For each mutant line, the data were analyzed using Genotype (+/+ vs. −/−) × DHPG ANOVA, with repeated measures on the DHPG factor (four levels).

#### 2.6.2 Depolarization-Induced Dopamine and Glutamate Release

To relate the distinct neurochemical phenotypes of Homer1a (Park et al., 2013) versus Homer1 (Szumlinski et al., 2004, 2005a) mutants to the excitability of dopamine and glutamate terminals within the NAC, we compared depolarization-induced neurotransmitter release by locally infusing high K+ solutions (50 and 100 mM; Sigma-Aldrich, St. Louis, MO, United States) through the microdialysis probe in 1-h intervals (e.g., He and Shippenberg, 2000). To conserve animal numbers, this microdialysis experiment was conducted 3–4 days following the DHPG study described in Section 2.6.1 above, using the other side of the head. The data were analyzed separately for each mutant line using a Genotype × K + ANOVA, with repeated measures on the K+ factor (three levels).

#### 2.6.3 Glutamate No Net-Flux Procedures

Pan-Homer1 deletion reduces basal extracellular glutamate content within the NAC of mice (Szumlinski et al., 2005a) and prior conventional in vivo microdialysis studies suggested lower basal extracellular glutamate content within the NAC also of Homer1a−/− animals (Park et al., 2013). However, estimates of basal neurotransmitter levels assessed under conventional microdialysis procedures are subject to differences in probe recovery (Parsons and Justice, 1992). Thus, we quantified the effects of Homer1a deletion upon the basal extracellular glutamate content within the NAC using no net-flux procedures (Szumlinski et al., 2004, 2005a, 2017; Lominac et al., 2005; Ary et al., 2013). For this, increasing concentrations of glutamate (2.5, 5, and 10 µM) were infused through the probe in ascending order for 1 h each. Linear regression analyses were then conducted on the plot of the average net-flux of glutamate at each glutamate concentration versus the concentration of glutamate infused through the probe and the point of no net-flux (y = 0; estimate of basal extracellular levels of glutamate), as well as the slope of regression lines (estimate of glutamate clearance), were determined and analyzed using independent samples t-tests. A follow-up experiment determined how an intra-NAC AAV-Homer1a infusion altered the genotypic differences in our dependent measures in this assay. This follow-up study employed identical no net-flux procedures as those employed in the original genotypic comparison, with the exceptions that the Homer1a+/+,−/− mice were infused intra-NAC with either AAV-control or -Homer1a, a minimum of 3 weeks prior to probe insertion and the data were analyzed using an AAV (control vs. Homer1a) × Genotype (+/+ vs. −/−) ANOVA.

#### 2.6.4 Cocaine-Induced Sensitization of Dopamine and Glutamate within the NAC

To determine whether or not Homer1a within the NAC actively regulates the development of cocaine-induced dopamine and glutamate sensitization in this region, Homer1a+/+ and Homer1a−/− mice were infused with AAV-control or -Homer1a and then conventional in vivo microdialysis procedures were conducted a minimum of 3 weeks later. Low fecundity in our Homer1a colony at the time of study forced us to conserve animal numbers and thus, microdialysis procedures were conducted in the same AAV-infused mice as those tested for cocaine-induced locomotor sensitization/place-preference (see section 2.3). In order to accommodate both paradigms, mice first underwent a microdialysis session to assay the effects of AAV infusion upon the neurochemical response to an acute injection of 30 mg/kg cocaine (i.p.). Following a 3-h equilibration period, baseline sampling was conducted over an hour and then mice were injected with cocaine and samples collected for 3 h thereafter. At the end of this first microdialysis session, the probe was removed and mice were allowed to recover for 2 days prior to the 10-day place-conditioning procedure (consisting of four alternating injections of saline and 30 mg/kg cocaine). One to 2 days following the post-conditioning test, a second microdialysis session was conducted using the opposite side of the head to assay for cocaine-induced neurochemical sensitization.

In contrast to our prior study (Park et al., 2013), the Homer1a+/+,−/− mice in this study were treated with a total of six injections of 30 mg/kg cocaine. Due to technical

issues associated with subject attrition and sample loss due to technical difficulties with the HPLC, not all mice were tested for cocaine-induced neurochemical changes during both microdialysis sessions. Thus, in order to include all of the remaining the mice tested, Injection Number was treated as a between-subjects variable. As group differences were not observed for baseline glutamate levels on either injection, the data were expressed as a percent of baseline values for each group and to facilitate visualization of group differences in the magnitude of the cocaine-induced rise in dopamine and glutamate, the area under the curve (AUC) was employed in the statistical analyses of the results (Lominac et al., 2012; Shin et al., 2016). Thus, for both baseline neurotransmitter levels and the AUC for cocaine-induced changes in neurotransmitter levels, the data were analyzed using a Genotype × AAV × Injection Number between-subjects ANOVA.

### 2.7 High Pressure Liquid Chromatography

The high pressure liquid chromatography (HPLC) system and procedures for the electrochemical detection of glutamate and dopamine in the dialysate of mice, as well as the chromatographic analysis of the data, were identical to those described previously by our group (Lominac et al., 2012, 2014; Ary et al., 2013; Szumlinski et al., 2017). Each HPLC system consisted of a Coularray detector, a Model 542 autosampler and two Model 582 solvent delivery systems (ESA/Thermo-Fisher), with a detection limit of 0.01 fg/sample (20 µl/sample onto column). For analysis of dopamine, the MD-TM mobile phase was employed (ESA/Thermo-Fisher), and neurotransmitters in 30 µl from each 50 µl sample were separated using a MD-150 mm × 3.2 mm column (ESA/Thermo-Fisher). An ESA/Thermo-Fisher 5014B analytical cell was used for the detection of monoamines (oxidation and reduction electrode potentials of +220 and −150 mV, respectively). For glutamate, the mobile phase consisted of 3.5% acetonitrile (v/v), 22% methanol (v/v), 100 mM NaPO4, pH = 6.75. A reversed phase column (50 mm × 3.0 mm Capcell PAK; Shiseido, Tokyo, Japan) was used to separate the amino acids, and precolumn derivatization with o-phthaladehyde (2.7 mg/ml) of the 20 µl from each 50 µl sample was performed using an ESA Model 540 autosampler (ESA/Thermo-Fisher). Glutamate was detected using an electrochemical analytical cell with an oxidizing potential of +550 mV. Glutamate and dopamine content in each sample were analyzed by peak height and compared with an external standard curve for quantification (glutamate standards: 2.5, 5.0, 10 mM; dopamine standards: 1.25, 2.5, 5 nM).

#### 2.8 General Statistical Approaches

As detailed above, the majority of data were analyzed using ANOVAs, with repeated measures as appropriate for the experimental design. Significant interactions were deconstructed along the relevant factors, and significant main effects were followed by least significant differences (LSDs) post hoc tests or t-tests, as appropriate for the number of comparisons. α = 0.05 for all analyses.

## 3 RESULTS

### 3.1 Negative Affect

Exposure to a novel environment induces Homer1a expression within forebrain (Vazdarjanova et al., 2002) and pan-Homer1 KO mice exhibit robust signs of hyper-emotionality across a variety of behavioral paradigms (Szumlinski et al., 2004, 2005a; Lominac et al., 2005). Thus, we first assayed Homer1a+/+ and Homer1a−/− mice for behavioral signs of negative affect using a comparable behavioral test battery as that employed in our previous work. As detailed below, the results do not support a consistent role for Homer1a induction in the manifestation of anxiety- or depressive-like behaviors in mice.

#### 3.1.1 Elevated Plus Maze

When tested in the elevated plus maze test, no main effects or interactions with sex were detected (p > 0.05 for all variables); thus, the data for male and female mice were collapsed for data presentation. Homer1a+/− mice exhibited a significant increase in open arm entries, relative to both Homer1a+/+ and Homer1a−/− mice (**Figure 1A**) [Genotype effect: F(1,79) = 4.50, p = 0.01; LSD post hoc tests]. A similar pattern of genotypic differences was observed for the time spent in the open arms, but the result was shy of statistical significance (**Figure 1B**; Genotype effect: p = 0.1).

#### 3.1.2 Novel Object Test

In the novel object test assay, females exhibited lower behavioral indices of anxiety than males, as indicated by a significant increase in both the time in object contact [Sex effect: F(1,59) = 18.91, p < 0.0001], and number of object contacts [Sex effect: F(1,59) = 36.50, p < 0.0001]. No sex difference was observed for the latency to first make contact with the novel object (p = 0.31) and, as illustrated in **Figure 1C** (females) and **Figure 1D** (males), genotypic differences in exploratory behavior were not observed for either sex (p > 0.20 for all variables).

#### 3.1.3 Porsolt Swim Test

No sex differences were observed for any of the variables measured during Porsolt Swim testing (Sex effects and interactions, p's > 0.05). Thus, the data were collapsed across sex for presentation. Homer1a deletion reduced the latency to first float during both the 15-min exposure session, as well as during the 5-min re-exposure session conducted 24 h later (**Figure 1E**), with both Homer1a+/− and Homer1a−/− mice exhibiting a shorter latency to float, compared to controls [Test effect: p = 0.08; Genotype effect: F(2,51) = 6.5, p = 0.003; Genotype × Test: p = 0.93]. However, no genotypic differences were observed regarding the total floating during either session (**Figure 1F**) [Test effect: F(1,51) = 3.93, p = 0.05; Genotype effect and Genotype × Test: p > 0.31].

#### 3.1.4 Saccharin Preference

Although the results of the Porsolt swim test suggested greater depressive-like behavior in Homer1a−/− mice, we failed to detect genotypic differences in the initial or average preference for a palatable saccharin solution over water (for both variables,

FIGURE 1 | Anxiety-like behavior in Homer1a+/+, Homer1a+/−, and Homer1a−/− mice. When compared in an elevated plus maze test, Homer1a+/− mice exhibited relatively lower anxiety as indicated by greater (A) number of open arm entries and (B) time spent in the open arms. While no sex differences in anxiety-like behavior were observed on the elevated plus maze, females (C) exhibited greater anxiety than males (D), in the novel object test, but no genotypic differences were observed. No sex differences were apparent in the Porsolt Swim test, however, Homer1a deletion increased (E) the latency to first float, without influencing the total amount of floating (F). The data represent the mean ± SEM of the number of mice indicated in parentheses. ∗p < 0.05 vs. +/+; +p < 0.05 vs. +/+ and –/– (LSD post hoc tests).

Genotype effects, p > 0.25), with the average preference exhibited by Homer1a+/+, Homer1a+/−, and Homer1a−/− mice being 43.1 ± 3.80, 50.90 ± 2.49, and 45.11 ± 4.48%, respectively.

#### 3.1.5 Habituation to a Novel Environment

As the above data argue an inconsistent role for Homer1a in mediating the behavioral response to an acute mild stressor, we next determined whether Homer1a deletion might impair the ability to habituate to the repeated presentation of a mild stressor using a simple locomotor habituation paradigm. No significant main effects of, or interactions with, the Sex factor were observed (p's > 0.15). While there were trends for reduced overall locomotor activity in Homer1a+/− and Homer1a−/− mice, this genotypic difference was not significant nor were there genotypic differences in the extent to which animals habituated with repeated testing (data not shown) [Session effect: F(4,208) = 23.41, p < 0.0001; Genotype effect and Genotype × Session, p > 0.05]. Further, no genotypic differences were observed for the change in the latency to enter the center zone (data not shown) [Session effect: p = 0.09; Genotype × Session, p > 0.05] or in the number of entries into the center zone of the activity monitors with repeated testing (data not shown) [Session effect: F(4,52) = 5.33, p < 0.0001; Genotype × Session: p > 0.95].

### 3.2 Sensorimotor Processing

Pan-Homer1 knock-out mice exhibit marked deficits in sensorimotor-gating (Szumlinski et al., 2004, 2005a; Lominac et al., 2005). Thus, we determined the effects of Homer1a deletion upon acoustic startle and pre-pulse inhibition of acoustic startle (PPI). Again, we failed to detect a sex difference in behavior (for all variables, no Sex effect or interactions, p > 0.30), thus, the data were collapsed across sex for presentation.

#### 3.2.1 Acoustic Startle during PPI Testing

Relative to Homer1a+/+ littermates, both Homer1a+/− and Homer1a−/− mice exhibited reduced startle responsiveness to the various acoustic stimuli (**Figure 2A**) [Genotype effect: F(2,79) = 21.95, p < 0.0001; Tone effect: F(6,474) = 84.60, p < 0.0001; Genotype × Tone: F(12,474) = 14.4, p < 0.0001]. Relative to controls, lower startle-responding to the 110 dB tone was observed in both Homer1a+/− and Homer1a−/− mice during the habituation phase (hab110), when it was presented alone (st110) or when it was preceded by either the 74 or the 90 dB pre-pulse (p's < 0.007; LSD post hoc tests). Moreover, both Homer1a+/− and Homer1a−/− mice exhibited lower spontaneous activity than Homer1a+/+ animals in the absence of any acoustic stimuli (st0; p = 0.006).

#### 3.2.2 PPI

While the 74 dB pre-pulse failed to inhibit acoustic startle in any genotype (p > 0.25), a genotypic difference in PPI was observed in the presence of the 90 dB pre-pulse (**Figure 2B**) [Genotype effect: F(2,79) = 4.59, p = 0.01]. Further, a significant impairment in PPI was observed at the 90 dB prepulse in Homer1a−/− mice, relative to controls (p = 0.003), with a modest impairment observed in Homer1a+/− mice (p = 0.09).

#### 3.2.3 Acoustic Acuity

Although the tone-dependency of the startle response exhibited by Homer1a+/− and Homer1a−/− mice during PPI testing (**Figure 2A**) argued that Homer1a deletion does not impair the ability to discriminate between certain acoustic stimuli, the unexpected blunting of the startle-responsiveness of the mutant mice prompted a test for a primary hearing deficit. For this, male Homer1a+/+, Homer1a+/−, and Homer1a−/− mice were randomly presented with acoustic stimuli ranging in intensity from 0 to 125 dB. In this cohort, startle amplitude was also intensity-dependent (**Figure 2C**) [Tone effect: F(8,152) = 82.29, p < 0.0001]; however, although the results of the statistical

analyses indicated a significant interaction between genotype and tone [F(8,152) = 2.36, p = 0.004], post hoc comparisons at each dB level failed to reveal genotypic differences in startle responsiveness.

### 3.3 Spatial Learning and Memory

Pan-Homer1 deletion markedly impairs learning and memory on several assays (Lominac et al., 2005; Szumlinski et al., 2005a; Gerstein et al., 2012) and intracranial manipulations of Homer1a expression affect spatial memory (e.g., Klugmann et al., 2005; Celikel et al., 2007; Gerstein et al., 2012, p. 213). Thus, we trained male mice to locate a hidden platform in a Morris water maze task and then assessed for long-term spatial memory in a probe trial, conducted 24 h following the 4th acquisition session. Surprisingly, genotypic differences in behavior were not detected during the acquisition phase of testing, either across trials within days, or across days of training (**Figure 2D**) [distance to locate platform, Trial effect: F(3,225) = 5.18, p = 0.003; Day effect: F(3,225) = 33.50, p < 0.0001; no main effect of, or interactions with the Genotype factor; data not shown for time taken to locate platform, Trial effect: F(3,225) = 7.87, p < 0.0001; Day effect: F(3,225) = 34.46, p < 0.0001; no main effect of, or interaction with, the Genotype factor]. In contrast to the data for maze acquisition, marked genotypic differences were observed regarding recall of the platform location, when tested 24 h later (**Figure 2E**), with both Homer1a+/− and Homer1a−/− mice exhibiting impaired recall, as indicated by lower percent total distance and percent time spent in the SW quadrant, which formerly contained the hidden platform [for % Distance: Genotype effect: F(2,27) = 6.79, p = 0.004; for % Time: Genotype effect: F(2,27) = 5.82, p = 0.008; post hoc tests]. No genotypic difference in the distance swam (**Figure 2E**; p = 0.65) argues that the recall impairment exhibited by Homer1a mutants was unrelated to swimming ability.

### 3.4 Cocaine-Conditioned Place-Preference

Pan-Homer1 deletion increases sensitivity to the conditioned rewarding properties of cocaine (Szumlinski et al., 2004; Lominac et al., 2005). Thus, we characterized the dose-response function for cocaine-induced place-conditioning in male and female Homer1a+/+,+/−,−/− mice.  Despite the large sample sizes employed in this experiment, we did not detect an effect of Homer1a deletion upon the dose-response function for cocaine-induced place-preference (**Figure 3**; no Genotype effect or Genotype × Side interactions, p's > 0.35). These data argue little role for Homer1a induction in either the associative learning/memory or incentive motivational processes involved in the development and expression of cocaine-conditioned reward.

#### 3.5 DHPG-Induced Glutamate Release

Although it appeared as if Homer1a deletion elevated baseline glutamate in the study of DHPG-induced glutamate release (**Figure 4A**; DHPG = 0 µM), the genotypic difference was not significant (t-test: p = 0.19). However, Homer1a deletion prevented the capacity of DHPG to stimulate glutamate release within NAC (**Figure 4A**) [Genotype × Dose: F(3,36) = 26.02, p < 0.0001]. DHPG dose-dependently elevated glutamate in Homer1a+/+ mice [DHPG effect: F(3,15) = 68.31, p < 0.0001], while no DHPG-induced rise was apparent in Homer1a−/− mice (DHGP effect: p = 0.70). In contrast to Homer1a deletion, a comparison of baseline glutamate indicated lower glutamate levels in Homer1−/− versus Homer1+/+ mice [t(16) = 2.36, p = 0.02]. Further, pan-Homer1 deletion blunted, but did not block, the capacity of DHPG to increase NAC glutamate (**Figure 4B**) [Genotype × DHPG: F(3,51) = 4.65, p = 0.006], as evidenced by a significant DHPG-induced rise in glutamate in

both genotypes [DHPG effect, for Homer1+/+: F(3,24) = 15.00, p < 0.0001; for Homer1−/−: F(3,27) = 4.37, p < 0.01]. These data provide evidence for distinct effects of Homer1a and pan-Homer1 deletion upon mGlu1/5-dependent regulation of extracellular glutamate in vivo.

#### 3.6 Depolarization-Induced NAC Glutamate and Dopamine Release

In this study, both Homer1a−/− (**Figure 4C**) and pan-Homer1−/− (**Figure 4D**) mice exhibited significantly lower baseline glutamate levels than their wild-type controls [for Homer1a: t(13) = 2.39, p = 0.03; for pan-Homer1: t(14) = 3.02, p = 0.009]. In contrast, Homer1a−/− KO mice exhibited a modest, but statistically significant elevation, in basal dopamine levels (**Figure 4E**) [t(13) = 2.05, p = 0.045], whereas differences in basal dopamine were not apparent between Homer1+/+ and Homer1−/− mice (**Figure 4F**; t-test, p > 0.35).

Despite employing the same perfusate through the course of study, the effects of depolarization were more pronounced in the Homer1a versus the Homer1 study, which might reflect differences in the genetic background of the two mutant lines. Nevertheless, a marked genotypic difference in K+-stimulated glutamate release was apparent in the study of Homer1a mice [Genotype × K +: F(2,26) = 7.93, p = 0.002]; while, K+ stimulated a rise in glutamate in both genotypes (**Figure 4C**) [for Homer1a+/+: F(2,14) = 10.70, p = 0.002; for Homer1a−/−: F(2,12) = 6.01, p = 0.02], the magnitude of the rise observed in Homer1a−/− animals was considerably less than that observed in their controls. Although, Homer1−/− mice exhibited lower

FIGURE 4 | Comparison of the effects of Homer1a versus pan-Homer1 deletion upon the regulation of NAC glutamate and dopamine. Intra-NAC perfusion with increasing concentrations of the mGlu1/5 agonist DHPG failed to increase extracellular glutamate in Homer1a−/− mice (A), while Homer1−/− mice exhibited a moderate, but significant, DHPG-stimulated rise in NAC glutamate (B). Intra-NAC perfusion with high K+ solutions elevated NAC glutamate levels and this effect was markedly blunted in Homer1a−/− mice (C), but not in Homer1−/− mice, although Homer1−/− mice exhibited lower glutamate levels, overall (D). Homer1a−/− mice failed to exhibit K +-stimulated dopamine release (E), while dopamine release was unaffected in Homer1−/− mice (F). The data represent the mean ± SEM of the number of mice indicated in parentheses. ∗p < 0.05 vs. 0 µM; +p < 0.05 vs. Homer1a+/+ or Homer1+/+.

glutamate levels than Homer1+/+ animals, irrespective of the concentration of K+ perfused (**Figure 4D**) [Genotype effect: F(1,14) = 9.67, p = 0.008], Homer1 deletion did not alter the capacity of K+ to stimulate glutamate release within the NAC [K+ effect: F(2,28) = 13.08, p < 0.0001; Genotype × K +: p = 0.11].

In the case of dopamine, Homer1a deletion prevented K+ stimulated dopamine release within the NAC (**Figure 4E**) [Genotype × K +: F(2,38) = 4.57, p = 0.02; one-way ANOVAs: for Homer1a+/+, F(2,26) = 9.32, p = 0.001; for Homer1a−/−: p = 0.78]. In contrast, pan-Homer1 deletion had no impact upon K+-stimulated dopamine release in this region [K+ effect: F(2,40) = 6.86, p = 0.003; Genotype effect and interaction, p's > 0.70]. These data provide novel evidence that the selective deletion of Homer1a, but not the entire Homer1 gene, reduces

the excitability of both dopamine and glutamate terminals within the NAC.

#### 3.7 Glutamate No Net-Flux

Linear regression analyses of the plots of the no net-flux study (**Figure 5A**) confirmed lower NAC extracellular glutamate content in Homer1a−/− versus Homer1a+/+ mice (**Figure 5B**) [t(13) = 5.85, p = 0.03]. Linear analysis also indicated a significant reduction in the in vivo recovery of glutamate in Homer1a−/− mice relative to Homer1a+/+ controls, as indicated by differences in the slopes of the linear regressions (**Figure 5C**) [t(13) = 5.51, p = 0.04].

### 3.8 AAV-Homer1a Effects upon Genotypic Differences in Behavior in Drug-Naïve Animals

To examine an active role for nucleus accumbens Homer1a in the behavioral phenotype of Homer1a−/− mice, AAV constructs carrying either Homer1a cDNA (AAV-Homer1a) or a scrambled DNA control were infused into the nucleus accumbens of Homer1a+/+ and Homer1a−/− mice.

#### 3.8.1 Acoustic Startle and PPI

We first employed Tukey tests for planned comparisons to compare between Homer1a+/+ and Homer1a−/− mice infused with the control AVV to confirm replication of our prior genotypic difference in startle amplitude. Indeed, we replicated lower startle responsiveness in Homer1a−/− mice, specifically during habituation to the 110 dB stimulus (p = 0.04), in response to the 90 dB tone (p = 0.02), the 110 dB tone (p = 0.04) and in response to the 110 dB tone when preceded by the 74 or the 90 dB prepulse (both p's = 0.05). We did not observe a genotypic difference between AAV-controls at the 0 or 74 dB stimuli or when the 110 dB stimulation was preceded by the 90 dB prepulse (p's > 0.90). Planned comparisons were then conducted between AAV-control- and AAV-Homer1a-infused animals within each genotype. Despite what appeared to be a relatively selective effect of Homer1a over-expression upon the startle responsiveness of Homer1a−/− mice (**Figure 6A**), this statistical approach yielded

FIGURE 6 | The effects of AAV-mediated restoration of Homer1a expression in the NAC of Homer1a−/− mice upon sensorimotor function and depressive-like behavior. (A) Genotypic differences in acoustic startle were apparent only between Homer1a+/+ and Homer1a−/− mice infused with AAV-control (con). (B) Group differences were not observed for PPI. (C) Homer1a−/− mice exhibited a shorter latency to float in a 15-min Porsolt swim test and AAV-Homer1a (H1a) increased the latency to float in both genotypes. The data represent the mean ± SEM of the number of mice indicated in parentheses. ∗p < 0.05 vs. respective +/+; +p < 0.05 vs. respective AAV control.

no AAV-Homer1a effects in either genotype at any of the stimulus intensities (p's > 0.20), with the exception of a significant reduction in the response exhibited by Homer1a+/+ mice to the 90 dB tone (p = 0.02; st90 in **Figure 6A**). However, a comparison of the startle amplitude exhibited between Homer1a+/+ and Homer1a−/− mice infused intra-NAC with AAV-Homer1a indicated no significant genotypic differences at any of the stimulus intensities (p's > 0.40), arguing that AAV-Homer1a infusion into the NAC was sufficient to reverse the blunted sensorimotor-processing induced by Homer1a deletion.

A comparable analysis of PPI (**Figure 6B**) detected no group differences at the 74 dB prepulse (for all comparisons, p's > 0.70) or at the 110 dB prepulse (for all comparisons, p's > 0.10). Thus, we failed to replicate the PPI deficit observed in AAV-naïve mice (**Figure 2B**), nor did we find evidence to support an active role for NAC Homer1a in regulating the magnitude of PPI.

#### 3.8.2 Porsolt Swim Test

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Replicating the data in **Figure 1E**, Homer1a deletion reduced the latency to first exhibit floating [Genotype effect: F(1,29) = 15.93, p < 0.0001]; however, intra-NAC AAV-Homer1a infusion increased the latency first float, irrespective of genotype (**Figure 6C**) [AAV effect; F(1,29) = 5.00, p = 0.03; interaction: p = 0.48], with AAV-Homer1a partially reversing the behavior of Homer1−/− mice. In contrast, we did not replicate the genotypic difference in total floating nor did we observed any AAV effect upon total floating in this experiment (data not shown; Genotype × AAV ANOVA, all p's > 0.35). Further, AAV infusion did not impact the distance traveled during swim testing (data now shown; Genotype × AAV ANOVA, all p's > 0.55).

#### 3.9 AAV-Homer1a Effects upon Genotypic Differences in Cocaine-Induced Behavior

Although we failed to detect an effect of Homer1a deletion upon cocaine-conditioned reward (**Figure 3**), Homer1a deletion prevents the development of cocaine-induced behavioral sensitization (Park et al., 2013) and the effect of Homer1a overexpression within the NAC is not known. Thus, we employed our cocaine-induced place-conditioning procedures to determine the effects of intra-NAC AAV-Homer1a infusion upon spontaneous and cocaine-induced changes in locomotor activity, as well as place-conditioning, in Homer1a+/+ and Homer1a−/− mice.

#### 3.9.1 Locomotor Reactivity to Novelty and Habituation

A significant Genotype by AAV interaction was observed for the locomotor hyperactivity expressed when mice were allowed to explore the place-conditioning apparatus on the PreTest (**Figure 7A**) [Genotype × AAV: F(1,31) = 4.45, p = 0.04]. This interaction reflected genotypic differences between Homer1a+/+ and Homer1a−/− mice infused with AAV-control (p = 0.02), which was not apparent in mice infused with AAV-Homer1a (p = 0.70). When compared to their respective AAV-control, there was no detectable effect of AAV-Homer1a upon the novelty-induced locomotion of either Homer1a+/+ (p = 0.12) or Homer1a−/− mice (p = 0.18). No group differences were observed with respect to either the locomotor response to an acute saline injection (Genotype × AAV ANOVA, all p's > 0.65) or the change in saline-induced locomotion across the four saline-conditioning sessions (data not shown) [Saline Injection effect: F(3,84) = 4.69, p = 0.04; all other p's > 0.07].

#### 3.9.2 Cocaine-Induced Locomotion and Sensitization

Interestingly, the group differences in the acute locomotor response to an acute injection of 30 mg/kg cocaine was inverse

PreTest (A) and the distance traveled in response to an acute injection of 10 mg/kg cocaine (B). (C) Homer1a−/− controls failed to exhibit cocaine-induced sensitization and this phenotype was reversed by AAV-Homer1a infusion. (D) Group differences were not observed for the magnitude of a conditioned place-preference elicited by four pairings of 10 mg/kg cocaine. The data represent the mean ± SEM of the number of mice indicated in parentheses. ∗p < 0.05 vs. respective +/+; +p < 0.05 vs. respective AAV control.

of that observed for novelty-induced locomotion (**Figure 7A** vs. **7B**) [Genotype × AAV: F(1,28) = 5.30, p = 0.03], with significant genotypic differences observed between Homer1a+/+ and Homer1a−/− mice infused with AAV-control (p = 0.03), but not mice infused with AAV-Homer1a (p = 0.38). Further, we observed no effect of AAV-Homer1a upon acute cocaine-induced locomotion in either Homer1a+/+ (p = 0.13) or Homer1a−/− mice (p = 0.11).

In contrast to the locomotor response to acute cocaine, AAV-Homer1a infusion completely reversed the effects of Homer1a deletion upon the development of cocaine-induced locomotor sensitization [Genotype × AAV × Cocaine Injection: F(3,84) = 4.94, p = 0.003]. Consistent with prior results for AAV-naïve mice (Park et al., 2013), Homer1a+/+ mice infused with AAV-control exhibited robust sensitization across the four cocaine injections [F(3,21) = 15.23, p < 0.0001], as did Homer1a+/+ mice infused with AAV-Homer1a [F(3,21) = 12.37, p < 0.0001]. While cocaine-induced locomotion did vary as a function of repeated cocaine treatment in Homer1a−/− infused with AAV-control [F(3,21) = 4.33, p = 0.02], this reflected a decline, rather than an increase, in activity with repeated cocaine treatment. In contrast, Homer1a−/− mice infused with AAV-Homer1a exhibited an injection-dependent increase in cocaineinduced locomotion [F(3,21) = 8.36, p = 0.001], indicative of restored sensitization.

To facilitate visualization of the group differences in cocaineinduced sensitization, the data are presented as difference in locomotor activity from injection 1 to 4 of repeated treatment (**Figure 7C**). Analysis of these data further indicates a reversal of the cocaine-sensitization phenotype of Homer1a−/− mice [Genotype × AAV: F(1,31) = 4.21, p = 0.05], with Homer1a−/− AAV-control exhibiting a locomotor phenotype distinct from the other groups tested (p's ≤ 0.001). Although AAV-Homer1a infusion increased the magnitude of the cocaine-sensitized response in both genotypes, the AAV effect was significant only in Homer1a−/− mice (Homer1a+/+: p > 0.20; Homer1a−/−: p < 0.0001).

#### 3.9.3 Cocaine-Conditioned Place-Preference

Relative to their sensitized locomotor response to cocaine (**Figure 7C**), the magnitude of a cocaine-conditioned placepreference was highly variable (**Figure 7D**) and we detected no effect of genotype or AAV treatment upon the CPP Score exhibited by the mice when tested for compartment preference in a cocaine-free state (**Figure 8D**; Genotype × AAV ANOVA on CPP Score, p's > 0.45).

#### 3.10 AAV-Homer1a Effects on Basal and Cocaine-Stimulated Neurotransmission 3.10.1 Cocaine-Induced Glutamate Sensitization

An analysis of the average baseline glutamate levels obtained using conventional in vivo microdialysis procedures failed to detect any group differences (data not shown; Genotype × AAV × Injection Number ANOVA, p's > 0.15). As expected, the capacity of cocaine to increase NAC glutamate was injection-dependent [Injection Number effect: F(1,67) = 16.04, p < 0.0001]. However, the magnitude of the cocainesensitized glutamate response varied as a function of both genotype and AAV treatment [Genotype × AAV × Injection Number: F(1,67) = 8.36, p = 0.005]. Neither genotype nor AAV infusion influenced the glutamate response to acute cocaine (Genotype × AAV ANOVA: all p's > 0.45), while AAV-Homer1a infusion augmented the capacity of the 6th cocaine injection to increase glutamate response only in Homer1a−/− mice [Genotype × AAV: F(1,32) = −10.58, p = 0.003]. Indeed, all groups but the Homer1a−/− controls exhibited greater cocaine-induced glutamate release on the 6th versus the 1st cocaine injection (for Homer1a−/− AAV-control: p = 0.23; for all other groups, p's < 0.04).

#### 3.10.2 Cocaine-Induced Dopamine Sensitization

As observed for glutamate, there were no group differences in baseline levels of dopamine (data not shown; Genotype × AAV × Injection Number ANOVA, all p's > 0.06) and repeated cocaine treatment elicited a robust increase in dopamine that varied as a function of Genotype [Injection Number effect: F(1,67) = 21.76, p < 0.0001; Genotype × Injection Number: F(1,67) = 9.00, p = 0.004], but not AAV infusion (AAV × Injection Number: p = 0.15). Although the magnitude of the dopamine response to cocaine was greater following repeated treatment, the effects of

in Homer1a−/− mice upon basal and cocaine-stimulated neurotransmission. (A) Under conventional in vivo microdialysis procedures, repeated treatment with 30 mg/kg cocaine sensitized cocaine-induced glutamate release in both Homer1a+/+ infused intra-NAC with AAV, but only in Homer1a−/− mice infused with AAV-Homer1a (H1a). (B) Intra-NAC AAV-Homer1a infusion increased the magnitude of the cocaine-elicited rise in dopamine in Homer1a−/− mice and facilitated the expression of dopamine sensitization with repeated cocaine treatment. The sample sizes in (B) are presented in (A). When determined using glutamate no net-flux procedures, intra-NAC AAV-Homer1a reversed the low basal glutamate phenotype of Homer1a−/− mice (C), but did not alter the slope of linear regressions in either genotype (D). The samples sizes in (D) are presented in (C). The data in (A–D) represent the mean ± SEM of the number of mice indicated in parentheses. ∗p < 0.05 vs. respective +/+; +p < 0.05 vs. respective AAV control; #p < 0.05 vs. Injection 1 (sensitization). (E) Representative images of HA-staining within the NAC of Homer1a+/+ (left) and Homer1a−/− mice (right) infused with AAV-Homer1a (20X magnification), illustrating comparable neuronal transduction around the site of microinjection/microdialysis probe insertion. Arrows in (E) represent the extent of the microdialysis probe membranes.

AAV infusion upon the genotypic differences in dopamine responsiveness were similar across the microdialysis sessions (**Figure 8B**) [Genotype × AAV: F(1,67) = 7.46, p = 0.008; threeway interaction: p = 0.21], with Homer1a−/− controls exhibiting a blunted dopamine response to cocaine, relative to their Homer1+/+ counterparts (p) and AAV-Homer1a augmenting the cocaine-induced rise in dopamine only in Homer1a−/− animals (for Homer1a+/+: p = 0.57; Homer1a−/−: p < 0.0001).

#### 3.10.3 No Net-Flux Glutamate

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A planned comparison of the genotypic difference in the x-intercept for mice infused with AAV-control replicated lower basal glutamate content in Homer1a−/− mice (p < 0.0001). We also detected a significant Genotype × AAV interaction for the x-intercept (**Figure 8C**) [F(1,30) = 14.08, p = 0.001] that reflected an AAV-Homer1a-induced increase in basal glutamate content in Homer1a−/− (p < 0.0001), but not in Homer1a+/+ animals (p = 0.65). A comparable analysis of the slope of the linear regressions indicated that we did not replicate the genotypic difference in the extraction fraction in AAV-controls (**Figure 8D**; p = 0.67). Further, we did not observe any effect of AAV infusion upon this measure of glutamate release/reuptake in the NAC (Genotype × AAV: all p's > 0.10). Thus, Homer1a actively regulates basal extracellular glutamate content with in the NAC without influencing release/reuptake.

#### 4 DISCUSSION

The induction of the IEG Homer isoform Homer1a is theorized to play a critical role in synaptic rearrangement and neuroplasticity relevant to various cognitive and behavioral processes, as well as pathophysiology in mental disease (cf. Shiraishi-Yamaguchi and Furuichi, 2007; Szumlinski et al., 2008a; Fagni, 2012; Marton et al., 2015). Despite its initial characterization over 20 years ago (Brakeman et al., 1997; Kato et al., 1998), we still know relatively little regarding the functional relevance of Homer1a induction in vivo. Herein, we employed an AAV strategy and extend prior results for Homer1a−/− mice (Park et al., 2013) by demonstrating that Homer1a within the NAC actively regulates cocaineinduced behavior, glutamate and dopamine sensitization, as well as basal extracellular glutamate levels in this region. Moreover, we demonstrate that NAC Homer1a expression actively regulates sensorimotor processing and sensorimotorgating. We show also that Homer1a induction is necessary for normal recall of spatial memory and serves to inhibit depressive-like behavior in certain behavioral paradigms, but is not required to manifest anxiety-like behavior or cocaineconditioned reward. Lastly, we demonstrate the importance of Homer1a induction for mGlu1/5 function within the NAC, as well as the excitability of both glutamate and dopamine terminals therein, and provide novel in vivo evidence that the NAC neurochemical phenotype produced by Homer1a deletion is distinct from that produced by deletion of the entire Homer1 gene.

Our current knowledge concerning the behavioral relevance of distinct Homer1 isoforms can be derived from a relatively short-list of behavioral phenotyping studies of: pan-Homer1−/− mice (Szumlinski et al., 2004, 2005a; Lominac et al., 2005; Jaubert et al., 2007; Gerstein et al., 2012; Wagner et al., 2014, 2015), transgenic mice over-expressing Homer1a within the striatum (Tappe and Kuner, 2006) and transgenic mice incapable of Homer1a induction (Inoue et al., 2009; Park et al., 2013; present study). This line of interrogation is complemented and augmented by a number of reports describing the effects of site-specific manipulations of different Homer1 isoforms in the brain of wild-type rodents, as well as Homer-related mutants (Ghasemzadeh et al., 2003; Klugmann et al., 2005; Lominac et al., 2005; Szumlinski et al., 2006; Ary et al., 2013; Gerstein et al., 2013; Wagner et al., 2013, 2014, 2015; Banerjee et al., 2016; **Figure 7**). However marked procedural variations across these studies render it difficult to compare outcomes directly. This being said, the behavioral testing procedures employed in the present study of Homer1a−/− mice were highly comparable, and sometimes identical, to those used in our earlier behavioral characterization of pan-Homer1−/− mice (Szumlinski et al., 2004, 2005a; Lominac et al., 2005), facilitating interpretation of disparities and similarities in behavioral phenotypes across the two mutant lines. A side-by-side comparison of the effects of Homer1a versus pan-Homer1 deletion upon spontaneous behaviors is provided in **Table 1**. As is apparent from this table and the extant literature, pan-Homer1−/− mice exhibit robust and complex behavioral anomalies (Szumlinski et al., 2004, 2005a; Lominac et al., 2005; Jaubert et al., 2007; Gerstein et al., 2012; Wagner et al., 2013, 2014, 2015). In contrast, the behavioral phenotype of Homer1a−/− mice is surprisingly similar to their wild-type controls in a variety of assays (Inoue et al., 2009; Park et al., 2013, **Table 1**), although some behavioral anomalies were observed in the present study that are discussed below.

### 4.1 Preventing Homer1a Induction Produces an Affective Phenotype That Is Distinct from That Produced by Homer1 Deletion

Pan-Homer1−/− mice exhibit hyper-emotionality across a variety of paradigms (Szumlinski et al., 2004, 2005a; Jaubert et al., 2007; Wagner et al., 2014, 2015). In stark contrast, the anxiety-like behavior exhibited by Homer1a−/− herein was indistinguishable from that of wild-type mice, although Homer1+/− mutants did exhibit signs of hypo-anxiety on the elevated plus maze (**Figures 1A,B**). The present results are in line with the results of a study by Inoue et al. (2009), in which the Homer1a deletion did not alter anxiety-like behavior on the elevated plus-maze, open field or light–dark box tests (Inoue et al., 2009). These data for Homer1a-null mutants are interesting in light of evidence that transgenic mice over-expressing Homer1a within striatum increases anxiety signs in mice, which could be reversed upon conditional deletion of Homer1a (Tappe and Kuner, 2006). Taken together, the above findings from constitutive mutant mice argue that Homer1a over-expression is sufficient, but not necessary, for the manifestation of anxiety-like behavior.

However, other data in the literature argue that Homer1a induction exerts an "anxiolytic" or "anti-depressant" effect. For example, AAV-mediated Homer1a over-expression within the basolateral amygdala reduces the manifestation of fearconditioning, as well as social interaction (Banerjee et al., 2016), while AAV-mediated restoration of Homer1a to the prefrontal cortex of pan-Homer1−/− mice reverses their hyper-anxious and depressive-like phenotype (Lominac et al., 2005). Consistent

TABLE 1 | Comparison of the behavioral phenotypes observed in Homer1a−/− versus pan-Homer1−/− mice.

Summary of the results of comparable studies examining the effects of Homer1a or Homer1 deletion (KO) upon behavior. > Denotes greater behavioral response in wild-type (WT) than KO; < denotes less behavioral response in WT than KO; = denotes no genotypic difference; ≥denotes inconsistencies in study outcomes, with reports of either no genotypic difference or greater behavioral response in WT than KO mice; ≤ denotes inconsistences in study outcomes, with reports of either no genotypic difference or less behavioral response in WT than KO. Highlighted rows indicate phenotypic similarities between Homer1a−/− and Homer1−/− mice. 1Jaubert et al. (2007); 2Szumlinski et al. (2005a); 3Lominac et al. (2005); 4Park et al. (2013).

with these latter data, both Homer1a+/− and Homer1a−/− mice exhibited a reduced latency to float in the Porsolt swim test in the present study (**Figure 1E**). In fact, this particular response to a stressor was the only emotion-related phenotype expressed in common by Homer1a−/− and pan-Homer1−/− mice (see **Table 1**). Moreover, AAV-mediated Homer1a overexpression within the NAC increased the float latency in both Homer1a+/+ and Homer1a−/− mice (**Figure 6C**), arguing that Homer1a induction within both the cell body and terminal regions of the corticoaccumbens projections actively gates a depressive-like phenotype – a finding in line with associations between variants in the Homer1 gene and major depressive disorder (e.g., Rietschel et al., 2010; Strauss et al., 2012; Rao et al., 2017). However, it is noteworthy that Homer1a deletion did not produce a "pro-depressive" effect in our saccharin preference test. In fact, a review of the extant literature suggests that site-specific Homer1a manipulations induce more robust and consistent effects upon negative affect (Lominac et al., 2005; Tappe and Kuner, 2006; Banerjee et al., 2016) than Homer1a deletion (Inoue et al., 2009; **Figure 1** and **Table 1**). This raises the possibility that Homer1a induction within distinct brain regions may exert opposing roles in regulating affective responding that are masked under constitutive inhibition of IEG induction.

### 4.2 Prevention of Homer1a Induction Produces a Cognitive Phenotype That Is Distinct from That Produced by Homer1 Deletion

Homer1a induction is theorized to facilitate the rearrangement of the synaptic architecture of excitatory glutamate synapses necessary for metaplasticity and learning (e.g., Hu et al., 2010; Fagni, 2012; Marton et al., 2015). Although associative learning is intact in pan-Homer1−/− mice (Szumlinski et al., 2004), this mutant exhibits deficits several other forms of learning and memory, including simple habituation (Lominac et al., 2005; Szumlinski et al., 2005a), spatial learning/memory (Lominac et al., 2005; Szumlinski et al., 2005a; Jaubert et al., 2007), and instrumental learning (Szumlinski et al., 2005a; Wagner et al., 2014). Moreover, pan-Homer1−/− mice are deficient in long-term depression (Ronesi and Huber, 2008) and long-term potentiation (Gerstein et al., 2012), while Homer1a−/− mice fail to exhibit homeostatic AMPA receptor scaling within cortex (Hu et al., 2010). In contrast to pan-Homer1−/− mice (Szumlinski et al., 2005a), Homer1a-null mutants exhibited no observable deficits in the acquisition phase of a Morris water maze (**Figure 2D**), arguing that the induction of Homer1a is not necessary for the consolidation

or recall of the information pertaining to the platform location.

However, akin to pan-Homer1−/− mice (Szumlinski et al., 2005a), both Homer1a+/− and Homer1a−/− animals spent less time in the quadrant of the pool that formerly contained the hidden platform on the probe test (**Figure 2E**). While this result might reflect impaired spatial memory recall, such an interpretation is inconsistent with their intact learning of the maze location during the acquisition phase of this experiment. As no genotypic differences were observed in the amount of floating behavior during Porsolt swim testing (**Figure 1F**) or in the total distance traveled during the probe trial (**Figure 2E**), it is unlikely that Homer1a deletion altered swimming capacity during the memory probe test. Alternatively, the reduced time spent in the platform quadrant might reflect the deployment of an alternative platform search or escape strategy in Homer1a mutant mice. Indeed, pan-Homer1−/− mice employ a "chaining" strategy to successfully navigate a water-version of the radial arm maze (Lominac et al., 2005; Szumlinski et al., 2005a) and AAV-mediated restoration of Homer1c, not Homer1a, to the PFC of pan-Homer1−/− mice reverse their deficits in spatial working memory (Lominac et al., 2005). Further, hippocampal restoration of Homer1c reverses the spatial memory impairments exhibited by pan-Homer1−/− (Gerstein et al., 2012), as well as aged mice (Gerstein et al., 2013) and hippocampal overexpression of Homer1a impairs the acquisition and/or recall of spatial memories (Klugmann et al., 2005; Celikel et al., 2007). Thus, it is clear that a more concerted research effort is required to understand more precisely how Homer1a induction within specific neurocircuits contributes to spatial and nonspatial learning of relevance to the etiology and treatment of attentional, learning and memory disorders (Wells et al., 2015).

### 4.3 Preventing Homer1a Induction Profoundly Impairs in Vivo Regulation of NAC Neurotransmitter Levels

Since their initial discovery, the glutamatergic consequences of Homer1 vs. Homer1a deletion have been studied extensively in vitro, with a focus on the regulation of post-synaptic signal transduction mechanisms (cf. Shiraishi-Yamaguchi and Furuichi, 2007; Worley et al., 2007; Fagni, 2012; Marton et al., 2015). However, transfection of hippocampal neuronal cultures with constitutively expressed Homer isoforms can augment, while transfection with Homer1a reduces, presynaptic neuronal activity (Sala et al., 2001) and a more recent study demonstrated opposing roles for CC- and IEG-Homer1 isoforms in regulating mGlu1/5-stimulated Ca2+ entry and glutamate release from astrocytes both in vitro and in situ (Buscemi et al., 2017). Further, CC- and IEG-Homer1 isoforms exert opposing effects upon mGlu1/5-dependent generation of anandamide (Jung et al., 2007), which is well-characterized to inhibit glutamate release via activation of CB1 receptors (e.g., Johnson and Lovinger, 2016). Indeed, we have reported several anomalies with respect to in vivo measures of glutamate function in the brains of pan-Homer1−/− mice, notably, reduced NAC and elevated PFC basal extracellular glutamate content (Szumlinski et al., 2004, 2005a), and an altered capacity of acute cocaine to elevate extracellular glutamate levels within both the NAC (increased in pan-Homer1−/−; **Table 2**) (Szumlinski et al., 2004) and PFC (decreased in pan-Homer1−/−) (Lominac et al., 2005; Szumlinski et al., 2005a) that suggest a critical role for Homer1 proteins in regulating presynaptic aspects of glutamate transmission.

Herein, we report that, akin to Homer2−/− mice (Szumlinski et al., 2004), pan-Homer1−/− mice also exhibit a blunted capacity of the Group1 mGlu receptor agonist DHPG to stimulate glutamate release within the NAC (**Figure 4B**), which might reflect a "de-scaffolding" of mGlu1/5 receptors within neurons and/or astrocytes in this region (Brakeman et al., 1997; Tu et al., 1998, 1999; Buscemi et al., 2017). This being said, Homer1a−/− mice also exhibited blunted DHPG-stimulated glutamate release (**Figure 4A**), as well as reduced basal extracellular glutamate content within the NAC (**Figure 5B**). These results were rather unexpected given that prior transgenic work argued a more critical role for CC-Homer1 versus Homer1a expression in regulating extracellular glutamate levels in vivo (Lominac et al., 2005), as well as glutamate release in vitro (Buscemi et al., 2017) and Homer1a−/− mice exhibit increased cell surface expression of mGlu5 in situ and in vitro (Hu et al., 2010). While the source of the extracellular glutamate remains to be determined (i.e., neuronal, glial, or both), the capacity of DHPG to augment NAC glutamate levels in freely moving animals can be blocked by co-infusion of an mGlu1, but not mGlu5, inhibitor (Swanson et al., 2001). By extension then, the failure of both pan-Homer1−/− and Homer1a−/− mice to exhibit a DHPG-induced rise in NAC extracellular glutamate likely relates to a deficit in mGlu1-dependent, rather than mGlu5-dependent, signaling, with the DHPG phenotype of pan-Homer1−/− mice likely reflecting incapacity to induce Homer1a.

DHPG-induced glutamate release within the NAC is also activity-dependent in vivo (Swanson et al., 2001). However, while Homer1 deletion does not alter the extraction fraction index of glutamate clearance/reuptake within the NAC (Szumlinski et al., 2005a), the extraction fraction was lower in Homer1a−/− mice than wild-type controls (**Figure 5C**). This difference in extraction fraction was driven primarily by genotypic differences in glutamate flux at concentrations below y = 0 (**Figure 5C**), suggesting reduced glutamate release, rather than increased glutamate reuptake in Homer1a−/− animals (Parsons and Justice, 1992). Although Homer1a−/− mice do not exhibit changes in the frequency of miniature excitatory post-synaptic potentials within cortical pyramidal neurons (Hu et al., 2010), Homer1a−/− mice exhibited robust deficits in high K +-stimulated release of both dopamine and glutamate within the NAC (**Figures 4C,E**), which is a finding in-line with their blunted dopamine response to acute cocaine and their failure to exhibit both dopamine and glutamate sensitization with repeated cocaine treatment (Park et al., 2013; **Figures 8A,B**). In contrast, depolarization-induced glutamate or dopamine release was intact within the NAC of pan-Homer1−/− mice, despite their lower extracellular glutamate levels (**Figures 4D,F** and **Table 2**).

#### TABLE 2 | Comparison of the cocaine phenotype of Homer1a−/− versus pan-Homer1−/− mice.

Summary of the results of comparable studies examining the effects of Homer1a or Homer1 deletion (KO) upon cocaine-induced changes in behavior and neurochemistry. > Denotes greater response in wild-type (WT) than KO; < denotes less response in WT than KO; = denotes no genotypic difference; ≥ denotes inconsistencies in study outcomes, with reports of either no genotypic difference or greater response in WT than KO mice. Highlighted rows indicate phenotypic similarities between Homer1a−/− and Homer1−/− mice. 1Szumlinski et al. (2004).

This result indicates that pan-Homer1 deletion does not grossly impair the excitability of either dopamine or glutamate terminals within this region – a finding consistent with the fact that cocaine can elevate the NAC levels of both neurotransmitters in Homer1−/− mice (Szumlinski et al., 2004). Importantly, AAVmediated restoration of Homer1a to the NAC of Homer1a−/− mice reversed both the anomalies in basal glutamate, as well as the blunted cocaine responsiveness of dopamine and glutamate observed in Homer1a−/− animals, while NAC Homer1a over-expression in Homer1+/+ mice was without any overt effect on our neurochemical measures (**Figure 8**). It remains to be determined whether or not virally mediated Homer1a expression incorporates into the post-synaptic density in a manner akin to endogenous protein to influence neurotransmission. Nevertheless, the present neurochemical data argue that Homer1a induction within the NAC actively regulates, but is not sufficient to augment, basal extracellular glutamate, as well as dopamine and glutamate responsiveness within this region. These data provide confirmatory evidence of a novel role for this IEG Homer1 isoform in regulating at least two neurotransmitters systems within a brain region highly implicated in incentive motivational and attentional processing.

### 4.4 NAC Homer1a Induction Actively Regulates Cocaine-Induced Behavioral Sensitization But Not Conditioned Reward

As reported previously (Park et al., 2013), the failure of Homer1a−/− mice to develop cocaine-induced neurochemical sensitization within the NAC (**Figures 8A,B**) is associated with a lack of locomotor sensitization in these animals (**Figure 7C**). Herein, we extend this prior work by demonstrating that AAV-mediated restoration of NAC Homer1a reversed the sensitization phenotype of Homer1a−/− mice, without significantly affecting spontaneous or cocaine-induced changes in locomotor activity within Homer1a+/+ controls (**Figure 7**). While we previously observed no overt effects of Homer1a deletion upon spontaneous locomotion or acute cocaine-induced locomotor-activity (Park et al., 2013), Homer1a−/− GFPcontrols exhibited blunted novelty-induced locomotion (**Figure 7A**), but greater locomotor activity in response to an acute cocaine injection (**Figure 7B**), which could relate to surgical/AAV procedures employed herein. Nevertheless, the striking parallels between the effects of manipulating Homer1a induction upon behavioral and neurochemical sensitization within the NAC observed to date argue a facilitatory role for Homer1a induction in the development of dopamine/glutamate plasticity that sensitizes drug-induced psychomotor activity.

The capacity of cocaine to augment NAC Homer1a expression (Brakeman et al., 1997; Ghasemzadeh et al., 2009b; Park et al., 2013) is reported to develop tolerance with repeated drug experience (Ghasemzadeh et al., 2009b). However, repeated cocaine exposure also reduces the NAC expression of constitutively expressed Homer1 and Homer2 proteins (e.g., Swanson et al., 2001; Ary and Szumlinski, 2007; Ben-Shahar et al., 2009; Ghasemzadeh et al., 2009a, 2011; Knackstedt et al., 2010; Loweth et al., 2014). A cocaine-induced reduction in the relative expression of CC-Homers is predicted to bias intracellular signaling within the NAC in favor of the dominant negative actions of the IEG isoforms, which we have now confirmed, herein, to be necessary for the development of drug-sensitized behavior. Consistent with this notion, pan-Homer1 deletion (Szumlinski et al., 2004; Lominac et al., 2005) or anti-sense oligonucleotide-mediated knock-down of Homer1 expression within the NAC augments the acute locomotor-stimulatory effects of cocaine (Ghasemzadeh et al., 2003). Conversely, restoration of the CC-Homer2b isoform to the NAC of cocaine Homer2−/− mice (Szumlinski et al., 2004) or over-expression of either Homer1c or Homer2b within the NAC of cocainesensitized rats (Szumlinski et al., 2006) prevents their sensitized locomotor and glutamate responses to cocaine. Given the association between polymorphisms in the human Homer1 with disorders characterized by preservative behaviors (e.g., addiction, autism, and schizophrenia) (Norton et al., 2003; Dahl et al., 2005; Kelleher et al., 2012), it will be important to understand more precisely the mechanisms through which Homer1a modulates the capacity of cocaine, as well as other

TABLE 3 | Comparison of the NAC neurochemical phenotype of Homer1a−/− versus pan-Homer1−/− mice.

Summary of the results of comparable studies examining the effects of Homer1a or Homer1 deletion (KO) upon in vivo measures of basal glutamate function. > Denotes greater response in wild-type (WT) than KO; < denotes less response in WT than KO; = denotes no genotypic difference; ≥ denotes inconsistencies in study outcomes, with reports of either no genotypic difference or greater response in WT than KO mice. Highlighted rows indicate phenotypic similarities between Homer1a−/− and Homer1−/− mice. 1Szumlinski et al. (2005a); 2Szumlinski et al. (2004).

psychotomimetic drugs, to induce neuroplasticity within corticostriatal circuits that underpin enduring psychomotor hyperactivity.

Our prior studies of pan-Homer1−/− and Homer2−/− mice also demonstrated active and necessary roles for CC-Homer isoform expression within the NAC in regulating more complex forms of drug-induced associative and instrumental learning (Szumlinski et al., 2004, 2005b, 2008b; Lominac et al., 2005; **Table 3**). Indeed, disruption of Homer1-dependent binding within the NAC core subregion prevents both cue- and cocaineprimed reinstatement of drug-seeking (Wang et al., 2013), although AAV-mediated over-expression of either Homer1c or Homer2b within this subregion does not prevent the incubation of cue-elicited cocaine-seeking in rat models of drug selfadministration (Loweth et al., 2014). Given the robust effects of Homer1a deletion upon cocaine-induced behavioral and neurochemical sensitization within the NAC, we were very surprised by the absolute lack of genotypic differences upon the capacity of cocaine to elicit a conditioned place-preference in mice (**Figures 3**, **7D**). The cocaine doses and place-conditioning procedures employed in this study are sensitive to the effects of other transgenic manipulations of Homer and/or mGlu5 function (Szumlinski et al., 2004; Ary et al., 2013; Park et al., 2013). In fact, the dose-response function for cocaineinduced place-conditioning in both pan-Homer1−/−, as well as Homer2−/−, mice is shifted to the left of wild-type controls, indicating that deletion of either CC-Homer isoform increases behavioral sensitivity to cocaine-conditioned reward (Szumlinski et al., 2004). Important for data interpretation, the negative results for place-conditioning were derived from the same Homer1a+/+ and Homer1a−/− that exhibit blunted cocaineinduced behavioral and neurochemical sensitization (AAV-naïve, Park et al., 2013; AAV-infused, **Figures 7**, **8**) and thus, the disparate findings across the different dependent measures cannot reflect cohort effects.

Although both transgenic and pharmacological evidence indicates that a cocaine-conditioned place-preference can develop independent of a dopamine-sensitized state within the NAC (e.g., Szumlinski et al., 2004, 2007), in our experience and to the best of our knowledge, the Homer1a−/− mouse is the first example in which the magnitude of drug-conditioned reward in mice is independent of a glutamate-sensitized state within the NAC (e.g., Szumlinski et al., 2005a,b, 2008b, 2017; Penzner et al., 2008). Thus, while increasing NAC glutamate is sufficient to augment the magnitude of drug-induced place-conditioning (e.g., Szumlinski et al., 2017), NAC glutamate hypersensitivity does not appear to be required for this form of drug-related learning. Whether or not Homer1a deletion impacts more complex drug-related instrumental learning remains to be determined.

The NAC receives major glutamate input from the PFC and local manipulations of both CC- and IEG-Homer expression can regulate extracellular glutamate, as well as the cocaine responsiveness of glutamate within the PFC (Lominac et al., 2005; Szumlinski et al., 2005a; Ary et al., 2013). In fact, mimicking cocaine-induced changes in the expression of different Homer isoforms within PFC (Ary and Szumlinski, 2007; Ben-Shahar et al., 2009; Gould et al., 2015), by increasing Homer2 expression or reducing Homer1c expression, produces a number of glutamatergic adaptations within the NAC of cocainenaïve mice that resemble those observed in cocaine-experience rodents (Ary et al., 2013). Importantly, mimicking the cocaineinduced imbalance in CC-Homer isoforms within PFC is also sufficient to potentiate a cocaine-conditioned place-preference (Ary et al., 2013). Conversely, Homer1c over-expression within ventral PFC prevents cocaine-primed reinstatement in a rat selfadministration model (Gould et al., 2015). As withdrawal from repeated cocaine augments the glutamate responsiveness to drugassociated cues (Shin et al., 2016) and prior work indicates that Homer1a induction is required for normal homeostatic scaling of glutamatergic transmission within cortex (Hu et al., 2010), it will be important in future work to characterize the importance of Homer1a induction for the gating of corticofugal glutamate output from PFC subregions and the executive control the PFC exerts over subcortical structures regulating incentive motivation, affect and learning. It is clear that Homer1a function is complex, strengthening or weakening synaptic connectivity in manner that depends upon on-ongoing cellular activity and the specific intracellular signaling pathways engaged (e.g., Park et al., 2013; Marton et al., 2015). Given the interpretational difficulties associated with studying a constitutive mutant model, new animal models are required that are capable of acute upand down-regulation of this isoform within specific neuronal and glial populations, to gain a deeper understanding of how Homer1a induction bi-directionally influences synaptic strength of relevance to the etiology of human neuropsychiatric disease.

### AUTHOR CONTRIBUTIONS

fnbeh-11-00208 October 31, 2017 Time: 16:30 # 18

MCD, MW, CMR, KDL, and KKS conducted the experiments. MW, KL, and KKS analyzed the data. GvJ and MK supplied the AAVs. J-HH and PFW supplied the KO mice. MCD, PFW, and KKS composed the manuscript. All authors edited the manuscript.

#### REFERENCES

#### FUNDING

This work was supported, in part, by a Young Investigator Award from Brain and Behavior Research Foundation (formerly NARSAD), and funds from the University of California, Santa Barbara to KKS, as well as NIDA grants DA 024038 to KKS and NIDA DA10309 to PFW.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Datko, Hu, Williams, Reyes, Lominac, von Jonquieres, Klugmann, Worley and Szumlinski. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Immediate-Early Genes Modulation by Antipsychotics: Translational Implications for a Putative Gateway to Drug-Induced Long-Term Brain Changes

Andrea de Bartolomeis\*, Elisabetta F. Buonaguro, Gianmarco Latte, Rodolfo Rossi, Federica Marmo, Felice Iasevoli and Carmine Tomasetti

*Laboratory of Molecular and Translational Psychiatry and Unit of Treatment Resistant Psychosis, Section of Psychiatry, Department of Neuroscience, Reproductive Sciences and Odontostomatology, University School of Medicine "Federico II", Naples, Italy*

An increasing amount of research aims at recognizing the molecular mechanisms involved in long-lasting brain architectural changes induced by antipsychotic treatments. Although both structural and functional modifications have been identified following acute antipsychotic administration in humans, currently there is scarce knowledge on the enduring consequences of these acute changes. New insights in immediate-early genes (IEGs) modulation following acute or chronic antipsychotic administration may help to fill the gap between primary molecular response and putative long-term changes. Moreover, a critical appraisal of the spatial and temporal patterns of IEGs expression may shed light on the functional "signature" of antipsychotics, such as the propensity to induce motor side effects, the potential neurobiological mechanisms underlying the differences between antipsychotics beyond D2 dopamine receptor affinity, as well as the relevant effects of brain region-specificity in their mechanisms of action. The interest for brain IEGs modulation after antipsychotic treatments has been revitalized by breakthrough findings such as the role of early genes in schizophrenia pathophysiology, the involvement of IEGs in epigenetic mechanisms relevant for cognition, and in neuronal mapping by means of IEGs expression profiling. Here we critically review the evidence on the differential modulation of IEGs by antipsychotics, highlighting the association between IEGs expression and neuroplasticity changes in brain regions impacted by antipsychotics, trying to elucidate the molecular mechanisms underpinning the effects of this class of drugs on psychotic, cognitive and behavioral symptoms.

Keywords: Arc, BDNF, Homer1a, clozapine, haloperidol, schizophrenia, bipolar disorders, cognition

#### INTRODUCTION

There is growing interest in unraveling the cellular mechanisms putatively involved in long-term changes in brain architecture and function following antipsychotic administration (Ahmed et al., 2008; Ho et al., 2011; Cannon et al., 2015; Vita et al., 2015; Yue et al., 2016; Emsley et al., 2017). In vivo human studies have pointed out that volumetric and functional changes may be detected after

#### *Edited by:*

*Amelia Gallitano, University of Arizona, United States*

#### *Reviewed by:*

*Clive R. Bramham, University of Bergen, Norway Gregg Stanwood, Florida State University, United States*

> *\*Correspondence: Andrea de Bartolomeis<EMAIL_ADDRESS>
*Received: 30 May 2017 Accepted: 22 November 2017 Published: 11 December 2017*

#### *Citation:*

*de Bartolomeis A, Buonaguro EF, Latte G, Rossi R, Marmo F, Iasevoli F and Tomasetti C (2017) Immediate-Early Genes Modulation by Antipsychotics: Translational Implications for a Putative Gateway to Drug-Induced Long-Term Brain Changes. Front. Behav. Neurosci. 11:240. doi: 10.3389/fnbeh.2017.00240*

acute antipsychotic treatments (Emsley et al., 2015, 2017). However, the long-term consequences of these acute changes remain still elusive.

Immediate-early genes (IEGs) may represent a significant candidate to explore how acute antipsychotics administration may set the molecular scenario for long-term changes.

New insights in IEGs expression following acute or chronic antipsychotic administration in preclinical models may help to fill the gap between primary molecular responses to antipsychotic administration and putative long-term synaptic changes (**Figure 1**). Recent observations are opening new avenues in our understanding of how antipsychotics work and strongly challenge the old idea that significant changes in synaptic plasticity may be caused by prolonged treatments. Indeed, multiple lines of evidence demonstrated that in vivo antipsychotic treatment may significantly impact the architecture of the synapse, as well as the re-arrangement of gene expression of scaffolding and adaptor proteins after acute exposure to the drugs. In line with this view, haloperidol acute administration has been shown to reduce dendritic spines size, possibly through a beta-adducin-mediated mechanism (Engmann et al., 2016). Moreover, the acute administration of typical and atypical antipsychotics has been demonstrated to re-arrange the topography of Homer1a gene expression in cortical and subcortical brain regions (Buonaguro et al., 2017b).

Acute antipsychotics administration has been demonstrated to impact signal-transduction pathways in specific brain regions with significant implications for long-term treatment (De Bartolomeis et al., 2013b, 2015a). For instance, acute i.v. infusion at therapeutic doses of haloperidol may trigger changes in the volume of the striatum (Tost et al., 2010), this effect being consistent with the rapid and transient IEGs induction by acute dopamine D2 receptors (D2Rs) blockade.

IEGs activation after antipsychotics acute administration could be pivotal to dissect primary molecular and cellular events that may prime the long-term effects of antipsychotic treatment. Similarly, multiple studies have pointed out the significant functional changes in cortical and subcortical networks after acute administration of antipsychotics (Emsley et al., 2015, 2017).

At the same time, new exciting discoveries in early gene functions have reinvigorated the research on the role of IEGs in the brain, thanks also to novel techniques, such as the following: serial two-photon tomography (STP) for automated whole-brain histology using fluorescent reporters (Ragan et al., 2012); light sheet fluorescence microscopy (LSFM) coupled with tissue clearing for imaging IEG expression in the intact brain (Renier et al., 2016); optogenetics for selectively activate target neurons (Bepari et al., 2012).

Despite the relevance of the issue, the role of IEGs in antipsychotics action has not been reviewed recently and a comprehensive analysis is still lacking.

Herein, starting from the major IEGs proven to be induced by antipsychotics and from their involvement in brain functions believed to be translationally relevant for schizophrenia as well as for antipsychotic mechanism of action, we will review the following issues:

Moreover, we will consider:

### BACKGROUND: ANTIPSYCHOTICS, IEGs, AND BRAIN CHANGES

#### IEGs Relevance for Synaptic Plasticity

IEGs are a heterogeneous class of genes that are rapidly and transiently activated by a large number of stimuli, including environmental (i.e., light/dark phase changes, exposure to behavioral stressors such as intruder animals, learning session during acquisition tasks), pharmacological, and physical stimuli (Perez-Cadahia et al., 2011; Sauvage et al., 2013). IEGs represent a primary response to cellular perturbation, which is a standing process that is activated at the transcriptional level and occurs in the absence of de novo protein synthesis. IEGs are dynamically regulated by different forms of synaptic activity underlying information processing and storage, therefore they are excellent candidates involved in both Hebbian and homeostatic plasticity (Hu et al., 2010; Hayashi et al., 2012; Shin et al., 2012). Several studies demonstrated, indeed, that long-term forms of synaptic plasticity—such as long-term potentiation (LTP) require new production of intracellular macromolecules, whereas short-term synaptic plasticity processes do not (Kandel, 2001; Hayashi et al., 2012). IEGs expression occurring promptly after stimuli is considered a fundamental step for the establishment of synaptic plasticity, since synaptic plasticity changes may be prevented when mRNA synthesis is blocked early after the induction of a stimulus (Lanahan and Worley, 1998). Thus, IEGs may be considered as "gateway" genes controlling synaptic plasticity and may underlie processes like learning and memory formation.

IEGs encode a large number of proteins with different functions, such as transcription factors (e.g., c-Fos, Egr1, NGFI-B), postsynaptic proteins (e.g., Norbin, Homer 1a, Arc) and signaling molecules (e.g., RSG2, CaMKII).

The induction of an IEG is one of the earliest intracellular mechanism mediating the cellular response to external stimuli (Lanahan and Worley, 1998). According to these view, IEGs induction may be considered a recent activity marker, and its assessment may be used to determine when specific neural populations are activated, making possible to assess the extent of antipsychotics spatial and temporal impact on neural plasticity in different brain areas.

FIGURE 1 | Representative molecular imaging of *Homer1a* IEG expression by acute antipsychotics administration. Molecular imaging of IEGs expression may represent a tool to investigate topographic distribution of antipsychotic-mediated acute and long-term molecular effects within brain Regions of Interest (ROIs). Here we show a representative depiction of *Homer1a* IEG expression by different antipsychotics acutely administered in rodents. The autoradiographic film images of *Homer1a* mRNA detected by means of *in situ* hybridization histochemistry in coronal brain sections have been extracted from different studies carried on by our laboratory (Polese et al., 2002; Ambesi-Impiombato et al., 2007; Tomasetti et al., 2007, 2011; Iasevoli et al., 2009, 2010a,b, 2011; De Bartolomeis et al., 2015a) and representatively placed side by side in order to outline gene expression topography after treatments with haloperidol (HAL), olanzapine (OLA), sertindole (SERT), amisulpiride (AMS), risperidone (RISP), clozapine (CLO), and different doses of ziprasidone (4 mg/kg, ZIP4; 10 mg/kg, ZIP10), aripriprazole (12 mg/kg, ARI12; 30 mg/kg, ARI30), quetiapine (15 mg/kg, QUE15; 30 mg/kg, QUE30). *Homer1a* is a postsynaptic effector of plastic synaptic changes mainly mediated by dopamine and glutamate-dependent signaling pathways. Therefore, in this case, IEG molecular imaging may also provide putative information on antipsychotic-triggered changes in synaptic plasticity. ROIs: AC, Anterior Cingulate Cortex; M2, Medial Agranular Cortex; M1, Motor Cortex; SS, Somatosensory Cortex; I, Insular Cortex; dmCP, Dorso Medial Caudate-Putamen; dlCP, Dorso Lateral Caudate-Putamen; vlCP, Ventro Lateral Caudate-Putamen; vmCP, Ventro Medial Caudate-Putamen; Core, Nucleus Accumbens; Core, Shell, Nucleus Accumbens, Shell. Red, significant gene induction as compared to the respective control (*p* < 0.05); Dark red, significant gene induction as compared to the respective control (*p* < 0.001).

## IEGs: A Putative Gateway for Antipsychotic-Induced Brain Changes

Antipsychotic drugs are the mainstay of pharmacological treatment for schizophrenia, and their use has been expanded for the treatment of bipolar disorder and, in some cases, for pervasive disorders of the autistic spectrum (Geddes and Miklowitz, 2013). All antipsychotics share a variable degree of antagonism, or partial agonism, at D2 dopamine receptors (D2Rs) and both therapeutic and motor side effects of either typical or atypical antipsychotic drugs have been proven to directly depend on the occupancy of D2Rs (Seeman, 2002; Ginovart and Kapur, 2012). However, besides the dynamics of D2Rs binding by different antipsychotics, emerging evidence demonstrates that the study of the downstream signaling elicited by these compounds may help to better understand the mechanisms of action implicated in their clinical effects (De Bartolomeis et al., 2013a; Iasevoli et al., 2013). Additionally, dissecting the molecular basis of antipsychotic actions may shed light on new avenues of investigation to bypass the critical issues related to receptor pharmacodynamics, such as D2Rs down- or up-regulation and D2Rs supersensitivity, which have been considered among the potential reasons of antipsychotic treatment resistance (Seeman, 2002; Nnadi and Malhotra, 2007; Seeman and Seeman, 2014; Oda et al., 2015).

In the last decades, several studies on the effects of typical and atypical antipsychotics on brain IEGs expression have been carried out, trying to unravel the bases of regional neuronal response to pharmacological stimuli (Dragunow, 1990; Miller, 1990; Young et al., 1998; Semba et al., 1999; Beaudry et al., 2000; Kovacs et al., 2001; Cochran et al., 2002), as well as to shed light on the molecular mechanisms implicated in antipsychotic actions (Deutch et al., 1991, 1995). Transcriptional fingerprint of IEGs, and their functionally related molecules, has progressively emerged as a potential methodology to explore temporal and functional brain regions recruitment by antipsychotics and psychotomimetic compounds (Gonzalez-Maeso et al., 2003; Tomasetti et al., 2007; Sakuma et al., 2015). Moreover, IEGs have been found to be modulated also by other psychotropic drugs, such as antidepressants (De Foubert et al., 2004; Alme et al., 2007; Molteni et al., 2008; Calabrese et al., 2011), mood stabilizers (De Bartolomeis et al., 2012), as well as by the combination of antipsychotics and antidepressants or mood stabilizers, which has been demonstrated to differentially induce IEGs patterns of expression as compared to the compounds when individually administered (Dell'aversano et al., 2009; Tomasetti et al., 2011).

#### LITERATURE RESEARCH METHODOLOGY

As a first step, we carried out multiple searches on Pubmed, Scopus, and ISI Web of Knowledge using as a reference the following keywords (we reported in parentheses the results obtained on Pubmed for the search conducted on May 2017): Immediate Early Genes AND brain (2335); Immediate Early Genes AND antipsychotics (84); Immediate early genes AND antipsychotics AND brain (74). Successively, we searched by the name of each single IEG or related gene of interest, together with the keywords "brain" AND/OR "antipsychotics." The name of the IEGs or related genes searched were: Arc/Arg, BDNF, cfos, fos, c-Jun, jun, Egr1, Delta-fos, Narp1, NPAS-4, Homer1, Homer2, Homer3, Nor1, Nurr, Nurr1, NGFI-B/Nur77, Nerve Growth Factor Inducible-B, NR4A.

A "parallel search" was conducted using as key words the combination the following ones: antipsychotics AND acute effects, antipsychotics AND brain volume, antipsychotics AND cortical thickness, antipsychotics AND acute AND PET, antipsychotics AND acute AND fMRI, antipsychotics AND brain changes.

For the above-mentioned first search (keywords: Immediate Early Genes AND brain), each abstract retrieved was considered for coherence of the subject with the content of the review by two independent co-authors. If the text of the abstract was coherent with the review, the full text was considered and the references double-checked for potential new articles of relevance. All the articles retrieved with the second search (Immediate-early genes AND antipsychotics) and the third search (Immediateearly genes AND antipsychotics AND brain), as well as the articles retrieved by the search for single early gene name, were considered for the full text. The results of the first search where then compared with the result of the other searches and with the one of the "parallel search."

### C-*fos*, 1*FosB*, C-*Jun*: MAPPING THE NEURAL ACTIVITY IN RESPONSE TO ANTIPSYCHOTICS, OLD AND NEW FINDINGS

Synaptic plasticity processes occurring in response to neural activity are mediated by complex programs of gene expression controlled by transcription factors (TFs; Beckervordersandforth et al., 2015; Ortega-Martinez, 2015; Ehrlich and Josselyn, 2016).

Antipsychotics have been demonstrated to differentially impact IEGs encoding neural TFs, thus inducing a significant reprogramming in the expression of genes involved in synaptic plasticity (**Figure 2**; **Table 1**).

### *C-fos:* The Prototypical IEG C-fos Regulation by Dopamine

C-fos is a proto-oncogene encoding for a TF that is induced in response to multiple stimuli, included neural activity (Durchdewald et al., 2009). In resting conditions, the product of the c-fos gene, the Fos protein, is expressed in small amounts in the brain.

C-fos transcription may be activated in response to many different extracellular signals, including growth factors and neurotransmitters, such as dopamine. Several early studies demonstrated that transcriptional regulation after dopaminergic stimuli, such as amphetamine/cocaine administration, is a pivotal mechanism by which neurons may respond to environmental adaptations (De Bartolomeis et al., 2013b). The phosphorylation of the cAMP response element binding protein (CREB) is crucial to couple dopamine stimulation to the IEG transcription. Indeed, signals starting at dopamine receptors may promote CREB phosphorylation, which in turn regulates c-fos transcription. When translated, the Fos protein may dimerize with members of Jun family in order to start the formation of the Activator Protein-1 heterocomplex (AP-1), which in turn may trigger the expression of genes involved in cell proliferation and differentiation, as well as in activity-stimulated synaptic rearrangements (Herrera et al., 1990).

Based on its fast induction dynamics, c-fos expression has been widely used to characterize the different topographic patterns of neural activation following treatments with different antipsychotics (Nguyen et al., 1992; Merchant et al., 1994).

#### C-fos in Schizophrenia and Its Modulation by Antipsychotics

Recent human studies have pointed out that specific polymorphisms of c-fos gene may be either negatively or positively associated to schizophrenia, since decreased Fos protein blood levels may be found in schizophrenia patients (Boyajyan et al., 2015), thus reinforcing the possibility of an implication of this IEG in schizophrenia pathophysiology and, possibly, in its treatment.

Early studies observed that typical and atypical antipsychotics may induce different patterns of c-fos activation in cortical and subcortical brain regions. Indeed, typical antipsychotics, such as haloperidol, may induce c-fos expression in the dorso-lateral

regions of the striatum, as well as in the nucleus accumbens and in the lateral septum. Atypical antipsychotics, such as clozapine, were found to induce c-fos expression in prefrontal cortex and medial striatum (Robertson et al., 1994). Since the dorso-lateral striatum has been implicated in motor control (Balleine and O'doherty, 2010), it has been suggested that the liability of an antipsychotic drug to induce extrapyramidal side-effects (EPSEs) might be predicted by its propensity to induce c-fos expression in the motor circuits of the striatum (Robertson and Fibiger, 1996). On the other hand, the induction of c-fos expression in prefrontal cortex and limbic striatum by atypical antipsychotics (e.g., clozapine; Robertson et al., 1994) has been potentially correlated with the ability of these compounds to impact, at least in part, brain circuitry implicated in the pathophysiology of negative symptoms of schizophrenia, based on the hypothesis explaining negative symptoms with a potential hypo-frontality in schizophrenia patients (Weinberger and Berman, 1996). However, this observation should nowadays be discussed with caution, considering recent advances in molecular characterization of old and novel antipsychotics, as well as the latest results on the real effect size of atypical antipsychotics on negative symptoms (Kantrowitz, 2017).

Typical and atypical antipsychotics differentially enhance cfos expression in the two histological compartments of the striatum, striosome, and matrix. Indeed, typical antipsychotics induce c-fos at a similar extent in the striosome and in the matrix, while most atypical antipsychotics preferentially induce c-fos in the striosome (Hiroi and Graybiel, 1996; Bubser and Deutch, 2002). It is noteworthy that no difference in the striosome/matrix ratio (SMR) has been found for typical antipsychotics between dorso-lateral caudate-putamen and dorso-medial caudate-putamen, while clozapine showed a

#### TABLE 1 | Detection of IEGs expression evaluation in rodents after antipsychotics administration.

#### *(Continued)*

#### TABLE 1 | Continued

↑*, Gene expression is up-regulated;* ↓*, Gene expression is down-regulated; NAc, Nucleus accumbens; PFC, Prefrontal cortex, FC Frontal cortex.*

significantly higher SMR in dorso-lateral than in the dorsomedial region of the caudate-putamen (Bubser and Deutch, 2002). Numerous studies have correlated the matrix with motor behavior and stimulus-response memory consolidation, while the striosome has been related to reward mechanisms (White and Hiroi, 1998). Thus, the striosome/matrix architecture of the striatum has been proposed as a morphological substrate for a modular reinforcement-learning model (Amemori et al., 2011). Moreover, it has been suggested that the striosome may be linked to cognition, since it receives prominent inputs from association cortex (Bubser and Deutch, 2002).

Differences in the putative clinical profile of typical and atypical antipsychotics may be inferred by their specific spatial pattern of c-fos induction.

D2Rs blockade by antipsychotics has been demonstrated to relieve the inhibition of adenylyl cyclase and activate the PKA, which in turn is responsible for the phosphorylation of CREB. Phospho-CREB may then interact with the cAMP response element (CRE) site in the promoter region the c-fos gene (Benito and Barco, 2015). Considering that all the antipsychotics, with few exceptions, are multireceptor-binding drugs, it is conceivable that other receptors beyond D2Rs could be responsible and/or contribute to c-fos activation.

 In fact, although antipsychotics show specific topographical patterns of c-fos induction according to their typical/atypical characteristics, the expression of c-fos in neurons has been described to be coupled to multiple different extracellular stimuli, hence it is difficult to attribute a specific c-fos "fingerprint profile" to each antipsychotic compound. C-fos induction may help to detect where and when a brain area is activated by a certain compound, but it gives little information on which is the specific intracellular pathway stimulated by this compound. Thus, the data obtained by c-fos induction in response to antipsychotics need to be integrated with by other IEGs induction with a more direct function in the synapse.

### 1*FosB:* An IEG with a Dual Function

1FosB is a splicing variant of the FosB gene, a member of the FRA family (Fos Related Antigens). Depending on its expression kinetics, the role of 1FosB can be either of transcription activator or repressor, with lower levels leading to short-term gene repression and higher levels leading to long-term gene activation (Nestler, 2015).

Among the main target genes of 1FosB there are: metabotropic Glutamate Receptors subtype 2 (mGluR2), Dynorphin, the nuclear factor kB (NFkB) and c-fos.

1FosB gene is rapidly induced in the dorsal striatum and nucleus accumbens dynorphin-expressing Medium-sized Spiny Neurons (MSNs)—two neural populations closely involved into reward and addiction—in response to addictive drugs such as cocaine and amphetamines (Maze and Russo, 2010). 1FosB induction is reported to not undergo tolerance, thus its accumulation in those brain regions is quite stable after repeated administrations. Moreover, unlike other IEGs, 1FosB levels in striatum and nucleus accumbens are quite stable across the time, and remain up-regulated for weeks after the initial stimulus (Nestler, 2005).

#### 1FosB Modulation by Antipsychotics

1FosB expression has been also studied in response to antipsychotic drugs. Indeed, chronic treatment with haloperidol may enhance the expression of 1FosB in the ventral, medial, and dorso-lateral aspects of the striatum (Rodriguez et al., 2001). Clozapine, on the other hand, may induce 1FosB-like immunoreactivity not only in the ventral striatum but also in the prefrontal cortex and lateral septum, with a weaker impact on dorso-lateral striatum, whereas risperidone and olanzapine only weakly induce 1FosB in striatum (Vahid-Ansari et al., 1996; Atkins et al., 1999). Given the long-term standing of 1FosB activation in response to dopaminergic drugs, this IEG has been proposed as a specific marker to identify neurons undergoing prolonged activation in chronic paradigms (Dietz et al., 2014).

#### *C-Jun*

#### C-Jun Regulation and Schizophrenia Preclinical Modeling

C-Jun is a TF that dimerizes with Fos family members to form the AP-1 complex. As an IEG, c-Jun plays a pivotal role in neuronal apoptosis and neurons survival (Jochum et al., 2001). Various extracellular stimuli may activate the JNK (c-jun kinase)/C-Jun cascade, including stress, ischemia, and stroke, seizures, learning and memory, axonal injury (Raivich and Behrens, 2006). Interestingly, a potential indirect role for c-Jun in a preclinical model of schizophrenia pathophysiology has been recently highlighted by investigating attentive function in mice haploinsufficient for Map2k7 (Map2k7+/− mice). Specifically, Map2k7 encodes for MKK7 (MAP kinase kinase 7), which is responsible for the activation of JNK. The reduction of Map2k7 function has been found to be associated to cognitive deficits in mice (Openshaw et al., 2017).

Moreover, putative links between c-Jun and schizophrenia are suggested in preclinical models by several findings showing that both psychotomimetic and antipsychotic drugs modulate c-Jun levels in brain regions implicated in schizophrenia. Indeed, c-Jun expression is affected by Nmethyl-D-aspartate (NMDA) receptor antagonists such as MK-801 (Gerlach et al., 2002) mimicking a preclinical model of schizophrenia.

#### C-Jun Modulation by Antipsychotics

C-Jun expression has been demonstrated to be modulated by antipsychotics. Chronic treatment with clozapine or haloperidol induces long-lasting c-Jun expression in the rat forebrain and basal ganglia even after a washout period (Kontkanen et al., 2002). A recent proteomic quantification analysis has demonstrated that chronic haloperidol administration in rodents may modulate the expression of 216 proteins in hippocampus, including c-Jun N-terminal kinase signaling (Schubert et al., 2016). In contrast, acute haloperidol or clozapine treatment have been shown to exert no effects on c-Jun expression, although both these treatments produce clear changes in the expression of several other IEGs, including c-fos and other Jun-family members (MacGibbon et al., 1994). These data suggest that antipsychotic drugs may play different roles in modulating apoptosis-related molecules, and are coherent with the findings suggesting that typical and atypical antipsychotics may differentially affect putative neuroprotection (Jarskog, 2006; Nandra and Agius, 2012).

Thus, taken together, the findings reviewed until now may suggest that:

### NUCLEAR RECEPTORS (NUR) SUPERFAMILY: ANTIPSYCHOTICS MODULATION OF DOPAMINERGIC NEURODEVELOPMENTAL FACTORS

### The Nur Superfamily: Role in Dopamine System Development and Modulation by Antipsychotics

Nurr1, NGFI-B/Nur77 (Nerve Growth Factor Inducible-B) and Nor1 (neuron-derived orphan receptor-1) are members of the NR4A (Nuclear Receptors 4A) subgroup of nuclear orphan receptors superfamily, which includes a wide variety of TFs, such as retinoid hormone receptor, steroid, and thyroid hormone receptor (Law et al., 1992; Maxwell and Muscat, 2006). All the three Nur members share overlapping sequences and play essential roles in the development of the dopaminergic system. The three members of NR4A subgroup have been described to respond to several physiological and physical stimuli, such as prostaglandins, stress, hormones, neurotransmitters, membrane depolarization, and magnetic fields (Katagiri et al., 1997; Tetradis et al., 2001; Kagaya et al., 2005) in an IEG-like fashion. Thus, the immediate-early response of NR4A genes to environmental stimuli is an essential feature of these nuclear receptors, which has been extensively studied with regard to their correlation with dopaminergic system (Campos-Melo et al., 2013).

Nurr1 is mainly expressed in the central nervous system, especially in midbrain dopaminergic neurons of the substantia nigra and the ventral-tegmental area (VTA; Backman et al., 1999). Several studies have reported that Nurr1 plays an essential role in the development and differentiation of dopaminergic neurons of the midbrain. Given its role in dopamine neurons development, Nurr1 has been implicated in neuropsychiatric disorders in which dopamine system is dysfunctional, such as schizophrenia. Recent studies reported that Nurr1 gene may be considered as a possible candidate to explore the dysfunctional gene-environment interaction that is considered to be at the basis of these disorders. Indeed, two missense mutations in the gene have been found in schizophrenia patients (Buervenich et al., 2000) and may be directly related to their impaired cognitive performances (Ancin et al., 2013).

Heterozygous deletion of Nurr1 gene in mice has been recently considered as a possible animal model of schizophrenia, since these animal display elevated dopamine levels in basal ganglia (Moore et al., 2008), and characteristic dysfunctional behaviors resembling psychotic symptoms in humans (Rojas et al., 2007).

Similarly, also Nur77 has been implicated in the pathophysiology of schizophrenia. Indeed, reduced levels of Nur77 have been detected in prefrontal cortex of post-mortem schizophrenia patients (Xing et al., 2006). Moreover, single nucleotide polymorphisms of Nur77 gene have been associated with elevated risk of tardive dyskinesia (TD) in schizophrenia patients (Novak et al., 2010).

Given the direct correlation between Nur IEGs and the development of dopaminergic system, a large body of evidence has been set up on the regulation of NR4A IEGs in response to dopamine manipulation.

#### *Nur* IEGs Modulation by Antipsychotics

The first studies with antipsychotics demonstrated that Nur IEGs response to these drugs may resemble that of c-fos in rat brain. For instance, typical and atypical antipsychotics induce differential patterns of Nur IEGs expression. Indeed, acute haloperidol administration pronouncedly increases Nur77 expression in dorso-lateral striatum, whereas clozapine induces this gene preferentially in prefrontal cortex and in the shell of the nucleus accumbens (Beaudry et al., 2000). Moreover, haloperidol selectively increases Nur77 dorsolateral striatal expression in enkephalin-containing neurons, which are MSN neurons mostly expressing D2Rs, whose up-regulation has been correlated to extrapyramidal symptoms induced by neuroleptics. In addition, the same report showed that chronic haloperidol administration provokes a further increase in Nur77 dorso-lateral striatal expression, whereas chronic clozapine reduces Nur77 gene expression below basal values in prefrontal and accumbal areas. To confirm the role of Nur77 in acute neuroleptic-induced EPSEs, later studies demonstrated that in Nur77-deficient mice haloperidol-induced acute catalepsy was completely abolished, as well as the Nur77 mRNA overexpression in enkephalinpositive neurons (Ethier et al., 2004). Therefore, similarly to c-fos expression, Nur IEGs modulation by antipsychotics may be used as a tool to dissect the propensity of a neuroleptic drug to induce extrapyramidal side effects.

Some other significant similarities with c-fos have been shown in the regulation of NR4A members expression by antipsychotics. Indeed, Maheux and coworkers demonstrated that Nur IEGs may be induced by typical antipsychotics selectively in striatal areas that control motor functions, whereas atypical antipsychotics induced Nur IEGs expression in limbic areas (Maheux et al., 2005). This induction pattern tightly correlates with D2Rs affinity by each antipsychotic in striatum and with D2/D3Rs affinity in the nucleus accumbens. The same research group further demonstrated that selective serotonergic and adrenergic drugs may modulate haloperidol-induced Nur IEGs expression, suggesting that also serotonin neurotransmission may take part into the differential patterns of regulation of these genes by typical and atypical antipsychotics (Maheux et al., 2012).

There are substantial differences in response to antipsychotics between the different members of Nur family: both Nur77 and Nor1 are de novo induced in dopamine neurons and striatal areas, whereas Nurr1 is basally expressed in VTA and substantia nigra and its expression is enhanced by antipsychotics (Eells et al., 2012).

Regarding the mechanisms involved in Nur regulation by antipsychotics drugs and D2Rs antagonists, it has been demonstrated that, at least for Nur-77 and Nor-1, the induction/increase in mRNA are depending by both mitogenassociated and extracellular signal-regulated kinases (MEK) and Protein Kinase C (PKC) in the case of Nurr-77 and by PKC only in the case of Nor-1 (Bourhis et al., 2008).

Hence, Nur IEGs modulation by antipsychotics appears to provide a complementary information as compared to c-fos expression patterns, thereby contributing to shed further light on the impact of these drugs not only on brain areas that are targeted by dopamine neurons, but also on areas in which dopamine neurons localize. Thus, although still elusive, the analysis of Nur IEGs modulation by antipsychotic drugs may be a further tool to dissect the mechanisms of action of these compounds on dopamine systems, as well as it may help to further clarify the molecular mechanisms by which these drugs alter locomotor activity in animal models and in humans.

### MODULATION BY ANTIPSYCHOTICS OF IEGs INVOLVED IN SYNAPTIC PLASTICITY: PUTATIVE TARGETS FOR COGNITIVE DEFICITS IN PSYCHOSIS

Abnormal synaptic plasticity may account for several cognitive and behavioral processes that are dysfunctional in schizophrenia. Cognitive impairment, indeed, is a striking clinical aspect of psychotic illnesses, is detectable before the onset of other symptoms, and it is considered among the best predictors of long-term lifetime functioning (Green, 1996).

Since long-term neural plasticity requires protein synthesis, IEGs expression could be considered as a necessary step in synaptic architecture remodeling. For several IEGs involved in these processes, a role in the pathophysiology of psychosis has been proposed. In the following section, we will summarize how antipsychotic therapy may impact these genes expression.

### *Egr1*: How Antipsychotics Impact Synaptic Processes Underpinning Memory and Learning

#### Egr1 Regulation and Involvement in Synaptic Plasticity

The Egr-family consists of four highly homologous zinc-finger TFs: Egr1 (Early growth response gene 1, also named NGFI-A, zif-268, Krox 24), Egr2 (Krox 20), Egr3 (PILOT), and Egr4 (NGFI-C).

Egr1 is an immediate-early gene (IEG) coding for a TF and is constitutively expressed in the cortex, amygdala, striatum, nucleus accumbens, hippocampus, and cerebellum (Beckmann and Wilce, 1997). Several stimuli have been demonstrated to induce Egr1 overexpression in these areas, such as seizures, ischemia, stress, and drug administration (Hughes and Dragunow, 1995). Many of these stimuli share the common feature of elevating intracellular calcium (Ca2+; Ghosh et al., 1994). Pharmacological stimuli provoking massive Ca2+ influx in neurons have been described to increase Egr1 expression (Shirayama et al., 1999; Zhou et al., 2009; Gangarossa et al., 2011). Egr1 activity has been related to the transcription of other IEGs involved in synaptic plasticity, above of all Arc (Penke et al., 2011). In turn, Egr1 expression may be under the control of other genes, such as the Brain-Derived Neurotrophic Factor (BDNF; Robinet and Pellerin, 2011) and other proteins involved in intracellular signaling (Lam et al., 2009).

Egr1 synaptic action has been related to neural plasticity, in particular to synaptic processes leading to memory consolidation and behavioral adaptations (Davis et al., 2003; Okada et al., 2015) Intriguingly, dysfunctions in the postsynaptic machinery deputed to control memory and learning processes have been recently related to cognitive impairment in major neuropsychiatric disorders, such as schizophrenia (Grant, 2012). In paradigms of instrumental learning, Egr1 is markedly induced in frontal and cingulate cortices (Hernandez et al., 2006; Snyder et al., 2012). Furthermore, Egr1 plays a pivotal role in maintaining the late phase of LTP in hippocampus, in dorsal caudate-putamen (Gill et al., 2007), and in the retrosplenial cortex (Amin et al., 2006). Egr1, along with BDNF, also appears to play a role in retrieval-dependent plasticity, a mechanism accounting for the modification of previously consolidated memories being recalled (Lee, 2010). Moreover, it has been shown that Egr1, together with BDNF (Barnes et al., 2012), Homer1a and Arc, has a relevant role in the mechanisms of the initial consolidation, reconsolidation and extinction of fear- and anxiety-related memory (Lonergan et al., 2010; Maddox et al., 2011; Cheval et al., 2012).

Dopamine and glutamate systems have been implicated in synaptic processes involved in memory consolidation [e.g., longterm Potentiation (LTP) and long-term depression (LTD)], and several studies reported that Egr1 expression may be modulated by stimuli affecting either glutamate or dopamine neurotransmission (Li et al., 2016).

#### Egr1 Modulation by Antipsychotics

Several antipsychotics (e.g., clozapine) modulate synaptic proteins related to memory formation in hippocampus and improve cognitive tasks in animal models of pharmacological NMDA receptor hypofunction (Ozdemir et al., 2012). With regard to dopamine, Egr1 gene and protein expression are modulated by both acute and chronic antipsychotic treatments in preclinical settings (Wheeler et al., 2014; De Bartolomeis et al., 2015a).

Clinical studies have reported an abnormal regulation of Egr1 in schizophrenia patients compared to normal controls. Specifically, post-mortem gene expression studies and in vivo plasma detection have demonstrated that Egr1 is downregulated in prefrontal cortex of schizophrenia patients in a fashion that is directly correlated with decrease in GAD1 (glutamate decarboxylase 1, the enzyme that is responsible for GABA production), and plasma levels were reduced, therefore supporting the view that Egr1 may be a potential biomarker of the disease (Kimoto et al., 2014). Interestingly, an association of SNPs in Egr3 and Arc with schizophrenia has been proposed as a biological pathway of environmentally responsive, synaptic plasticity-related, schizophrenia risk genes (Huentelman et al., 2015).

Given the potential role of this IEG in schizophrenia pathophysiology and treatment, it is to mention that early studies have also compared Egr1 modulation by antipsychotics to the modulation of the other well-known IEG c-fos, showing that these two IEGs, although similarly impacted by typical and atypical antipsychotics in cortex and striatum, displayed some substantial differences. Haloperidol has been reported to induce both cfos and Egr1 expression in striatum, whereas clozapine may induce Egr1 but not c-fos expression in the same region. Both antipsychotics may induce the expression of both these genes in nucleus accumbens (Nguyen et al., 1992; MacGibbon et al., 1994).. Subsequent findings further demonstrated a differential response of Egr1 from c-fos also in chronic antipsychotics administration paradigms. Egr1, indeed, is robustly downregulated in locus coeruleus and prefrontal cortex of olanzapine chronically administered rats, whereas c-fos expression remains up-regulated (Verma et al., 2007). Moreover, chronic haloperidol may increase cortical Egr1 expression, whereas it decreases c-fos expression in this area (Verma et al., 2007).

Targeted experiments have demonstrated that Egr1 modulation by antipsychotic drugs may be directly related to the synaptic functions of drug-associated memory consolidation. For instance, high D2R-blocking antipsychotics (e.g., sulpiride) may prevent the increase in Egr1 expression induced by acute cocaine administration in striatum, but not in the cortex (Daunais and McGinty, 1996). The modulation of Egr1 and other activity-regulated genes such as Arc and Npas4 has been studied in rodents after the administration of the novel antipsychotic lurasidone, which is characterized by a multi-receptor profile and particularly by a potent 5-HT7 receptor antagonism, considered beneficial for mood and cognition (Luoni et al., 2014b).

Finally, Egr1 levels of expression have been recently investigated in preclinical settings exploring new therapeutic strategies in schizophrenia beyond current antipsychotic drugs (Gentzel et al., 2015).

However, it should be considered that the dopaminergic regulation of Erg1 is even more complicated by the action of multiple pathways in reciprocal interplay with dopaminergic system. Recent evidence, indeed, demonstrated that mu-opioid receptors have also a major implication in psychostimulantinduced sensitization (Shen et al., 2010) and antipsychotic drugsseem unable to prevent methamphetamine-induced striatal overexpression of Egr1 in mu-opioid receptors knock-out animals (Tien et al., 2010).

Putting together the findings reviewed therein, the role of Egr1 in antipsychotics action appears relevant mainly because it is directly implicated in specific signaling and neuronal plasticity programs related to memory, cognition and executivelike functions in preclinical models. This is a major point, since there is a need to explore new compounds in schizophrenia that may address dysfunctions in the domains of cognitive and negative symptoms, which are poorly affected by currently available antipsychotic agents.

### *Arc/Arg3.1*: Modulation of Long-Term Activity-Dependent Synaptic Efficacy by Antipsychotic Treatments

#### Arc Regulation and Involvement in Synaptic Plasticity: Relevance for Schizophrenia

As other IEGs, Arc (activity-regulated cytoskeletal-associated protein) also referred to as Arg3.1 (activity-regulated gene homolog 3.1), is expressed at low levels in neurons, especially in the hippocampus. However, Arc levels are relatively higher in cortex, and are directly linked to NMDA receptor activation (Link et al., 1995). Arc shows unique features, since its mRNA may be induced together with other IEGs by neural activation (i.e., single seizures), but differently from other mRNAs—such as Egr1—that remain in the neuron soma, Arcis rapidly translocated to dendritic spines (Wallace et al., 1998). The activity-dependent translocation of Arcrequires NMDA receptor activation (Steward and Worley, 2001). Moreover, Arc protein is selectively produced in dendritic spines near the activation site, even in the presence of protein-synthesis inhibitors, thereby indicating that Arc mRNA owns a unique intrinsic signal that permits the activityrelated targeting to stimulated dendrites (Bramham et al., 2010; Steward et al., 2014). The activity-dependent regulation of Arc expression has been extensively studied, thus leading to several findings reporting its involvement in synaptic plasticity and its implication in memory and learning processes, which have been demonstrated to be altered in schizophrenia. Experiencerelated stimuli may potently increase Arc expression in brain areas involved in memory consolidation (Lyford et al., 1995). Indeed, the exploration of new environments strongly induces Arc expression in hippocampus and cortex (Vazdarjanova et al., 2002). Consistently, Arc knock-out animals fail to form longterm memories for learning tasks, displaying impaired LTP and LTD, but unaltered short-term memory (Plath et al., 2006). A role for Arc in synaptic scaling, a form of homeostatic synaptic plasticity, has been postulated (Gao et al., 2010).

Hence, Arc functions seem to be required for synaptic plasticity processes starting at multiple neurotransmitter receptors, such as alpha-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid (AMPA), NMDA, dopamine, serotonin, acetylcholine, and adrenaline receptors (Chowdhury et al., 2006; Rial Verde et al., 2006).

Dopamine and glutamate are considered among the principal neurotransmitters implicated in memory and learning processes, as well as in schizophrenia pathophysiology in which aberrant salience has been reported. Dopamine has been demonstrated to be pivotal for working memory in rodents and in nonhuman primates (Castner et al., 2004; Rinaldi et al., 2007). Arc response to memory and learning stimuli may account for a direct involvement of this IEG into dopaminergic-dependent mechanisms of memory consolidation, as well as for its implication in dopamine-glutamate subcellular interactions that control synaptic plasticity processes thought to be dysfunctional in the pathophysiology of schizophrenia (Eastwood, 2004; Grant, 2012; Yin et al., 2012). To confirm the role of Arc in synaptic processes of dopamine-dependent memory formation, it has been demonstrated that amphetamines may modulate Arc learning-induced expression in hippocampus (Wiig et al., 2009). Bloomer and coworkers have reported a combined effect of NMDA receptors and D1Rs on Arc expression in hippocampal neurons (Bloomer et al., 2008), as well as a dramatic reduction in Arc expression by dopamine agonists when NMDA receptor blockers are concurrently administrated, thus confirming the role of dopamine-glutamate correct interaction in memory consolidation and the crucial functions of Arc in this process. Arc gene has been reported to be affected by de novo mutations in schizophrenia patients and it is part of an enriched gene set characterized by rare disruptive mutations contributing to the genetic risk for schizophrenia (Fromer et al., 2014; Purcell et al., 2014). Further evidence of an implication of Arc in schizophrenia pathophysiology comes from preclinical studies (Manago and Papaleo, 2017). Disruption of Arc produces deficits in sensorimotor gating, cognitive functions, social behaviors, and amphetamine-induced psychomotor responses in mice that are reminiscent of some features of psychosis (Manago et al., 2016).

#### Arc Modulation by Antipsychotics

Arc expression in response to antipsychotic challenges has been extensively investigated, in order to shed further light on the molecular mechanisms involved in antipsychotic-mediated modulation of the synaptic plasticity processes putatively disrupted in schizophrenia.

Early works demonstrated that Phencyclidine (PCP)-induced Arc overexpression in prefrontal cortex and nucleus accumbens may be inhibited by pretreatment with clozapine, olanzapine, and risperidone, but not by haloperidol (Nakahara et al., 2000), thus suggesting that Arc modulation may be useful to dissect typical from atypical antipsychotics impact on psychotomimetic drug-induced synaptic dysfunctions. Subsequently, acute administration of both typical and atypical antipsychotics was demonstrated to induce Arc gene expression in striatum, with haloperidol showing a more prolonged effect on Arc induction than olanzapine (Fumagalli et al., 2009). These results have been directly correlated to the degree of D2R blockade induced by each antipsychotic, since a selective D2Rs antagonist, such as raclopride, may induce Arc striatal expression and reduce its cortical expression, whereas a selective D2Rs agonist, such as quinpirole, may reduce Arc striatal expression and has no effects on its cortical expression. Notably, Fumagalli et al. (2009) further demonstrated that a prolonged treatment with haloperidol and olanzapine markedly reduced Arc striatal expression, as well as only olanzapine may reduce the expression of the gene in the cortex.

With regard to cortical and subcortical expression of Arc, work from our laboratory demonstrated that Arc gene expression may be induced by haloperidol, but not by sertindole in the striatum, thus further suggesting that Arc modulation may be tightly related to the tuning of dopamine neurotransmission exerted by each antipsychotic (Iasevoli et al., 2010b). Sertindole shows a milder D2Rs impact than haloperidol in the striatum, with a quite absent blockade of D2 autoreceptors (Valenti and Grace, 2010). The involvement of serotonin 5-HT2A receptors has been demonstrated in Arc cortical modulation, with NMDA receptor function being relevant in these effects, thereby suggesting a crucial role of Arc in synaptic rearrangements induced by combined serotonin-dopamine-glutamate stimuli in the cortex by antipsychotics (Pei et al., 2004).

Arc has been shown to be responsive to acute (with significant increase of the transcript in striatum) and chronic (with prevalent gene expression increases in prefrontal cortex and hippocampus and decreases in striatum) lurasidone treatment, suggesting a region-specific fingerprint of Arc induction (Luoni et al., 2014b). An intra-striatal specificity of Arc activation was detected after acute administration of amisulpride (35 mg/kg) with prevalent increase of the transcript in the medial caudateputamen compared to more pronounced induction in dorsal caudate-putamen by haloperidol (0.8 mg/kg; De Bartolomeis et al., 2013b). Again, a region-specific induction of Arc protein was detected in the shell of the nucleus accumbens by clozapine (20 mg/kg) compared to haloperidol (1 mg/kg; Collins et al., 2014). Finally, Arc has been instrumental also for exploring the brain region effect of innovative treatment approaches in schizophrenia, such as augmentation strategies to antipsychotics (i.e., minocycline in combination with haloperidol; Buonaguro et al., 2017b).

Direct evidence exist that Arc induction can be responsible for increasing the density and for reducing the width of dendritic spine possibly by a mechanism involving AMPA endocytosis (Peebles et al., 2010).

These data globally suggest that Arc is involved in the neural plasticity mechanisms induced by antipsychotics in distinct brain regions. Arc modulation may be demonstrated to occur before the timing necessary to observe the therapeutic-like effects commonly observed during antipsychotic therapies, possibly suggesting that Arc is potentially relevant in establishing the correct synaptic rearrangements underlying antipsychotic effects. Finally, the recent discovery of Arc subdomains similar to the domain of HIV capsid and its involvement in rapid synaptic functions (possibly derived from the ancestral viral origin) deranged in schizophrenia (Zhang et al., 2015), make this IEG of relevant interest for studying specifically "fast" synaptic changes during antipsychotic treatment.

#### *Homer1a*: IEG-Mediated Activity-Dependent Postsynaptic and Architecture Rearrangements in Response to Antipsychotic Treatment The Homer Family and Its Regulation by Dopamine-Glutamate Interaction