To express optogenetic or fluorescent proteins in glutamatergic neurons, adeno-associated virus (AAV) vectors were produced by and purchased from the University of Pennsylvania vector core (Philadelphia, PA) and injected into *CaMKIIa-Cre* mice. In this work we used AAVs for the *Cre*-inducible expression of the excitatory optogenetic probe *Channelrho dopsin* (AAV2/9.EF1a.DIO.hChR2(H134R)-EYFP.WPRE.hGH; Addgene #20298), inhibitory optogenetic probe *Archaerhodopsin* (AAV2/9.flex.CBA.Arch-GFP.W.SV40; Addgene #22222), fluorescent protein *tdTomato* (AAV2/1.CAG.FLEX.tdTomato.WPRE. bGH; Allen Institute #864) and *GFP* tagged Synaptophysin (AAV2/9.CMV.FLEX.Synaptophysin-Venus.WPRE.hGH; plasmid kindly provided by Anton Maximov, PhD, Department of Molecular and Cellular Neuroscience, The Scripps Research Institute). For stimulation of excitatory vmPFC terminals in the DRN of *GAD2-tdTomato* or *Pet1-tdTomato* mice we used an AAV for the *CaMKIIa-*driven expression of *Channelrhodopsin* fused to *YFP* (AAV2/9.CaMKII.ChR2-YFP.SV40; Stanford) (Mattis et al., 2012).

For viral injections, mice were anesthetized with isofluorane and stereotaxically injected unilaterally in the prelimbic region of the vmPFC (from Bregma, in mm: +1.8 AP, +0.8 ML, −2.7 DV, 15◦ angle) with 0.5μl of virus. Viral yields (in GC) were 3*.*54 × 1012 for *ChR2-YFP,* 6*.*962 × 1011 for *Arch-GFP*, 2*.*049 × 1012 for *tdTomato* and 4*.*347 × 1012 for *CaMKIIa-ChR2-YFP*. Social defeat began 4 weeks post-surgery for non-cannulated mice to allow time for recovery and viral expression.

For *in vivo* optical stimulation, precut guide cannulae (Plastics One, Roanoke, VA) targeting the DRN (from Lambda, in mm: 0.0 AP, +0.8 ML, −3.3 DV, 15◦ angle) were secured to the skull using stainless steel skull screws and acrylic cement. A fitted dustcap dummy was secured atop the guide cannula and mice were placed

# Optogenetic modulation of descending prefrontocortical inputs to the dorsal raphe bidirectionally bias socioaffective choices after social defeat

# *Collin Challis 1,2, Sheryl G. Beck2,3 and Olivier Berton1,2\**

*1 Department of Psychiatry, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA*

*2 Neuroscience Graduate Group, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA*

*3 Department of Anesthesiology, Children's Hospital of Philadelphia and University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA*

#### *Edited by:*

*Mary K. Lobo, University of Maryland School of Medicine, USA*

#### *Reviewed by:*

*Daniel W. Wesson, Case Western Reserve University, USA Xiao-Dong Wang, Zhejiang University, China*

#### *\*Correspondence:*

*Olivier Berton, Department of Psychiatry, Center for Neurobiology and Behavior, University of Pennsylvania Perelman School of Medicine, 125 S. 31st Street, Philadelphia, PA 19104, USA e-mail: bertonol@ mail.med.upenn.edu*

It has been well established that modulating serotonin (5-HT) levels in humans and animals affects perception and response to social threats, however the circuit mechanisms that control 5-HT output during social interaction are not well understood. A better understanding of these systems could provide groundwork for more precise and efficient therapeutic interventions. Here we examined the organization and plasticity of microcircuits implicated in top-down control of 5-HT neurons in the dorsal raphe nucleus (DRN) by excitatory inputs from the ventromedial prefrontal cortex (vmPFC) and their role in social approach-avoidance decisions. We did this in the context of a social defeat model that induces a long lasting form of social aversion that is reversible by antidepressants. We first used viral tracing and *Cre*-dependent genetic identification of vmPFC glutamatergic synapses in the DRN to determine their topographic distribution in relation to 5-HT and GABAergic subregions and found that excitatory vmPFC projections primarily localized to GABA-rich areas of the DRN. We then used optogenetics in combination with *cFos* mapping and slice electrophysiology to establish the functional effects of repeatedly driving vmPFC inputs in DRN. We provide the first direct evidence that vmPFC axons drive synaptic activity and immediate early gene expression in genetically identified DRN GABA neurons through an AMPA receptor-dependent mechanism. In contrast, we did not detect vmPFC-driven synaptic activity in 5-HT neurons and *cFos* induction in 5-HT neurons was limited. Finally we show that optogenetically increasing or decreasing excitatory vmPFC input to the DRN during sensory exposure to an aggressor's cues enhances or diminishes avoidance bias, respectively. These results clarify the functional organization of vmPFC-DRN pathways and identify GABAergic neurons as a key cellular element filtering top-down vmPFC influences on affect-regulating 5-HT output.

**Keywords: dorsal raphe, ventromedial prefrontal cortex, serotonin, optogenetics, electrophysiology, depression and anxiety disorders, social perception, social defeat**

# **INTRODUCTION**

The capacity to detect and interpret the affective state of others using non-verbal social cues (e.g., facial expression, vocal prosody, posture, body movement, and olfactory cues) is a necessary survival skill shared by many animal species (Chang et al., 2013; Oliveira, 2013). It allows individuals to anticipate harmful intentions of others and adapt through rapid approach or avoidance decisions (O'Connell and Hofmann, 2012). The capacity to conduct social-cognitive appraisal is also a determining aspect of human social competence (Todorov, 2008; Volman et al., 2011) and dysfunction of the neural systems that mediate socioaffective decisions are thought to contribute to excessive reassurance-seeking behaviors and social withdrawal, which are two symptomatic dimensions shared across several affective disorders, including major depression, and social phobia (Heuer et al., 2007; Seidel et al., 2010; Derntl et al., 2011; Stuhrmann et al., 2011; Cusi et al., 2012; Moser et al., 2012).

Serotonin (5-HT) is a neurotransmitter system that plays an evolutionarily conserved role in regulating affiliative and antagonistic behaviors (Canli and Lesch, 2007; Dayan and Huys, 2009; Rogers, 2011). Increases in 5-HT output, such as resulting from treatment with SSRI antidepressants, have consistently been shown to positively bias socioaffective appraisals and facilitate social affiliation and dominance in human and animals (Raleigh et al., 1991; Knutson et al., 1998; Tse and Bond, 2002; Bond, 2005; Harmer and Cowen, 2013). In contrast, 5-HT depletion facilitates socially defensive behaviors and aggression (Young and Leyton, 2002; Munafo et al., 2006). The fact that the output of ascending 5-HT neurons located in the dorsal raphe nucleus (DRN) is under top-down control by multiple forebrain areas (Peyron et al., 1998; Freedman et al., 2000; Chiba et al., 2001; Celada et al., 2002; Lee et al., 2003; Vertes, 2004) suggests a potentially key role for DRN afferent systems in the modulation of socioaffective responses. Studies conducted *in vivo* in anesthetized rodents combining electrical stimulation of the ventromedial prefrontal cortex (vmPFC) and extracellular recordings in the DRN demonstrated the rapid inhibition of putative 5- HT neurons (Varga et al., 2001; Celada et al., 2002). Parallel

dorsal raphe nucleus using juxtacellular labelling methods. *Eur. J. Neurosci.* 25, 119–126. doi: 10.1111/j.1460-9568.2006.05276.x

prefrontal-amygdala connectivity while viewing facial signals of aggression. *Biol. Psychiatry* 71, 36–43. doi: 10.1016/j.biopsych.2011.07.033

**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.

*Received: 09 December 2013; paper pending published: 03 January 2014; accepted: 29 January 2014; published online: 17 February 2014.*

*Citation: Challis C, Beck SG and Berton O (2014) Optogenetic modulation of descending prefrontocortical inputs to the dorsal raphe bidirectionally bias socioaffective choices after social defeat. Front. Behav. Neurosci. 8:43. doi: 10.3389/fnbeh.2014.00043 This article was submitted to the journal Frontiers in Behavioral Neuroscience.*

*Copyright © 2014 Challis, Beck and Berton. 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.*

# Optogenetics reveals a role for accumbal medium spiny neurons expressing dopamine D2 receptors in cocaine-induced behavioral sensitization

**Shelly Sooyun Song1† , Byeong Jun Kang1† , Lei Wen2,3,4† , Hyo Jin Lee1 , Hye-ri Sim1 , Tae Hyong Kim1 , Sehyoun Yoon1 , Bong-June Yoon1 , George J. Augustine2,3,4 and Ja-Hyun Baik1\***

1 Department of Life Sciences, Molecular Neurobiology Laboratory, School of Life Sciences and Biotechnology, Korea University, Seoul, South Korea

2 Center for Functional Connectomics (CFC), KIST, Seoul, South Korea

3 Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore

4 Institute of Molecular and Cell Biology, Singapore, Singapore

#### **Edited by:**

Anton Ilango, Leibniz Institute for Neurobiology, Germany

#### **Reviewed by:**

Simona Cabib, Università "Sapienza", Roma, Italy Vincent Pascoli, Geneva University, Switzerland

#### **\*Correspondence:**

Ja-Hyun Baik, Department of Life Sciences, Molecular Neurobiology Laboratory, School of Life Sciences and Biotechnology, Korea University, Nokji Bldg, Rm 402, Anam-dong, Sungbuk-gu, Seoul, 136-713, South Korea e-mail<EMAIL_ADDRESS>†These authors have contributed

equally to this work.

Long-lasting, drug-induced adaptations within the nucleus accumbens (NAc) have been proposed to contribute to drug-mediated addictive behaviors. Here we have used an optogenetic approach to examine the role of NAc medium spiny neurons (MSNs) expressing dopamine D2 receptors (D2Rs) in cocaine-induced behavioral sensitization. Adeno-associated viral vectors encoding channelrhodopsin-2 (ChR2) were delivered into the NAc of D2R-Cre transgenic mice. This allowed us to selectively photostimulate D2R-MSNs in NAc. D2R-MSNs form local inhibitory circuits, because photostimulation of D2R-MSN evoked inhibitory postsynaptic currents (IPSCs) in neighboring MSNs. Photostimulation of NAc D2R-MSN in vivo affected neither the initiation nor the expression of cocaine-induced behavioral sensitization. However, photostimulation during the drug withdrawal period attenuated expression of cocaine-induced behavioral sensitization. These results show that D2R-MSNs of NAc play a key role in withdrawal-induced plasticity and may contribute to relapse after cessation of drug abuse.

**Keywords: optogenetics, medium spiny neurons, dopamine D2 receptors, cocaine, drug addiction**

#### **INTRODUCTION**

Dopamine (DA) signaling is associated with reward expectation and goal-directed behavior (Wise, 2004; Goto and Grace, 2005; Berridge, 2007). One of the well-known pathologies of dopaminergic disorders is drug addiction (Robinson and Berridge, 1993, 2003). Following repeated exposure to addictive substances, adaptive changes occur at the molecular and cellular level in the DA mesolimbic pathway; these can lead to drug dependence, which is a chronic, relapsing disorder in which compulsive drug-seeking and drug-taking behaviors persist despite their serious negative consequences (Thomas et al., 2008; Baik, 2013). Characterization of the modifications that take place in the mesolimbic dopaminergic system is thus key to understanding drug addiction.

Dopamine D1 receptors (D1R) and D2 receptors (D2R) are highly expressed in the medium spiny neurons (MSNs) of the striatum. It has been suggested that long-lasting drug-induced adaptations in the ventral striatum, better known as the nucleus accumbens (NAc), contribute to the development of addiction as well as drug-seeking and relapse behaviors (Lobo and Nestler, 2011; Smith et al., 2013). Dopaminergic cell bodies from the ventral tegmental area mostly innervate the NAc. Over 95% of the cells within the NAc are MSNs, which receive excitatory inputs from four major brain regions: the prefrontal cortex, the ventral subiculum of the hippocampus, the basolateral amygdala, and the thalamus (Sesack and Grace, 2010; Lüscher and Malenka, 2011). MSNs within the NAc can be divided into two major subpopulations: direct pathway MSNs that express D1Rs and project directly to midbrain DA areas, and indirect pathway MSNs that express D2Rs and project to the ventral pallidum (Kreitzer and Malenka, 2008; Sesack and Grace, 2010; Lüscher and Malenka, 2011; Smith et al., 2013). Because MSN are GABAergic, activation of MSNs neurons will inhibit their downstream targets which are also GABAergic (Chevalier and Deniau, 1990). Therefore, activation of D1R-MSNs will excite midbrain DA neurons, which then contributes to the regulation of reward-related behaviors (Lüscher and Malenka, 2011; Bocklisch et al., 2013).

Recent studies using genetically engineered mice that express Cre recombinase in a cell-type specific manner have revealed different roles for D1R-MSNs and D2R-MSNs in cocaine addiction behaviors. Such mice enable genetic targeting of specific toxins, optogenetic probes or DREADD (designer receptors exclusively activated by a designer drug) to selectively manipulate D1R-MSN or D2R-MSN. This approach has led to some consensus about the role of MSNs in addictive behaviors: D1R-MSNs apparently promote addictive behaviors, while no specific role (or an inhibitory role) in the development of drug-induced addictive behaviors has been suggested for D2R-MSNs (Hikida et al., 2010; Lobo et al., 2010; Ferguson et al., 2011; Bock et al., 2013). Cocaine exposure apparently induces synaptic modification and alterations in gene expression in both MSN populations (Lobo et al., 2010; Lobo and Nestler, 2011; Grueter et al., 2013). Although it appears that D1R-MSNs and D2R-MSNs play opposing roles in cocaine-mediated addictive behaviors, the precise role of D2R-MSNs is not clear.

Previously it has been shown that D2R knockout (KO) mice display normal cocaine-mediated behavioral sensitization and cocaine-seeking behaviors, with only a slight decrease in sensitivity caused by the absence of D2R (Baik et al., 1995; Chausmer et al., 2002; Sim et al., 2013). However, exposure to stress during drug withdrawal suppresses expression of cocaine-induced behavioral sensitization as well as cocaine-seeking and relapse behaviors in D2R KO mice (Sim et al., 2013). Specific knockdown of D2R in the NAc does not affect basal locomotor activity, nor cocaine-induced behavioral sensitization, but does confer the ability of stress to inhibit expression of cocaine-induced behavioral sensitization (Sim et al., 2013). These findings strongly suggest that blockade of D2R in the NAc does not prevent cocaine-mediated behavioral sensitization. Rather, it appears that D2R in the NAc play a distinct role in regulation of the stresstriggered synaptic modifications during withdrawal that lead to an increase in cocaine-seeking and relapse behaviors (Sim et al., 2013).

Here we have used optogenetics to further evaluate the role of NAc D2R-MSNs in cocaine-induced behavioral sensitization. Using brain slices, we find that photostimulation of D2R-MSNs activates local inhibitory circuits within NAc involving neighboring MSNs. Photostimulation of NAc D2R-MSNs *in vivo* affects neither the initiation nor the expression of cocaineinduced behavioral sensitization. However, repetitive activation of NAc D2R-MSNs during the drug withdrawal period attenuates cocaine-induced addictive behavior. Our results show that D2R-MSNs of NAc play a key role in withdrawal-induced plasticity and may contribute to relapse after cessation of drug abuse.

# **MATERIALS AND METHODS**

## **MICE**

D2-Cre BAC transgenic mice on a C57Bl/6 background were obtained from MMRRC (Mutant Mouse Regional Resource Centers, B6.FVB(Cg)-Tg(Drd2-cre)ER44Gsat/Mmucd). In behavioral experiments, littermates lacking the D2-Cre transgene were used as controls for the D2-Cre mice. Mice were maintained in a specific pathogen-free barrier facility under constant conditions of temperature and humidity, and on a 12-h light, 12-h dark schedule. Animal care and handling were performed in accordance with standards approved by the Institutional Animal Care and Use Committees of Korea University and KIST.

## **VIRUS VECTOR PREPARATION**

pAAV-EF1a-DIO-hChR2(H134R)-EYFP-WPRE was generously provided by Karl Deisseroth (Stanford Univ.). For preparation of AAV, HEK293T cells were grown in DMEM media with antibiotics and FBS. The day before transfection, four plates beyond 90% confluence from 10-cm dishes were plated onto five 15-cm dishes and incubated for 18–22 h or until 60 to 70% confluence. HEK293T cells were transfected with pAAV-DIO-ChR2-EYFP, pAAV-DJ and pHelper using jetPEI transfection reagent (QBiogene). The DNA/DMEM/PEI cocktail was vortexed and incubated at room temperature for 20 min. After incubation, the transfection mixture was added to each 15 cm dish. Transfected cells were harvested 48 h after transfection and incubated with 0.5% sodium deoxycholate (Sigma; D6750) and 50 units/ml of benzonase nuclease (Sigma; E1014) at 37◦C for 1 h. After removing cellular debris by centrifuging at 3000 × g for 15 min, the supernatant was filtered through a 0.45 mm PVDF filter (Millipore). Purification of AAV- DJ particles was performed using HiTrap heparin affinity columns (GE Healthcare). For concentration of AAV, Amicon ultra-15 centrifugal filter units with a 100,000 molecular weight cutoff were used. Concentrated virus aliquoted and frozen for storage at −80◦C. The final viral concentrations was 3∼6 × 1012 virus particles per ml for each AAV.

## **STEREOTAXIC INJECTION AND OPTICAL FIBER PLACEMENT**

Animals were anesthetized by i.p. injections of 1.6 µl of Zoletil and 0.05 µl of xylazine (Rompun, Bayer) per gram of body weight and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA). For injection of viruses, a 31-gauge syringe needle was used to bilaterally infuse 2 µl of virus into NAc at an angle of 0◦ (AP +1.7; ML ±1.3; DV −4.5) at a rate of 0.1 ul/min. The needle was left in place for 10 min after injection before being slowly withdrawn. The fiber-optic cannula for implantation consisted of a zirconia ferrule (1.25 mm in diameter and 4.5 mm long) and flat tip of an optical fiber (200 µm in diameter). The implantation of the fiber-optic cannula into NAc for illumination of D2-MSNs was performed immediately after injection of viruses. The coordinates for implantation of the fiber-optic cannula were an angle of 0◦ (AP +1.7; ML ±1.35; DV −4.2) for targeting NAc. To help anchor the optical fiber, two screws were anchored into the skull to the rear of the implantation site of the fiber-optic cannula. To fix the fiberoptic cannula on the skull, C&B Superbond (Sun Medical) was applied to the surface of the skull around the base of the cannula. Once the C&B Superbond hardened, the cannula was released from the holder and dental cement (Poly-F, Dentsply) was applied around the cannula and screws. To close the incision around the cannulation site, Vetbond tissue adhesive (3 M, 7003449) was used. After implantation, mice were given subcutaneous injection of antibiotics (Enrofloxacin, 5 mg/kg, q 12 h) and analgesia (Carprofen, 5 mg/kg, q 24 h) for 3 consecutive days.

## **IN VIVO PHOTOSTIMULATION**

A 200 µm patch cord was connected to the external portion of the chronically implantable optical fiber using a sleeve. Optical fibers were attached through an FC/PC adaptor to a blue laser diode (473 nm, MBL-III 473-150 mW), and light pulses were generated through a stimulator (BNC 575). For photostimulation of ChR2-expressing neurons, the stimulation paradigm was 20 Hz frequency, 5 ms pulse duration and 2–5 mW of light power. Light power emitted from the patch cord was measured using a power meter (PM100D) with a S121C light sensor.

## **BEHAVIORAL ANALYSIS**

Behavioral experiments were performed with male D2-Cre mice at 11–13 weeks of age, with the exception of mice subjected to electrophysiological analysis which were 5–6 weeks of age. Agematched D2-Cre and Cre negative control mice were injected with virus and housed individually and allowed to acclimate to the cage until the behavioral test. For each manipulation, mice were transferred to the experimental room 60 min before the onset of the experiment to allow for habituation and to reduce stress (brightness of the experimental room was 70 lux). Each experimental apparatus was cleaned with 70% ethanol between experiments to remove any potential odor cues.

## **COCAINE SENSITIZATION**

For initiation of cocaine sensitization, mice were habituated to saline injections (i.p.) for 3 consecutive days and then injected with saline or cocaine (15 mg kg−1 , i.p) for 5 consecutive days. Mice were injected intraperitoneally (i.p.) with either cocaine hydrochloride (Johnson Mattney, Edinburgh, UK) dissolved in saline (0.9% NaCl) or saline with a 30 G needle. Immediately after each injection, mice were tested for horizontal locomotor activity in an open-field chamber for 30 min. For measurement of the effect of photostimulation on the initiation and expression of sensitization (**Figure 5**), mice were given blue light illumination bilaterally through dual fiber-optic patch cords onto the NAc for four 3-min periods during 30 min sessions in home cages. Patch cords from the fiber-optic cannula located on the mouse skull was removed and mice were given at least 10 min rest. Mice were then injected with either cocaine or saline (coc 1dcoc 5d). After initiation of sensitization, cocaine was withdrawn for 14 days without any injection of saline. During this withdrawal period, no photostimulation was applied. The expression of behavioral sensitization to cocaine was then determined by injection of a challenge dose of the drug (10 mg kg−1 , i.p.) after photostimulation of the NAc as illustrated in **Figure 5A**. To measure the effect of photostimulation during the cocaine withdrawal period (**Figure 6**), mice were subjected to the same protocol for sensitization as described above (for **Figure 5**) except photostimulation was given. After initiation of cocaine sensitization, photostimulation was applied to the NAc daily for 1 h during the total withdrawal period of 14 days. After 14 days of withdrawal, all groups of mice were injected with the challenge dosage of cocaine, (10 mg kg−1 ).

#### **IMMUNOFLUORESCENCE AND CONFOCAL LASER MICROSCOPY**

For immunofluorescence, mice were anesthetized with Zoletil (Virbac, 1.6 µl/g, intraperitoneally) and 0.05 µl/g Rompun (Bayer) and perfused with filter-sterilized 0.1 M PBS followed by fixation using 4% paraformaldehyde/PBS solution (Sigma). The brain was then removed and post-fixed for 4 h with icecold fixative as above. The brains were then dehydrated in 30% sucrose/0.1 M PBS for 2 days. Brains were then frozen and 40-µmthick consecutive coronal sections were prepared on a cryostat (Leica CM 1900, Germany). Sections (40 µm) were blocked for 1 h in 0.1 M PBS containing 5% normal goat serum and 0.2% Triton X-100 and incubated with rabbit polyclonal anti-D2R (1:500, Millipore, AB5084P) at 4◦C overnight. After washes with PBS containing 0.2% Triton X-100, samples were incubated at RT for 1 h with Alexa Fluor 568 goat anti-rabbit IgG (1:500; Molecular Probes, Eugene, OR, USA) and 0.2 µl/ml 4, 6-diamidino-2 phenyl-indole HCl (DAPI; Sigma, St. Louis, MO, USA) in PBS containing 1% normal goat serum and 0.2% Triton X-100. As a negative control, samples were incubated with DAPI and the secondary antibody only. Sections were examined on a C1 Plan Apo × 40/1.4 water confocal laser scanning system (LSM 700, Zeiss, Berlin, Germany).

## **ELECTROPHYSIOLOGY AND PHOTOSTIMULATION IN NUCLEUS ACCUMBENS SLICES**

Mice were used for experiments 4 weeks after virus injection, to achieve optimal expression of ChR2-EYFP. Mice were then anesthetized and decapitated for preparation of acute brain slices. The brain was quickly removed and immediately placed in ice-cold cutting solution containing (in mM) 250 Sucrose, 26 NaHCO3, 10 D-Glucose, 3 Myo-inositol, 2.5 KCl, 2 Na-pyruvate, 1.25 NaH2PO4, 0.5 Ascorbic acid, 1 Kynurenic acid and 7 MgCl2 which was bubbled with 95% O2/5% CO2 (pH = 7.4). Coronal brain slices (250 µm thick) containing the NAc were prepared using a vibratome (Leica VT 1200 S) and were then incubated in gassed artificial cerebrospinal fluid (ACSF) containing (in mM): 11 D-glucose, 125 NaCl, 25 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 1.25 MgCl2 and 2.5 CaCl2 at 34◦C for 1 h before recording. Slices were then transferred to a submersion recording chamber in which O2 saturated ACSF solution was continuously superfused. Cells in NAc and VTA were visualized using a 2-photon microscope (Olympus FV1000 MPE, Tokyo, Japan) equipped with a 25X water immersion lens and infrared DIC optics. Whole-cell patch clamp recordings were obtained from NAc cells with a Multiclamp 700B amplifier and Digidata 1440A digitizer (Molecular Devices, LLC). Data were sampled using pCLAMP 10.2 software and further analyzed using Clampfit 10.2 software (Molecular Devices, LLC). Patch electrodes with resistances between 3–5 M were filled with an internal solution containing (in mM): 130 K-gluconate, 2 NaCl, 2 MgCl2, 20 HEPES, 4 Na2ATP, 0.4 Na3GTP, 0.5 EGTA and 10 Na2- phosphocreatine, with pH adjusted to 7.3 using 1 N KOH. Bicuculline (10 µM) was bathapplied to brain slice to block GABA receptors in a subset of experiments.

NAc cells expressing ChR2-EYFP were photostimulated by a LED light source (460 ± 27 nm, UHP-Mic-LED-460, Prizmatix). Blue light from the LED was further filtered and attenuated by a filter cube equipped with an excitation filter (470–495 nm); flashes of light (10 ms duration, 0.0366–0.354 mW/mm2 ) were delivered to the brain slice via the 25X objective lens at frequencies of 5–40 Hz. In a subset of experiments, photocurrents were measured in ChR2-expressing cells in response to 2 s duration light flashes.

## **STATISTICAL ANALYSIS**

Data are presented as means ± s.e.m. and were analyzed with the two-tailed Student's *t*-test, or with two-way analysis of variance followed by Bonferroni's *post hoc* test. A *P*-value of <0.05 was considered statistically significant.

background figure, 1 mm: insert, 200 µm.  **(C)** Confocal image of neurons in a living NAc slice.

structure (C2). aca: anterior part of anterior commissure; NAc: nucleus accumbens core. **(D)** ChR2-mediated photocurrent (black traces) evoked by light flashes (470 nm, blue traces at bottom) of various intensities.

(Continued)

#### **FIGURE 1 | Continued**

**(E)** Relationship between peak amplitude of photocurrents and light intensity. Points indicate mean ± S.E.M (n = 4), while the curve is fitted with the Hill equation. **(F)** Representative membrane potential responses to trains of light flashes (10 ms, 0.35 mW/mm2 ) at the indicated frequencies. **(G)** Relationship between light intensity and probability of light-evoked action potentials (APs) during trains of light flashes, as in F (n = 5). Curves are fitted with the Hill equation.

### **RESULTS**

## **SELECTIVE PHOTOSTIMULATION OF MEDIUM SPINY NEURONS IN NUCLEUS ACCUMBENS**

To determine the role of NAc D2R-MSNs in cocaine-mediated addictive behaviors, we used an optogenetic approach to stimulate NAc D2R neurons. To selectively control the activity of D2R-MSNs in NAc by light, viral vectors coding AAV-DIO-ChR2-EYFP were stereotaxically injected into the NAc of D2R-Cre BAC transgenic mice. 4 weeks after viral injection, robust expression of ChR2-EYFP was observed in the NAc (**Figure 1A**). The specificity of ChR2 expression in D2R-MSNs was confirmed by immunofluorescence confocal analysis: expression of YFPtagged ChR2 was co-localized with D2R in NAc (**Figure 1B**), showing that ChR2 was expressed in D2R-expressing neurons in NAc.

Although such an approach has been used in other studies (e.g., Lobo et al., 2010), the details of virus injection procedures will vary from one lab to another, making it important to document optogenetic control under our specific experimental conditions. We assessed the functional expression of ChR2 by making whole-cell patch clamp recordings from MSNs in NAc slices. MSNs were identified by: (1) a relatively hyperpolarized resting membrane potential (RMP), typically more negative than −80 mV; (2) a regular pattern of AP firing in response to applied current pulses; (3) long latency to firing of the first AP during a current pulse; (4) absence of a voltage "sag" during hyperpolarization caused by a hyperpolarization-activated cation current (Ih); and (5) relatively small size of their cell bodies (Chang and Kitai, 1985; O'Donnell and Grace, 1993; Le Moine and Bloch, 1996; Taverna et al., 2008). Blue light (470 nm) was applied over the entire field of view (0.78 mm2 ) while voltage-clamping the MSNs at a holding potential of −69 mV. Some MSNs expressed ChR2, evident as YFP fluorescence in their somata (arrows in **Figures 1C1,C3**). Such neurons exhibited substantial photocurrents, with brighter light stimuli eliciting larger photocurrents (**Figure 1D**). The relationship between peak photocurrent amplitude and light intensity (**Figure 1E**) had a half-maximal light sensitivity of 0.054 ± 0.0023 mW/mm2 and a maximal peak amplitude of 1.16 ± 0.16 nA (mean ± s.e.m., *n* = 4).

Under current-clamp conditions, MSNs expressing ChR2 fired APs reliably in response to trains of light pulses (10 ms duration; **Figure 1F**). Under these conditions, light intensities greater than 0.1 mW/mm2 were sufficient to evoke APs (**Figure 1G**, *n* = 5). APs were reliably evoked at photostimulation frequencies up to 20 Hz, while at 40 Hz the light-induced responses summed to cause a sustained depolarization that was less effective at evoking APs (**Figures 1F,G**).

**circuits. (A)** Confocal image of a live NAc slice, showing a dye-filled neuron that does not express ChR2 and a neighboring cell (arrowhead) that expressed ChR2 and could be photostimulated. **(B)** IPSC (black trace) induced by a light pulse (10 ms duration, 0.35 mW/mm2 ) at a holding potential of −60 mV. The IPSC was blocked by bath application of bicuculline (red trace). **(C)** IPSC evoked by light pulses (10 ms, 0.35 mW/mm2 ) when the membrane potential was clamped at 10 mV increments from −99 mV to −39 mV. **(D)** Voltage dependence of light-induced IPSCs. The relationship between IPSC amplitude and membrane potentials (n = 6) could be fit by a straight line and had an estimated reversal potential of −81 mV. **(E)** Four different classes of responses of NAc neurons to photostimulation.

## **PHOTOSTIMULATION OF D2R-MSNs DRIVES LOCAL INHIBITORY CIRCUITS**

To investigate the consequences of D2R-MSNs activity on local circuits in NAc, we photostimulated presynaptic MSN expressing ChR2 while measuring postsynaptic responses in ChR2-negative MSNs (**Figure 2A**). The neuron shown in **Figure 2A** does not express ChR2, as indicated by the absence of EYFP fluorescence as well as the absence of short-latency photocurrents like those shown in **Figure 1D**. However, when the postsynaptic MSNs were held as a potential of −69 mV, 10 ms duration light flashes evoked outward currents after a latency of 9.0 ± 0.42 ms (**Figure 2B**, *n* = 15). To determine the nature of these responses, the postsynaptic membrane potential was varied between −99 mV to −39 mV, while a light flash was applied (**Figure 2C**). Lightinduced responses varied with membrane potential (**Figure 2D**, *n* = 6) and reversed their polarity at −81 ± 3.4 mV. Given that the equilibrium potential for chloride ions is −80 mV under our ionic conditions, the light-induced outward currents could be due to chloride flux mediated by postsynaptic GABAA receptors. To

# Optogenetics reveals a role for accumbal medium spiny neurons expressing dopamine D2 receptors in cocaine-induced behavioral sensitization

**Shelly Sooyun Song1† , Byeong Jun Kang1† , Lei Wen2,3,4† , Hyo Jin Lee1 , Hye-ri Sim1 , Tae Hyong Kim1 , Sehyoun Yoon1 , Bong-June Yoon1 , George J. Augustine2,3,4 and Ja-Hyun Baik1\***

1 Department of Life Sciences, Molecular Neurobiology Laboratory, School of Life Sciences and Biotechnology, Korea University, Seoul, South Korea

2 Center for Functional Connectomics (CFC), KIST, Seoul, South Korea

3 Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore

4 Institute of Molecular and Cell Biology, Singapore, Singapore

#### **Edited by:**

Anton Ilango, Leibniz Institute for Neurobiology, Germany

#### **Reviewed by:**

Simona Cabib, Università "Sapienza", Roma, Italy Vincent Pascoli, Geneva University, Switzerland

#### **\*Correspondence:**

Ja-Hyun Baik, Department of Life Sciences, Molecular Neurobiology Laboratory, School of Life Sciences and Biotechnology, Korea University, Nokji Bldg, Rm 402, Anam-dong, Sungbuk-gu, Seoul, 136-713, South Korea e-mail<EMAIL_ADDRESS>†These authors have contributed

equally to this work.

Long-lasting, drug-induced adaptations within the nucleus accumbens (NAc) have been proposed to contribute to drug-mediated addictive behaviors. Here we have used an optogenetic approach to examine the role of NAc medium spiny neurons (MSNs) expressing dopamine D2 receptors (D2Rs) in cocaine-induced behavioral sensitization. Adeno-associated viral vectors encoding channelrhodopsin-2 (ChR2) were delivered into the NAc of D2R-Cre transgenic mice. This allowed us to selectively photostimulate D2R-MSNs in NAc. D2R-MSNs form local inhibitory circuits, because photostimulation of D2R-MSN evoked inhibitory postsynaptic currents (IPSCs) in neighboring MSNs. Photostimulation of NAc D2R-MSN in vivo affected neither the initiation nor the expression of cocaine-induced behavioral sensitization. However, photostimulation during the drug withdrawal period attenuated expression of cocaine-induced behavioral sensitization. These results show that D2R-MSNs of NAc play a key role in withdrawal-induced plasticity and may contribute to relapse after cessation of drug abuse.

**Keywords: optogenetics, medium spiny neurons, dopamine D2 receptors, cocaine, drug addiction**

#### **INTRODUCTION**

Dopamine (DA) signaling is associated with reward expectation and goal-directed behavior (Wise, 2004; Goto and Grace, 2005; Berridge, 2007). One of the well-known pathologies of dopaminergic disorders is drug addiction (Robinson and Berridge, 1993, 2003). Following repeated exposure to addictive substances, adaptive changes occur at the molecular and cellular level in the DA mesolimbic pathway; these can lead to drug dependence, which is a chronic, relapsing disorder in which compulsive drug-seeking and drug-taking behaviors persist despite their serious negative consequences (Thomas et al., 2008; Baik, 2013). Characterization of the modifications that take place in the mesolimbic dopaminergic system is thus key to understanding drug addiction.

Dopamine D1 receptors (D1R) and D2 receptors (D2R) are highly expressed in the medium spiny neurons (MSNs) of the striatum. It has been suggested that long-lasting drug-induced adaptations in the ventral striatum, better known as the nucleus accumbens (NAc), contribute to the development of addiction as well as drug-seeking and relapse behaviors (Lobo and Nestler, 2011; Smith et al., 2013). Dopaminergic cell bodies from the ventral tegmental area mostly innervate the NAc. Over 95% of the cells within the NAc are MSNs, which receive excitatory inputs from four major brain regions: the prefrontal cortex, the ventral subiculum of the hippocampus, the basolateral amygdala, and the thalamus (Sesack and Grace, 2010; Lüscher and Malenka, 2011). MSNs within the NAc can be divided into two major subpopulations: direct pathway MSNs that express D1Rs and project directly to midbrain DA areas, and indirect pathway MSNs that express D2Rs and project to the ventral pallidum (Kreitzer and Malenka, 2008; Sesack and Grace, 2010; Lüscher and Malenka, 2011; Smith et al., 2013). Because MSN are GABAergic, activation of MSNs neurons will inhibit their downstream targets which are also GABAergic (Chevalier and Deniau, 1990). Therefore, activation of D1R-MSNs will excite midbrain DA neurons, which then contributes to the regulation of reward-related behaviors (Lüscher and Malenka, 2011; Bocklisch et al., 2013).

Recent studies using genetically engineered mice that express Cre recombinase in a cell-type specific manner have revealed different roles for D1R-MSNs and D2R-MSNs in cocaine addiction behaviors. Such mice enable genetic targeting of specific toxins, optogenetic probes or DREADD (designer receptors exclusively activated by a designer drug) to selectively manipulate D1R-MSN or D2R-MSN. This approach has led to some consensus about the role of MSNs in addictive behaviors: D1R-MSNs apparently promote addictive behaviors, while no specific role (or an inhibitory role) in the development of drug-induced addictive behaviors has

(**Figure 4B**). No such effects were observed in the control D2R-Cre(−) NAc-ChR2 mice (**Figures 4B,C**), indicating that the effects of photostimulation were caused by activation of ChR2, rather than possible non-specific effects such as heating of brain tissue. Therefore, our data indicated that photostimulation of D2R-MSNs in NAc elicited a decrease in locomotor activity.

These results established our ability to control the activity of D2R-MSNs within NAc *in vivo*. We next used this capability to examine the influence of D2R-MSN activity on behavioral sensitization to repeated administration of cocaine. Behavioral sensitization refers to the process that allows an initial exposure to psychostimulants, such as cocaine, to enhance the ability of subsequent drug exposures to stimulate locomotor activity. This process can be separated into initiation and expression phases: initiation describes the immediate neural events that induce behavioral sensitization (Vanderschuren and Kalivas, 2000; Sim et al., 2013), while expression is known to be a long-lasting form of behavioral plasticity that persists after drug withdrawal (Vanderschuren and Kalivas, 2000; Sim et al., 2013).

**FIGURE 5 | Effects of activation of D2-MSN during sensitization to cocaine. (A)** Experimental scheme for photo-stimulation of D2-MSNs during initiation and expression of sensitization to cocaine. Blue-light illumination (2∼5 mW, 5 ms, 20 Hz) was delivered for four 3-min periods during 40-min sessions before measuring locomotion as indicated by blue bars. Sal, saline; Coc, cocaine; IS, initation of sensitization; WD, withdrawal; ES, expression of sensitization. **(B)** The effects of photo-stimulation during the initiation of behavioral sensitization (IS) with five daily injections of either saline or cocaine (15 mgkg−1 , i.p.) and during the expression of behavioral sensitization (ES) induced by challenge dose of cocaine (10 mgkg−1 , i.p.) after 14 days of drug withdrawal (For coc 1d-5d: cocaine effect F(1,11) > 24.55, p < 0.05. For ES: cocaine effect in F(1,11) = 40.14, p < 0.0001; cocaine × D2-Cre interaction F(1,11) = 0.7147, p = 0.407, n = 6∼7). Two-way analysis of variance (ANOVA) post hoc test: \* P < 0.05, \*\* P < 0.01, \*\*\* P < 0.001 vs. saline-treated group). Data are means ± s.e.m. **(C)** Detailed time course of locomotor activity for D2-Cre(–) or D2-Cre(+) mice during ES to cocaine (10 mgkg–1, i.p.) following blue-light illumination. Arrows denote time of injection with either saline or cocaine (For 20–50 m: cocaine effect F(1,11) > 1.812, P < 0.1919, n = 6∼7). Two-way analysis of variance (ANOVA) post hoc test: \* P < 0.05, \*\* P < 0.01, \*\*\* P < 0.001 vs. saline-treated group). Data are means ± s.e.m.

## **BEHAVIORAL ANALYSIS**

Behavioral experiments were performed with male D2-Cre mice at 11–13 weeks of age, with the exception of mice subjected to electrophysiological analysis which were 5–6 weeks of age. Agematched D2-Cre and Cre negative control mice were injected with virus and housed individually and allowed to acclimate to the cage until the behavioral test. For each manipulation, mice were transferred to the experimental room 60 min before the onset of the experiment to allow for habituation and to reduce stress (brightness of the experimental room was 70 lux). Each experimental apparatus was cleaned with 70% ethanol between experiments to remove any potential odor cues.

## **COCAINE SENSITIZATION**

For initiation of cocaine sensitization, mice were habituated to saline injections (i.p.) for 3 consecutive days and then injected with saline or cocaine (15 mg kg−1 , i.p) for 5 consecutive days. Mice were injected intraperitoneally (i.p.) with either cocaine hydrochloride (Johnson Mattney, Edinburgh, UK) dissolved in saline (0.9% NaCl) or saline with a 30 G needle. Immediately after each injection, mice were tested for horizontal locomotor activity in an open-field chamber for 30 min. For measurement of the effect of photostimulation on the initiation and expression of sensitization (**Figure 5**), mice were given blue light illumination bilaterally through dual fiber-optic patch cords onto the NAc for four 3-min periods during 30 min sessions in home cages. Patch cords from the fiber-optic cannula located on the mouse skull was removed and mice were given at least 10 min rest. Mice were then injected with either cocaine or saline (coc 1dcoc 5d). After initiation of sensitization, cocaine was withdrawn for 14 days without any injection of saline. During this withdrawal period, no photostimulation was applied. The expression of behavioral sensitization to cocaine was then determined by injection of a challenge dose of the drug (10 mg kg−1 , i.p.) after photostimulation of the NAc as illustrated in **Figure 5A**. To measure the effect of photostimulation during the cocaine withdrawal period (**Figure 6**), mice were subjected to the same protocol for sensitization as described above (for **Figure 5**) except photostimulation was given. After initiation of cocaine sensitization, photostimulation was applied to the NAc daily for 1 h during the total withdrawal period of 14 days. After 14 days of withdrawal, all groups of mice were injected with the challenge dosage of cocaine, (10 mg kg−1 ).

## **IMMUNOFLUORESCENCE AND CONFOCAL LASER MICROSCOPY**

For immunofluorescence, mice were anesthetized with Zoletil (Virbac, 1.6 µl/g, intraperitoneally) and 0.05 µl/g Rompun (Bayer) and perfused with filter-sterilized 0.1 M PBS followed by fixation using 4% paraformaldehyde/PBS solution (Sigma). The brain was then removed and post-fixed for 4 h with icecold fixative as above. The brains were then dehydrated in 30% sucrose/0.1 M PBS for 2 days. Brains were then frozen and 40-µmthick consecutive coronal sections were prepared on a cryostat (Leica CM 1900, Germany). Sections (40 µm) were blocked for 1 h in 0.1 M PBS containing 5% normal goat serum and 0.2% Triton X-100 and incubated with rabbit polyclonal anti-D2R (1:500, Millipore, AB5084P) at 4◦C overnight. After washes with PBS containing 0.2% Triton X-100, samples were incubated at RT for 1 h with Alexa Fluor 568 goat anti-rabbit IgG (1:500; Molecular Probes, Eugene, OR, USA) and 0.2 µl/ml 4, 6-diamidino-2 phenyl-indole HCl (DAPI; Sigma, St. Louis, MO, USA) in PBS containing 1% normal goat serum and 0.2% Triton X-100. As a negative control, samples were incubated with DAPI and the secondary antibody only. Sections were examined on a C1 Plan Apo × 40/1.4 water confocal laser scanning system (LSM 700, Zeiss, Berlin, Germany).

## **ELECTROPHYSIOLOGY AND PHOTOSTIMULATION IN NUCLEUS ACCUMBENS SLICES**

Mice were used for experiments 4 weeks after virus injection, to achieve optimal expression of ChR2-EYFP. Mice were then anesthetized and decapitated for preparation of acute brain slices. The brain was quickly removed and immediately placed in ice-cold cutting solution containing (in mM) 250 Sucrose, 26 NaHCO3, 10 D-Glucose, 3 Myo-inositol, 2.5 KCl, 2 Na-pyruvate, 1.25 NaH2PO4, 0.5 Ascorbic acid, 1 Kynurenic acid and 7 MgCl2 which was bubbled with 95% O2/5% CO2 (pH = 7.4). Coronal brain slices (250 µm thick) containing the NAc were prepared using a vibratome (Leica VT 1200 S) and were then incubated in gassed artificial cerebrospinal fluid (ACSF) containing (in mM): 11 D-glucose, 125 NaCl, 25 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 1.25 MgCl2 and 2.5 CaCl2 at 34◦C for 1 h before recording. Slices were then transferred to a submersion recording chamber in which O2 saturated ACSF solution was continuously superfused. Cells in NAc and VTA were visualized using a 2-photon microscope (Olympus FV1000 MPE, Tokyo, Japan) equipped with a 25X water immersion lens and infrared DIC optics. Whole-cell patch clamp recordings were obtained from NAc cells with a Multiclamp 700B amplifier and Digidata 1440A digitizer (Molecular Devices, LLC). Data were sampled using pCLAMP 10.2 software and further analyzed using Clampfit 10.2 software (Molecular Devices, LLC). Patch electrodes with resistances between 3–5 M were filled with an internal solution containing (in mM): 130 K-gluconate, 2 NaCl, 2 MgCl2, 20 HEPES, 4 Na2ATP, 0.4 Na3GTP, 0.5 EGTA and 10 Na2- phosphocreatine, with pH adjusted to 7.3 using 1 N KOH. Bicuculline (10 µM) was bathapplied to brain slice to block GABA receptors in a subset of experiments.

NAc cells expressing ChR2-EYFP were photostimulated by a LED light source (460 ± 27 nm, UHP-Mic-LED-460, Prizmatix). Blue light from the LED was further filtered and attenuated by a filter cube equipped with an excitation filter (470–495 nm); flashes of light (10 ms duration, 0.0366–0.354 mW/mm2 ) were delivered to the brain slice via the 25X objective lens at frequencies of 5–40 Hz. In a subset of experiments, photocurrents were measured in ChR2-expressing cells in response to 2 s duration light flashes.

## **STATISTICAL ANALYSIS**

Data are presented as means ± s.e.m. and were analyzed with the two-tailed Student's *t*-test, or with two-way analysis of variance followed by Bonferroni's *post hoc* test. A *P*-value of <0.05 was considered statistically significant.

been suggested for D2R-MSNs (Hikida et al., 2010; Lobo et al., 2010; Ferguson et al., 2011; Bock et al., 2013). Cocaine exposure apparently induces synaptic modification and alterations in gene expression in both MSN populations (Lobo et al., 2010; Lobo and Nestler, 2011; Grueter et al., 2013). Although it appears that D1R-MSNs and D2R-MSNs play opposing roles in cocaine-mediated addictive behaviors, the precise role of D2R-MSNs is not clear.

Previously it has been shown that D2R knockout (KO) mice display normal cocaine-mediated behavioral sensitization and cocaine-seeking behaviors, with only a slight decrease in sensitivity caused by the absence of D2R (Baik et al., 1995; Chausmer et al., 2002; Sim et al., 2013). However, exposure to stress during drug withdrawal suppresses expression of cocaine-induced behavioral sensitization as well as cocaine-seeking and relapse behaviors in D2R KO mice (Sim et al., 2013). Specific knockdown of D2R in the NAc does not affect basal locomotor activity, nor cocaine-induced behavioral sensitization, but does confer the ability of stress to inhibit expression of cocaine-induced behavioral sensitization (Sim et al., 2013). These findings strongly suggest that blockade of D2R in the NAc does not prevent cocaine-mediated behavioral sensitization. Rather, it appears that D2R in the NAc play a distinct role in regulation of the stresstriggered synaptic modifications during withdrawal that lead to an increase in cocaine-seeking and relapse behaviors (Sim et al., 2013).

Here we have used optogenetics to further evaluate the role of NAc D2R-MSNs in cocaine-induced behavioral sensitization. Using brain slices, we find that photostimulation of D2R-MSNs activates local inhibitory circuits within NAc involving neighboring MSNs. Photostimulation of NAc D2R-MSNs *in vivo* affects neither the initiation nor the expression of cocaineinduced behavioral sensitization. However, repetitive activation of NAc D2R-MSNs during the drug withdrawal period attenuates cocaine-induced addictive behavior. Our results show that D2R-MSNs of NAc play a key role in withdrawal-induced plasticity and may contribute to relapse after cessation of drug abuse.

# **MATERIALS AND METHODS**

## **MICE**

D2-Cre BAC transgenic mice on a C57Bl/6 background were obtained from MMRRC (Mutant Mouse Regional Resource Centers, B6.FVB(Cg)-Tg(Drd2-cre)ER44Gsat/Mmucd). In behavioral experiments, littermates lacking the D2-Cre transgene were used as controls for the D2-Cre mice. Mice were maintained in a specific pathogen-free barrier facility under constant conditions of temperature and humidity, and on a 12-h light, 12-h dark schedule. Animal care and handling were performed in accordance with standards approved by the Institutional Animal Care and Use Committees of Korea University and KIST.

## **VIRUS VECTOR PREPARATION**

pAAV-EF1a-DIO-hChR2(H134R)-EYFP-WPRE was generously provided by Karl Deisseroth (Stanford Univ.). For preparation of AAV, HEK293T cells were grown in DMEM media with antibiotics and FBS. The day before transfection, four plates beyond 90% confluence from 10-cm dishes were plated onto five 15-cm dishes and incubated for 18–22 h or until 60 to 70% confluence. HEK293T cells were transfected with pAAV-DIO-ChR2-EYFP, pAAV-DJ and pHelper using jetPEI transfection reagent (QBiogene). The DNA/DMEM/PEI cocktail was vortexed and incubated at room temperature for 20 min. After incubation, the transfection mixture was added to each 15 cm dish. Transfected cells were harvested 48 h after transfection and incubated with 0.5% sodium deoxycholate (Sigma; D6750) and 50 units/ml of benzonase nuclease (Sigma; E1014) at 37◦C for 1 h. After removing cellular debris by centrifuging at 3000 × g for 15 min, the supernatant was filtered through a 0.45 mm PVDF filter (Millipore). Purification of AAV- DJ particles was performed using HiTrap heparin affinity columns (GE Healthcare). For concentration of AAV, Amicon ultra-15 centrifugal filter units with a 100,000 molecular weight cutoff were used. Concentrated virus aliquoted and frozen for storage at −80◦C. The final viral concentrations was 3∼6 × 1012 virus particles per ml for each AAV.

## **STEREOTAXIC INJECTION AND OPTICAL FIBER PLACEMENT**

Animals were anesthetized by i.p. injections of 1.6 µl of Zoletil and 0.05 µl of xylazine (Rompun, Bayer) per gram of body weight and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA). For injection of viruses, a 31-gauge syringe needle was used to bilaterally infuse 2 µl of virus into NAc at an angle of 0◦ (AP +1.7; ML ±1.3; DV −4.5) at a rate of 0.1 ul/min. The needle was left in place for 10 min after injection before being slowly withdrawn. The fiber-optic cannula for implantation consisted of a zirconia ferrule (1.25 mm in diameter and 4.5 mm long) and flat tip of an optical fiber (200 µm in diameter). The implantation of the fiber-optic cannula into NAc for illumination of D2-MSNs was performed immediately after injection of viruses. The coordinates for implantation of the fiber-optic cannula were an angle of 0◦ (AP +1.7; ML ±1.35; DV −4.2) for targeting NAc. To help anchor the optical fiber, two screws were anchored into the skull to the rear of the implantation site of the fiber-optic cannula. To fix the fiberoptic cannula on the skull, C&B Superbond (Sun Medical) was applied to the surface of the skull around the base of the cannula. Once the C&B Superbond hardened, the cannula was released from the holder and dental cement (Poly-F, Dentsply) was applied around the cannula and screws. To close the incision around the cannulation site, Vetbond tissue adhesive (3 M, 7003449) was used. After implantation, mice were given subcutaneous injection of antibiotics (Enrofloxacin, 5 mg/kg, q 12 h) and analgesia (Carprofen, 5 mg/kg, q 24 h) for 3 consecutive days.

## **IN VIVO PHOTOSTIMULATION**

A 200 µm patch cord was connected to the external portion of the chronically implantable optical fiber using a sleeve. Optical fibers were attached through an FC/PC adaptor to a blue laser diode (473 nm, MBL-III 473-150 mW), and light pulses were generated through a stimulator (BNC 575). For photostimulation of ChR2-expressing neurons, the stimulation paradigm was 20 Hz frequency, 5 ms pulse duration and 2–5 mW of light power. Light power emitted from the patch cord was measured using a power meter (PM100D) with a S121C light sensor.

#### **FIGURE 1 | Continued**

**(E)** Relationship between peak amplitude of photocurrents and light intensity. Points indicate mean ± S.E.M (n = 4), while the curve is fitted with the Hill equation. **(F)** Representative membrane potential responses to trains of light flashes (10 ms, 0.35 mW/mm2 ) at the indicated frequencies. **(G)** Relationship between light intensity and probability of light-evoked action potentials (APs) during trains of light flashes, as in F (n = 5). Curves are fitted with the Hill equation.

#### **RESULTS**

## **SELECTIVE PHOTOSTIMULATION OF MEDIUM SPINY NEURONS IN NUCLEUS ACCUMBENS**

To determine the role of NAc D2R-MSNs in cocaine-mediated addictive behaviors, we used an optogenetic approach to stimulate NAc D2R neurons. To selectively control the activity of D2R-MSNs in NAc by light, viral vectors coding AAV-DIO-ChR2-EYFP were stereotaxically injected into the NAc of D2R-Cre BAC transgenic mice. 4 weeks after viral injection, robust expression of ChR2-EYFP was observed in the NAc (**Figure 1A**). The specificity of ChR2 expression in D2R-MSNs was confirmed by immunofluorescence confocal analysis: expression of YFPtagged ChR2 was co-localized with D2R in NAc (**Figure 1B**), showing that ChR2 was expressed in D2R-expressing neurons in NAc.

Although such an approach has been used in other studies (e.g., Lobo et al., 2010), the details of virus injection procedures will vary from one lab to another, making it important to document optogenetic control under our specific experimental conditions. We assessed the functional expression of ChR2 by making whole-cell patch clamp recordings from MSNs in NAc slices. MSNs were identified by: (1) a relatively hyperpolarized resting membrane potential (RMP), typically more negative than −80 mV; (2) a regular pattern of AP firing in response to applied current pulses; (3) long latency to firing of the first AP during a current pulse; (4) absence of a voltage "sag" during hyperpolarization caused by a hyperpolarization-activated cation current (Ih); and (5) relatively small size of their cell bodies (Chang and Kitai, 1985; O'Donnell and Grace, 1993; Le Moine and Bloch, 1996; Taverna et al., 2008). Blue light (470 nm) was applied over the entire field of view (0.78 mm2 ) while voltage-clamping the MSNs at a holding potential of −69 mV. Some MSNs expressed ChR2, evident as YFP fluorescence in their somata (arrows in **Figures 1C1,C3**). Such neurons exhibited substantial photocurrents, with brighter light stimuli eliciting larger photocurrents (**Figure 1D**). The relationship between peak photocurrent amplitude and light intensity (**Figure 1E**) had a half-maximal light sensitivity of 0.054 ± 0.0023 mW/mm2 and a maximal peak amplitude of 1.16 ± 0.16 nA (mean ± s.e.m., *n* = 4).

Under current-clamp conditions, MSNs expressing ChR2 fired APs reliably in response to trains of light pulses (10 ms duration; **Figure 1F**). Under these conditions, light intensities greater than 0.1 mW/mm2 were sufficient to evoke APs (**Figure 1G**, *n* = 5). APs were reliably evoked at photostimulation frequencies up to 20 Hz, while at 40 Hz the light-induced responses summed to cause a sustained depolarization that was less effective at evoking APs (**Figures 1F,G**).

**circuits. (A)** Confocal image of a live NAc slice, showing a dye-filled neuron that does not express ChR2 and a neighboring cell (arrowhead) that expressed ChR2 and could be photostimulated. **(B)** IPSC (black trace) induced by a light pulse (10 ms duration, 0.35 mW/mm2 ) at a holding potential of −60 mV. The IPSC was blocked by bath application of bicuculline (red trace). **(C)** IPSC evoked by light pulses (10 ms, 0.35 mW/mm2 ) when the membrane potential was clamped at 10 mV increments from −99 mV to −39 mV. **(D)** Voltage dependence of light-induced IPSCs. The relationship between IPSC amplitude and membrane potentials (n = 6) could be fit by a straight line and had an estimated reversal potential of −81 mV. **(E)** Four different classes of responses of NAc neurons to photostimulation.

## **PHOTOSTIMULATION OF D2R-MSNs DRIVES LOCAL INHIBITORY CIRCUITS**

To investigate the consequences of D2R-MSNs activity on local circuits in NAc, we photostimulated presynaptic MSN expressing ChR2 while measuring postsynaptic responses in ChR2-negative MSNs (**Figure 2A**). The neuron shown in **Figure 2A** does not express ChR2, as indicated by the absence of EYFP fluorescence as well as the absence of short-latency photocurrents like those shown in **Figure 1D**. However, when the postsynaptic MSNs were held as a potential of −69 mV, 10 ms duration light flashes evoked outward currents after a latency of 9.0 ± 0.42 ms (**Figure 2B**, *n* = 15). To determine the nature of these responses, the postsynaptic membrane potential was varied between −99 mV to −39 mV, while a light flash was applied (**Figure 2C**). Lightinduced responses varied with membrane potential (**Figure 2D**, *n* = 6) and reversed their polarity at −81 ± 3.4 mV. Given that the equilibrium potential for chloride ions is −80 mV under our ionic conditions, the light-induced outward currents could be due to chloride flux mediated by postsynaptic GABAA receptors. To

background figure, 1 mm: insert, 200 µm.  **(C)** Confocal image of neurons in a living NAc slice.

structure (C2). aca: anterior part of anterior commissure; NAc: nucleus accumbens core. **(D)** ChR2-mediated photocurrent (black traces) evoked by light flashes (470 nm, blue traces at bottom) of various intensities.

(Continued)

# Nociception and pain: lessons from optogenetics

## **Fiona B. Carr and Venetia Zachariou\***

Fishberg Department of Neuroscience, Department of Pharmacology and Systems Therapeutics and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

#### **Edited by:**

Mary Kay Lobo, University of Maryland School of Medicine, USA

#### **Reviewed by:**

Philippe Seguela, Montreal Neurological Institute, Canada Feng Tao, Johns Hopkins University School of Medicine, USA

#### **\*Correspondence:**

Venetia Zachariou, Fishberg Department of Neuroscience, Department of Pharmacology and Systems Therapeutics and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, New York, NY 10029, USA e-mail<EMAIL_ADDRESS>The process of pain perception begins in the periphery by activation of nociceptors. From here nociceptive signals are conveyed via the dorsal horn of the spinal cord to multiple brain regions, where pain is perceived. Despite great progress in pain research in recent years, many questions remain regarding nociceptive circuitry and behavior, in both acute nociception and chronic pain states. Techniques that allow for selective activation of neuronal subpopulations in vivo can provide a better understanding of these complex pathways. Here we review the studies to date that have employed novel optogenetic tools to improve our understanding of the pain pathway at the peripheral, spinal and supraspinal levels.

**Keywords: pain, nociception, nociceptor, spinal cord, amygdala, prefrontal cortex**

## **CIRCUITS AND CELL TYPES INVOLVED IN PAIN**

Chronic pain represents a significant clinical problem affecting up to 20% of the general population (Breivik et al., 2006; Reid et al., 2011) and is a symptom of a variety of different underlying pathologies including arthritis, nerve injury, depression and cancer. An understanding of nociceptive mechanisms and the neurobiology of pain perception are necessary to better treat chronic pain conditions. Here we provide a brief overview of the circuitry and cell types involved, and review studies to date that have used optogenetic tools to manipulate these pathways.

Unlike some neurobiological disorders which can be attributed to specific brain regions, pain incorporates multiple components of the nervous system, at the peripheral, spinal, and supraspinal levels. The first components of the pain pathway are the peripheral nociceptors. These are a heterogeneous population, and can be classified in a variety of ways including myelination properties, expression of transducer molecules, peptidergic content and voltage gated sodium channel expression (Gold and Gebhart, 2010). Transgenic mice have been invaluable in demonstrating that nociceptor heterogeneity is of functional significance, with different nociceptors contributing to distinct nociceptive modalities such as heat and mechanical pain (Abrahamsen et al., 2008; Cavanaugh et al., 2009). Nociceptor subtypes also contribute in distinct ways to different forms of chronic pain such as inflammatory and neuropathic states (Cavanaugh et al., 2009; Seal et al., 2009; Minett et al., 2012). Activation of specific nociceptor subtypes in the intact system will be helpful in confirming modality specificity in pain, and determining appropriate peripheral targets for novel therapeutic strategies.

The dorsal horn of the spinal cord, where the majority of nociceptors terminate, provides the next level of complexity in pain processing. An exception to this are the trigeminal nociceptors, which innervate the head and facial areas and terminate in the medullary dorsal horn (Ren and Dubner, 1999). Interneurons are the most numerous population in the dorsal horn (Todd, 2010) and the balance between excitation and inhibition within the dorsal horn may be disrupted in chronic pain states. For instance, it is known that decreased GABAergic and glycinergic inhibition contribute to neuropathic pain hypersensitivity (Moore et al., 2002; Lu et al., 2013). In addition subtypes of glutamatergic interneurons may play distinct roles in acute, inflammatory and neuropathic pain states (Wang et al., 2013). To date our understanding of interneuron circuitry in the dorsal horn is based largely on electrophysiological and pharmacological approaches. Given the pivotal role of interneurons in the gate control theory of pain (Melzack, 1999), regional and cell type specific activation using optogenetics will be a powerful way to investigate this important circuitry.

The principal direct targets of dorsal horn projection neurons are the parabrachial area and thalamus (Gauriau and Bernard, 2002, 2004). From here a range of areas are activated including the somatosensory cortex, anterior cingulate, prefrontal cortex (PFC), and amygdala (Apkarian et al., 2005; Tracey and Mantyh, 2007; Baliki et al., 2013). It is important to note that chronic pain states are frequently accompanied by psychological symptoms such as anxiety and depression (Bushnell et al., 2013) and understanding the role of brain areas in both the sensory and affective components of pain is important for the development of novel treatment approaches. Another important aspect of pain circuitry to be considered is the top-down pain modulatory system, which allows higher brain structures to signal to the dorsal horn to regulate nociceptive processing. This may be either inhibitory or

facilitatory in nature, arises from a variety of brain structures and is relayed to the dorsal horn via the rostral ventromedial medulla (RVM; Ossipov et al., 2010). Optogenetic approaches will allow further investigation of these ascending and descending circuits in animal pain behavior (**Figure 1**).

#### **OPTOGENETIC STIMULATION OF NOCICEPTORS IN C. ELEGANS**

C. elegans was the first multicellular organism to be investigated using optogenetic methods (Nagel et al., 2005), and is one of the most useful simple organisms in the study of genetics and molecular biology. Many advances in neuroscience and behavior have also been made using this system. C. elegans is particularly useful in the study of nociception as it exhibits a clear and reproducible withdrawal behavior, involving a reversal and change in direction away from the noxious stimulus (Wittenburg and Baumeister, 1999; Tobin and Bargmann, 2004). Recently, optogenetic activation of C. elegans nociceptors has been used to characterize molecules associated with nociception (Husson et al., 2012). Channelrhodopsin 2 was expressed under the control of the F49H12.4 promoter, predominantly found within PVD nociceptive neurons. Stimulation of the worm with blue light therefore allowed activation of the nociceptive network, without activating other non-noxious mechanical sensing neurons. Interestingly, activation of these nociceptors is less susceptible to habituation than other sensory responses in C. elegans. The authors used this technique to screen for genes involved in mediating the nociceptive response in the PVD neuron, investigating the contribution of RNA interference for various target genes to the optogenetically induced nociceptive behavior. This led to the identification of a number of genes involved in nociceptive transmission in the organism, including voltage gated calcium channel subunits and melastatin-related transient receptor (TRPM) channels. This study highlights the potential of using optogenetically triggered behavior as a method to screen for novel nociceptive mediators, or even analgesics.

## **OPTOGENETIC STIMULATION OF SPECIFIC NOCICEPTOR POPULATIONS IN MICE**

Optogenetic stimulation of a subset of peripheral nociceptor has also been used as a tool to characterize the spinal cord circuitry associated with that nociceptor subtype (Wang and Zylka, 2009). Mas-related G protein coupled receptor D (Mrgprd) is a marker for a subset of non-peptidergic nociceptors in the rat and mouse. These neurons appear to be involved in mediating mechanical nociception, but not noxious thermal or cold stimuli (Cavanaugh et al., 2009). Although anatomically it is known that Mrgprd-positive fibers terminate largely in lamina II of the dorsal horn, the precise connectivity of this nociceptor subpopulation was not known. In contrast to the variety of molecular markers available to characterize nociceptor populations, relatively few markers are available to characterize neuronal populations in the dorsal horn. To characterize the dorsal horn neurons innervated by Mrgprd expressing primary afferents, Wang and colleagues used a combined optogenetic and electrophysiological approach (Wang and Zylka, 2009). Knock-in mice were generated to express channlerhodopsin-2 at the Mrgprd locus, allowing this nociceptor population to be selectively activated by blue light. Patch clamp recordings from lamina II dorsal horn neurons were made using a slice preparation and stimulation of Mrgprd expressing terminals by brief high frequency blue light application. This was sufficient to induce excitatory post synaptic potentials in 50% of the lamina II neurons recorded. The non-responsive lamina II neurons are therefore likely to be innervated by other afferents, such as peptidergic nociceptors, supporting the view that pain processing is modality specific at both the peripheral and spinal levels. Interestingly this preparation illustrates that stimulation of channelrhodopsin-2 on axon terminals is sufficient to evoke post synaptic activity, as the dorsal root ganglions (DRGs) were not attached. This study highlights the potential of optogenetics in understanding the complex pain related circuitry within the dorsal horn. Unlike traditional electrophysiological studies which use electrical stimulation of afferents or application of a noxious stimulus, the optogenetic approach allows precise stimulation a defined nociceptor population. This is of great interest for the understanding connectivity between peripheral nociceptors and neurons in the spinal cord.

Recently, an alternative approach has been used to explore the use of optogenetics in pain studies. *Nav1.8* encodes a voltage gated sodium channel expressed in nociceptors, and Nav1.8 expressing primary afferent neurons have been previously shown to contribute to inflammatory and neuropathic pain (Abrahamsen et al., 2008). Many studies have used the Nav1.8-Cre mice as a background to study the role of particular genes in nociception and pain-like behaviors (Stirling et al., 2005). This mouse model has now been used to express channelrhodopsin-2 specifically in nociceptors. Stimulation of the hindpaw with blue light was sufficient to induce a number of characteristic nocifensive behaviors, including paw withdrawal, licking, jumping and vocalizations, in freely moving mice. Therefore this study provides the first evidence that optical stimulation of peripheral nociceptors can trigger nocifensive behaviors in the awake and freely moving animal (Daou et al., 2013). Furthermore, c-Fos labeling of the dorsal horn following this pattern of optical activation indicated activation of both lamina I and lamina II dorsal horn neurons, suggesting that both peptidergic nociceptors and non-peptidergic nociceptors are activated. Interestingly, the authors found that optogenetic stimulation of Nav1.8 nociceptors is also capable of driving long term hypersensitivity to mechanical and thermal stimuli, mimicking aspects of chronic pain models in mice. As pain has an important affective component, the authors also demonstrated that blue light could drive conditioned place aversion in these animals.

In contrast to the approach taken by Zlyka et al. in which channelrhodopsin was inserted into the locus of a low abundance marker of a subset of nociceptors, the Nav1.8-Cre approach may be more useful for behavioral studies, and investigations of spinal and supraspinal activation, given that a greater number of nociceptors will be activated in this way.

#### **OPTOGENETIC STUDIES OF BRAIN REGIONS INVOLVED IN PAIN**

To date few studies have used optogenetic techniques to address the role of specific brain regions in pain behavior in rodents. One example has incorporated optogenetic stimulation to investigate the role of the central nucleus of the amygdala (CeA) in a mouse model of visceral pain (Crock et al., 2012). Visceral pain is a common complaint, and can be modeled in rodents by bladder or colorectal distension. In the study by Crock and colleagues, bladder distention was carried out, and the visceromotor response (VMR, elecromyographic response of the abdominal muscle) used as an index of visceral sensitivity. The amygdala is important in the affective dimensions of the pain response, and previous work had shown that the c-fos expression is increased in the CeA following noxious visceral stimulation (Han and Neugebauer, 2004). To explore this further, the right CeA was injected with herpes simplex virus (HSV) viral vector expressing channelrhodopsin-2. Optogenetic stimulation was carried out for 30 min before, during or after bladder distension. Optical stimulation before distension increased VMR sensitivity. This hypersensitivity was maintained for up to 15 min following the termination of the optical stimulation, providing direct evidence that neuronal activity in the CeA can lead to increased visceral nociception. A limitation of this study is the nature of the bladder distension protocol which requires the animals to be lightly anesthetized in order to perform measures of the VMR. Future studies to incorporate stimulation in awake, moving animals will provide further insights into the role of specific brain regions in the modulation of pain states.

Another important region involved in the modulation of pain is the PFC. As with the amygdala, this region is particularly associated with the affective component of the pain experience (Tracey and Mantyh, 2007). In a recent investigation, the PFC was injected with an adenoassociated virus (AAV), expressing channelrhodopsin-2 under the control of the calcium/calmodulin dependent kinase type II alpha (CaMKIIα) promoter (Ji and Neugebauer, 2012). This allows selective expression within pyramidal neurons only, which are the major excitatory population in the region, and avoids expression within inhibitory interneurons. Two distinct regions of the PFC were explored, the infralimbic (IL) and prelimbic (PL) areas. Following optogenetic stimulation of the IL region, extracellular recordings were made from pyramidal neurons in the IL and the nearby PL region with or without peripheral stimulation, and also following noxious mechanical stimulation of the knee joint. This approach helps provide a better understanding of circuitry in the PFC. Excitation of neurons in the IL region not only increases spontaneous activity of other neurons in this area, but also the evoked responses to noxious and non-noxious stimulation. Optical stimulation of the IL conversely led to a decrease in pyramidal neuron excitability in the PL, suggesting an inverse relationship between IL output and between PL activity (Ji and Neugebauer, 2012). This study is a good example of the type of analysis that can be achieved by combining optogenetic stimulation with electrophysiological recordings. Future studies may be useful to investigate the effects of chronic pain states, as opposed to acute noxious stimulation, on specific neuronal populations in the brain.

Recently we have applied an optogenetic approach to explore novel brain regions involved in opiate analgesia. Opioids are important clinically, however their use is limited through the development of tolerance and addiction (Ling et al., 2011). Understanding the brain regions and cell types involved in these processes are is important to help tackle the problem. We had previously demonstrated that regulator of G protein signaling 9-2 (Rgs9-2) is a negative modulator of morphine tolerance (Zachariou et al., 2003). In our recent study we demonstrated a potent role of Rgs9-2 in the nucleus accumbens (NAc, part of the brain reward center) in modulation of morphine tolerance. Taking advantage of an optogenetic strategy, we have showed that activation of Rgs9 expressing neurons in the NAc leads to the rapid development of analgesic tolerance in the hot plate test (Gaspari et al., 2014). Using bacterial artificial chromosome (BAC) lines expressing Cre recombinase, we were also able to selectively stimulate the two main NAc neuronal subpopulations: those expressing primarily D1 dopamine receptors (D1-type, direct pathway) and those enriched in D2 dopamine receptors (D2-type, indirect pathway). Activation of channelrhodopsin-2 in each of these neuronal subpopulation demonstrated that morphine tolerance is modulated by D1 type neurons, which is the population that mostly expresses mu opioid receptors (MORs; Cui et al., 2014). In accord with our earlier findings on a role of Rgs9-2 in the NAc in morphine tolerance, activation of D1-type but not D2 type neurons increases Rgs9-2 levels in the NAc (Gaspari et al., 2014).

## **OPTOPHARMACOLOGY AND NOVEL OPTORECEPTORS IN PAIN RESEARCH**

Optogenetic approaches require the expression of an exogenous light sensitive molecule in the target system. Although this has proved a very useful tool in basic neurobiological research, by definition this approach is of limited use clinically. An alternative approach is the use of "optopharmacology", that is administration of compounds that confer light sensitivity onto a specific cell type (Kramer et al., 2013). One recent example of this approach is the discovery of a novel small molecule, optovin, which confers light sensitivity on transient receptor potential cation channel, member A1 (TRPA1; Kokel et al., 2013). This ion channel is part of the transient receptor potential (TRP) family that act as transducers of noxious stimuli at the periphery. TRPA1 is activated by noxious cold and can also respond to chemical irritants such as formalin and mustard oil, and may also play a role in mediating mechanical nociception (Vay et al., 2012). The ability to optically stimulate peripheral fibers expressing TRPA1 is therefore of experimental interest to investigate the role of these neurons in animal and human pain sensation. Optovin was identified by screening a library of compounds in zebrafish embryos, which are normally insensitive to light. This molecule was found to confer sensitivity to violet light, and the effect was mediated by sensory neurons, as a spinalized preparation retained the response. A subset of mouse DRG cells also responded to optovin treatment, in a light dependent manner. These were identified as TRPA1 expressing nociceptors. Using zebrafish homozygous for TRPA1 it was demonstrated that this ion channel is required for the effects of optovin. Expression of human TRPA1 in cell line derived from human embryonic kidney (HEK) cells also conferred sensitivity to optovin. The significance of this study lies in the ability to modulate the pain sensing TRPA1 neurons, without the need for genetic manipulations to confer this sensitivity. This approach may be useful for research and treatment of pain in the future. In particular, such an approach would allow selective optical control of subpopulations of peripheral nociceptors without the need for genetic manipulations.

Another approach has been the recent development of a photoactive MOR, the main receptor mediating the analgesic actions of morphine (Barish et al., 2013). This study designed a recombinant receptor, containing the optically active component of the rhodopsin molecule and the parts of the mu opioid receptor responsible for MOR receptor protein and G protein signaling. In a HEK cell system, this receptor is activated by light and has the ability to activate endogenous MOR receptor intracellular signaling pathways. Further development would be required, but expression of this mutant MOR receptor could be a useful way to overcome the side effects of MOR mediated analgesia, including analgesic tolerance, which is associated with alterations in MOR signal transduction pathways. Future work will determine if selective activation of the MOR in the periphery by light application prevents the CNS problems in patients requiring long term morphine treatment.

## **FUTURE DIRECTIONS IN PAIN OPTOGENETICS**

The power of optogenetics lies in the ability to achieve regional and cell type specific neuronal activation. These methods provide an unprecedented opportunity to probe the complexities of the pain pathway at the peripheral, spinal and supraspinal levels. Among the most exciting developments in the field to date is the ability to produce pain-like behaviors in transgenic mice by optical stimulation of nociceptors in the skin (Daou et al., 2013). This allows direct investigation of nociceptor activation in awake, freely moving animals, avoiding the stimulation of nonnociceptive neurons and other cell types in the periphery. The stimulus is also under precise temporal control, and avoids the need for using other artificial stimuli such as chemical irritants. Future studies to target specific subtypes of nociceptor will lead to greater advances in our understanding of modality specificity, and contribution of nociceptor subtypes to different forms of chronic pain.

Somewhat surprisingly, to date no studies have taken advantage of these tools within the dorsal horn however this may reflect technical challenges in inserting optogenetic fibers into the spinal cord. Considering the importance of this component of the pain pathway, however, it is likely that optogenetics will also provide valuable new insights into this complex circuitry. Interneuron based optogenetic experiments have been performed within the ventral horn of the spinal cord, to investigate the contribution of particular subsets to locomotor activity (Dougherty et al., 2013). It is feasible that a similar approach could be employed in the dorsal horn, given our increasing anatomical and molecular knowledge of interneuron populations (Todd, 2010).

As described, a small number of studies have explored brain areas in pain using optogenetics, however many other areas of the pain matrix remain to be studied in this way. Both brain studies to date have relied either on electrophysiological recordings (Ji and Neugebauer, 2012) or behavioral outcomes in the anesthetized animal (Crock et al., 2012). One of the most important directions to be explored will be the combination of optogenetic brain stimulation with behavior in the awake animal.

The studies described here are only the beginning of what we expect to be fruitful and informative exploration of pain circuits using optogenetic tools. It is likely that subsequent studies will move beyond these initial proof of concept studies, and use optogenetic tools to tackle unanswered questions regarding pain circuitry.

#### **REFERENCES**

**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.

*Received: 05 December 2013; accepted: 18 February 2014; published online: 25 March 2014.*

*Citation: Carr FB and Zachariou V (2014) Nociception and pain: lessons from optogenetics. Front. Behav. Neurosci. 8:69. doi: 10.3389/fnbeh.2014.00069*

*This article was submitted to the journal Frontiers in Behavioral Neuroscience.*

*Copyright © 2014 Carr and Zachariou. 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.*

# Optogenetic dissection of amygdala functioning

# **Ryan T. LaLumiere\***

Department of Psychology, University of Iowa, Iowa City, IA, USA

## **Edited by:**

Mary Kay Lobo, University of Maryland School of Medicine, USA

#### **Reviewed by:**

Christa McIntyre, University of Texas, USA Joshua Johansen, RIKEN Brain

#### Science Institute, Japan **\*Correspondence:**

Ryan T. LaLumiere, Department of Psychology, University of Iowa, 11 Seashore Hall E, Iowa City, IA 52242, USA e-mail<EMAIL_ADDRESS>
Studies of amygdala functioning have occupied a significant place in the history of understanding how the brain controls behavior and cognition. Early work on the amygdala placed this small structure as a key component in the regulation of emotion and affective behavior. Over time, our understanding of its role in brain processes has expanded, as we have uncovered amygdala influences on memory, reward behavior, and overall functioning in many other brain regions. Studies have indicated that the amygdala has widespread connections with a variety of brain structures, from the prefrontal cortex to regions of the brainstem, that explain its powerful influence on other parts of the brain and behaviors mediated by those regions. Thus, many optogenetic studies have focused on harnessing the powers of this technique to elucidate the functioning of the amygdala in relation to motivation, fear, and memory as well as to determine how the amygdala regulates activity in other structures. For example, studies using optogenetics have examined how specific circuits within amygdala nuclei regulate anxiety. Other work has provided insight into how the basolateral and central amygdala nuclei regulate memory processing underlying aversive learning. Many experiments have taken advantage of optogenetics' ability to target either genetically distinct subpopulations of neurons or the specific projections from the amygdala to other brain regions. Findings from such studies have provided evidence that particular patterns of activity in basolateral amygdala (BLA) glutamatergic neurons are related to memory consolidation processes, while other work has indicated the critical nature of amygdala inputs to the prefrontal cortex and nucleus accumbens (NA) in regulating behavior dependent on those downstream structures. This review will examine the recent discoveries on amygdala functioning made through experiments using optogenetics, placing these findings in the context of the major questions in the field.

**Keywords: basolateral amygdala, central amygdala, memory, consolidation, fear, anxiety, channelrhodopsin**

Despite its relatively small size in the human brain, the amygdala influences a wide variety of neural functions, especially those related to emotion and memory. As a result, considerable research has targeted this structure and investigated how it regulates different processes and interacts with other brain regions to do so. The development of optogenetics over the past decade has opened new doors for investigations into neural circuits, both on a systems-circuitry level and on a microcircuitry level, and into how those circuits govern behavior. The amygdala, with its rich history of research in neuroscience, has been a ripe target for optogenetic investigations and, thus, many studies have applied this technique to address deeper issues regarding amygdala functioning, providing new avenues of research into "old" questions about the amygdala. Because optogenetics permits stimulation and inhibition of specific projection pathways through illumination of opsin-expressing axons and axon terminals (LaLumiere, 2011; Yizhar et al., 2011), many studies have used this approach to explore specific circuits. Moreover, because opsin expression can be genetically targeted to distinct cell types, many studies have also taken advantage of this capability to control activity in specific classes of neurons to determine their functional roles. This review will explore the recent studies that have used optogenetics to

better understand the function of the amygdala in the brain and behavior, with a particular focus on how these studies have elucidated better understandings of the systems regulating emotion, fear, and memory as well as of the functional neural circuits that involve the amygdala.

## **AMYGDALA AND EMOTION**

Perhaps the oldest line of research on the amygdala's role in the brain originates with how the amygdala regulates emotion and emotional output. Indeed, early work showed that amygdala lesions in monkeys produced the now-classic Kluver-Bucy syndrome (Kluver and Bucy, 1937; Weiskrantz, 1956). The symptoms of the syndrome involved profound alterations in the monkeys' emotional behavior, especially those involving fear-based behavior. These early findings have led to a considerable number of studies that have expounded upon this function for the amygdala. Studies in humans have confirmed the findings on amygdala lesions, as selective amygdala lesions also appear to produce deficits in emotion-related behaviors, especially those regarding fear (Adolphs et al., 1994, 2005).

Thus, it is not surprising that studies have harnessed the power of optogenetics to develop an improved understanding for how the amygdala influences emotional behavior. In a series of studies, Kay Tye et al. have made several inroads into understanding amygdala functioning in anxiety and anxiety-related behavior. Tye et al. (2011) used an optogenetic approach to distinguish the roles of the basolateral amygdala (BLA) inputs to the medial vs. lateral portions of the central amygdala (CEA) in such behavior in mice. Indeed, previous work had suggested that the medial CEA and its inputs from the BLA drive anxiety and/or fear-related behaviors, whereas BLA inputs to the lateral CEA provide feedforward inhibition of the medial CEA (Paré et al., 2004; Ciocchi et al., 2010). To distinguish these pathways, Tye et al. (2011) transduced BLA neurons with either the depolarizing cation channel channelrhodopsin-2 (ChR2) or the hyperpolarizing chloride pump halorhodopsin and illumination was provided to the BLA terminals in the lateral CEA. The expression of the opsins was limited to the pyramidal neurons of the BLA through the use of a CaMKIIα promoter. The results indicated that stimulation and inhibition of the BLA terminals in the lateral CEA reduced and increased, respectively, anxiety in the mice, as measured in different tasks. Moreover, the findings strongly suggested that this effect was due to feedforward inhibition of the medial CEA. Illumination of the BLA cells themselves did not produce the same behavioral effect and sometimes produced the opposite effect. With low light levels, illumination of the terminals did not produce reliable antidromic propagation of action potentials back to the BLA, largely eliminating the concern that such propagation could be responsible for the behavioral effect, though it is clear that optical stimulation of axon terminals can produce reliable antidromic propagation to cell bodies (Jennings et al., 2013). These results not only provided a clearer understanding of the amygdala circuits driving behavior but illustrated the importance of targeting specific downstream projection targets, as stimulating or inhibiting specific projections may produce rather different effects than might be found with stimulating or inhibiting the entire structure.

Since this initial work, Tye et al. have extended their focus on anxiety by examining the projections from the BLA to the ventral hippocampus in the regulation of anxiety-related behaviors (Felix-Ortiz et al., 2013). Previous studies had indicated that the BLA projects to the ventral hippocampus and that the ventral hippocampus is also involved in anxiety (Pitkänen et al., 1995, 2000; Bannerman et al., 2003). Using an open-arm plus maze and an open-field chamber, Felix-Ortiz et al. found that inhibition of BLA afferents to the ventral hippocampus, via activation of halorhodopsin, decreased anxiety-related behaviors. Similarly, stimulation of such afferents, using 20 Hz light pulses to activate ChR2, increased anxiety-related behaviors. Importantly, control experiments demonstrated that the effects of the BLA terminal stimulation were not due to antidromic stimulation of the BLA itself or orthodromic stimulation of fibers of passage. Moreover, electrophysiology analysis suggested that the effects of stimulating BLA inputs to the ventral hippocampus were mediated through local circuit mechanisms involving both direct activation of principal cells in the hippocampus and indirect recruitment of inhibitory neurons. In other work, Felix-Ortiz and Tye (2014) examined optical stimulation and inhibition of the BLA axon terminals in the ventral hippocampus during tests of social behavior. In a resident-juvenile-intruder test, stimulation of these terminals, using 20 Hz light pulses, reduced social interactions of the resident, whereas inhibition of these terminals increased such interactions, suggesting that activation of this pathway increased anxiety in the animals whereas silencing of this pathway produced the opposite effect. The effects with stimulation were blocked with microinjections of glutamate receptor antagonists into the ventral hippocampus, indicating that the results were due to stimulation of BLA axon terminals and the concomitant release of glutamate from those terminals. Similar behavioral effects with stimulation were observed with a three-chamber sociability test. Together, the studies by Tye et al. have produced a wealth of knowledge regarding how the amygdala, and especially the BLA, influences anxiety-related behavior through different outputs to other regions. Moreover, the findings have also demonstrated the potential for using optogenetics to develop an improved understanding of the circuits underlying behavior.

Other work has focused on how inputs from the hypothalamus regulate amygdala activity and fear-related behaviors. Previous work had indicated that oxytocin exerts its effects on a variety of behaviors, at least in part, through activation of oxytocin receptors in the CEA (Viviani et al., 2011). However, while oxytocin neurons are located in the hypothalamus, it has not been clear whether the oxytocin is released via dendritic mechanisms and then spreads passively to the amygdala or is released via classic axonal mechanisms (Landgraf and Neumann, 2004). To address this issue, Knobloch et al. (2012) transduced hypothalamic oxytocin neurons using an oxytocin promoter. Optical stimulation of these neurons' axonal terminals in the CEA produced oxytocindependent effects on CEA neuronal activity, indicating that the hypothalamic neurons are able to directly release oxytocin from their axon terminals. In particular, optical stimulation appeared to increase neuronal activity in the lateral CEA and inhibit activity in medial CEA neurons. Anatomical analysis found that the hypothalamic inputs terminated in the lateral, but not medial, CEA. A separate experiment providing stimulation during a test for contextual fear conditioning found an attenuation of freezing behavior with the optical stimulation. Indeed, the overall evidence from this work suggests that the hypothalamic oxytocin neuronal input to the CEA makes synaptic contacts on the lateral, rather than medial, aspect of the CEA, consistent with the function of the lateral CEA in inhibiting medial CEA output and reducing the expression of fear and fear-related behavior. On a larger level, these findings contribute to our understanding of how specific inputs, and even genetically distinct inputs, to the amygdala regulate both behavior and local circuit activity.

## **AMYGDALA AND MEMORY**

Other work has focused on the use of optogenetics to understand the relationship between the amygdala and memory, especially aversive learning such as fear conditioning. One of the earliest uses of optogenetics in studies of the amygdala examined whether optogenetic stimulation of the lateral amygdala, combined with tones, produces fear conditioning (Johansen et al., 2010). In these experiments, the authors targeted the pyramidal cells of the lateral amygdala. These early findings confirmed that different frequencies of light produced robust firing in lateral amygdala neurons, though with the early version of ChR2 used, the fidelity of the neurons' responding to 50 Hz light pulses was not as good as that found in response to 20 Hz light pulses. Nonetheless, with 20 Hz stimulation, there was a strong c-fos response in the neurons. Optical stimulation, paired with a tone, produced an increase in freezing both during training and in a later retention test. However, the authors noted that the levels of freezing were considerably lower than had been found in previous studies, an effect that the authors suggest indicates that other mechanisms must also be involved in order to produce full fear conditioning. These findings contributed to previous work showing that activity in the lateral amygdala is critical for the normal development and retention of tone fear conditioning. Moreover, through the use of optogenetics, these experiments were able to selectively target the pyramidal cells while providing temporally precise stimulation, setting the stage for future experiments to build on these early results and develop a clearer understanding of the amygdala's role in fear conditioning and memory.

Our own work has used an optogenetic approach to examining the role of the BLA in modulating memory consolidation for inhibitory avoidance, a similar aversive learning task (Huff et al., 2013). Prior work suggested that BLA activity in the gamma frequency range (35–45 Hz) is important for synchronizing activity in downstream structures and promoting the consolidation of learning (Bauer et al., 2007; Popescu et al., 2009). However, such work has depended on physiological recordings, which cannot determine whether driving activity in that range alters memory consolidation. Therefore, in our experiments, the BLA pyramidal neurons were transduced with ChR2(E123A), a "ChETA" version of channelrhodopsin that permits high-fidelity responding to light pulses up to 200 Hz (Gunaydin et al., 2010; Yizhar et al., 2011). We found that stimulating the BLA pyramidal cells with bursts of gamma-frequency light pulses (40 Hz) for 15 min immediately after inhibitory avoidance training enhanced retention 2 days later (Huff et al., 2013). Stimulation with bursts of 20 Hz pulses did not produce a significant effect on retention. Previous work has shown that other types of posttraining stimulation (e.g., electrical) of the amygdala produce an inverted-U curve with regard to retention (Gold et al., 1975), but it is not known whether optical stimulation of the BLA also produces such an effect. In another groups of rats, we inhibited BLA neuronal activity immediately after training via activation of the outward proton pump ArchT and found that 15 min of neuronal inhibition, but not 1 min, impaired retention of the learning (Huff et al., 2013). These findings indicated that BLA stimulation in the gamma frequency range enhances memory consolidation and, critically, have provided a set of effective parameters for using optogenetic approaches to modulate memory consolidation that we are continuing to use in new experiments addressing BLA interactions with efferent brain regions.

Other work has investigated a subpopulation of BLA neurons to examine its role in fear conditioning (Jasnow et al., 2013). Specifically, Jasnow et al. targeted the glutamatergic pyramidal cells found in the BLA by driving ChR2 expression with the Thy1 promoter, which limits expression to a specific subpopulation of glutamatergic cells in the BLA and other forebrain regions (Sugino et al., 2006). Optical stimulation of this specific class of BLA neurons during tone fear conditioning impaired retention of the learning while having no effect on the expression of freezing itself during the conditioning (Jasnow et al., 2013). Moreover, optical stimulation of the neurons paired with the tone alone during extinction training enhanced the retention of the extinction learning, again without having any effect on the freezing itself during the extinction training. Generally, it has been thought that activity in the lateral amygdala and medial CEA drive the expression of fear, but electrophysiological characterization of this subpopulation suggests that it shunts activity in lateral amygdala neurons and inhibits activity of medial CEA neurons. Indeed, optical activation of this neuronal subpopulation had no effect on the acute expression of fear but, rather, appeared to influence consolidation specifically for memories that *oppose* fear conditioning.

Recent work has also investigated the interactions of distinct subpopulations within the BLA with efferent targets in relation to fear conditioning. Prior work has noted the existence of neurons within the basal nucleus of the BLA that are responsive to cues associated with footshocks and other neurons that are responsive to cues previously associated with footshocks that have been extinguished. Senn et al. (2014) investigated whether such neurons also show distinct projection patterns to the medial prefrontal cortex (mPFC). To perform a functional investigation of these subpopulations of neurons, retrogradely transported viruses containing Cre-recombinase were injected into either the prelimbic (PL) or infralimbic (IL) cortex while a conditional viral vector expressing the opsins in a Cre-dependent manner was injected into the BLA. As a result, the opsins were selectively expressed in the IL-projecting neurons or the PLprojecting neurons, enabling illumination of the entire BLA to only stimulate or inhibit the specific subpopulation of neurons. Consistent with previous work suggesting a dichotomy between the dorsal regions of the mPFC (PL) and the ventral regions (IL) with regard to fear conditioning (Peters et al., 2009), the authors demonstrated that BLA neurons projecting to the PL are activated by unextinguished cues whereas those projecting to the IL are activated by extinguished cues. Moreover, inhibition of IL-projecting neurons during cue extinction training produced a significant impairment in the retention of the extinction learning, compared to stimulating such neurons. Conversely, inhibition of the PL-projecting neurons during such training enhanced the retention of the extinction learning, compared to stimulating such neurons. While much previous research has focused on the mPFC inputs to the BLA in regulating fear extinction and expression, these findings provide evidence that BLA inputs to distinct mPFC regions also differentially influence such behaviors.

## **AMYGDALA AND REWARD AND ADDICTION**

Considerable evidence indicates that the amygdala is involved in reward and addiction-related behaviors (Everitt et al., 1999; See et al., 2003; Di Ciano and Everitt, 2004). As part of understanding the precise role played by the BLA in such behaviors, Stuber et al. (2011) examined the pathway from the BLA to the nucleus accumbens (NA) core in modulating and driving reinforcement of operant behavior. Although previous evidence had suggested that the BLA could directly influence dopamine release in the NA (Howland et al., 2002), a key mechanism for reinforcement, it was not clear whether activity in the BLA inputs to the NAcore could actually reinforce operant behavior or was necessary for reward seeking. Stuber et al. found that mice engaged in an operant behavior (nose pokes) in order to receive optical stimulation (burst of 20 Hz light pulses) of the BLA axon terminals in the NAcore. Similarly, optical inhibition, via activation of halorhodopsin, of the BLA terminals decreased responding for a sucrose reward. Strikingly, optical stimulation of mPFC inputs to the NAcore did not produce the same effects. Other work has shown that optical stimulation of BLA inputs to the NAshell are also reinforcing (Britt et al., 2012), though, in contrast to the mPFC inputs to the NAcore, stimulation of mPFC inputs or ventral hippocampal inputs to the NAshell was also found to be reinforcing. Of particular interest in this study was that the authors used the ability to selectively control different glutamatergic inputs to the NAshell, via illumination of opsin-transduced axons from different regions, to determine the degree of plasticity in the connections following cocaine exposure. Together, these findings illustrate not only the increasing evidence for a role for BLA glutamatergic inputs to the NA in regulating reinforcement and reward but also that, depending on the part of the NA targeted, other glutamatergic inputs do not necessarily produce identical results.

Other studies have indicated that, consistent with its role in different forms of associative learning, the BLA is a key part of the neural circuit that drives drug-seeking behavior triggered by drug-associated cues (See et al., 2003). However, as the BLA projects to both the PL and the NAcore, both of which are considered critical components of a general drug-seeking circuit (McFarland and Kalivas, 2001; McFarland et al., 2004; LaLumiere and Kalivas, 2008), it has not been clear which pathway is responsible for driving cue-induced drug-seeking. Recent work by Stefanik and Kalivas (2013) has used optogenetics to investigate this issue. The BLA was transduced with ArchT. Inhibition of BLA terminals in either the PL or NAcore reduced cue-induced reinstatement of cocaine-seeking, indicating that activity in both pathways is obligatory for associative cue-driven cocaine-seeking.

# **CIRCUITRY OF THE AMYGDALA AND INTERACTIONS WITH OTHER BRAIN REGIONS**

A number of studies have used optogenetic approaches to develop a better understanding of the functional connections between the amygdala and other brain regions. For example, Li et al. (2012) have investigated the role of kappa opioid receptor signaling in the bed nucleus of the stria terminalis (BNST). Patch-clamp recordings in the BNST provided evidence that activation of kappa opioid receptors inhibits GABAergic transmission via presynaptic mechanisms. As the CEA provides an important GABAergic input to the BNST, one that has been implicated as a critical pathway in the central stress system (Jasnow et al., 2004; Walker and Davis, 2008), the authors used optogenetics to target and control activity in this pathway. The results indicated that kappa opioid receptor activation inhibited GABAergic transmission in this pathway specifically, a result that would have been difficult to demonstrate using other techniques.

Other research has used optogenetics to delineate precisely how the amygdala influences activity in other regions. Luna and Morozov (2012) contrasted the microcircuitry of BLA inputs vs. anterior piriform inputs to the posterior piriform cortex. Although both structures were found to innervate deep pyramidal cells of the posterior piriform, the BLA and anterior piriform connected with different kinds of interneurons. Specifically, the BLA produced strong connections with fast-spiking interneurons, whereas the anterior piriform had its strongest synapses on irregular-spiking interneurons. As these different classes of interneurons synapse on different regions of the pyramidal cells (somatic vs. distal dendritic), the feedforward inhibition from BLA vs. anterior piriform inputs would be expected to have profoundly different effects on the likelihood of spiking in the principal cells of the posterior piriform.

Several studies have used optogenetics to understand amygdala function and interactions in combination with a variety of other techniques, an approach that will likely dominate much future work as optogenetics affords significant advantages in our ability to gain deeper knowledge regarding neural circuitry. For example, experiments have focused on the well-known connections between the BLA and the mPFC. The reciprocal connections between these regions appear to be involved in a wide variety of behavioral and higher cognitive functions. Yet, the mPFC receives inputs from many other structures and the distinctions among the connections formed by these inputs have not been clear. Therefore, Little and Carter (2012) investigated how BLA, ventral hippocampal, midline thalamus, and contralateral mPFC inputs to the layer two pyramidal neurons of the mPFC make synaptic connections. The authors used optogenetics to target specific pathways by transducing the efferent structures with ChR2 and providing illumination to their axonal terminals in the mPFC. Moreover, they combined their optogenetic manipulations with two-photon microscopy in order to determine the functional connections on a subcellular level. The results from this study indicate that the different regions do, in fact, make different subcellular connections. The BLA appears to make synaptic connections much closer to the soma, relative to other regions, especially the thalamic inputs. Additionally, the BLA inputs target spines of an "intermediate" size, along with ventral hippocampal inputs, in contrast to the thalamic inputs to the large spines and the contralateral mPFC inputs to the small spines. As both the size of the spine and the distance from the soma govern the relative strengths of the inputs, these findings shed light on how different regions influence local circuit activity in other regions. In a follow-up study, Little and Carter (2013) extended their findings, again using optogenetics combined with two-photon microscopy. In this case, their findings indicated that BLA inputs to the mPFC were considerably stronger on mPFC neurons that innervated the BLA, compared to mPFC neurons that provide inputs to the contralateral mPFC. Together, these findings have contributed to a deeper understanding for how the BLA and mPFC interact and, critically, provide a foundation for understanding how BLA inputs to the mPFC may regulate PFC activity and PFCdependent functions.

Work has examined other brain regions' inputs to the amygdala. In a recent study, Carter et al. (2013) found evidence of a circuit involving projections from the parabrachial nucleus in the brainstem to the CEA that suppresses appetite. After genetically identifying and targeting neurons in the parabrachial nucleus to determine their ability to suppress appetite in mice, the authors then transduced these cells with ChR2 and provided illumination to downstream targets. Although stimulating parabrachial axon terminals in the BNST had no effect on food intake, stimulation between 20–40 Hz of the axon terminals in the lateral CEA reduced food consumption. In contrast to many studies that have used 20 Hz stimulation but consistent with our own work (e.g., Huff et al., 2013), Carter et al. found that 20 Hz stimulation did not produce as strong a behavioral effect as found with the 30 and 40 Hz stimulation. By utilizing both the genetic targeting ability of combining optogenetics with transgenic mice and by targeting the axon terminals, these findings provide a significant step forward in understanding how genetically distinct neuronal populations connect with different regions in the brain and, in turn, regulate appetite-related behavior.

Optogenetic studies have also targeted specific interneuron populations in the BLA to understand local circuits. Chu et al. (2012) examined how dopamine influences parvalbumin-positive interneurons in the BLA and, thereby, influence principal cell activity. This issue is of importance because previous work has shown that dopamine influences BLA activity and modulates memory consolidation (Bissiere et al., 2003; LaLumiere et al., 2004, 2005). Prior work has found that D2 receptor activation in the BLA suppresses feedforward inhibition, thereby providing a gating mechanism for synaptic plasticity in the amygdala (Bissiere et al., 2003), while other work has also found that dopamine disinhibits the BLA via inhibition of intercalated cells in a D1 receptor-dependent manner (Marowsky et al., 2005). Using a Cre line of transgenic mice, Chu et al. were able to target ChR2 expression to the parvalbumin-positive cells of the BLA, which are believed to be the major class of interneurons in the structure. The authors then demonstrated that dopamine selectively reduced GABAergic transmission to principal cells, but not to other interneurons, and that this occurred in a presynaptic D2 receptordependent mechanism. These findings provide additional confirmation of the critical role of dopamine, and especially D2 receptors, in modulating BLA activity. Moreover, these findings were demonstrated in a specific subclass of interneurons, an important issue as other work has suggested that different stimuli influence different subtypes of interneurons in the BLA (Bienvenu et al., 2012).

## **CONCLUSIONS**

As a heterogeneous collection of nuclei with a variety of influences on neural functioning and behavior, the amygdala has been the subject of countless neurobiological studies. Our ability to investigate this structure and its role in the brain and behavior has been significantly altered by the development of optogenetics. The studies reviewed here and listed in **Table 1** have provided an understanding for the kinds of questions that this approach can address with regard to the amygdala and, critically, have provided roadmaps for continuing research into amygdala functioning. The findings from these studies indicate that the local circuitry in the amygdala regulates a number of emotion-related behaviors and have produced a clearer picture for how this circuitry works. Other work has begun providing glimpses into how the BLA influences activity in efferent structures, even delineating the local

microcircuits to determine the precise differences between BLA inputs to the region compared to other inputs. Moreover, other studies presented here have demonstrated that specific frequencies of BLA activity differentially influence behavior and memory consolidation. These findings will prove invaluable as neuroscience builds a deeper understanding of the full connectivity of the nervous system on a functional level. All of this work and the findings from these experiments, however, would not have been possible without the temporal, spatial, and/or genetic precision afforded by optogenetics. As future studies utilizing optogenetic approaches build upon these early findings, we should expect considerable advancement in our understanding of amygdala functioning in behavior and in interactions with other brain systems.

#### **ACKNOWLEDGMENTS**

The author would like to acknowledge NIH support through grants MH097111 (Ryan T. LaLumiere) and DA034684 (Ryan T. LaLumiere).

## **REFERENCES**

amygdala. *J. Neurosci.* 33, 15333–15342. doi: 10.1523/JNEUROSCI.2385-13. 2013

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

*Received: 03 February 2014; accepted: 13 March 2014; published online: 26 March 2014.*

*Citation: LaLumiere RT (2014) Optogenetic dissection of amygdala functioning. Front. Behav. Neurosci. 8:107. doi: 10.3389/fnbeh.2014.00107*

*This article was submitted to the journal Frontiers in Behavioral Neuroscience*.

*Copyright © 2014 LaLumiere. 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*.

# *Ex vivo* dissection of optogenetically activated mPFC and hippocampal inputs to neurons in the basolateral amygdala: implications for fear and emotional memory

#### *Cora Hübner 1,2†, Daniel Bosch1†, Andrea Gall 1, Andreas Lüthi 3 and Ingrid Ehrlich1 \**

*1 Hertie Institute for Clinical Brain Research and Centre for Integrative Neuroscience, University of Tuebingen, Tuebingen, Germany*

*2 Graduate School of Neural and Behavioral Sciences, IMPRS, Tuebingen, Germany*

*3 Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland*

#### *Edited by:*

*Anton Ilango, National Institutes of Health, USA*

#### *Reviewed by:*

*Pankaj Sah, Queensland Brain Institute, Australia Francisco Sotres-Bayon, Universidad Nacional Autónoma de México, Mexico*

#### *\*Correspondence:*

*Ingrid Ehrlich, Hertie Institute for Clinical Brain Research and Centre for Integrative Neuroscience, Otfried-Mueller-Str. 25, 72076 Tuebingen, Germany e-mail: ingrid.ehrlich@ uni-tuebingen.de*

*†These authors have contributed equally to this work.*

Many lines of evidence suggest that a reciprocally interconnected network comprising the amygdala, ventral hippocampus (vHC), and medial prefrontal cortex (mPFC) participates in different aspects of the acquisition and extinction of conditioned fear responses and fear behavior. This could at least in part be mediated by direct connections from mPFC or vHC to amygdala to control amygdala activity and output. However, currently the interactions between mPFC and vHC afferents and their specific targets in the amygdala are still poorly understood. Here, we use an *ex-vivo* optogenetic approach to dissect synaptic properties of inputs from mPFC and vHC to defined neuronal populations in the basal amygdala (BA), the area that we identify as a major target of these projections. We find that BA principal neurons (PNs) and local BA interneurons (INs) receive monosynaptic excitatory inputs from mPFC and vHC. In addition, both these inputs also recruit GABAergic feedforward inhibition in a substantial fraction of PNs, in some neurons this also comprises a slow GABAB-component. Amongst the innervated PNs we identify neurons that project back to subregions of the mPFC, indicating a loop between neurons in mPFC and BA, and a pathway from vHC to mPFC via BA. Interestingly, mPFC inputs also recruit feedforward inhibition in a fraction of INs, suggesting that these inputs can activate dis-inhibitory circuits in the BA. A general feature of both mPFC and vHC inputs to local INs is that excitatory inputs display faster rise and decay kinetics than in PNs, which would enable temporally precise signaling. However, mPFC and vHC inputs to both PNs and INs differ in their presynaptic release properties, in that vHC inputs are more depressing. In summary, our data describe novel wiring, and features of synaptic connections from mPFC and vHC to amygdala that could help to interpret functions of these interconnected brain areas at the network level.

**Keywords: medial prefrontal cortex, hippocampus, amygdala, conditioned fear, optogenetics**

## **INTRODUCTION**

Emotional information is processed in distinct neural circuits. Salient emotions such as fear and anxiety are among those most intensely investigated, because resulting behaviors can be easily evoked and studied in the laboratory and the underlying brain areas are highly conserved among mammalian species from mice to man (Ledoux, 2000; Phelps and Ledoux, 2005). The most powerful models used to date to elucidate the neural circuits and mechanisms that control fear are classical Pavlovian fear conditioning and extinction of acquired fear (Maren, 2001; Ehrlich et al., 2009; Herry et al., 2010; Pape and Pare, 2010). Fear conditioning involves pairing of a previous neutral stimulus (CS) with an aversive stimulus (US), such that a CS-US association is formed. During extinction training, the CS is repeatedly presented without the US, which leads to a decrease in the learned fear response. Many lines of evidence suggest that fear and extinction learning create two distinct memory traces, and which memory is retrieved depends on the retrieval context (Bouton et al., 2006; Myers and Davis, 2007; Quirk and Mueller, 2008).

Although the amygdala is one of the most important brain areas for mediating fear and its extinction, the medial prefrontal cortex (mPFC) and hippocampus (HC), structures that are reciprocally connected to the amygdala, are implicated in aspects of acquisition, consolidation and retrieval of fear and extinction memories (Myers and Davis, 2007; Quirk and Mueller, 2008; Maren, 2011). Projections from mPFC to amygdala originate from layers 2 and 5 of different subregions, including the prelimbic (PL) and infralimbic (IL) areas and form asymmetric synapses (Pinto and Sesack, 2000, 2008). Tracing studies are not completely consistent regarding target nuclei in the amygdala. Projections from PL appear to target mainly the basal nucleus of the basolateral amygdala (BA) and portions of the capsular subdivision of the central amygdala (CeC), whereas IL projections are generally less dense and target large parts of the amygdaloid complex including the intercalated cells, and more densely a specialized lateral part of the CeC, and the ventromedial part of the LA as well as the magnocellular division of the BA (McDonald et al., 1996; Vertes, 2004; Pinard et al., 2012). Moreover, PL and IL receive amygdala projections originating mainly from the BA (Conde et al., 1995; Hoover and Vertes, 2007). Recently it has been shown that BA neurons projecting to IL and PL have opposing roles in expression of fear following extinction learning (Senn et al., 2014). Together, this raises the possibility that IL and PL may interact with the amygdala by virtue of their reciprocal connections to influence the outcome of fear and extinction learning. Projections from HC to amygdala originate in the temporal subiculum and the adjacent part of CA1. Subicular projections are dense in the accessory basal (AB) and medial part of BA, but moderate in LA and light in central amygdala, while CA1 projections to the amygdala mainly terminate in the BA, with lighter projections to LA and AB (Canteras and Swanson, 1992; Pitkanen et al., 2000). The ventral HC (vHC) is thought to contribute contextual information following extinction learning either via direct amygdala projections or indirectly by strong projections to the mPFC, which subsequently projects to the amygdala (Hoover and Vertes, 2007; Pape and Pare, 2010; Orsini et al., 2011).

Systems-level studies started to elucidate specific functions of mPFC and hippocampal regions and their interactions with the amygdala in fear learning, fear expression and extinction of fear (Maren and Quirk, 2004; Pape and Pare, 2010; Maren, 2011). For example, synchronization of activity in amygdala-hippocampalprefrontal cortical circuits plays a critical role in anxiety, acquired fear, extinction learning, and fear discrimination (Seidenbecher et al., 2003; Lesting et al., 2011; Likhtik et al., 2014), but the underlying connectivities of neurons and microcircuits are still incompletely understood. The mPFC appears to play a double role in high and low fear states. Activation of the IL suppresses fear by suppressing amygdala output possibly via intercalated cells and central amygdala inhibition (Quirk et al., 2003; Paré et al., 2004; Maren, 2011), and/or via local BLA interneurons (Rosenkranz and Grace, 2001, 2002). The PL is thought to excite the amygdala to increase fear output during fear expression and renewal (Vidal-Gonzalez et al., 2006; Orsini et al., 2011; Sierra-Mercado et al., 2011). However, *in vivo* recordings of neuronal responses in the BLA during mPFC stimulation have yielded conflicting results about amygdala activation (Rosenkranz and Grace, 2001, 2002; Likhtik et al., 2005). Also, few data are available on how hippocampal activity influences BLA activity (Maren and Fanselow, 1995; Hobin et al., 2003; Maren and Hobin, 2007). It has been proposed that both hippocampal and PL projections to the BA mediate context-dependent fear renewal (Orsini et al., 2011), but if and how these inputs converge in the BA has not been studied.

Thus, a key open question that will guide our understanding and interpretation of systems-level functions and mechanisms, is to decipher the functional connectivities in amygdalahippocampal-prefrontal circuits including innervation of distinct cell types, delineation of similarities or differences in synaptic input properties, and the recruitment of specific microcircuits. Here, we use an *ex vivo* optogenetic approach to study the properties of mPFC and vHC inputs to specific subtypes of BA neurons and describe distinct wiring principles and synaptic properties between these three structures.

# **MATERIALS AND METHODS**

# **ANIMALS**

For all experiments, we used adult male mice (8–12 week old at time of slice recordings) of the following lines: C57BL/6J (Harlan, Netherlands), glutamate decarboxylase 67 (GAD67)–green fluorescent protein (GFP) transgenic mice (Tamamaki et al., 2003) backcrossed to C57BL/6J, and Parvalbumin-Cre (PV-Cre, Jackson stock 008069) mice crossed to Ai14 reporter mice (Jackson stock 007914) that were backcrossed to C57BL/6J. All experimental procedures were in accordance with the EU directive on use of animals in research and approved by the Regierungspraesidium Tuebingen, state of Baden-Wuerttemberg, Germany.

## **STEREOTACTIC INJECTIONS**

Four to six week old mice were maintained under isoflurane anesthesia, fixed in a stereotactic frame (Stoelting, USA) and injected bilaterally in either the mPFC or ventral hippocampus or a combination of both at the following coordinates from bregma (in mm). mPFC: posterior 1.9, lateral ±0.3, ventral −2.1; ventral hippocampus: posterior −3.1, lateral ±3.4, ventral −3.8. Pressure injections were performed using glass capillaries (1B150F-4, WPI, Germany) attached to a Toohey Spritzer (Toohey Company, USA). For mPFC inputs, the mPFC was injected either with 0.5μl of rAAV-CAG-hChR2(H134R) mCherry (serotype 2/1 or 2/9, Penn Vector Core, USA) alone or with a 0.5μl mix of rAAV-hSyn.hChR2(H134R)-eYFP (serotype 2/9, Penn Vector Core, USA) and red retrobeads (Lumafluor, USA). For hippocampal inputs, the ventral hippocampus was injected with 0.5μl rAAV-hSyn.hChR2(H134R)-eYFP and the mPFC was injected with 0.4μl red retrobeads. In all cases, viral preps were diluted such that they had comparable titers (1 × 1012 GC/ml). Retrobeads were dialyzed against 0.32 M sucrose prior to use to avoid osmotic damage of the tissue. Four to six weeks postinjection, amygdala slices were prepared for slice recordings.

## **SLICE RECORDINGS**