Supplementary Methods. the ventrolateral orbitofrontal cortex (VLO) and basolateral amygdala (BLA). AAV8-CaMKII-HAhM
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1 Supplementary Materials, Zimmermann et al. Supplementary Methods Surgery. AAV5-CaMKII-HA-hM 4 D(Gi)-IRES-mCitrine or AAV5-CaMKII-GFP was infused into the ventrolateral orbitofrontal cortex (VLO) and basolateral amygdala (BLA). AAV8-CaMKII-HAhM 4 D(Gi)-IRES-mCitrine or AAV8-CaMKII-GFP was infused into the dorsolateral striatum (DLS). All viral vectors were purchased from the UNC Viral Vector Core. Mice were anesthetized with Ketamine/Dexdomitor (75 mg/kg/1 mg/kg, i.p.). Mice were placed in a digitized stereotaxic frame (Stoelting), the head shaved, scalp incised, skin retracted, and head leveled. Two burr holes were drilled, and viral vectors were infused bilaterally. VLO coordinates were AP+2.6, ML+1.2, DV-2.8, 0.5µl/side. BLA coordinates were AP-1.4, ML±3.0, DV-4.9, 0.25 µl/side. DLS coordinates were AP+0.5, ML+/-2.7, DV-3.5, 0.4 µl/side. Infusions were delivered 0.05 µl/minute, with the needle left in place for 5 additional minutes. The scalp was sutured and mice revived with Antisedan (1 mg/kg, i.p.). Mice were allowed >2 weeks for recovery before behavioral training or physiological experiments. Histology. Mice used in behavioral tests were deeply anesthetized and transcardially perfused with chilled 4% paraformaldehyde. Brains were rapidly removed and stored in chilled 4% paraformaldehyde for 24 hours, followed by 30% sucrose for >48 hours. The brains were then sectioned at 55µm on a microtome held at -15 C; sections were stored in cryoprotectant. Every third section was mounted and coverslipped using Vectashield Mounting Media. GFP (controls) or mcitrine (Gi-DREADDs) was imaged on a Nikon Eclipse inverted microscope. Electrophysiological recordings. Slice preparation 1
2 350µm sections containing the VLO were obtained as described (Rainnie, 1999). Briefly, mice were anesthetized with isoflurane, and brains were rapidly dissected and immersed in a 4 C 95-5% oxygen/carbon dioxide oxygenated cutting solution, made up of (in mm): NaCl (130), NaHCO3 (30), KCl (3.5), KH2PO4 (1.1), MgCl2 (6.0), CaCl2 (1.0), glucose (10), and supplemented with kynurenic acid (2.0). VLO-containing sections were cut using a Leica VTS Vibratome (Leica Microsystems Inc.) and maintained at 37 C in oxygenated cutting solution for at least 50 minutes prior to being transferred to artificial cerebrospinal fluid (ACSF), made up of: NaCl (130), NaHCO3 (30), KCl (3.5), KH2PO4 (1.1), MgCl2 (1.3), CaCl2 (2.5), and glucose (10). Slices were maintained in ACSF at least 30 minutes prior to recording. Patch clamp recording Patch clamp recordings and LTP induction were conducted as previously described (Li et al., 2011). Briefly, following tissue preparation, 350µm brain slices containing the VLO were transferred to a submersion-type recording chamber mounted on a fixed stage Leica DMLFS microscope (Leica Microsystems Inc.) and continuously perfused by gravity-fed oxygenated ACSF at 32 C with a flow rate of 1-2ml/minute. Neurons were identified using differential interference contrast (DIC) optics and infrared (IR) illumination with an IR sensitive CCD camera (Orca ER). Thin-walled borosilicate glass patch electrodes, which had a resistance of 4-6MΩ were filled with (in mm): K-gluconate (130), KCl (2), HEPES (10), MgCl2 (3), K-ATP (2), NaGTP (0.2), and phosphocreatine (5), adjusted to ph 7.3 with KOH, and having an osmolarity of mosm. AAV-transfected VLO neurons were first identified as being either GFP- or mcitrinepositive using fluorescence microscopy. Neurons were then visualized for recording using DIC microscopy in combination with a 40x water immersion objective and displayed in real time on a computer monitor. Data acquisition and analysis were performed using a MultiClamp700B amplifier in conjunction with pclamp10.0 software and a DigiData 1320A AC/DA interface 2
3 (Molecular Devices). Whole cell patch clamp recordings were obtained, and recorded voltages were low-pass filtered at 5kHz and digitized at 10-20kHz. Excitatory postsynaptic currents (EPSCs) onto VLO neurons were evoked by placing a concentric bipolar stimulation electrode (FHC) approximately 500µm from the recorded neuron, close to the fiber tract of the forceps minor of the corpus callosum immediately adjacent to the VLO. To isolate evoked EPSCs, 50µM picrotoxin was added to the patch solution to block GABA A currents exclusively in the recorded neuron. Slices were continuously perfused with oxygenated ACSF (32 C) containing the selective GABA B receptor antagonist, CGP36742 (5µM). This recording configuration allowed for stable recording of isolated EPSCs without contamination from epileptiform, recurrent EPSCs. EPSCs (adjusted to 30% of maximal response) were evoked at 0.05Hz, a 10-minute baseline period was recorded, and recordings continued for up to 40 minutes after LTP induction. Initial EPSC amplitudes were normalized to the average of the baseline EPSC amplitude. To induce LTP, a 5x high frequency stimulation (HFS) LTP protocol was employed, consisting of 5 trains of 100Hz stimulation delivered for 1 second, with a 20-second interval between each train. As previously reported, this form of LTP is NMDA-receptor-dependent (Woo et al., 2003). During EPSC measurement, a negative DC holding current was injected into the neurons to maintain the membrane potential at -70mV, except during HFS when the potential was adjusted to -60mV to facilitate spike firing. To calculate the paired-pulse ratio (PPR), two EPSCs were evoked with an interstimulus-interval of 50ms, and PPR was calculated as (eepsc 1 /eepsc 2 ), where eepsc 1 and eepsc 2 represent the amplitude of the first and the second eepsc, respectively. PPR was measured before, immediately after, as well as 10 minutes and 30 minutes after 5xHFS. Clozapine-N-Oxide (CNO) application and assessment of intrinsic membrane properties 3
4 To examine the effects of CNO application on the intrinsic membrane properties of VLO neurons, and their response to low frequency (0.05 Hz) afferent stimulation, neurons were first held at -60 mv and a series of transient hyperpolarizing and depolarizing current injection steps were applied and the voltage response measured, as previously described (Rainnie, 1999). CNO (100µM) was then applied in the ACSF for a minimum of 10 minutes using continuous gravity-fed bath application at a flow rate of 2ml/min, during which time the evoked EPSC amplitude was continuously monitored. In our experience, complete turnover of the bath ACSF is achieved in ~1 minute. Immediately prior to the initiation of the LTP stimulation protocol, the intrinsic membrane properties of VLO neurons were measured again in the presence on CNO to compare with pre-cno membrane properties. Final concentration of DMSO was <0.1%. 4
5 Supplementary Figure 1 Supplementary figure 1: Activation of Gi-DREADDs in the VLO increases the threshold for LTP induction. (a) The forceps minor of the corpus callosum was stimulated, and recordings were collected from nearby Gi-DREADD-infected VLO neurons, identified by the expression of mcitrine (inset). (b) No apparent differences in the resting membrane input resistance or action potential firing rate during application of CNO (100µM) were detected. While somewhat surprising, we note that our recordings lasted only ~10 minutes. In some reports, CNO-induced reductions in fepsc amplitude with CaMKII-driven Gi-DREADDs are delayed following CNO application relative to, for example, elevations induced by excitatory Gq-DREADDs; meanwhile, effects on LTP are rapid (e.g., Lopez et al., 2016). In other words, the brevity of our recording period could account for the lack of effect on intrinsic membrane properties. Whether Gi-DREADD stimulation hyperpolarizes all neuron types has also been questioned (Li and Rainnie, 2014; Roth, 2016). (c) Next, evoked-epscs were recorded for 15 minutes prior to CNO application to establish a baseline measure of EPSC amplitude. Following a 10-minute bath application of CNO (100µM), GFP- or Gi-DREADD-infected neurons (1/mouse) were subjected to 5 bursts of HFS to induce 5
6 LTP. Gi-DREADD-expressing neurons showed significantly attenuated LTP magnitude [interaction F (10,170) =3.7,p<0.001]. Time of CNO application is marked by the blue horizontal line. Symbols represent means+sems, **p<0.001 following interactions. (d) Normalized EPSC amplitudes 30 minutes following HFS are also shown. Each symbol represents one neuron. 6
7 Supplementary Figure 2 Supplementary figure 2: Preference scores associated with appetitive conditioning tests reported in the main text. Response rates during the choice tests in the main text were converted to preference scores by dividing the responses associated with the nondegraded contingency by those associated with the degraded contingency. Values >1 reflect a preference for the response most closely aligned with reinforcement, i.e., a goal-directed response strategy that reflects new learning regarding response-outcome contingencies. Scores of 1 reflect chance-level responding. Scores were compared against 1 (no preference) by 1-sample t-test. The p values are indicated within each bar, with asterisks noting values <
8 (a) When the VLO was inactivated prior to conditioning, all mice generated a preference for the response most likely to be reinforced, indicated by values >1. (b) When the VLO was inactivated following response-outcome contingency degradation, inactivated mice initially generated a preference, but this preference decayed by test 2. (c) BLA inactivation had rapid effects, causing chance-level responding at the equivalent of test 1 in our VLO experiments above. Impairments were detected following 1 and 10 mg/kg CNO. 8
9 Supplementary Works Cited Li C, Dabrowska J, Hazra R, Rainnie DG (2011) Synergistic activation of dopamine D1 and TrkB receptors mediate gain control of synaptic plasticity in the basolateral amygdala. PLoS ONE. 6:e Li C, Rainnie DG (2014) Bidirectional regulation of synaptic plasticity in the basolateral amygdala induced by the D1-like family of dopamine receptor and group II glutamate receptors. J Physiol 592: Lopez AJ, Kramar E, Matheos DP, White AO, Kwapis J, Vogel-Ciernia A, Sakata K, Espinoza M, Wood MA (2016) Promoter-specific effects of DREADD modulation on hippocampal synaptic plasticity and memory formation. J Neurosci. 36: Rainnie DG (1999) Serotonergic modulation of neurotransmission in the rat basolateral amygdala. J Neurophysiol. 82: Roth BL (2016) DREADDs for neuroscientists. Neuron 89: Woo NH, Duffy SN, Abel T, Nguyen PV (2003) Temporal spacing of synaptic stimulation critically modulates the dependence of LTP on cyclic AMP-dependent protein kinase. Hippocampus. 13:
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