Nociceptin/Orphanin FQ (N/OFQ) and its receptor (NOP) make up a neuropeptide observations indicate that NOP actions vary not only by brain region and neuron subpopulation, 89 but also by structural localization within a neuron.
Electrophysiology: Most experiments were completed in tissue from male Sprague 93 Dawley rats, p22 -p36, except mechanism experiments which were completed in tissue from 94 adult rats (>200g). Rats were anesthetized with isoflurane, and brains were removed. The 95 brains were submerged in Ringer's solution containing (in mM): 119 NaCl, 2.5 KCl, 1.3 MgSO4, 96 1.0 NaH2PO4, 2.5 CaCl2, 26.2 NaHCO3, and 11 glucose saturated with 95% O2-5% CO2 and 97 horizontal brain slices (150 µm thick) containing the VTA were prepared using a Vibratome 98 (Leica Instruments, Nussloch, Germany). Slices were and allowed to recover at 35°C for at 99 least 1 hr before recordings were initiated. The same Ringer's solution was used for cutting, 100 recovery, and recording. 127 or pressure injected (2 min) only after a 5 min stable baseline was achieved. Responses were such as the desensitization experiments, were completed with bath application. To test that 133 observed N/OFQ-mediated effects were specific to NOP, the selective NOP antagonist BTRX-134 246040 (10 or 100 nM) was applied for 10 min prior to N/OFQ. As there was no statistical 135 difference in the mean amplitude of response for bath application and pressure injection the 136 results were combined for the analysis.
For iontophoresis experiments, the holding current was set to -50 mV to increase the 138 driving potential for Cl -. GABA (100 mM, pH adjusted to 4.9 with 37% HCl) was prepared daily 139 and the GABA-containing pipette was positioned approximately 50 μm away from the recorded 140 neuron. Negative retention current (approximately -35 nA) was applied to the GABA pipette,
interrupted by positive ejection current pulses (100 ms) once every 30 s, with the intensity 142 adjusted so that the response amplitude was in the range of 100-300 pA. 
−4.0 from skull surface), the pACC (AP, 1.6; ML, ± 0.6; V, −3.5), or the NAc (AP, +1.5; ML, ± 152 0.8; V, −6.7). Neuro-DiI (7% in ethanol; Biotium) was slowly injected, 50.6 nL per side. Animals 153 were allowed to recover for 5 to 7 days while the retrograde tracer transported back to the cell 154 bodies. On the day of recording, the experimenter was blind to the location of retrograde tracer 155 injection (mPFC, pACC, or NAc) and slices were prepared as above. Projection neurons were 156 chosen by selecting cells observed as labeled using epiflorescent illumination. All injection sites 157 were histologically confirmed by a third party blind to the electrophysiology results to avoid bias.
N/OFQ responses were analyzed prior to unblinding. Animals with improper injection 159 placements or significant diffusion outside of the target region were rejected.
Immunohistochemistry: Slices were pre-blocked for 2 h at room temperature in PBS with 161 0.2% BSA and 5% normal goat serum, then incubated at 4°C with a rabbit anti-TH polyclonal 162 antibody (1:100; EMD Millipore, RRID: AB_390204). Slices were then washed thoroughly in 163 PBS with 0.2% BSA before being agitated overnight at 4°C with Cy5 anti-rabbit secondary 164 antibody (1:100; Jackson ImmunoResearch Labs Inc., West Grove, PA, RRID: AB_2534032) 165 and FITC streptavidin (6.5 µL/mL). Sections were rinsed and mounted on slides using Bio-Rad To evaluate our within cell statistical comparisons to identify "responsive" vs "nonresponsive" neurons, in particular to test the possibility that drift might contribute to some of our identified drug effects, we conducted a sliding window analysis on a subset of our drug responses (all TH positive neurons tested with bath application of 10 nM N/OFQ). Further, any increase in statistically significant sliding windows during drug washout compared to the static baseline would suggest underlying Iholding drift. We compared all 4 minute windows from pre-drug application through drug washout to a fixed "baseline" window (the 4 min preceding the onset of the drug). To create the windows, Iholding of each recording was binned into 30 second intervals and assigned a bin number (1, 2, 3 Ö n). The "baseline" 8 bin (4 min) window was compared with the target 4 min window by way of a student's unpaired t-test. The P value and significance of the comparison was then corrected using the Bonferroni method for multiple comparisons. The alignment of the sliding window was then increased by a single bin and the comparison repeated, resulting in an array that represents all significant 4 minute intervals for each drug effect. The resulting arrays were plotted as a histogram representing, at the initial bin time of the sliding window, the proportion of recordings in which this calculation was significantly different from the fixed baseline target window (A). In the neurons previously classified as "responsive" by a single "baseline" compared to "drug" window comparison, the rising left edge of the histogram begins to plateau around the 4 th minute of drug application, consistent with the plateau of the mean effects across all cells reported in Figure 1D . Further, consistent with washout reversal of N/OFQ effects in most but not all neurons, the proportion of significant bins falls off as soon as N/OFQ application was terminated. That both the rise and fall of the frequencies of significant windows are time locked to the drug application suggests the response classification scheme is reliable. In neurons previously classified as "non-responsive" only one neuron had any significant windows, with 3 sliding window locations where this analysis yielded P < 0.05, suggesting that there was not systematic drift in these "non-responsive" neurons. In addition, a scatter plot (B) indicates the maximum number of consecutive significant sliding windows for each cell analyzed, because a well-behaved change in Iholding in response to the drug application should be detected in consecutive sliding windows. This graph shows that 8/12 neurons that were classified as "responsive" have more consecutive sliding windows different from baseline than the maximum found in "non-responsive" neurons. This analysis was conducted using a custom script created in Python (available at https://osf.io/c8gu7/?view_only=63ea4c0623b54e46a4efaccc450a89c6).
; 51% of all neurons tested, 44/86; mean response magnitude = 15 ± 2 pA; Fig. 1D ). In 229 many cases the holding current returned to baseline during N/OFQ washout, as in Fig 
To confirm responses were due to activation of the NOP, we tested whether these 
We also observed a subpopulation of neurons that responded to N/OFQ application with The mean ± SEM time course across neurons with inward currents shows the onset of this response is time locked to the initiation of drug application (n = 13). C: Across all VTA neurons from control rats that were tested for 10 nM N/OFQ responses, 19% responded with a significant inward current. D: Grey dots indicate each neuron showing a negative change, both significant and not significant, in holding current with N/OFQ application (grey dots include all neurons with a change < 0 pA; median shown in white dots; black bars show 25 and 75 percentiles. Significant inward currents were observed at 10 nM, while higher concentrations only generated outward currents (see Fig. 1E ). E: Inward currents were observed in both immunocytochemically identified TH(+) and TH(-) neurons. F: Locations of VTA recordings show that neurons that responded to N/OFQ with inward and outward currents were intermixed.
Given the inconsistencies in the reports of behavioral effects of NOP agonists and 262 antagonists, we tested whether N/OFQ causes rapid NOP desensitization at moderate doses.
We observed a concentration-dependent diminished response to a second application of 264 N/OFQ when the first application of N/OFQ was ³ 100 nM (n = 12 neurons from 12 rats; paired 265 t-test p = 0.00003; Fig. 4A,B) . This is consistent with NOP desensitization, and observed in both 266 TH(+) and TH(-) neurons (Fig. 4B ). In contrast, following administration of 10 nM N/OFQ, no 267 difference in response was observed between the first and second applications (n = 10 neurons 268 from 8 rats; paired t-test p = 0.13; Fig. 4C,D) . Therefore, desensitization occurs at moderate were mostly prevented by this treatment (Fig. 5C ). This raised the possibilities that the outward 294 currents are via an inhibition of AMPAR signaling, via an increase in GABAAR signaling, or via a 295 non-GIRK-dependent effect of a substance released by action potential activity in the slice. We 
Zheng and colleagues also reported larger average outward currents compared to our dataset 333 and did not observe desensitization with repeated applications of 300 nM N/OFQ, inconsistent 334 with our findings here. As a positive control to test that 100 µM BaCl2 was sufficient to block K + 335 mediated effects in our preparation, and in an attempt to resolve these discrepancies, we 336 completed additional recordings in the SNc, just lateral to the VTA (Fig. 5I ). First, we tested if neurons that were retrogradely labeled by tracer injections into mPFC, pACC, or medial NAc 355 (Fig. 6A,B) . Recordings were conducted with the investigator blinded to the injection site. Half of NAc-projecting VTA neurons (7/14) responded to N/OFQ with outward currents (9 372 ± 1 pA, 7 responsive neurons from 7 rats; Fig. 6D ). No inward currents were observed in this 373 projection. Of the 7 NAc-projecting neurons that responded to N/OFQ, 2 were confirmed TH(+) 374 and 3 were TH(-) (Fig. 6D) . Together, these data indicate that similar N/OFQ inhibitory effects 375 occur in VTA neurons that project to mPFC and NAc, but these effects are opposed to those on 376 VTA projections to pACC, many of which responded to N/OFQ with an inward current.
ICV or intra-VTA N/OFQ decreases dopamine levels in the NAc (Murphy et al., 1996;  380 Murphy and Maidment, 1999) . Consistent with this result, we found that N/OFQ directly inhibits 381 a subset of the VTA dopamine NAc-projecting somata. N/OFQ may also inhibit dopamine 382 release in the NAc at the terminals; to test if NOPs on dopamine terminals in the NAc also 383 contribute to an N/OFQ-induced decrease in NAc dopamine levels, we used FSCV to detect 384 changes in stimulated dopamine release in NAc slices (Fig. 7A) . In tissue from control SD rats 385 (9 rats), we stimulated dopamine release with a bipolar electrode. In a second set of animals, to 386 limit stimulation to dopaminergic axons, we expressed ChR2 in Th::Cre rats and stimulated with 387 470 nm light pulses (9 rats). In these preparations, repeated electrical, and especially optical, We used FSCV in acute, coronal slices containing the NAc to test for N/OFQ effects on terminal release of dopamine. Dopamine release was evoked in slices from control rats with bipolar electrodes locally in the NAc. Recordings were made on the NAc shell-core border. Alternatively, to limit stimulation to dopamine fibers, Th::Cre rats were injected with (AAV2-Ef1a-DIO-hChR2(H134R)-mCherry) in the VTA at least 4 weeks prior to recordings, during which 470 nm light pulses were used to stimulate dopamine release. A: Example color plots of FSCV measurement of electrically evoed dopamine release. Inset, top: background subtracted cyclic voltammogram at peak of putative dopamine release. Inset, bottom: locations of FSCV recordings in schematic of coronal section of rat brain AP: +1.5 mm (Paxinos and Watson, 1997 
We also found that NOP activation signals through the canonical GIRK pathway in the 
Kappa opioid receptor activation, on the other hand, has no effect on the cell bodies of NAc- affect NAc-projecting dopamine neurons will depend upon the brain region of peptide release.
There is also evidence for dopamine release in the NAc that is independent of action potential why such an ongoing break on learning and memory by N/OFQ contributes to normal behavioral 544 adaptation, and whether dopamine or other VTA outputs play a role, remains to be determined.
One provocative possibility is that it is this degradation of working memory function that is the 546 primary mechanism underlying the lack of place conditioning in response to central N/OFQ, 547 rather than that this treatment is affectively neutral. This interpretation is consistent with work
showing that N/OFQ blocks opioid induced conditioned place preference yet has no effect on 549 opioid self-administration as well (Sakoori and Murphy, 2004; Walker et al., 1998) .
The results of this study extend our understanding of the NOP system biology and 551 provide considerations for additional investigation into NOP function within limbic circuits.
These findings clarify that strong NOP desensitization occurs in neurons at moderate 553 concentrations of the endogenous agonist N/OFQ. Importantly, not only does the nature of the 554 NOP response vary with the projection target of VTA neurons, but the NOP function is largely 555 sequestered to the somatodendritic compartment of VTA dopamine neurons that project to the 556 NAc, demonstrating two different kinds of circuit level organization of this receptor system.
Building on this groundwork, future studies of these VTA circuits during different behavioral 558 states and tasks related to motivation and cognition will help to elucidate the differences 559 between the normal and dysfunctional NOP-N/OFQ system, improving the potential for 560 therapeutic targeting. 561 562