bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
In vivo detection of optically-evoked opioid peptide release
Ream Al-Hasani1,2,3*, Jenny-Marie T. Wong7*, Omar S. Mabrouk7,8 Jordan G. McCall1,2,3,5,
Gavin P. Schmitz1,3, Kirsten A. Porter-Stransky9, Brandon J. Aragona9, Robert T.
Kennedy7,8#, Michael R. Bruchas1,4,5,6#
1Department of Anesthesiology Division of Basic Research, Washington University
School of Medicine, St. Louis, MO, USA; 2Departments Pharmaceutical and
Administrative Sciences, St. Louis College of Pharmacy, St. Louis, MO, USA; 3Center for
Clinical Pharmacology, Washington University School of Medicine and St. Louis College
of Pharmacy, St. Louis, MO, USA, 4Department of Neuroscience, Washington University
School of Medicine, St. Louis, MO, USA, 5Washington University Pain Center,
Washington University School of Medicine, St. Louis, MO, USA; 6Department of
Anesthesiology and Pain Medicine, Center for the Neurobiology of Addiction, Pain, and
Emotion, University of Washington; 7Department of Chemistry, University of Michigan,
Ann Arbor, Michigan, USA. 8Department of Pharmacology, University of Michigan, Ann
Arbor, Michigan, USA 9Department of Psychology, University of Michigan, Ann Arbor,
Michigan, USA. *Co-first author. # Co-last author
To whom correspondence should be addressed:
Michael R Bruchas, PhD
Professor
Department of Anesthesiology and Pain Medicine
Center for the Neurobiology of Addiction, Pain, and Emotion
University of Washington
Email Address: [email protected]
1 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Robert Kennedy, PhD
Chair, Chemistry Department
Hobart Willard Distinguished University Professor
Professor of Chemistry
Professor of Pharmacology
Department of Chemistry
University of Michigan
Ann Arbor, MI 48109-1055
Email: [email protected]
Ream Al-Hasani, PhD
Assistant Professor
Department of Pharmaceutical and Administrative Sciences
St. Louis College of Pharmacy
Department of Anesthesiology
Washington University in St. Louis
Center for Clinical Pharmacology
Washington University School of Medicine
660 S. Euclid Ave. | Box 8054
St. Louis, MO 63110
Email: [email protected]
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1 Abstract
2 Though the last decade has seen accelerated advances in techniques and technologies
3 to perturb neuronal circuitry in the brain, we are still poorly equipped to adequately dissect
4 endogenous peptide release in vivo. To this end we developed a system that combines in
5 vivo optogenetics with microdialysis and a highly sensitive mass spectrometry-based
6 assay to measure opioid peptide release in freely moving rodents.
7
8 Introduction
9 Neuropeptides are the largest class of signaling molecules in the central nervous
10 system where they act as neurotransmitters, neuromodulators, and hormones1. In vivo
11 neuropeptide detection is technically challenging as neuropeptides can be rapidly
12 cleaved by peptidases and undergo posttranslational modifications. To further
13 complicate detection, neuropeptides are found at orders of magnitude lower
14 concentrations compared to classical neurotransmitters (i.e. glutamate, GABA, and
15 the biogenic amines) and adsorb to a variety of surfaces during sample handling2,3.
16
17 For decades, neuropeptides have been assayed with immunoaffinity-based techniques
18 due to their high sensitivity4,5. However, selectivity remains a concern due to poor antibody
19 specificity for full length vs. truncated peptides that contain identical binding epitopes.
20 Nanoflow liquid chromatography-mass spectrometry (nLC-MS) is a powerful alternative
21 for peptide detection because it provides high sensitivity and specificity without the need
22 for an immunocapture step. To this end, nLC-MS has been used successfully to detect a
23 number of endogenous peptides derived from intracranial sampling techniques such as
24 microdialysis6.
25
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26 Opioid peptides are prominent neuromodulators for regulating motivated behaviors.
27 Though understanding their endogenous release properties is critical for dissecting the
28 neural circuits that mediate these behaviors it has been extremely difficult to reliably
29 achieve thus far. The reason primarily being that opioid peptides are very similar in
30 structure and origin making selective detection of peptides and their subtype fragments
31 virtually impossible. All opioid peptides, except nociceptin share a common N-terminal
32 Tyr-GlyGly-Phe signature sequence, which interacts with opioid receptors. The
33 preproenkephalin gene encodes several copies of the pentapeptide Met-enkephalin and
34 one copy of Leu-enkephalin. The preprodynorphin gene also encodes multiple opioid
35 peptides, including dynorphin A, dynorphin B, and neo-endorphin. The preprodynorphin-
36 derived peptides all contain the Leu-enkephalin N-terminal sequence. To further
37 complicate things the enkephalins preferentially bind to the d over µ opioid receptor
38 whereas dynorphin preferentially binds to the k opioid receptor.
39
40 Despite these complexities some studies have reliably detected opioid peptides in rat
41 models7–12, however, it has not been previously possible to reliably detect evoked
42 neuropeptide release in a cell-type selective manner using transgenic mouse models.
43 However, with the advent of optogenetic approaches allowing researchers to spatially
44 target and manipulate neuronal firing patterns the specific neuronal properties of
45 neuropeptide release can now more likely be empirically determined.
46
47 We and others have recently shown that discrete targeting of the kappa opioid system in
48 the nucleus accumbens (NAc) can modulate both rewarding and aversive behaviors13,14.
49 We showed that photostimulation of dynorphin (dyn) cells in the ventral nucleus
50 accumbens shell (vNAcSh) elicited robust aversive behavior, while photostimulation of
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51 dorsal NAcSh dyn (dNAcSh) cells induced a place preference that was positively
52 reinforcing, however, no successful measurements of optogenetically-evoked
53 neuropeptide release in vivo have been reported to date. Furthermore, few reports have
54 extensively investigated regional distinctions within the NAc shell and no reports to our
55 knowledge have successfully measured optogenetically-evoked neuropeptide release in
56 vivo.
57
58 To quantify evoked-neuropeptide release in anatomically and behaviorally distinct regions,
59 we pursued a method to reliably detect neuropeptides in the nucleus accumbens during
60 photostimulation of opioid-containing neurons. We developed a custom optogenetic-
61 microdialysis (opto-dialysis) probe that simultaneously provides photostimulation with the
62 ability to sample local neurochemical release in awake, freely moving mice. We
63 established a targeted nLC-MS method to analyze the opioid peptides dyn (fragment
64 dynorphin A1-8) and enkephalins (leu- and met-), as well as dopamine, GABA and
65 glutamate. We applied these techniques to precisely investigate regional differences
66 between the vNAcSh and dNAcSh neuropeptide release dynamics. This system allows
67 quantification of neuropeptide release while directly controlling cell-type selective neuronal
68 firing in the NAcSh.
69
70 Results
71 To establish a quantitative assay, we used a custom synthesized isotopically labeled dyn
13 15 72 (DYN*, YGGFLRRI with isotope C6 N1-leucine) with a mass shift of +7 and a +3.5 m/z
73 shift (+2 charge state) as an internal standard (IS) to account for variability during the LC
74 injection, surface adsorption, and ionization efficiency. High concentration injections of
75 DYN* were fully resolved by the mass spectrometer and did not result in cross talk with
76 endogenous dyn (Fig. 1a & b), demonstrating that the addition of labeled DYN* does not
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77 contribute to or interfere with the endogenous dyn signal, while maintaining a similar
78 retention time. To further validate the use of DYN* as an internal standard when detecting
79 opioid peptides we prepared a linear calibration curve with a mixture of dyn, Leu-
80 Enkephalin (LE) and Met-Enkephalin (ME) standards spiked with DYN* and detected all
81 four compounds within 5 min following loading and desalting the sample on the column.
82 Four distinct peaks are shown in the reconstructed ion chromatographic trace, confirming
83 the separation and reliable detection of all three opioid peptides with isotopically labeled
84 DYN* in one sample (Fig. 1c). The addition of the DYN* isotope to our assay further
85 improves quantification by improving relative standard deviation for repeated injections of
86 standards (Fig.1d-f). Ratios of dyn, LE, and ME to a consistent DYN* were used for
87 calibrations and analysis of standards for quantitative analysis for all experiments shown
88 here. No effect of dialysate matrix for any targeted analyte was observed (Supplementary
89 Fig. 1g-j).
90
91 For in vivo detection in freely moving mice, we developed a customizable microdialysis
92 probe (i.e. customizable length, depth and sampling area) with an integrated fiber optic to
93 locally sample proximal to the site of photostimulation in the brain (Fig. 2a,
94 Supplementary Fig. 1a-e). Traditional dialysis probes incorporate inlet and outlet tubing
95 encased in a semi-permeable membrane enclosed with epoxy, and further encased in a
96 stiff cannula for rigidity and robustness. To minimize the size of the opto-dialysis probe we
97 did not include the final external casing and instead took advantage of the natural rigidity
98 of the fiber optic to support the dialysis inlet-outlet assembly (Fig. 2a, Supplementary
99 Fig. 1a-e). This resulted in a maximal probe diameter of 480 μm. To maximize
100 concentrations of peptides entering the probe, we used a 60 kDa molecular weight cutoff,
101 polyacrylonitrile membrane with a slight negative charge for optimal peptide recovery
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102 (AN69, Hospal, Bologna Italy) 2,4 and showed we can measure changes in peptide stock
103 concentration within the 15 min fraction collection time (Fig. 2b).
104
105 To evoke and measure in vivo peptide release we injected AAV5-EF1a-DIO-ChR2-eYFP
106 in to either the vNAcSh or the dNAcSh of preprodynorphin-IRES-cre (dyn-cre) mice and
107 implanted the custom opto-dialysis probes 3 weeks later (Fig. 3a). Following recovery
108 artificial cerebrospinal fluid (aCSF) was perfused through the device at 0.8 μL/min and
109 fractions were collected on ice every 15 min, generating 12 μL volumes, 2 μL of which
110 were aliquoted and used for a small molecule detection assay. Three baseline fractions
111 were collected prior to a fraction capturing 15 min of 10 Hz (10 ms pulse width)
112 photostimulation, and six additional fractions were collected following photostimulation. As
113 a positive control for detecting dynamic changes in vivo by nLC-MS and to establish
114 sampled neuron responsivity to stimuli, we infused 100 mM K+ aCSF at the end of each
115 collection experiment. The influx of K+ ions causes depolarization, resulting in vesicular
116 exocytosis, which is expected to evoke a large increase in peptide concentration. Mice
117 were included in the study if 100 mM K+ stimulation resulted in a positive increase in
118 analytes at the end of the experiment (Supplementary Fig. 2a-c) and if correct anatomical
119 probe placement and viral expression were confirmed (Supplementary Fig. 3a and b).
120 In the current study 81.1% (15/18) of the mice were included in the analysis.
121
122 A significant increase in dyn was detected in dyn-cre positive mice during photostimulation
123 in both the vNAcSh (interaction effect; t=3.941, p<0.001) and dNAcSh (interaction effect;
124 t=3.012, p=0.003), compared to control mice (Fig. 3b). Interestingly, in the vNAcSh there
125 was also a sustained increase in dyn after photostimulation (interaction effect; t = 2.499,
126 p = 0.014) (Fig. 3b). Dyn release during photostimulation was also significantly higher in
127 the vNAcSh compared to the dNAcSh (interaction effect; t=2.749, p=0.007) and post
8 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
128 stimulation (interaction effect; t=2.806, p=0.006) (Fig. 3b). Photostimulation of vNAcSh
129 dyn neurons was previously shown to cause aversive behavior, consistent with early
130 pharmacological studies which linked dyn with negative emotional states13,15. Here we
131 demonstrate sufficiently discrete dynorphin detection to measure different levels of peptide
132 in two regions of the NAc shell separated by 1mm.
133
134 We simultaneously detected robust release of LE and ME in both the vNAcSh and
135 dNAcSh. During photostimulation of dyn-containing cells in the dNAcSh, LE levels were
136 significantly elevated compared to controls (interaction effect; t=5.384, p<0.0001) (Fig.
137 3c). In contrast, no changes in LE were detected during photostimulation of dyn-containing
138 cells in the vNAcSh (Fig. 3c). Converse to this, we observed a significant increase in ME
139 during photostimulation of dyn-containing cells in the vNAcSh (comparing cre+ and cre-,
140 interaction effect; t=2.824, p=0.006). However, the same effect was not significant in the
141 dNAcSh (comparing cre+ and cre-, t=1.78, p=0.053) (Fig. 3d). Importantly, we observed
142 a significant change in ME in the dNAcSh when compared to its baseline (t=1.94, p=0.033)
143 and there was no significant difference between the effect of photostimulation in ventral
144 and dorsal (i.e. both are increased). However, the lack of a significance between Cre+ and
145 Cre- in dNAcSh is likely due, at least in part, to the fact the baseline levels of opioid
146 peptides in dNAcSh are higher than the vNAcSh (7.114 pM versus 2.71 pM, respectively).
147 Though data is represented as % of baseline the range of absolute dialysate concentration
148 detected for dyn was 0.28-0.44 pM in dNAcSh and 0.13-0.28 pM in vNAcSh; LE 1.39-3.28
149 pM in dNAcSh and 1.30- 2.24 pM in vNAcSh; ME 6.19-8.69 pM in dNAcSh and 2.57-4.11
150 pM.
151
152 To determine if peptide release corresponded with small molecule neurotransmitter
153 release, we applied a benzoyl chloride (BzCl) derivatization LC-MS method to monitor 3
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154 small molecules, dopamine, GABA and glutamate in dialysis samples16,17. Interestingly,
155 dopamine increased in the dNAcSh during photostimulation (interaction effect; t=5.007,
156 p<0.0001), which persisted following stimulation (interaction effect; t=2.081, p=0.039)
157 (Fig. 3e). Levels of GABA also increased in the vNAcSh following photostimulation
158 (t=2.363, p=0.020) and had a prolonged response (t=4.744, p<0.0001) (Supplementary
159 Fig. 4a). There were no significant changes in glutamate levels during photostimulation in
160 either vNAcSh or dNAcSh (Supplementary Fig. 4b).
161
162 Discussion
163 The data show that photostimulation of dyn-containing cells in the vNAcSh and dNAcSh
164 results in a detectable increase in dyn release, which is greater in the vNAcSh. This result
165 correlates with previous behavioral data demonstrating that dyn release likely drives a
166 region dependent preference and aversion behavior13. We observed a significant increase
167 in LE and dopamine during photostimulation in the dNAcSh. Importantly, we previously
168 observed a preference behavior when photostimulating dyn cells in the dNAcSh and these
169 data suggest that the preference may be related, at least in part, to the detected increases
170 in dopamine and LE. Furthermore, many studies have shown that levels of both dopamine
171 and LE increase during preference or reward18–20. Importantly, using this method it is not
172 possible to know whether LE is derived from pDyn or pENK but in future experiments we
173 can combine this with conditional knockout approaches to selectively delete the peptides
174 in specific cell types, so that we can later determine what happens to these signals in the
175 absence of expression of particular peptides in D1 vs D2 cells and subregions. In addition,
176 we cannot preclude interactions between MSN cell types, specifically that optogenetic
177 activation of D1 cells could impart changes on D2 cell activity thereby releasing Met-Enk,
178 indirectly, and/or via indirect circuits outside the NAc. In addition, dopamine release via
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179 D1 MSN stimulation, could be via two mechanisms 1) via indirect suppression of D1
180 projections back to VTA GABA neurons21, which acts via GABA-B to disinhibit VTA DA
181 neurons. This would then result in dopamine release in the NAc following D1 stimulation.
182 Furthermore, an alternative, presynaptic regulation of DA release from VTA to NAc
183 projections is also plausible, and recently been investigated by multiple groups22,23.
184
185 The observed changes in GABA in both vNAcSh and dNAcSh are as predicted due to the
186 fact that dyn-containing medium spiny neurons are GABAergic. The observed increase in
187 ME is intriguing, as ME is not released from the same cells as dynorphin. There has been
188 some evidence to suggest that enkephalins play a role in regulating disruptions in
189 homeostasis following chronic exposure to drugs of abuse24,25. Specifically, increases in
190 enkephalin release during drug withdrawal has been shown through peptide tissue
191 content, mRNA levels and microdialysis of extracellular enkephalins in a number of brain
192 regions including the nucleus accumbens26. Concurrently, studies have measured
193 increases in dynorphin expression during withdrawal27–31. It seems possible therefore that
194 when we are stimulating the release of dynorphin (perhaps mimicking some aspects of
195 withdrawal), levels of met-enkephalin increase in response to this, as a homeostatic
196 mechanism to relieve this withdrawal-like state. This release profile is unique to the
197 vNAcSh suggesting that this region is critical in the regulation of hedonic homeostasis.
198 Though we have previously shown that dyn mRNA expression is similar in the vNAcSh
199 and dNAcSh13, it would be ideal to also verify the expression pattern and distribution of
200 the enkephalins in the vNAcSh and dNAcSh. Finally, it should be noted that this detection
201 technique maintains the poor temporal resolution of peptide and other microdialysis
202 approaches. Therefore, conclusions regarding the differential transmitter and peptide
203 release profiles should be tempered by the long time course of collection.
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204
205 The ability to detect multiple peptides and small molecules in vivo with high sensitivity, as
206 shown here demonstrates the potential of this method. It is, however, important to
207 acknowledge limitations and future considerations with this methodology. Due to the
208 structural similarity between ME/LE and dyn, we included DYN* as our only internal
209 standard. An ideal assay would include isotopically labeled internal standards for each
210 analyte of interest, however, the addition of analytes to detect in the ion trap reduces
211 points per peak and complicates the chromatogram.
212
213 In this current method the 3 peaks of interest, LE/ME/dyn, all appear in the chromatogram
214 in close proximity between 4-5.5 minutes, which may not seem ideal. A way to resolve this
215 would be to flatten out the solvent gradient to better resolve the peaks
216 chromatographically, however the length of the run would be longer, therefore reducing
217 overall throughput. Here it takes 18 minutes from injection-to-injection and it is important
218 to note that the peaks, while not fully resolved in time, were fully resolved by mass-to-
219 charge ratio, so the separation was sufficient in this case. It is important to consider this
220 delicate balance going forward to detect more peptides in an extended experimental
221 paradigm and for concurrent detection of other peptides within individual samples.
222
223 Here we only detected dyn1-8, LE and ME, but as mentioned in the introduction there are
224 many more fragments that could be involved or altered that we have not measured. We
225 primarily focused on dyn1-8 as this was the fragment that was the most reliable to detect
226 in developing this methodology. Since we were at the early stages of method development
227 it was crucial to limit variability where possible. The limitations to in vivo detection of
228 neuropetides by microdialysis with LC-MS has been thoroughly explored and are largely
229 due to absorption and lack of adequate recovery2. In this current method organic modifiers
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230 are not added to the perfusate for dialysis or to the sample in the vial, as we validated that
231 there was no peptide carryover/stickiness in the LC-MS lines without such agents.
232 However, it has been shown that the addition of an organic modifier, like acetonitrile, to a
233 sample could be optimized for each peptide fragment and improve quantitation by
234 reducing stickiness to the vial and LC-MS fluidic pathway2. This approach, however,
235 appears to be dependent on each individual peptide.
236
237 This new approach, we describe here allows for detection of cell-type specific evoked in
238 vivo neuropeptide release within neural circuits to be observed during freely moving
239 behavior. Neuropeptide biology remains a challenging territory in neuroscience. Methods
240 to detect in vivo release of endogenous peptides are limited, preventing the full
241 understanding of their properties and dynamics. The method described here is a first step
242 in closing this gap in our understanding of neuropeptide-containing circuits during
243 behavior.
244 245 Materials and Methods 246 247 Animals and surgical procedure
248 Adult male C57BL/6 mice (Envigo, 5–6 weeks of age) were used for initial experiments to
249 determine perfusion media conditions and effects of probe design. For optogenetic
250 studies, adult male preprodynorphin-IRES-cre (dyn-Cre) mice were used. Mice were
251 unilaterally injected with 300 nL of AAV5-EF1a-DIO-ChR2-eYFP (Washington University
252 in St. Louis, Hope Center Viral Vector Core, viral titer 2 × 1013 vg/mL) into either the
253 dNAcSh or vNAcSh and were allowed to recover from surgery at Washington University
254 in St. Louis 1 week prior to shipment. Mice were then shipped to, and acclimated for 2-3
255 weeks at, the University of Michigan before probe implantation.
256
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257 Chemicals
258 Dynorphin A1-8 (abbreviated dyn) and LE were purchased from American Peptide
259 (Sunnyvale, CA); ME was purchased from Sigma Aldrich (St. Louis, MO). Isotopically
13 15 260 labeled leucine ( C6 N1-leucine) was used to create an isotopically labeled dynorphin A
261 1-8 internal standard (DYN*) through the University of Michigan’s protein synthesis core.
262 Water, methanol, and acetonitrile for mobile phases are Burdick & Jackson HPLC grade
263 purchased from VWR (Radnor, PA). All other chemicals were purchased from Sigma
264 Aldrich (St. Louis, MO) unless otherwise noted. Artificial cerebrospinal fluid (aCSF)
265 consisted of 145 mM NaCl, 2.67 mM KCl, 1.4 mM CaCl2, 1.01 mM MgSO4, 1.55 mM
266 Na2HPO4, and 0.45 mM Na2H2PO4 adjusted to pH 7.4 with NaOH. Ringer solution
267 consisted of 148 mM NaCl, 2.7 mM KCl, 2.4mM CaCl2, and 0.85 mM MgCl2 adjusted pH
268 to 7.4 with NaOH. In experiments that used high K+ ringer solution NaCl was adjusted to
269 48 mM and KCl was adjusted to 100 mM, all other chemicals remained the same.
270
271 Fabrication of opto-dialysis probe
272 Fabrication of the optogenetic fiber optic probe was made as previously described13,32,33.
273 Fabrication of the microdialysis probe and opto-dialysis probe are described in the
274 supplemental information.
275
276 In vivo microdialysis
277 Mice were group housed in temperature and humidity controlled rooms with 12 h light/dark
278 cycles with access to food and water ad libitum. Both the Washington University in St.
279 Louis and University of Michigan Unit for Laboratory Animal Medicine approved animal
280 procedures and they were in accordance with the National Institute of Health Guidelines
281 for the Care and Use of Laboratory Animals. All experiments were conducted within the
282 guidelines of Animal Research Reporting in vivo Experiments. Surgical procedures for
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283 inserting probes were similar to those previously described8,13,32,34. Briefly, mice were
284 anesthetized in an induction chamber with 5% isoflurane prior to surgical procedures and
285 placed in a Model 963 stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA)
286 equipped with a mouse ear and bite bar. Mice were maintained under anesthesia with 1-
287 2% isoflurane during cannulation procedures. A custom-made 1 mm polyacrylonitrile
288 membrane (Hospal AN69) concentric probe, was inserted into either the dNAcSh
289 (stereotaxic coordinates from bregma: +1.3 anterior-posterior [AP], ±0.5 medial-lateral
290 [ML], -4.5 mm dorsal-ventral [DV]) or vNAcSh (stereotaxic coordinates from bregma: +1.3
291 [AP], ±0.5 [ML], -5.0 mm [DV]). Light power from a 473-nm laser was measured at the
292 membrane to ensure satisfactory light power (defined as ≥ 5 mW at a distance of 1 mm
293 from the end of fiber optic) before implantation of microdialysis probes integrated with fiber
294 optic (opto-dialysis probe). Implanted probes were secured using two bone screws and
295 dental cement. Mice were allowed to recover 24 h with free access to food and water prior
296 to baseline collection for microdialysis studies.
297
298 For microdialysis studies, the fiber optic was connected to the laser via a tether running
299 through a Ratturn (Bioanalytical Systems, Inc.) along with microdialysis perfusion lines.
300 Microdialysis probes were flushed for 1 h using a Fusion 400 syringe pump (Chemyx,
301 Stafford, TX USA) at a flow rate of 2 μL/min. The flow rate was lowered to 0.8 μL/min and
302 flushed for an additional 1 h prior to fraction collection. Microdialysis fractions were
303 collected every 15 min, resulting in a 12 μL sample. 2 μL of sample was removed for BzCl
304 derivatization12 to monitor small molecules, and the remaining 10 μL was spiked with 1.1
305 μL of 100 pM isotopically labeled DYN* (10 pM final concentration) and was used for
306 peptide analysis.
307
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308 When experiments were completed, mice were euthanized and perfused with
309 paraformaldehyde. Brains were extracted to confirm probe placement and virus
310 expression by histology. Mice with verified virus expression and correct probe placement
311 were included in the data set.
312
313 Optogenetic stimulation
314 On the day of the experiment, mice were connected to a laser via a tether alongside the
315 microdialysis perfusion lines. An Arduino UNO was programmed and connected to the
316 473-nm laser to provide stimulation frequency of 10 Hz, 10-ms pulse width. The laser was
317 manually operated and turned on and off during a single 15 min fraction after 3 baseline
318 collections. Six additional fractions were collected after the photostimulation, followed by
319 two fractions with high 100 mM K+ ringer solution for a total of 12 fractions.
320
321 Peptide assays with nanoflow LC-MS
322 An assay was developed to monitor opioid peptides (dyn, LE, and ME) using nanoflow
323 LC-MS. Capillary columns and electrospray ionization emitter tips were prepared in-
324 house8,34. Capillary columns were prepared using a 10 cm length of 50/360 μm (inner
325 diameter/outer diameter) fused silica capillary packed with 5 μm AltimaTM C18 particles to
326 a bed length of 3.5 cm. The column was connected to a fused silica ESI emitter tip using
327 a Teflon connector.
328
329 5 μL samples were injected onto the capillary column. An Agilent 1100 HPLC pump (Santa
330 Clara, CA) was used to deliver the elution gradient containing water with 0.1% FA for
331 mobile phase A and mobile phase B was MeOH with 0.1% FA delivered as initial 0% B; 1
332 min, 30 %B; 4 min, 50% B; 4.1 min 100% B; 7 min, 100% B; 7.1 min, 0%B; and 10 min,
333 0% B. The capillary column was interfaced to a linear ion trap (LTQ XL, Thermo Scientific),
16 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
334 operating in positive mode. The MS2 pathway for opioid peptides dyn, LE, and ME were
335 detected using m/z values of 491 à 435, 556 à 397, and 574 à 397 respectively.
13 15 336 Isotopically labeled DYN* (+7 mass shift, isotopically labeled C6 N1-leucine) internal
337 standard was detected using m/z values of 495 à 438. In vitro recovery of a 1 mm probe
338 for dyn, LE, and ME were 12 ± 2%, 13 ± 3%, and 13 ± 2%.
339
340 Fresh dyn, LE, and ME standards, spiked with DYN* for the opioid assay were prepared
341 daily. Standards were analyzed with nLC-MS at 0.01, 0.05, 0.1, 1, 10, 20, 50, and 100 pM
342 concentrations in triplicate to determine linearity, reproducibility, and limits of detection.
343 Opioid analytes were normalized to DYN*. Limits of detection for dyn, LE, and ME were
344 0.2 ± 0.04, 0.5 ± 0.3, and 0.6 ± 0.4 pM, respectively, in 5 μL and were determined each
345 day of experimentation. Average carry over across all experiments for dyn, LE, and ME
346 were 0.8 ± 0.03%, 0.3 ± 0.3%, and 0.3 ± 0.2% determined by running the
347 highest calibration point immediately followed by a blank and integrating the peak area
348 across the same time. Mice that had average basal levels above the limits of detection
349 and had appropriate probe placement and virus expression were used for the study.
350
351 Small molecule analysis using benzoyl chloride (BzCl) LC-MS
352 For small molecule analysis, dialysate samples were derivatized with BzCl and analyzed
353 by LC-MS16,17. This BzCl assay targeted dopamine, GABA and glutamate. 2 µL dialysate
354 were aliquoted from the peptide samples and were derivatized with 1.5 µL sodium
355 carbonate, 100 mM; 1.5 µL BzCl, 2% (v/v) BzCl in acetonitrile; 1.5 µL isotopically labeled
356 internal standard mixture diluted in 50% (v/v) acetonitrile containing 1% (v/v) sulfuric acid,
357 and spiked with deuterated ACh and Ch (C/D/N isotopes, Pointe-Claire, Canada).
358 Derivatized samples were analyzed using Thermo Fisher Accela UHPLC system
359 interfaced to a Thermo Fisher TSQ Quantum Ultra triple quadrupole mass spectrometer
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360 fitted with a HESI II ESI probe, operating in multiple reaction monitoring. 5 µL samples
361 were injected onto a Phenomenex core-shell biphenyl Kinetex HPLC column (2.1 mm x
362 100 mm). Mobile phase A was 10 mM ammonium formate with 0.15% formic acid, and
363 mobile phase B was acetonitrile. The mobile phase was delivered an elution gradient at
364 450 µL/min as follows: initial, 0% B; 0.01 min, 19% B; 1 min, 26% B; 1.5 min, 75% B; 2.5
365 min, 100% B; 3 min, 100% B; 3.1 min, 5% B; and 3.5 min, 5% B. Thermo Xcalibur
366 QuanBrowser (Thermo Fisher Scientific) was used to automatically process and integrate
367 peaks. Each peak was visually inspected to ensure proper integration.
368
369 Assessment of probe placement
370 Mice were anesthetized with pentobarbital and transcardially perfused with ice-cold 4%
371 paraformaldehyde in phosphate buffer (PB). Brains were dissected, post-fixed for 24 h at
372 4°C and cryoprotected with solution of 30% sucrose in 0.1M PB at 4°C for at least 24 hr,
373 cut into 30 µm sections and processed for Nissl body staining. Sections were washed
374 three times in PBS and blocked in PBS containing 0.5% Triton X-100 (G-Biosciences) for
375 1 hr. This was followed by a 1-h incubation with fluorescent Nissl stain to allow
376 visualization of cell bodies (1:400, Neurotrace, Invitrogen). Sections were then washed
377 three times in PBS, followed by three 10-min rinses in PB and mounted on glass slides
378 with Hard set Vectashield (Vector Labs) for episcope microscopy. Correct regional
379 expression of the AAV5-DIO-ChR2-eYFP was verified in addition to placement of the opto-
380 dialysis probe in either the vNAcSh or dNAcSh, which are represented on hit maps.
381
382 Statistical analyses
383 The University of Michigan Center for Statistical Consultation and Research helped design
384 a linear mixed model analysis appropriate for this study using SPSS Statistics software.
385 The linear mixed model analysis was chosen to account for variations within and between
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386 mice and to account for missing data points within individual animals following sample loss
387 or mechanical failure of the instrument. The linear mixed model was used to determine
388 differences in basal conditions, effect of photostimulation relative to basal conditions, and
389 prolonged effects after photostimulation relative to basal conditions. Linear mixed models
390 were used to compare between genotypes within each region sampled, and between
391 regions within genotypes. In vitro data was represented as mean ± SD and the in vivo
392 was represented as mean ± SEM. In all cases significance was defined as p ≤ 0.05.
393
394 Acknowledgements:
395 This work is supported by NIDA R01 DA033396 (M.R.B.), NIDA K99 DA038725 (R.A.),
396 NIMH F31 MH101956 (J.G.M.), WUSTL DBBS (J.G.M.) and NIBIB R01 EB003320
397 (R.T.K). We thank the Bruchas Laboratory and Kennedy Laboratory for valuations
398 discussions and support. We especially thank Shanna Resendez and Curtis Austin from
399 the Aragona Laboratory for helpful discussion and technical assistance. We thank Karl
400 Deisseroth (Stanford) for the channelrhodopsin-2 (H134) construct, Bradford Lowell
401 (Harvard) and Michael Krashes (NIDDK) for the preprodynorphin-IRES-cre mice. We also
402 thank The WUSTL HOPE Center viral vector core (NINDS, P30NS057105) and the
403 University of Michigan Center for Statistical Consultation and Research (CSCAR,
404 consultant Yumeng Li) and Luke Ziolkowski from the McCall Laboratory at Washington
405 University who assisted with statistical analysis.
406
407 Author Contributions: R.A and J.G.M performed viral injections. R.A, J.T.W, O.S.M,
408 J.G.M made opto-dialysis probes and performed surgical implant procedure. R.A, J.T.W,
409 O.S.M, J.G.M, K.A.P-S performed behavioral expts. J.T.W, O.S.M performed nano LC-
410 MS analysis. R.A and G.P.S processed brain tissue. B.J.A facilitated discussions and
19 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
411 provided resources. R.A, J.T.W, O.S.M, J.G.M, R.T.K, M.R.B contributed to experimental
412 design, resources, and manuscript preparation.
413
414 Competing Interests: Michael Bruchas, PhD is a co-founder of Neurolux, Inc, a company
415 that is making wireless optogenetic probes. None of the work in this manuscript used these
416 devices or is related to any of the company’s activities, but we list this information here in
417 full disclosure.
20 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
418 419 References 420 421 1. Wotjak, C. T., Landgraf, R. & Engelmann, M. Listening to neuropeptides by
422 microdialysis: echoes and new sounds? Pharmacol. Biochem. Behav. 90, 125–134
423 (2008).
424 2. Zhou, Y., Wong, J.-M. T., Mabrouk, O. S. & Kennedy, R. T. Reducing adsorption to
425 improve recovery and in vivo detection of neuropeptides by microdialysis with LC-
426 MS. Anal. Chem. 87, 9802–9809 (2015).
427 3. Maes, K. et al. Improved sensitivity of the nano ultra-high performance liquid
428 chromatography-tandem mass spectrometric analysis of low-concentrated
429 neuropeptides by reducing aspecific adsorption and optimizing the injection solvent.
430 J. Chromatogr. A 1360, 217–228 (2014).
431 4. Maidment, N. T., Brumbaugh, D. R., Rudolph, V. D., Erdelyi, E. & Evans, C. J.
432 Microdialysis of extracellular endogenous opioid peptides from rat brain in vivo.
433 Neuroscience 33, 549–557 (1989).
434 5. Maidment, N. T., Siddall, B. J., Rudolph, V. R., Erdelyi, E. & Evans, C. J. Dual
435 determination of extracellular cholecystokinin and neurotensin fragments in rat
436 forebrain: microdialysis combined with a sequential multiple antigen
437 radioimmunoassay. Neuroscience 45, 81–93 (1991).
438 6. Van Wanseele, Y., De Prins, A., De Bundel, D., Smolders, I. & Van Eeckhaut, A.
439 Challenges for the in vivo quantification of brain neuropeptides using microdialysis
440 sampling and LC-MS. Bioanalysis 8, 1965–1985 (2016).
441 7. DiFeliceantonio, A. G., Mabrouk, O. S., Kennedy, R. T. & Berridge, K. C. Enkephalin
442 surges in dorsal neostriatum as a signal to eat. Curr. Biol. CB 22, 1918–1924 (2012).
21 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
443 8. Mabrouk, O. S., Li, Q., Song, P. & Kennedy, R. T. Microdialysis and mass
444 spectrometric monitoring of dopamine and enkephalins in the globus pallidus reveal
445 reciprocal interactions that regulate movement. J. Neurochem. 118, 24–33 (2011).
446 9. Lam, M. P. & Gianoulakis, C. Effects of corticotropin-releasing hormone receptor
447 antagonists on the ethanol-induced increase of dynorphin A1-8 release in the rat
448 central amygdala. Alcohol Fayettev. N 45, 621–630 (2011).
449 10. Lam, M. P. & Gianoulakis, C. Effects of acute ethanol on corticotropin-releasing
450 hormone and β-endorphin systems at the level of the rat central amygdala.
451 Psychopharmacology (Berl.) 218, 229–239 (2011).
452 11. Lam, M. P., Marinelli, P. W., Bai, L. & Gianoulakis, C. Effects of acute ethanol on
453 opioid peptide release in the central amygdala: an in vivo microdialysis study.
454 Psychopharmacology (Berl.) 201, 261–271 (2008).
455 12. Lam, M. P., Nurmi, H., Rouvinen, N., Kiianmaa, K. & Gianoulakis, C. Effects of acute
456 ethanol on beta-endorphin release in the nucleus accumbens of selectively bred
457 lines of alcohol-preferring AA and alcohol-avoiding ANA rats. Psychopharmacology
458 (Berl.) 208, 121–130 (2010).
459 13. Al-Hasani, R. et al. Distinct Subpopulations of Nucleus Accumbens Dynorphin
460 Neurons Drive Aversion and Reward. Neuron 87, 1063–1077 (2015).
461 14. Castro, D. C. & Berridge, K. C. Opioid hedonic hotspot in nucleus accumbens shell:
462 mu, delta, and kappa maps for enhancement of sweetness ‘liking’ and ‘wanting’. J.
463 Neurosci. Off. J. Soc. Neurosci. 34, 4239–4250 (2014).
464 15. Bals-Kubik, R., Ableitner, A., Herz, A. & Shippenberg, T. S. Neuroanatomical sites
465 mediating the motivational effects of opioids as mapped by the conditioned place
466 preference paradigm in rats. J. Pharmacol. Exp. Ther. 264, 489–495 (1993).
22 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
467 16. Song, P., Mabrouk, O. S., Hershey, N. D. & Kennedy, R. T. In Vivo Neurochemical
468 Monitoring Using Benzoyl Chloride Derivatization and Liquid Chromatography–Mass
469 Spectrometry. Anal. Chem. 84, 412–419 (2012).
470 17. Wong, J.-M. T. et al. Benzoyl chloride derivatization with liquid chromatography–
471 mass spectrometry for targeted metabolomics of neurochemicals in biological
472 samples. J. Chromatogr. A 1446, 78–90 (2016).
473 18. Olds, M. E. Reinforcing effects of morphine in the nucleus accumbens. Brain Res.
474 237, 429–440 (1982).
475 19. Spanagel, R., Herz, A. & Shippenberg, T. S. The effects of opioid peptides on
476 dopamine release in the nucleus accumbens: an in vivo microdialysis study. J.
477 Neurochem. 55, 1734–1740 (1990).
478 20. Bruijnzeel, A. W. Kappa-opioid receptor signaling and brain reward function. Brain
479 Res. Rev. 62, 127–146 (2009).
480 21. Edwards, N. J. et al. Circuit specificity in the inhibitory architecture of the VTA
481 regulates cocaine-induced behavior. Nat. Neurosci. 20, 438–448 (2017).
482 22. Lemos, J. C. et al. Enhanced GABA Transmission Drives Bradykinesia Following
483 Loss of Dopamine D2 Receptor Signaling. Neuron 90, 824–838 (2016).
484 23. Dobbs, L. K. et al. Dopamine Regulation of Lateral Inhibition between Striatal
485 Neurons Gates the Stimulant Actions of Cocaine. Neuron 90, 1100–1113 (2016).
486 24. Kreek, M. J. & Koob, G. F. Drug dependence: stress and dysregulation of brain
487 reward pathways. Drug Alcohol Depend. 51, 23–47 (1998).
488 25. Shoblock, J. R. & Maidment, N. T. Enkephalin release promotes homeostatic
489 increases in constitutively active mu opioid receptors during morphine withdrawal.
490 Neuroscience 149, 642–649 (2007).
23 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
491 26. Nylander, I., Vlaskovska, M. & Terenius, L. The effects of morphine treatment and
492 morphine withdrawal on the dynorphin and enkephalin systems in Sprague-Dawley
493 rats. Psychopharmacology (Berl.) 118, 391–400 (1995).
494 27. Isola, R., Zhang, H., Tejwani, G. A., Neff, N. H. & Hadjiconstantinou, M. Dynorphin
495 and prodynorphin mRNA changes in the striatum during nicotine withdrawal. Synap.
496 N. Y. N 62, 448–455 (2008).
497 28. McCarthy, M. J., Zhang, H., Neff, N. H. & Hadjiconstantinou, M. Nicotine withdrawal
498 and kappa-opioid receptors. Psychopharmacology (Berl.) 210, 221–229 (2010).
499 29. Przewłocka, B., Turchan, J., Lasoń, W. & Przewłocki, R. Ethanol withdrawal
500 enhances the prodynorphin system activity in the rat nucleus accumbens. Neurosci.
501 Lett. 238, 13–16 (1997).
502 30. Rylkova, D., Shah, H. P., Small, E. & Bruijnzeel, A. W. Deficit in brain reward
503 function and acute and protracted anxiety-like behavior after discontinuation of a
504 chronic alcohol liquid diet in rats. Psychopharmacology (Berl.) 203, 629–640 (2009).
505 31. Lindholm, S., Ploj, K., Franck, J. & Nylander, I. Repeated ethanol administration
506 induces short- and long-term changes in enkephalin and dynorphin tissue
507 concentrations in rat brain. Alcohol Fayettev. N 22, 165–171 (2000).
508 32. McCall, J. G. et al. CRH Engagement of the Locus Coeruleus Noradrenergic System
509 Mediates Stress-Induced Anxiety. Neuron (2015). doi:10.1016/j.neuron.2015.07.002
510 33. Sparta, D. R. et al. Construction of implantable optical fibers for long-term
511 optogenetic manipulation of neural circuits. Nat. Protoc. 7, 12–23 (2012).
512 34. Patterson, C. M. et al. Ventral Tegmental Area Neurotensin Signaling Links the
513 Lateral Hypothalamus to Locomotor Activity and Striatal Dopamine Efflux in Male
514 Mice. Endocrinology 156, 1692–1700 (2015).
515 516 517
24 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. a b DYN* c 100 100 LE Dynorphin 1-8 0.10 Dyn IS 0.05 ME 50 50 Leu-Enkephalin 0.00 4.5 5.0 Normalized Intensity (%) Intensity Normalized Normalized Intensity (%) Met-Enkephalin 0 0 0 2 4 6 8 10 0 1 2 3 4 5 6 7 8 9 10 Time (min) Time (min) d e f DYN LE ME 1.4 1.4 1.4
1.2 1.2 1.2
1.0 1.0 1.0
0.8 0.8 0.8 Normalized Signal Normalized Signal Normalized Signal 0.6 0.6 0.6 Peak Area Peak Area Peak Area Peak Area Peak Area Peak Area DYN* DYN* DYN* Peak Area Peak Area Peak Area
g DYN* h DYN i LE j ME 5 5 3 1.0
4 4 0.8 2 3 3 0.6
2 2 0.4 1 1 1 0.2 Peak Area (x 10,000) Peak Peak Area (x10,000) Peak Peak Area (x10,000) Peak Peak Area (x 10,000) Peak 0 0 0 0.0 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 Spiked Concentration (pM) Spiked Concentration (pM) Spiked Concentration (pM) Spiked Concentration (pM)
Figure 1 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
518 Figure 1: (a) Chemical structures of Dynorphin A 1-8, Met-Enkephalin and Leu-
519 Enkephalin (b) Isotopically labeled dynorphin as an internal standard for quantitative
520 analysis. High concentration injections of DYN* did not show significant traces of
521 endogenous dyn (inset trace). A 500 pM sample of DYN* was injected while monitoring
522 both the endogenous dyn (491 à 435 m/z) and isotopically labeled DYN* (495 à 438
523 m/z) mass-to-charge transitions. (c) Nano LC-MS chromatograms of 100 pM standards.
524 Reconstructed ion chromatogram of ME, dyn, DYN*, and LE. (d) Addition of isotopically
525 labeled DYN* results in better quantification of dyn A1-8, (e) Leu-Enkephalin and (f) Met-
526 Enkephalin. (g-j) No effect of ionization suppression from the matrix, as shown for DYN*,
527 Dyn A1-8, Leu-Enkephalin and Met-Enkephalin, respectively. Bulk dialysate was collected
528 and spiked with known amounts of standard. This resulted in a linear response that
529 corresponded with the signal increase from the original sample analyte plus the additional
530 analyte, showing no effect of ionization suppression from the matrix. Four replicates per
531 sample; data shown as average ± SD.
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25 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. b a 150 DYN LE Inlet ME 100 Fiber optic
Outlet 50 Normalized % Normalized Fiber optic ferrule 1 mm Microdialysis Probe 0 0 2 4 6 8 Fraction
Figure 2 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
540 Figure 2: (a) Images of the optodialysis probe. (b) Trace of an in vitro step change from
541 100 pM of DYN, LE, and ME stock solution to a solution of 1 nM Dyn and 400 pM LE and
542 ME. The arrow indicates the first fraction in which the peptide was expected to change.
543 Data was normalized to fraction 4, the fraction expected to reflect elevated concentration
544 stock change. Data shown as average ± SD, n=4 probes.
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26 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a aCC-BY-NC-ND 4.0 International license.
Connect Reduce Baseline Photo Post High K+ Equilibrate Flow stimulation stimulation NAc NAc Flow (0.8ul/min: 1-3 4 5-10 11-12 (2ul/min: 2h) 1h) (45min) Viral Injection Opto-dialysis probe (15 min) (75min) (30min) AP 1.34 AAV5-DIO-ChR2-eYFP Implant Fractions (15 min, 12ul) 2 uL for BzCl derivatization 10uL+1.1 uL DYN*
Day 1 Day 22 (Opto-Dialysis) b DYN d ME vNAcSh dNAcSh vNAcSh dNAcSh 400 400 200 * 200 * * 300 * 300 150 150
200 200 * 100 100 % Baseline % Baseline % Baseline % Baseline 100 100 50 50 0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10 Fraction Fraction Fraction Dyn-Cre - Dyn-Cre + Dyn-Cre - Dyn-Cre + Dyn-Cre - Dyn-Cre + Dyn-Cre - Dyn-Cre + c LE e DA 300 300 300 * 300 * 250 250 * 200 200 200 200
150 150 100 100 % Baseline % Baseline % Baseline % Baseline 100 100
50 50 0 0 0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10 Fraction Fraction Fraction Fraction Dyn-Cre - Dyn-Cre + Dyn-Cre - Dyn-Cre + Dyn-Cre - Dyn-Cre + Dyn-Cre - Dyn-Cre +
Figure 3 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
565 Figure 3: (a) Timeline of experimental procedure outlining viral injection, probe
566 implantation and dialysate collection. (b) Extracellular opioid peptide release shown as %
567 baseline in vNAcSh dyn1-8, (left panel, n=8), dNAcSh dyn1-8 (right panel, n=6). (c) vNAcSh
568 Leu-Enkephalin (left panel, n=4), dNAcSh Leu-Enkephalin (right panel, n=6). (d) vNAcSh
569 Met-Enkephalin (left panel, n=7) and dNAcSh Met-Enkephalin (right panel, n=7). (e) Small
570 molecules simultaneously collected and shown as % baseline in vNAcSh Dopamine (left
571 panel, n=7), dNAcSh Dopamine (right panel, n=7).
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27 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
a b
Push fiber optic down into holder
Apply a drop of thin Glue glue to secure
Push inlet/outlet up into holder
c d e Outlet Outlet Inlet Inlet
Slide inlet/outlet covers down and use thin glue to secure Y- Glue Glue joint Glue Use epoxy and cover the joint
Slide pipette tip up and over the joint
Supplemental Figure 1 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
591 Supplemental Figure 1: Step by step illustration of the custom-made integrated
592 optogenetic-dialysis probes measure peptides and small molecules in freely moving
593 animals. Also see supplementary protocol.
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28 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Ventral Dorsal a 8000 DYN 3000
6000 2000 4000
1000 Baseline (%) 2000 Baseline (%)
0 0 Dyn Cre - Dyn Cre + Dyn Cre- Dyn Cre+ b LE 10000 5000
8000 4000
6000 3000
4000 2000 Baseline (%) Baseline (%) 2000 1000
0 0 Dyn Cre - Dyn Cre + Dyn Cre - Dyn Cre + c 10000 ME 4000 8000 3000 6000 2000 4000 Baseline (%) Baseline (%) 1000 2000
0 0 DynCre - Dyn Cre + Dyn Cre - Dyn Cre +
Supplemental Figure 2 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
617 Supplemental Figure 2: In vivo K+ depolarization. Reliable depolarisation following K+
618 was detected for (a) vNAcSh dyn1-8, (left panel, n=8), dNAcSh dyn1-8 (right panel, n=6). (b)
619 vNAcSh Leu-Enkephalin (left panel, n=4), dNAcSh Leu-Enkephalin (right panel, n=6). (c)
620 vNAcSh Met-Enkephalin (left panel, n=7) and dNAcSh Met-Enkephalin (right panel, n=7).
621 Data show as mean ± SEM.
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29 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
a vNAcSh b dNAcSh
Supplemental Figure 3 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
643 Supplemental Figure 3: Hits maps showing placement of opto-dialysis probes (a)
644 vNAcSh, closed purple circles represent correct hits, open circles represent misses. (b)
645 dNAcSh, closed green circles represent correct hits, open circles represent misses.
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30 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. a GABA 250 250
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100 100 Baseline (%) Baseline (%) 50 50
0 0 0 2 4 6 8 10 0 2 4 6 8 10 Fraction Fraction Dyn-Cre - Dyn-Cre + Dyn-Cre - Dyn-Cre + b GLUT 250 250
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0 0 0 2 4 6 8 10 0 2 4 6 8 10 Fraction Fraction Dyn-Cre - Dyn-Cre + Dyn-Cre - Dyn-Cre +
Supplemental Figure 4 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
667 Supplemental Figure 4: Small molecules simultaneously collected and shown as %
668 baseline in (a) vNAcSh GABA (left panel, n=6), dNAcSh GABA (right panel, n=6). (b)
669 vNAcSh Glutamate (left panel, n=7) and dNAcSh Glutamate (right panel, n=7).
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31 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
714 Supplemental Protocol: Opto-dialysis probe fabrication 715 716 Materials
717 • Inlet = 40/100 (i.d./o.d.) fused silica capillary (5 cm)
718 • Outlet = 75/150 fused silica capillary (6 cm)
719 • Holder = 450/670 fused silica capillary (5 mm)
720 • Inlet cover = 150/360 fused silica capillary (8 mm), covered with 30G thin wall
721 Teflon tubing (7mm)
722 • Outlet cover = 180/360 fused silica capillary (10 mm), covered with 30G thin wall
723 Teflon tubing (9 mm)
724 • Microdialysis semi-permeable membrane (material and pore size dependent on
725 peptides analyzed, we used a 60 kDa molecular weight cutoff, polyacrylonitrile
726 membrane with a slight negative charge (AN69, Hospal, Bologna Italy))
727 • Fiber optic (total length 14 mm) with attached ferrule, this can be adjusted to
728 appropriate length depending on region of interest. (See citations in main text)
729 • Glue: Thin cyanoacrylate glue, gel superglue, five minute epoxy, glue accelerator
730 Instructions
731 1. Place the inlet and outlet capillaries side-by-side and slide the outlet capillary down
732 equal to the length of the desired active area (i.e. if you want to make a 1 mm
733 probe, offset the outlet 1mm below the inlet).
734 2. Using a needle, apply thin liquid glue to adhere capillaries together. Only apply the
735 least amount of glue needed
736 3. Carefully slide a piece of microdialysis membrane over both the inlet and outlet,
737 avoiding pinching and kinking of membrane. Cut excess membrane off. Cutting the
738 membrane at a 45° angle can help if it is problematic to slide over both capillaries
32 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
739 4. Mix a small amount of 5-minute epoxy, and let slightly harden to a consistency that
740 is able to hold stiff peaks. Once the correct consistency is achieved, use a clean
741 hypodermic needle and carefully fill the end of the membrane to create a complete
742 glue plug. Note: If you use the epoxy too early you will get a large glue plug. If you
743 use the epoxy too late, it will be too stiff and not fill the tip of the membrane
744 completely, leaving you with a hole for perfusion media to leak out of. Be careful
745 not to get epoxy on the exterior of the membrane. Let the glue plug dry for 2-3 h
746 before proceeding.
747 5. Once the glue plug is dry, gently tap/move the membrane down the capillary until
748 the inlet is ~100 μm away from the glue plug.
749 6. Mix a small amount of 5-minute epoxy and carefully apply a small amount around
750 the entire backside of the membrane. You should see the glue fill the back. Let dry
751 overnight. If you apply too much, the glue can wick up the membrane and clog the
752 outlet. If you are careful you can create an even seal around the entire membrane
753 7. Slide the 450/670 capillary holder over the back side of the inlet/outlet assembly,
754 followed by sliding the fiber optic into the backside of the capillary holder. The fiber
755 optic is rigid and cannot handle much angled force.
756 8. Position the fiber optic so the tip is adjacent to the outlet, keeping the capillary
757 holder is as close as possible to the ferrule.
758 9. Carefully apply the thin cyanoacrylate glue to the secure the capillary holder to the
759 inlet/outlet assembly and fiber optic together. Careful not to apply too much glue.
760 10. Take 30G thin walled Teflon tubing and slide over the inlet and outlet cover
761 capillaries until all but ~ 1mm of capillary is covered.
762 11. Use a sharp razor blade and gently score the Teflon closest to the exposed
763 capillary. This scoring will help glue to hold Teflon in place.
33 bioRxiv preprint doi: https://doi.org/10.1101/393603; this version posted August 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
764 12. Slide the inlet and outlet covers over the respective inlet and outlet and secure with
765 thin cyanoacrylate (applied to the joint side/exposed capillary side of inlet/outlet
766 cover)
767 13. Use a large plastic pipette tip, and cut a small piece off the pipette tip to use as a
768 junction cover.
769 14. Use gel glue and apply around the entire joint.
770 15. Carefully slide the pipette tip over the membrane/fiber optic and up to the joint with
771 glue.
772 16. Apply some glue accelerator to the joint. Let the joint dry, for a few minutes and
773 reinforce with 5-minute epoxy around the inlet/outlet covers and fiber optic.
774 17. Cut excess inlet and outlet capillaries as close to the inlet and outlet covers right
775 before use.
776 777
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