Chemogenetic modulation of cholinergic interneurons reveals their regulating role on the direct and indirect output pathways from the striatum

Accepted Manuscript Chemogenetic modulation of cholinergic interneurons reveals their regulating role on the direct and indirect output pathways from the striatum

Patrick Aldrin-Kirk, Andreas Heuer, Daniella Rylander Ottosson, Marcus Davidsson, Bengt Mattsson, Tomas Björklund PII: DOI: Reference:

S0969-9961(17)30236-X doi:10.1016/j.nbd.2017.10.010 YNBDI 4049

To appear in:

Neurobiology of Disease

Received date: Revised date: Accepted date:

7 June 2017 22 September 2017 11 October 2017

Please cite this article as: Patrick Aldrin-Kirk, Andreas Heuer, Daniella Rylander Ottosson, Marcus Davidsson, Bengt Mattsson, Tomas Björklund , Chemogenetic modulation of cholinergic interneurons reveals their regulating role on the direct and indirect output pathways from the striatum. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ynbdi(2017), doi:10.1016/j.nbd.2017.10.010

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ACCEPTED MANUSCRIPT

Chemogenetic modulation of cholinergic interneurons reveals their regulating role on the direct and indirect output pathways from the striatum

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Authors

Patrick Aldrin-Kirk1,2, Andreas Heuer1,2, Daniella Rylander Ottosson2,3, Marcus Davidsson1,2, Bengt Mattsson1,2,3 and Tomas Björklund1,2

Molecular Neuromodulation, Department of Experimental Medical Science, Lund

University, 221 84 Lund, Sweden

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Affiliations

Wallenberg Neuroscience Center, Lund University, 221 84 Lund, Sweden

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Developmental and Regenerative Neurobiology, Department of Experimental

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Medical Science, Lund University, 221 84 Lund, Sweden

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Corresponding Author

Tomas Björklund, Molecular Neuromodulation, Lund University, BMC A10 22184, Lund Sweden. E-mail: [email protected]

Running title Modulation of cholinergic neurons in Parkinson’s disease

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ACCEPTED MANUSCRIPT Keywords L-DOPA induced dyskinesias, Parkinson’s disease, animal models, chemogenetics, DREADD, cholinergic interneurons, AAV, transgenic rats, indirect pathway, direct

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pathway

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ACCEPTED MANUSCRIPT Abstract The intricate balance between dopaminergic and cholinergic neurotransmission in the striatum has been thoroughly difficult to characterize. It was initially described as a seesaw with a competing function of dopamine versus acetylcholine. Recent technical

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advances however, have brought this view into question suggesting that the two systems work rather in concert with the cholinergic interneurons (ChIs) driving dopamine release. In this study, we have utilized two transgenic Cre-driver rat lines, a choline acetyl transferase ChAT-Cre transgenic rat and a novel double-transgenic

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tyrosine hydroxylase TH-Cre/ChAT-Cre rat to further elucidate the role of striatal ChIs in normal motor function and in Parkinson’s disease. Here we show that selective and

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reversible activation of ChIs using chemogenetic (DREADD) receptors increases locomotor function in intact rats and potentiate the therapeutic effect of L-DOPA in the

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rats with lesions of the nigral dopamine system. However, the potentiation of the LDOPA effect is accompanied by an aggravation of L-DOPA induced dyskinesias (LIDs).

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These LIDs appear to be driven primarily through the indirect striato-pallidal pathway

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since the same effect can be induced by the D2 agonist Quinpirole. Taken together, the results highlight the intricate regulation of balance between the two output pathways

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from the striatum orchestrated by the ChIs.

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ACCEPTED MANUSCRIPT Introduction Parkinson’s disease (PD) is a neurodegenerative disorder characterized by loss of midbrain dopaminergic (DA) neurons of the substantia nigra pars compacta (SNpc) (Ehringer and Hornykiewicz, 1960; Marsden, 1990). The subsequent depletion of

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striatal DA leads to prominent motor deficits including the cardinal PD symptoms of akinesia, rigidity, tremor, gait and posture instability (Marsden, 1990). Pharmacological therapy through the treatment with 3,4-dihydroxyphenyl-L-alanine (L-DOPA) considerably alleviates the severe motor symptoms seen in PD patients (Cotzias et al.,

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1969; Marsden, 1990). Although initially very effective, motor complications in the form of L-DOPA induced dyskinesia (LID) typically occur within a few years of treatment,

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followed by a narrowing of the therapeutic window with time (Nyholm, 2007). The development of LIDs is associated with a broad range of changes in striatal

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neurotransmission (Jenner, 2008). Several recent reports have implicated elevated striatal cholinergic neurotransmission to play a role in the motor complications induced

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by chronic L-DOPA therapy (Ding et al., 2011; Lim et al., 2014; Shen et al., 2015; Won et

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al., 2014). The induction of LIDs has been linked to enhanced ERK phosphorylation of striatal cholinergic interneurons (ChIs) following repeated L-DOPA treatment, and

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selective ablation of ChIs has been shown to attenuate LIDs in dyskinetic parkinsonian mice (Ding et al., 2011; Won et al., 2014). Furthermore, pharmacological studies have also implicated both muscarinic and nicotinic receptors in LIDs (Bordia et al., 2016; Ding et al., 2011; Quik et al., 2013). Recent studies have further shown that activation of ChIs selectively using optogenetics is sufficient to drive DA release in the intact striatum and Nc. Accumbens (Cachope et al., 2012; Threlfell et al., 2012) with striatal activation also linked to aggravated LIDs (Bordia et al., 2016).

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ACCEPTED MANUSCRIPT ChIs are large, tonically active aspiny neurons located throughout the striatum and are the main source of striatal acetylcholine (Woolf and Butcher, 1981). Although comprising only about 1 % of striatal neurons, ChIs project widely throughout the striatum with high axonal density, each ChI giving rise to, on average, 500 000 axonal varicosities in the rat striatum, underlining the importance of cholinergic

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neurotransmission in striatal function (Bolam et al., 1984; Contant et al., 1996). The ChI influence striatal function through activation of muscarinic and nicotinic receptors on post-synaptic neurons. The expression pattern of these receptors is complex in most striatal afferents and efferents, especially in the medium spiny

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projection neurons. All five muscarinic receptors (M1-M5) are expressed in the dorsal

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striatum but they differ significantly in expression between the two output pathways (expressing the D1 or D2 dopamine receptor). Through single-cell rt-PCR it has been

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shown that in the direct (D1) pathway, 87% express M1, 70% express M4 and 9% express the M5 receptor (Yan et al., 2001). Similarly, in the indirect (D2) pathway 80%

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express the M1 receptor, 50% the M4 receptor, 15% the M3 receptor and 5% the M5 receptor. The M2 receptor is not expressed in any efferent neurons from the striatum. Of

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note is also that the levels of the receptors vary significantly with e.g., the M4 receptor

2001).

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being five-fold higher expressed in the D1 compared to the D2 neurons (Yan et al.,

In order to examine the role of ChIs in the intact striatum as well as in the context of LIDs with high selectivity, we used Cre-dependent viral expression of chemogenetic designer receptors exclusively activated by designer drug (DREADD) receptors (Armbruster et al., 2007; Guettier et al., 2009) in transgenic rats expressing the Cre recombinase under the control of the choline acetyltransferase promotor (ChAT-Cre

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ACCEPTED MANUSCRIPT rats) (Witten et al., 2011). This approach allowed for highly selective, non-invasive (post-surgery) modulation of ChI activity both in the intact system and in a DA depleted rat model of LIDs. Our results indicate that ChIs indeed play a modulatory role for striatal output, revealed as an enhancement of striatal DA release in the intact striatum and an increase in LIDs induced by chronic L-DOPA treatment through modulation of

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striatal D2-bearing medium spiny neurons (MSNs). However, this study also highlights the complexities of this modulatory capacity with an effect on the direct pathway that drives the system in the opposite direction. Taken together these data present a function

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of ChIs as a regulator of balance between the direct and indirect output pathways.

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ACCEPTED MANUSCRIPT Materials and methods Experimental procedure To evaluate the role of the ChIs in modulating the intact basal ganglia circuitry, transgenic Long Evans ChAT-Cre rats (n=16) were used. In order to further dissect the

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interaction between the ChIs and striatal dopaminergic release, 16 ChAT-Cre/TH-Cre double transgenic rats were generated through breeding homozygous TH-Cre males with ChAT-Cre homozygous females. To assess the modulatory role of ChIs in LIDs, transgenic Long Evans ChAT-Cre rats (n=30) and wild type siblings (n=10) received a

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unilateral 6-OHDA lesions of the medial forebrain bundle (MFB) through stereotactic

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surgery.

Adult female ChAT-Cre rats with an intact DA system were infused with and AAV-8

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vector containing either hM3Dq + rM3Ds DREADDs (n=8) or with a vector containing KORD (n=8) into the striatum. The ChAT-Cre/TH-Cre double transgenic rats (n=14)

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were infused with an AAV-8 containing the hM3Dq DREADD construct in the striatum and an AAV-6 containing the ChR2-IRES-YFP channel rhodopsin construct in the SNpc.

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In a third experiment, following a 6-OHDA lesion, the rats were assessed using the

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corridor and cylinder tests to include animals only with complete lesions and severe behavior impairment. The ChAT-Cre animals were balanced into three groups based on LID scores so that Ctrl and ChI inhibition (KORD) groups displayed high LIDs and the ChI activation (Dq/Ds) group displayed moderate LIDs. Stereotactic infusion was then performed into the right striatum using AAV-8 vectors containing DREADD constructs expressing either hM3Dq + KORD (n = 10) or hM3Dq + rM3Ds (n = 10). The third group of 6-OHDA lesioned, wild type, Long Evans rats were used as controls, matched to the hM3Dq + KORD with regards to dyskinesia severity and infused with a AAV-8 GFP vector 7

ACCEPTED MANUSCRIPT (n=10). All animals were evaluated in a battery of motor performance tests and dyskinesia scoring test with or without DREADD mediated modulation of ChIs. Euthanasia in all animal groups was conducted post in vivo electrochemical recordings or just prior to ex vivo electrophysiological patch clamping.

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Animals Adult, female, wild type Long Evans, ChAT-Cre transgenic, Sprague Dawley TH-Cre transgenic and ChAT-Cre/TH-Cre double transgenic rats (225-250g) were housed in standard laboratory cages with ad libitum access to food and water, under a 12:12h

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dark-light cycle in temperature and humidity controlled rooms. All experimental procedures performed in this study were approved by the local ethics committee in the

AAV Vector production

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with national and EU regulations.

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Malmo/Lund region “Malmö/Lunds regionala djurförsöksetiska nämnd” in accordance

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Double-floxed Inverted Orientation (DIO) AAV-8 vectors, containing the hSyn-DIO-

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rM3Dq-HA, hSyn-DIO-rM3Ds-mCherry, hSyn-DIO-KORD-IRES-mCitrine and AAV-6 hSynDIO-ChR2-IRES-YFP constructs, flanked 3’ by the Woodchuck hepatitis virus post-

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transcriptional regulatory element (WPRE) and terminated with a SV40 derived polyadenylation sequence, were produced by dual-plasmid, calcium precipitation transient transfection of HEK-293 cells (Grimm et al., 1998). The DIO (also called “flex” approach) is a vector design where two pairs of Cre-recombinase target sites (loxP) are inserted into the vector genome flanking the gene of interest. The gene itself is placed in reverse compared to the promoter and no gene expression is achieved without Crerecombinase. When Cre-expressing cells are transduced, the recombinase flips the gene

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ACCEPTED MANUSCRIPT into the expressing direction and it is locked in place by a second recombinase reaction. (For more information see Schnutgen et al. (2003)). Cells and supernatant were then purified by iodixanol gradient centrifugation and anion exchange chromatography (Zolotukhin et al., 2002). Viral titers were quantified to range between 4E12 and 1.2E13

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GC/ml using qPCR with primers against the AAV ITR sequences.

Surgical procedures

Rats were deeply anesthetized using an i.p. injection of fentanyl/dormitor mixture prior to all surgeries and placed in a stereotactic frame with the tooth bar individually

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adjusted for flat skull. Targeting coordinates for all infusions were performed in relation to the animals bregma. The animals then received small burr hole through the skull and

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infused with solutions containing either 6-OHDA or viral vectors unilaterally into the brain using a pulled glass capillary (60-80 µm i.d. and 120-160 µm o.d.) attached to a

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25µl Hamilton syringe connected to an automated infusion pump. All surgical interventions were performed on the right hemisphere of the brain and thus affect the

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left side of the body most severely in motor tests. For unilateral 6-OHDA lesions of the

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MFB, 3 µl of 6-OHDA (Cl-salt, Sigma Aldrich) was diluted in 0.02% ascorbic acid and infused at a concentration of 3.5 µg/µl (free base weight) at the following coordinates:

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AP= -4.4; ML = -1.1; DV -7.8 with an infusion rate of 0.3µl/min (Carta et al., 2007; Ungerstedt and Arbuthnott, 1970). AAV-8 viral vectors were injected into the striatum at two infusion sites with two deposits/site at the following coordinates and volumes: Rostral injection site (2.5+1 µl): AP= +1.0; ML= -2.5; DV= -5.0/-4.0. Caudal injection site: AP= -0.4; ML= -3.0; DV= -5.0/-4.0 (Cederfjall et al., 2012). AAV-6 viral vectors injected into the SNpc at a single site used the following coordinates: AP= -5.3; ML= 1.1; DV= -7.2.

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ACCEPTED MANUSCRIPT All viral vector solutions were injected with an infusion rate of 0.4 and 0.2 µl/min for the striatum and SNpc respectively.

Tissue preparation and immunohistochemistry All animals were sacrificed between 12 and 15 weeks post initial surgery following

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amperometry measurements by sodium pentobarbital overdose (Apoteksbolaget). Following recordings, the rats were trans-cardially perfused with 150 ml physiological saline solution followed by 250 ml of ice cold 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH = 7.4). The rat brains were then removed and posed-fixed for 2

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hours before being moved to buffered sucrose (30%) for cryoprotection for at least 24 hours. The brains were then frozen in dry ice and cut into coronal sections with a

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thickness of 35 µm using a sliding microtome (HM 450, Thermo Scientific). The brain sections were collected in 8 series and stored in a 0.5M sodium phosphate buffer, 30%

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glycerol and 20 % ethylene glycol anti-freeze solution at -20°C.

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For immunohistochemical analysis, using a standard free-floating protocol, tissue sections were washed (3x) with TBS (pH 7.4) and incubated for one hour in 3 % H2O2 in

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0.5 % TBS Triton solution in order to quench endogenous peroxidase activity and to increase tissue permeability. Following another washing step, the sections were blocked

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in 5 % bovine serum and incubated for one hour and subsequently incubated with primary monoclonal antibodies overnight. ChIs were identified through staining for choline acetyl transferase (goat anti-ChAT AB144P, 1:500). hM3Dq DREADDs were identified though HA-tag (mouse anti-HA MMS101R, 1:2000) and rM3Ds was identified through mCherry (goat anti-mCherry LSC204207, 1:1000). KORD and channelrhodopsin-2 were identified through GFP (chicken anti-GFP AB13970, 1:20000). Following overnight incubation, the primary antibodies 10

ACCEPTED MANUSCRIPT were first washed off using a TBS washing step and then incubated with secondary antibodies for two hours in TBS solution. For 3, 30-diaminobenzidine (DAB) immunohistochemistry, biotinylated anti-mouse (BA-2001, Vector Laboratories 1:250), anti-goat (705-065-147, Jackson ImmunoResearch 1:250) and anti-chicken (BA-9010, vector laboratories 1:250) secondary antibodies were used. Following incubation and

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another TBS wash step the brain sections were incubated with the ABC-kit (Vectorlabs) in order to amplify the staining intensity through streptavidin-peroxidase conjugation and followed by a DAB in 0.01% H2O2 color reaction. For immuno-fluorescence Alexa conjugated secondary antibodies (Alexa anti-chicken Thermo scientific A11039 1:250

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and Cy3 anti mouse Jackson Immunoresearch 715-165-150 1:250) were used.

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iDISCO tissue clearing

Animals used for iDISCO tissue clearing were perfused using 150 ml physiological saline

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solution followed by 250 ml of ice cold 2% PFA in 0.1 M phosphate buffer (pH = 7.4) and post fixed in 2 % PFA for one hour. iDISCO tissue clearing was performed according to

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Renier et al. (2014). Following dissection, the hemisphere that had received the viral

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infusions was dehydrated in methanol/H2O with progressively higher methanol concentration. The concentrations of methanol used was 20, 40, 60, 80 and 100 %

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methanol and each dehydration step was performed for one hour. The samples were then incubated overnight in 66 % DCM and 33 % methanol at room temperature and then washed twice in 100 % methanol for one hour and then chilled to 4 °C. Thereafter, they were incubated in freshly prepared 5 % H2O2 and kept overnight at 4 °C. Following overnight incubation, the samples were rehydrated in methanol/H2O with progressively lower methanol concentration. The concentrations of methanol used was 80, 60, 40 and 20 % methanol and finally PBS with each rehydration step being performed for 30

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ACCEPTED MANUSCRIPT minutes. After two washes for one hour in PTx.2 at room temperature. Following washing the samples were incubated in permeabilization solution for 48 hours at 37 °C and then washed in PTx.2 solutions twice for 30 minutes. Following washing, the samples were incubated in in blocking solution made up from PTwH/5 % DMSO/ 3 % donkey serum for 72 hours and then washed twice in PTwH solution for one hour.

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Following washing the samples were incubated with the primary antibodies (mouse anti-HA MMS-101R, 1:200, chicken anti-GFP AB13970, 1:100) in PTwH/5 % DMSO/ 3 % donkey serum for seven days. The samples were then washed four times in PTwH for one hour and incubated with secondary antibodies (Cy3 anti-chicken 703-165-155

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Jackson immunoresearch, 1:100 and alexa 647 anti-mouse 715-605-151 Jackson immunoresearch 1:100) for five days. Following four rounds of washing in PTwH for

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one hour, the samples were dehydrated in methanol/H2O with progressively higher methanol concentration. The concentrations of methanol used were 20, 40, 60, 80 and

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100 % methanol and each dehydration step was performed for one hour at room

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temperature. The samples were then incubated in 66 % DCM mixed with 33 % methanol for three hours followed by 100 % DCM 2x15 minutes at room temperature. Finally, the

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samples were transferred and incubated in DBE until analysis.

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Light sheet imaging

Tissue cleared samples were imaged in a sagittal orientation using a sCMOS-5.5-CL3 camera equipped light sheet microscope (Ultramicroscope II, LaVision Biotec) with a 2x/0.5 objective lens (MVPLAPO 2x) with a 6-mm working distance dipping cap. All imaging used the Imspector-Pro219 software and scanned continuously with a step size of 10 µm at 3.2x magnification (7988x9472 pixels). Post imaging visualization utilized the Arivis Vision 4D v.2.12.3 software.

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ACCEPTED MANUSCRIPT Abnormal involuntary movements (AIMs) Amplitude and severity of axial, forelimb and orolingual dyskinesias in response to LDOPA, D1 or D2 agonists was assessed using a well characterized abnormal involuntary movement (AIM) rating scale (Cenci and Lundblad, 2007). Following habituation for 10

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minutes in transparent cages, animals were injected with either L-DOPA (9mg/kg high dose s.c.), (3mg/kg mid dose s.c.), CNO (3mg/kg s.c.), SalB (10mg/kg s.c.), SKF38393 (2.5 mg/kg s.c.) or Quinpirole (0.05mg/kg s.c.). The animals were scored for a minimum of three hours, or until the dyskinetic behavior subsided, with each animal being scored for

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one minute every 20 minutes.

Rotational behavior

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Rotational behavior was used to evaluate the effect of unilateral ChI activation in conjunction with amphetamine, a potent inducer of endogenous striatal DA release. Rats

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were placed in bowls and fixed in a harness connected to an automated rotometer

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modelled after the design of Ungerstedt and Arbuthnott (1970) and recorded using the AccuScan Instruments recording software. Before the rotation test, the rats were

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allowed to habituate in the rotometer bowls for 10 minutes prior to injection. Following

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habituation, the animals were recorded for a total of 130 minutes following CNO injection (3 mg/kg s.c). Amphetamine (2.5 mg/kg i.p) and SalB (10 mg/kg s.c.) was injected 45 minutes into the rotation recording, allowing the CNO time to activate DREADD expressing ChIs.

Stepping test Unilateral lesion of the midbrain dopaminergic neurons produces a distinct phenotype that includes; forelimb akinesia, exploratory forelimb asymmetry and unilateral visio-

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ACCEPTED MANUSCRIPT spatial neglect. In order to assess the potency of the MFB 6-OHDA lesion and the effect of ChI activation on motor performance, we investigated the forelimb akinesia using the stepping task (Schallert et al., 1979), modified to a side-stepping test by Olsson et al. (1995) at four weeks following the 6-OHDA lesion. The rats were initially trained to be held by the researcher whilst making adjusting forelimb steps as the animal was moved

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sideways across a flat surface at a constant speed for a total distance of 90 cm. The number of adjusting steps was counted by the researcher for both forehand and backhand limb steps and compared to the number of steps counted on the intact forelimb (ipsilateral to the lesion). Once a stable baseline was achieved the animals were

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injected with CNO (10mg/kg (s.c.), L-DOPA 1mg/kg (low dose s.c), a combination both CNO and L-DOPA or a vehicle (saline) and tested for 3 consecutive trials. The researcher

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was blinded to both animal group and treatment.

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Cylinder test

Unilateral 6-OHDA lesioned animals exhibit a strong asymmetry in forelimb exploration

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as described previously (Bjorklund et al., 2010). In order to determine the potency of

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the 6-OHDA lesions as well as investigate the effect on forelimb exploration when activating ChIs, animals were placed in a glass cylinder (20 cm in diameter) and

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recorded with a digital video camera in reduced light conditions, in order to stimulate an exploratory response. Two perpendicular mirrors were placed behind the cylinder, permitting a clear recording of the animals from all angles within the cylinder. The animals were recorded for at least 30 paw touches on the walls of glass cylinder, or for a maximum of five minutes following injection of CNO (10mg/kg), L-DOPA (1mg/kg), a combination of CNO and L-DOPA or a vehicle (saline). The animals were tested at 70 minutes’ post injection of CNO. The paw touches on the cylinder wall was then scored

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ACCEPTED MANUSCRIPT post hoc from the recordings by a blinded researcher, with the score expressed as percentage of ipsilateral (right) or contralateral (left) touches out of the number of total paw touches.

Corridor test

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The potency of the 6-OHDA lesion was assessed by unilateral visuospatial neglect in the corridor test, as described previously (Dowd et al., 2005). The rats were placed inside a corridor (1500 x 70 x 230 mm) with ten pairs of adjacent food bowls distributed evenly throughout the corridor with each food bowl being filled with 5-10 sugar pellets.

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Retrievals from the food bowl was scored as each time the rats poked their nose into a unique bowl. Repeated nose pokes into the same bowl without retrievals from any other

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bowl was not scored. The rats were tested until 20 retrievals were scored, or for a maximum of five minutes. Prior to testing the rats were trained in the corridor for

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several days until a stable baseline of retrievals was achieved. Before each test the rats were habituated in an empty corridor without any food bowls for five minutes. The rats

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were then scored by percentage ipsilateral (right) and contralateral (left) retrievals out

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of the total number of retrievals by a blinded researcher.

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Drug preparation

6-OHDA (Cl-salt, Sigma-Aldrich) was prepared in sterile 0.9 % saline with the addition of 0.02 % ascorbic acid in order to prevent oxidization and finally vortexed until fully dissolved. d-Amphetamine was prepared at 1 mg/ml in sterile 0.9 % saline solution and vortexed until fully dissolved. L-DOPA was dissolved in sterile saline at 9 mg/ml together with Benserazide at 15 mg/ml and vortexed until fully dissolved. The L-DOPA and Benserazide mix was then injected s.c. at 3 mg/kg or 9 mg/kg for the L-DOPA or at

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ACCEPTED MANUSCRIPT 15 mg/kg for the Benserazide. d-Amphetamine was then injected i.p. at 2.5 mg/kg. CNO (Toronto Research Chemicals) was pre-diluted in pure DMSO (Sigma) (2% of final volume) and vortexed until fully dissolved into a clear yellow solution. The CNO solution was then diluted in sterile 0.9 % saline to 3 mg/ml and injected s.c. at 3 mg/kg. SalB (Cayman Chemical Company) was prepared in pure DMSO at 30 mg/ml and vortexed

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extensively until fully dissolved. Animals were then injected s.c. at 10 mg/kg. D1 agonist SKF38393 (sigma) was dissolved in sterile saline at 1.5 mg/ml and vortexed until fully dissolved. SKF38393 was then injected s.c. at 1.5 mg/kg. D2 agonist Quinpirole (sigma) was dissolved in sterile saline at 0.05 mg/ml and vortexed until fully dissolved. Animals

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were then injected i.p. at 0.05 mg/kg.

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Stereological quantification

The number of GFP positive cholinergic neurons in the striatum and TH positive

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neurons in the substantia nigra were quantified using the Stereo Investigator software suite (version 11 MBF Bioscience) with a 25 x magnification lens. The total numbers of

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cells were estimated according to the optical fractionator (West, 1999) and the

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coefficient of error was calculated according to Gundersen and Jensen (1987), and

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values ≤ 0.05 were accepted.

Amperometry recordings Electrochemical recordings were performed according to our previous protocol (AldrinKirk et al., 2016). DA release was measured from the rodent brain at the tip of a Nafion® coated carbon fiber electrode (SF1 A, Quanteon, L.L.C. Nicholasville, KY, USA) by applying high speed chronoamperometry. The recordings were made using a FAST-16 (Quanteon) setup as reported previously (Aldrin-Kirk et al., 2016; Lundblad et al., 2012).

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ACCEPTED MANUSCRIPT A square wave potential of 0.55 V; 0.0 V resting potential was applied vs an Ag/AgCl reference electrode and all recordings used a sampling rate of 2Hz. Each electrode used for the recordings was calibrated immediately before use in vivo. Linearity and sensitivity was calibrated by addition of three 2 µM increments of DA in 0.1M PBS. All electrodes had a linearity correlation of > 0.98, a selectivity of DA over ascorbic acid of >

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1000:1, and a limit of detection below 0.01 µM. Two glass micropipettes (outer diameter ≈ 25µm) were mounted in close proximity (50-100µm) to the tip of the electrode. The glass pipettes were loaded with either 120M KCl or 100µM CNO and connected to a micro pressure injection system (Picospitzer, Aldax, Alvsjo, Sweden). Injection volumes

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were estimated by measuring the KCl solution movement inside the pipette and were set to about 100 nL. Additionally, a 50µM optical fiber cable was mounted equidistant on

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the opposite side of the recording electrode. The optical fiber cable was connected to a blue laser and the energy release was calibrated individually before each recording. In

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vivo electrochemical recordings were performed under 1.5% Isoflurane anesthesia with

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the recording electrode/pipette/optical fiber assembly being lowered into the striatum at the following coordinates relative to bregma: AP: +0.8; ML: -3.2 and DV: -4.0 (from

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Dura) with the incisor bar set at -4.5mm (flat skull). After establishing a baseline for the

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electrode in the cortex (DV: -1.0 mm) DA released by either KCl, CNO, or laser light at different settings were recorded and no significant change in baseline DA levels were observed during any of the stimulations. For analysis of the light induced DA release the light artefacts observed during the cortical baseline measurements were subtracted from the signal obtained from recording in the striatum. To establish optical stimulation of DA release in combination with chronoamperometric recordings a series of pilot measurements were conducted (Supplemental Figure 1). A wide range of stimulation parameters were tested including stimulus duration (10s to 300s), stimulus intensity

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ACCEPTED MANUSCRIPT (0.625mW to 19mW) and frequency (25Hz pulse and constant illumination). Although there was variability in the minimum amount of energy necessary to induce DA release (2.5mW to 19.1mW) these can be explained by differences in distance from the optical fiber to the recording electrode, the transduction/expression of the opsin, the exact location of recording, etc. For the recordings presented in Figure 3, the parameters

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optimized for the strongest DA release were utilized. Those parameters were 120 seconds of continuous laser light at 13.0 mW power. Each individual observation was replicated in at least three animals prior to inclusion in the results.

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Electrophysiology

Patch-clamp electrophysiology was performed on coronal striatal brain slices from TH-

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Cre/ChAT-Cre transgenic rats who had received a unilateral injection of AAV-8-DIOhM3Dq-mCherry into the dorsal striatum. Rats were killed more than 4-wks post AAV-

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injection by an overdose of pentobarbital and brains were rapidly taken out and coronally cut on a vibratome at 275 mm in ice-cold artificial cerebrospinal fluid (ACSF).

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After 30 min of acclimatization in 34°C, slices were transferred to a recording chamber

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and submerged in a continuously flowing ACSF solution gassed with 95% O2 and 5% CO2 at 28 °C. The composition of the Krebs solution for slice recording was 126 mM NaCl, 2.5

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mM KCl, 1.2 mM NaH2PO4-H2O, 1.3 mM MgCl2-6H2O, and 2.4 mM CaCl2-6H2O. All recordings were made using Multiclamp 700B (Molecular Devices), and signals were acquired at 10kHz using pClamp10 software and a data acquisition unit (Digidata 1440A, Molecular Devices). Input resistances and injected currents were monitored throughout the experiments. Borosilicate glass pipettes (3–7 MOhm) for patching were filled with the following intracellular solution (in mM): 122.5 potassium gluconate, 12.5 KCl, 0.2 EGTA, 10 Hepes, 2 MgATP, 0.3 Na3GTP and 8 NaCl adjusted to pH 7.3 with KOH.

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ACCEPTED MANUSCRIPT DREADDs positive ChIs were identified with their red fluorescence and size (Fig. 3A) and patched in cell-attached configuration mode for discharge rate (n of cells = 7). Spontaneous firing activity was recorded in voltage-clamp mode first without and then with CNO added to the bath. CNO was freshly prepared in 10% DMSO and 90% saline and added at the concentration 10M. Discharge rates were compared in control (ACSF)

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and CNO condition with a paired t-test.

Statistics

Data analysis, plotting and statistics were conducted in R Statistical Computing Platform

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(version 3.2.1) and SPSS version 23. Statistical tests included Bonferroni corrected paired Student’s t-test when only two states/time points were compared and one-way

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ANOVA or two-way mixed model repeated measures ANOVA when three or more groups/states/time points were compared. In the latter case this was followed by

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Levene’s Homogeneity test. This determined the choice of post-hoc test to either Dunnett’s T3 (when Levene’s fail) or Tukey’s HSD. For assays where the results

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displayed a skewed, non-normal, distribution, the ANOVA was replaced by the Kruskal-

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Wallis test followed by Bonferroni corrected all pair comparison using the MannWhitney U test. Unless otherwise noted, data in figures is presented as the arithmetic

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mean plus and/or minus one standard error of the mean (SEM). Comparisons were considered significant when the multiple comparison corrected p-value was less than 0.05.

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ACCEPTED MANUSCRIPT Results AAV serotype selection in ChAT-Cre transgenic rats reveals that AAV-8 has a strong tropism for striatal ChIs We have found that AAV serotypes differ in spread and transduction efficiency between

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distinct neuronal populations in the rodent brain. In order to transduce the striatal ChIs with high efficiency, we validated AAV serotypes 1, 2, 5, 8 and 9 in intact ChAT-Cre BACtransgenic rats (Witten et al., 2011) for their ability to transduce the ChI cell populations in the striatum (Figure 1A-E). The tested AAVs contained a Cre-inducible (flexed or DIO)

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GFP construct and the titers were adjusted to 1x1012 GC/ml. As shown in Figure 1, AAV8 was consistently the most efficient for all tested parameters, followed by AAV-9. The

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histological analysis revealed that AAV-8 transduced a significantly higher number of ChIs compared to AAV serotypes 1 (p=0.005), 2 (p=0.019) and 5 (p=0.036) (Figure 1F).

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AAV-8 also displayed a significantly higher GFP expression compared to AAV serotypes

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1 (p=0.007), 2 (p=0.017) and 5 (p=0.028), as shown by optical densitometry (OD) analysis of the transduced striatal area (Figure 1G). Consistently, AAV-8 also displayed

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significantly higher optical density within the entire striatum (Figure 1H) compared to

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AAV-1 p=0.004) and AAV-2 (p=0.01). Although displaying a strong trend, AAV-8 did not transduce a significantly higher striatal volume compared to any of the higher vectors (Figure 1I). AAV-8 was not significantly different from AAV-9 in regards to the number of GFP+ neurons or in measured OD but consistently showed a higher mean in all analyzed parameters in the striatum. Taken together these results indicate that AAV-8 is the preferred AAV serotype for transducing cholinergic cell populations in the striatum. In order to confirm that AAV-8 could be used to transduce most of the striatal ChIs with DREADDs, we used an AAV-8 vector containing a Cre-inducible hM3Dq DREADD 20

ACCEPTED MANUSCRIPT construct and injected it into two sites of the striatum of intact ChAT-Cre transgenic rats. Histological analysis of these animals revealed that the hM3Dq DREADD was efficiently expressed in most ChIs throughout the striatum (Figure 1J and Figure 7C below).

Modulation of striatal ChIs using DREADDs reveal a novel modulatory function on

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motor performance in intact ChAT-Cre transgenic rats. Chemogenetics is a powerful tool that allows non-invasive and reversible in vivo modulation of activity in genetically defined neuronal populations. In order to elucidate the functional input of striatal ChIs on motor performance and behavior, the first

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experimental setup (Figure 2A) used chemogenetic AAV-8 DREADD vectors in intact ChAT-Cre transgenic rats. To achieve the desired neuronal modulatory control, we

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injected AAV-8 vectors containing either a mix of hM3Dq and rM3Ds DREADDs (expressed in separate AAV genomes) driving neuronal activation or a single AAV-8

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vector containing the KORD (Kappa-opioid receptor DREADD) receptor promoting inhibition of the striatal ChIs. The mix of hM3Dq and rM3Ds was based on experiments

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in the dopamine system showing an additive effect of the combination compared to each

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DREADD alone (Aldrin-Kirk et al., 2016).

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To evaluate the motor phenotype induced by ChI modulation, forelimb akinesia and asymmetry was assessed using the stepping and cylinder tests respectively. Forelimb akinesia as assessed by the stepping test (Figure 2B) revealed that animals injected with the combination of hM3Dq/rM3Ds DREADDs displayed a significant increase (p=0.01) in the number of adjusting steps following treatment with the CNO DREADD ligand. Conversely, the experimental group of animals injected with the inhibitory KORD vector displayed a small but significant decrease (p=0.047) in the number of adjusting steps following the treatment with SalB compared to a saline injection in the same rats. 21

ACCEPTED MANUSCRIPT Forelimb asymmetry as assessed in the cylinder test however, was not significantly changed following treatment with DREADD ligands CNO or SalB (Figure 2C), although there was a trend for increased contralateral paw touches following CNO treatment while SalB treatment had the opposite trend. Next, we used amphetamine driven rotational locomotor behavior in conjunction with treatment of DREADD ligands CNO or

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SalB in order to investigate the striatal cholinergic function on exploratory behavior. Animals expressing the activating hM3Dq/rM3Ds DREADDs displayed increased contralateral rotation when combined with CNO treatment (Figure 2D) indicating a facilitation of amphetamine induced DA release by the striatal cholinergic system. In

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contrast, animals expressing the inactivating KORD displayed increased ipsilateral rotations after SalB treatment (Figure 2D), indicating suppressed amphetamine driven

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DA release. The rotational effects were not seen when animals were treated with apomorphine (Figure 2E), indicating that the ChIs modulate striatal output on a pre-

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synaptic dopaminergic terminal.

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In order to investigate the effect of DREADD mediated cholinergic stimulation, we then used electrophysiological measurement on ChIs expressing mCherry-hM3Dq DREADDs

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in ex vivo brain slices from intact ChAT-Cre transgenic rats. ChIs were identified through

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mCherry fluorescence (Figure 3A). Following immersion of the brain slice in CNO the firing rate of patched ChIs significantly increased to >10Hz indicating activation of ChIs by CNO (Figure 3B, C). To further elucidate the role of ChIs and the effect of striatal cholinergic signaling on striatal DA release we generated a novel ChAT-Cre/TH-Cre double transgenic rat by crossing ChAT-Cre and TH-Cre transgenic rats (Aldrin-Kirk et al., 2016). ChAT-Cre/THCre rats were injected with Cre-inducible AAV-vectors expressing channelrhodopsin-2

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ACCEPTED MANUSCRIPT (ChR2) in the SNpc and hM3Dq in the striatum, allowing for independent activation of both the midbrain DA neurons and striatal ChIs. Light stimulation in the striatum resulted in a strong but short release of striatal DA as expected by ChR2 activation on dopaminergic terminals (Figure 3D). Interestingly, striatal infusion of CNO acting on hM3Dq transduced ChIs resulted in robust and prolonged striatal DA release but with a

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lower amplitude compared to the light induced DA release. Of note is that the light intensity levels in general had to be very high to elicit robust neurotransmitter release from dopamine neurons and that the saturating levels varied significantly between animals (See Supplemental Figure 1 for details).

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DREADD modulation of striatal ChIs enhances L-DOPA mediated motor recovery and

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modulates LIDs in a unilateral 6-OHDA model of Parkinson’s disease The influence of striatal ChI modulation on LIDs was evaluated using an experimental

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setup that was designed to allow for assessment of chemogenetic modulation in the context of 6-OHDA induced DA depletion (Figure 4A). In this experiment, we again

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mixed two Cre-inducible activating DREADD AAV-8 vectors expressing either the

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hM3Dq or the rM3Ds DREADD. In a second group, we mixed the same hM3Dq vector with the DIO-KORD vector to allow for orthogonal activation and inhibition using

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separate ligands in the same animal as conducted previously (Aldrin-Kirk et al., 2016; Vardy et al., 2015). Following unilateral lesion of the medial forebrain bundle (MFB) using the 6-OHDA toxin, the animals were treated for 2 weeks with L-DOPA (9mg/kg), inducing LIDs. AIM scoring and motor performance tests were then conducted before and after the animals were transduced with AAV-8 DIO-hM3Dq/rM3Ds or DIOhM3Dq/KORD vectors. Neither of the DREADD vectors displayed any effect on the animal’s motor performance in the absence of CNO/SalB (Figure 4B, C). Importantly,

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ACCEPTED MANUSCRIPT although the L-DOPA administration was switched from a daily to a twice-weekly administration protocol after the induction phase, LIDs and motor performance remained stable in all experimental groups following viral transduction of the ChIs (Figure 4D). The lesion severity was confirmed port-mortem using histological analysis of the nigro-

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striatal dopamine system (Figure 4E). The TH+ immunoreactivity was reduced by more than 90% in the right striatum in all groups as assessed by densitometry compared to the intact, left striatum (Figure 4F) which was coupled to a near complete loss of TH+

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neurons in the substantia nigra (Figure 4G).

At seven weeks post AAV injection, the animals were re-tested in the stepping limb

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akinesia and cylinder limb asymmetry motor performance tests as well as scored for AIMs following L-DOPA and DREADD ligand treatment. Animals expressing

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hM3Dq/rM3Ds DREADDs displayed no significant changes in motor performance in either the stepping or cylinder tests following treatment with either CNO or low dose L-

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DOPA (1mg/kg) (Figure 5A, C). However, when hM3Dq/rM3Ds animals received a

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combined treatment of CNO together with a low dose of L-DOPA, forelimb limb use was significantly increased in the stepping test indicating decreased akinesia (Figure 5A).

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This shows a positive potentiation of the therapeutic potential of low dose L-DOPA that was not associated with any observed dyskinesias. The experimental animal group expressing the inhibitory KORD vector did not display any difference in motor impairment either in the forelimb akinesia stepping test (Figure 5B) or in the forelimb asymmetry cylinder test following treatment with SalB and low dose L-DOPA (Figure 5D). There was also no significant change in motor performance when combining treatment with SalB and a low dose of L-DOPA in either motor performance tests.

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ACCEPTED MANUSCRIPT For evaluation of the influence of the striatal ChIs on LIDs we used a previously established AIM scoring system evaluating amplitude and severity of limb, axial and orolingual AIMs (Cenci and Lundblad, 2007). In order to allow for time dependent effects on dyskinesia following treatment with DREADD ligands in combination with LDOPA, the AIMs were time binned into three phases, initial (0-60 min), mid (80-120

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min) and end phases (100 min to end of AIMs). CNO treatment was combined with either a treatment of mid dose (3mg/kg, Figure 5E) or high dose (9mg/kg, Figure 5G) LDOPA. The hM3Dq/rM3Ds experimental animal group displayed significantly increased AIMs at both mid (p=0.037) and end (p=0.043) phases following treatment with a

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combination of CNO and a mid-dose of L-DOPA relative to baseline AIMs (Figure 5E, I). AIMs were also significantly increased in the end phase (p=0.007) in the hM3Dq/rM3Ds

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experimental group following treatment with CNO in combination with a high dose of LDOPA (Figure 5G, I). KORD expressing animals did not display any significant change in

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AIMs relative to baseline at any of the time points following treatment with either mid

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KORD ligand SalB.

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(Figure 5F, J) or high (Figure 5H, j) doses of L-DOPA treatment in combination with

Striatal cholinergic input differentially modulates the D1 direct and D2 indirect

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pathways, revealing cholinergic increase in AIMs mediated by the D2 pathway The striatal direct (dopamine D1 receptor expressing) and indirect (D2 receptor expressing) pathways play key roles in motor function and in the induction of LIDs (Jenner, 2008). In order to evaluate ChI-mediated modulation of these pathways in the context of LIDs, we evaluated dyskinesia in the hM3Dq/rM3Ds experimental group following injection of CNO (3mg/kg) combined with either a D1 specific agonist SKF38393 (2.5 mg/kg s.c.) or a D2 specific agonist Quinpirole (0.05mg/kg s.c.).

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ACCEPTED MANUSCRIPT Consistent with previous reports, both agonists are capable of inducing robust AIMs through distinct pathways in 6-OHDA lesioned animals, with SKF38393 inducing AIMs similar to L-DOPA (Figure 6A) while Quinpirole induced lower AIMs consisting mainly of axial AIMs (Figure 6B). Following, co-treatment using CNO and the D1 specific agonist SKF38393 there was a non-significant trend towards reduced initial phase AIMs

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compared to baseline levels (p=0.051) while there was no change in mid or end phases (Figure 6A and C). In contrast, co-treatment with CNO and D2 specific agonist Quinpirole significantly increased AIMs by ≥ 3-fold in both the mid phase (p=0.015) and the end phase (p=0.028) while compared to baseline (Figure 6B and D). As a result, the duration

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of the dyskinetic response was prolonged by about 1hr (Figure 6B).

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High-resolution confocal microscopy in ChAT-Cre / TH-Cre double transgenic animals shows potential synapse formation between nigral DA neurons and striatal cholinergic

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neurons

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The generation of a novel double-transgenic Cre-driver line with the recombinase expressed both in dopaminergic and cholinergic neurons enabled not only orthogonal

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activation of substantia nigra and ChIs for in vivo recording of DA release as described above but also allowed for highly specific labelling of the two systems for imaging of

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their interaction. We utilized the tagged protein hM3Dq-HA and the bi-cistronic ChR2IRES-YFP to enable immunofluorescent labelling selectively to nigral dopaminergic cells, their projections into the dorso-lateral striatum and the synapse formation onto the ChIs in this region. Using the iDISCO clearing protocol, we were able to macroscopically visualize the transduced nigral DA cells in the substantia nigra together with the transduced ChIs in the striatum using light sheet microscopy (Figure 7A-B). To resolve the connectivity on a 26

ACCEPTED MANUSCRIPT synaptic level we then performed sequential scanning confocal imaging on coronal brain sections covering the dorsolateral striatum (Figure 7C-F’’). These images confirm excellent separation of YFP-positive ChR2 expression (in dopaminergic projections) from HA-positive hM3Dq expression in the ChIs (Figure 7C-D’’). Furthermore, the YFPpositive axon terminals appeared to tightly pass along the soma of the HA positive cells

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with many obvious varicosities and signs of synapses formed on both the soma and

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dendrites of the HA-positive neurons (Figure 7E-F’’).

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ACCEPTED MANUSCRIPT Discussion In this study, we have implemented genetic, chemogenetic and optogenetic tools in the rat to elucidate the functional contribution of ChIs in both normal motor function and in motor dysfunction in Parkinson’s disease. Using the Cre-inducible AAV-8 vectors, we

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have here shown that we can selectively target the pool of ChIs unilaterally in the rat striatum. This enabled us to selectively assess their involvement in motor function and compare that to the contralateral, unaffected side. What we found was that an increased activation of ChIs provides a positive bias for the animals´ utilization of the affected paw

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compared to the contralateral paw. This movement appears highly goal directed and is thus different to hyperkinetic, stereotypic movements induced by dopamine agonists.

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However, it is still likely that this increased movement is due to an increased probability of dopamine release in the activated striatum, as we showed through in vivo recordings

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that the selective activation of ChIs is inducing a significant dopamine release This has also been shown through other methods of ChI activation e.g., using optogenetics

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(Threlfell et al., 2012). Further evidence for the hypothesis that the increase in directed

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motor behavior after ChI activation requires availability of dopamine was observed in the rodent model of end–stage Parkinson’s disease. Using the MFB 6-OHDA lesion

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paradigm, we removed almost entirely the dopamine system in the right striatum and after this insult, the activation of the ChIs appears to not provide any increase in goaldirected motor function. However, with the addition of a sub-therapeutic dose of LDOPA (1 mg/kg) the ChI activation does result in a symptomatic relief as observed in the stepping test. Facilitation of L-DOPA pharmacotherapy is however a double-edge sword and if this is broadly potentiating the effects of the dopamine it would also increase the unwanted side effects of the drug in Parkinson’s disease, most prominently the L-DOPA

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ACCEPTED MANUSCRIPT induced dyskinesias. This is indeed the case as we see an increased duration of the LIDs after ChI activation at higher doses. When assessing this further we find that the potentiation of the LIDs is mainly through the indirect pathway and the trend on the direct pathway is in the opposite direction. This is striking in comparison to the apomorphine treatment in intact ChAT-Cre rats where we see no indications on

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lateralized behavior after activation of the ChIs. This suggests that a post-synaptic potentiation of dopamine receptors (such as that observed after dopamine denervation) is required for the ChI mediated potentiation the exact mechanism underlying this effect remains to be elucidated.

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What remains difficult to achieve is a robust behavior effect of DREADD mediated

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inhibition of ChIs in Parkinsonian rats. While we observed small but significant changes both in stepping performance and in amphetamine induced rotation after SalB delivery

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in KORD expressing intact ChAT-Cre rats, we did not observe any significant changes in AIMs of 6-OHDA lesioned rats. Based on the literature in mice using ablation and

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pharmacological inhibition of the ChIs (Ding et al., 2011; Won et al., 2014), we had expected a robust decrease in the LIDs in this group. We believe that there may be a

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number of reasons for this. A partial activation of a subset of neurons may be sufficient

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to induce an activation cascade that may give a strong behavior readout (similar to an epileptic seizure) and this may be what is occurring in many observations using activating DREADDs (Alexander et al., 2009). Inhibition of a subset of neurons is much less likely to induce such amplifying cascades and thus require a much more potent inhibition. While we through the confocal analysis post mortem we have seen that the KORD receptor is expressed throughout the striatal ChIs, it appears to express less well than activating DREADDs. We have observed similar but worse challenges in achieving robust expression from other variants of inhibitory chemogenetic receptors in ChIs, 29

ACCEPTED MANUSCRIPT including the hM4Di DREADD (Armbruster et al., 2007) and the ivermectin inducible chloride channel GluCl (Frazier et al., 2013) expressed under various promoters. Why these channels are so difficult to express in the ChI population is still unresolved. A second possible reason for the lack of behavior effect is the reversible short duration of the inhibition achieved by the SalB/KORD system. This inhibition peaks at 20 minutes

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post SalB administration and is totally reversed 50 minutes after (Vardy et al., 2015). To counteract this rapid turnover, we have in this study aimed to always inject the SalB 20 minutes prior to the behavior or the peak dose effect of L-DOPA but compared to the chronic effects of ChI ablation, this silencing may be too transient to result in plastic

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changes that could result in the change in LIDs and / or motor function elsewhere.

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Unified model of modulated striatal signaling pathways

The intrinsic balance in the basal ganglia circuit between the inhibitory indirect pathway

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and the direct excitatory pathway is carefully orchestrated by both identified and unknown modulatory systems (Jenner, 2008). To visualize the findings of this study in

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the light of prior knowledge of this circuitry, we have in Figure 8 generated schematic

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overviews of this circuit in each tested state. While DA is a key neuromodulator for shifts in this balance in the normal brain, it is evident that it does not completely handle this

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balance alone, but is dependent on signaling cascades both intrinsic and extrinsic to the striatum (Figure 8A) (Cachope et al., 2012; Threlfell et al., 2012). This additional regulation becomes more evident when the DA system is depleted and the DA is supplied through the conversion of the precursor, L-DOPA (Picconi et al., 2003). While the traditional picture of DA-acetylcholine interaction was drawn as a seesaw, it is now, in light of recent technical advances, seen as a carefully balanced interplay (Gerfen and Surmeier, 2011). However, due to the interdependence and interplay between the two

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ACCEPTED MANUSCRIPT systems, a holistic picture of the requirements for cholinergic neurotransmission in the striatum has been difficult to achieve both in the intact and the parkinsonian brain. In this study, we have for the first time utilized the ChAT-Cre transgenic rat line in combination with chemogenetic modulation to evaluate the contribution of the striatal cholinergic system to motor function. Using a combination of double-transgenic ChAT-

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Cre / TH-Cre rats, intact ChAT-Cre rats and 6-OHDA lesioned dyskinetic rats we have been able to obtain a better understanding of the intricate fine-tuning of the striatal circuitry that is performed by the ChIs. Moreover, the high tropism of the AAV-8 vector serotype, for transduction of forebrain cholinergic neurons allowed us to target with

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high precision the majority of the ChIs unilaterally in the rat striatum.

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In the second experiment, we utilized this gene delivery paradigm to assess the effect on motor performance elicited by hyper- versus hypo-activity of the ChIs in the intact rat

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brain (Figure 8B). In this setting, we observed a consistent potentiation of motor function in the contralateral paw after activation of the ChIs using the hM3Dq/Ds

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DREADDs and the opposite effect using the inhibitory KORD vector. Recent findings

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have shown that activation of ChIs can cause a robust induction of DA release facilitated through the nicotinic receptors (nAChRs) localized on the pre-synaptic DA terminals in

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the striatum (Cachope et al., 2012; Surmeier and Graybiel, 2012; Threlfell et al., 2012). The activation of ChIs by CNO is thus likely to stimulate DA release which in turn drives the dSPNs through the D1 (Golf coupled) receptor and reduces the iSPNs through the D2 (Gi coupled) receptor, thereby facilitating movement (Figure 8B). This hypothesis is further supported by the observation that amphetamine induced rotation could be modulated differently by activation versus inhibition of the ChIs. Activation of ChIs

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ACCEPTED MANUSCRIPT increased contralateral rotation (indicative of an increased ipsilateral release of DA) and inhibition of the ChIs induced rotation in the opposite direction. In order to obtain further support for this model of ChI function, we bred a cohort of double-transgenic rats to obtain Cre expression in both striatal ChIs and nigral DA neurons which made it possible to induce orthogonal activation of the two systems, i.e.,

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ChR2 expression in the nigral neurons and hM3Gq expression in the striatal ChIs. Using in vivo chronoamperometric recording, we were able to show that activation of ChIs can result in a robust DA release with an amplitude similar to the optically released DA but with significantly longer release kinetics. The extended duration of the response can be

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attributed to the slower kinetics of CNO injection versus optical stimulation (Threlfell et

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al., 2012). One concern with ChAT-Cre/TH-Cre double transgenic animals in this application could potentially be the occurrence of TH+ neurons in the striatum that may

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drive Cre expression in non-ChI neurons. However, in rats the expression of TH in striatal neurons in seen only after DA denervation (Xenias et al., 2015). In intact TH-Cre

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animals, we have not been able to induce transgene expression after injection of DIOAAV vectors in the striatum. Furthermore, if this would have been the case, a recent

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optogenetic study has shown that these TH+ striatal neurons do not have the capacity to

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release DA and do not release glutamate (Xenias et al., 2015). A rat model of end-stage Parkinson’s disease was achieved through the unilateral depletion of the nigrostriatal DA system by injection of the neurotoxin 6-OHDA into the medial forebrain bundle (MFB, Figure 8C). These animals remain L-DOPA responsive with symptomatic relief observed using 6mg/kg L-DOPA and above (Winkler et al., 2002) while the striatal DA concentration is depleted by more than 99 % (Bjorklund et al., 2010). However, animals with complete DA lesions are also very sensitive to DA

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ACCEPTED MANUSCRIPT fluctuations and develop abnormal involuntary movements already on 3mg/kg L-DOPA. Interestingly, the activation of ChIs could potentiate both the symptomatic effect of LDOPA (observed by a recovery in stepping performance on 1mg/kg L-DOPA) and aggravate LIDs. As illustrated in Figure 8D, propose that this potentiating effect is caused by acetylcholine acting on pre-synaptic inhibitory M2 receptors localized on

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glutamatergic afferents from cortex and thalamus and thereby reducing the excitatory drive on the dSPN and the iSPN. The net effect is that the L-DOPA induced effect of enhancing the agonistic dSPN pathway while reducing the antagonistic iSPN pathway is enhanced (Figure 8D). Of note is also that the dSPNs express besides the inhibitory M4

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receptor also the excitatory M1. Activation of this receptor through ChI activation may

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further amplify the output of the direct pathway.

To gather support for this hypothesis, we decided to dissect the actions of L-DOPA

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through the use of selective D1 and D2 agonists to induce AIMs. When combining the D1 agonist with ChI activation, there was no potentiation of the D1-induced dyskinesias. In

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fact, the trend was towards a decrease. This would fit the idea that the modulatory effect of acetylcholine is acting pre-synaptically on the glutamatergic afferents to the dSPN and

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iSPNs. In contrast to L-DOPA stimulation, the D1 agonist treatment does not suppress

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the activity of the iSPNs (which lack D1 receptors and the agonist does not act on the D2 receptors). The ChI activation would therefore also drive the iSPN through the M1 receptor and this is known to reduce D1-induced dyskinesias (cf. iSPN activation in LID (Alcacer et al., 2017)) (Figure 8E). Our results show that ChI-activation aggravates the mild dyskinesias induced by a D2 agonist (Alcacer et al., 2017), suggesting that the mechanisms underlying the potentiation of D2 agonist-induced dyskinesias are very similar to those driving the aggravated LIDs (cf. Figure 8D and F), namely through the activation of M1 receptors located on the dSPNs (Bernard et al., 1992; Yan et al., 2001) 33

ACCEPTED MANUSCRIPT in combination with symmetrical silencing of the pre-synaptic glutamatergic terminals through the M2 receptor. Of note is that a major fraction of the increase in LIDs after CNO administration is due to increases in Axial AIMs (the type of dyskinesias induced primarily by Quinpirole), further strengthening the conclusion that the aggravated LIDs are mediated through the indirect pathway.

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These data help to explain why modulation of ChI activity has a relatively modest effect on LIDs, as the effect on the direct pathway may counterbalance the one acting on the indirect pathway. It is tempting to speculate that this dual action aids to maintain a delicate and functionally optimal balance between the two systems. Such intrinsic

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regulatory function is further supported by the close interaction between the DA system

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and the ChIs. Our double-transgenic model offers a very useful tool for further studies of the interaction between those two systems.

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The data generated here using chemogenetics in transgenic rats are consistent with the optogenetic activation studies performed in transgenic ChAT-Cre mice. Bordia et al.,

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have recently found that LIDs in 6-OHDA lesioned ChAT-Cre mice are similarly

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aggravated using ChR2 induced activation of ChIs and that this effect was mediated through muscarinic receptors (Bordia et al., 2016). However, in this context it was

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highly pulse-duration dependent with only short light pulses having this effect. The ChI modulation of the dSPN pathway is complex due to the apparent expression of both inhibitory (M4) and excitatory (M1) muscarinic receptors on this pathway (Bernard et al., 1992; Yan et al., 2001; Ztaou et al., 2016). The study from Shen et al. (2015) has shown that a positive allosteric modulator (PAM) with affinity to the M4 receptor can reduce LIDs in 6-OHDA lesioned mice by up to 30%. This stand in contrast to the observation in this paper that activation of ChIs aggravates LIDs. This points to the

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ACCEPTED MANUSCRIPT possibility that summed activation from the D1-receptor activation by L-DOPA and the M1 activation by acetylcholine is strong enough to overcome the M4-mediated inhibition. It is also worth to note that the M4 receptor is expressed in 50% of the D2 expressing neurons of the indirect pathway (albeit at lower expression levels) and this may also contribute to the aggravated LIDs (Yan et al., 2001).

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The novel component of the current study is that we through the use of chemogenetics and the broadly validated rat model of LIDs in Parkinson’s disease have been able to study strikingly different effect of ChI activation on the direct and indirect pathways of the striatum. Despite the complexities of the cholinergic system is also a testament to

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the robustness of this modulatory effect that it can be replicated between different

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species and radically different modulation techniques (optogenetic and chemogenetic). Chemogenetic modulation has many advantages in behavior assessment in larger

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species in that it can be modulated without an optrode /optic fiber interfering with the animals’ behavior and broadly distributed neuronal populations such as the ChIs can be

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more readily targeted. However, the chemogenetics is clearly not without drawbacks and challenges. Very recently, it has been shown that CNO can be back-converted to

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Clozapine also in the rat and non-human primates after peripheral administration

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(MacLaren et al., 2016; Raper et al., 2017). Although clozapine itself has a much higher affinity to the DREADD receptors and may thereby contribute to the on-target effects observed in DREADD studies, it raises concerns about off-target effects. While this phenomenon was not known when the current study was designed and conducted, we believe that we have included sufficient controls both in the form of contralateral, noninjected control sides, CNO treatment in wild-type animals and CNO administration to the Dq/KORD animals etc., to confidently exclude that the data presented in this paper is influenced by an off-target effect of clozapine. Going forward, however, the field would 35

ACCEPTED MANUSCRIPT benefit from the development and utilization of new DREADD ligands not having this potential confounder (Chen et al., 2015). In hindsight, one experiment would however have gained from another internal control using CNO on non-DREADD animals and this is the experiments using the Quinpirole in combination with ChI activation where we observe a potentiation of the AIMs. Unfortunately, the only other study to date utilizing

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Quinpirole in combination with Chemogenetics (Alcacer et al., 2017) also failed to include such a control. However, earlier studies have shown that, at least in the case of induction of polydipsia, reduced prepulse inhibition (PPI) and retinal dysfunction induced by Quinpirole, Clozapine can revert the effects instead of potentiating them (De

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Carolis et al., 2010; Zawilska et al., 1996). Thus, we would argue that these data are

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conclusive but acknowledge that such added controls are warranted going forward. While we recognize that the model proposed here (see Figure 8) is a simplified model

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removing many of the intrinsic complexities of the striatal circuit of motor control, we believe that it can serve as a useful starting point for further explorations. Therapies

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targeting ChIs in PD may be beneficial for symptomatic relief but this study also shows that careful validation in relevant animal models are highly warranted as the outcome of

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an increased cholinergic drive may be both pro-dyskinetic and anti-dyskinetic.

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ACCEPTED MANUSCRIPT Funding This work was supported by grants from Parkinson’s Disease Foundation International Research Grant (PDF-IRG-1303); Swedish Research Council (K2014-79X-22510-01-1 and ÄR-MH-2016-01997 Starting grant); Swedish Parkinson Foundation; Swedish

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Alzheimer foundation; Crafoord foundation; The Bagadilico Linnaeus consortium; Schyberg foundation; Thuring foundation; Kocks foundation; Åke Wiberg foundation; Åhlén foundation; Magnus Bergvall foundation; Tore Nilsson foundation; The Swedish Neuro foundation; OE and Edla Johanssons foundation and the Lars Hierta foundation.

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TB is supported by Ass. Senior lectureship from the Bente Rexed foundation. AH is supported by a stipend from the Swedish society for medical research (SSMF).

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Additional Information

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Authors’ contributions

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The authors declare that they have no competing financial interests.

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TB and PAK designed the experiment; PAK, MD and BM performed the in vivo experiments and behavior assessments, AH performed and analyzed the in vivo

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recordings, DR performed and analyzed the electrophysiological recordings in acute slices; TB and PAK wrote the manuscript.

Acknowledgement The authors would like to thank Prof. Karl Deisseroth for supplying the ChAT-Cre BAC transgenic rat stain, the Vector Core at the University of North Carolina at Chapel

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ACCEPTED MANUSCRIPT Hill and Jenny G Johansson at Lund University for the AAV production and Ulla Jarl for

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iDISCO preparation and whole brain immunohistochemistry.

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ACCEPTED MANUSCRIPT Figure legends Figure 1 In vivo characterization of AAV serotype specific tropism and spread in ChAT-Cre

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transgenic rats. (A-E) Comparison of AAV serotype tropism for striatal ChIs after a single deposit AAV injection (2µl at 1E12 gc/ml) using Cre-inducible (DIO) GFP constructs in AAV-1 (A), AAV-2 (B), AAV-5 (C), AAV-8 (D) or AAV-9 (E) capsids. Brown staining is through DAB peroxidase precipitation reaction after immunohistochemistry against the GFP protein.

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The DIO (also called “flex” approach) is a vector design where the gene is placed in

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reverse compared to the promoter and no gene expression is achieved without Crerecombinase. When Cre-expressing cells are transduced, the recombinase flips the gene

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into the expressing direction. The use of AAV-8 and AAV-9 capsids resulted in higher expression and more favorable spread compared to AAV-1, AAV-2 and AAV-5 in the

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striatal ChIs. (F) Quantification of GFP+ ChIs in the striatum of ChAT-Cre transgenic rats injected with the candidate AAV serotypes AAV-1, AAV-2, AAV-5, AAV-8 and AAV-9

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containing a DIO-GFP construct. AAV-8 showed a significantly higher ability to transduce

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ChIs compared to AAV-1 (p=0.005), AAV-2 (p=0.019) and AAV-5 (p=0.036) but not compared to AAV-9. (G) Quantification of relative optical density within the viral transduction area of the striatum of ChAT-Cre transgenic rats injected with the same AAV serotypes containing a DIO-GFP construct. AAV-8 showed a significantly higher expression as measured by optical density compared to AAV-1 (p=0.007), AAV-2 (p=0.017) and AAV-5 (p=0.028) but not compared to AAV-9. (H) Quantification of relative optical density for the entire striatum of the ChAT-Cre rats injected with the various serotypes containing DIO-GFP. Again AAV-8 displayed a significantly higher 39

ACCEPTED MANUSCRIPT optical density in the entire striatum compared to AAV-1 (p=0.004) and AAV-2 (p=0.01). (I) Measured volume of the GFP+ transduced area within the striatum of ChAT-Cre rats injected with the various AAV serotypes. Although there was no significant difference between the serotypes, there was a strong trend for AAV-8 having a higher capacity to infect a larger striatal volume. (J) DAB staining against HA-tagged DREADD receptor

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hM3Dq in the striatum of ChAT-Cre transgenic rats delivered using the AAV-8 serotype. The AAV-8 vector injection, using two needle tracts, resulted in a broadly distributed expression hM3Dq DREADDs in striatal ChIs throughout the striatum (See Supplemental

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File 2 for detailed statistics).

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ACCEPTED MANUSCRIPT Figure 2 Effect of activation and inhibition of striatal ChIs on motor performance using AAV-DREADDs in intact ChAT-Cre rats. (A) Experimental timeline of Experiment 1, illustrating the major time points for all

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surgical procedures and behavioral tests for intact ChAT-Cre animals expressing either a combination of activating (hM3Dq/rM3Ds) or inhibitory (KORD) DREADDs delivered using Cre-inducible (DIO) AAV-8 vectors to the striatum. (B) Motor performance as assessed in the stepping test following treatment with saline (white), hM3Dq/rM3Ds

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ligand CNO (cyan) or KORD ligand SalB (grey). Animals expressing the activating DREADDs (hM3Dq/rM3Ds) significantly increased the number of contralateral steps in

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the stepping test following CNO treatment compared to saline (p=0.01). Animals expressing the inhibitory DREADD (KORD) displayed the opposite effect with a

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significant reduction in the number of contralateral steps in the stepping test compared to saline (p=0.047). 100% represents the same number of adjusting steps as the intact,

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contralateral paw. All tests are intra-animal tests and the dashed line in B-C represents

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perfect symmetry. (C) Motor performance as assessed in the cylinder test following treatment with saline (white), hM3Dq/rM3Ds ligand CNO (cyan) or KORD ligand SalB

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(grey). Similarly, to the stepping test, animals expressing the activating DREADDs (hM3Dq/rM3Ds) displayed a trend towards an increased proportion of touches with the contralateral paw following CNO treatment compared to saline. Animals expressing the inhibitory DREADD (KORD) displayed the opposite trend, with decreased touches using the contralateral paw following SalB treatment. However, neither change was statistically significant. (D) Motor performance as assessed by the amphetamine rotation test. Animals injected with the hM3Dq/rM3Ds combination of DREADDs (red) displayed

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ACCEPTED MANUSCRIPT contralateral rotations following a combinational treatment of amphetamine and CNO. Conversely, animals injected with the KORD vector (blue) displayed ipsilateral rotations when treated with a combination of amphetamine and SalB. (E) To assess post-synaptic changes, we administered the dopamine agonist apomorphine to animals after CNO or SalB administration and studies rotational bias in the rotation test. None of the groups

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displayed any rotational bias regardless of treatment (See Supplemental File 2 for

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detailed statistics).

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ACCEPTED MANUSCRIPT Figure 3 Electrophysiological slice recording of hM3Dq-induced firing of striatal ChIs and ChI-induced DA release characterized in vivo using TH-Cre/ChAT-Cre double transgenic rats.

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(A) DREADD expressing ChIs were identified by their red fluorescence were selected for electrophysiological recordings. (B) Spontaneous firing trace of cell-attached ChI in ACSF as control (upper trace) compared to firing rate following addition of 10M CNO (lower trace) with examples taken from the same cell. (C) Average discharge rate of the

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ChIs in ACSF and following addition of CNO (Bars) and individual cells (connected circles). Average firing rate increased from 1.67 ± 0.273 to 6.17 ± 1.916 n=7, *P<0.05.

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(D) Amperometry trace in the lateral striatum from single TH-Cre/ChAT-Cre double transgenic rat with CHR2 selectively expressed in DA neurons of the SNpc and hM3Dq

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DREADDs selectively in ChIs in the striatum. DA release evoked by potassium chloride (red) compared to 13 mW laser light (blue) and the addition of 100 µM CNO (green)

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with the combination of 13 mW laser light and addition of 100 µM CNO (purple). Laser

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power and pulse duration were carefully optimized in Supplemental Figure 1 (See

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Supplemental File 2 for detailed statistics)

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ACCEPTED MANUSCRIPT Figure 4 Timeline for Experiment 2 and validation of 6-OHDA lesion in ChAT-Cre animals with regards to motor behavior and stability of L-DOPA induced dyskinesias (LIDs).

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(A) Experimental timeline of Experiment 2, illustrating the major time points for all surgical procedures and behavioral tests for dyskinetic 6-OHDA lesioned ChAT-Cre animals expressing either a combination of activating (hM3Dq/rM3Ds) or inhibitory (KORD) DREADDs. (B) Motor performance of 6-OHDA lesioned ChAT-Cre rats in the

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stepping test. 6-OHDA lesioning resulted in a severe reduction in the number of contralateral steps in the stepping test in all experimental groups compared to intact

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animal performance (Figure 2B-C). (C) Motor performance of 6-OHDA lesioned ChATCre rats in the cylinder test. 6-OHDA lesioning resulted in a severe reduction in the

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proportion of contralateral paw touches in the cylinder test in all experimental groups. Importantly the stepping and cylinder test motor performance remained stable

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throughout the behavioral test phase, both before and after AAV DREADD injection. (D)

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Stability of LIDs in 6-OHDA lesioned ChAT-Cre rats, assessed using the AIM-scoring paradigm. 6-OHDA lesioning resulted in the development of dyskinesia in all

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experimental groups following two weeks of L-DOPA treatment. Induction of LIDs was conducted before AAV injection and animals were balanced into the treatment groups based on magnitude of LIDs. In order to ensure that any increase in dyskinesia could be detected using AIM scoring, the experimental group receiving the activating DREADD vectors (hM3Dq/rM3Ds) had a lower level of dyskinesia compared to the KORD and wild-type groups at baseline, prior to AAV injection. Critically, the AIM scoring remained stable throughout the behavioral test phase, both before and after AAV DREADD

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ACCEPTED MANUSCRIPT injections. (E-E´) Representative overview images of the forebrain (E) and the midbrain (E´) of a lesioned rat from the study stained using the DAB reaction against the TH protein. (F) Post mortem, the striatal dopaminergic innervation was quantified using densitometry of TH+ DAB precipitation which showed a significant degeneration (Solid line represents the intact rat striatum). (G) Through stereological cell counts of TH+

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neurons in the substantia nigra we confirmed that this degeneration was coupled to a similar loss of nigral DA neurons. (Lines represent intact substantia nigra and the

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normal variance.)

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ACCEPTED MANUSCRIPT Figure 5 Effect of activation and inhibition of striatal ChIs on motor performance and LDOPA induced dyskinesias (LIDs) in 6-OHDA lesioned ChAT-Cre rats using DREADDs.

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(A and B) Motor performance as assessed in the stepping test following treatment with saline (white), DREADD ligands CNO/SalB (cyan), low dose L-DOPA (1 mg/kg, black) or a combination of DREADD ligands and low dose L-DOPA (grey). (A) Animals expressing the activating DREADDs (hM3Dq/rM3Ds) did not significantly change the number of

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contralateral steps in the stepping test following low dose L-DOPA or CNO treatment. However, combining low dose L-DOPA with activation of the DREADD in ChIs

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significantly increased the number of contralateral steps compared to low dose L-DOPA alone (p=0.037). There was no difference in contralateral steps following all treatments

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in the wild-type experimental group. (B) Animals expressing the inhibitory DREADD (KORD) displayed no significant difference in contralateral steps following treatment

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with DREADD ligand, low dose L-DOPA or a combination of low dose L-DOPA and

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DREADD ligand SalB. (C and D) Motor performance as assessed in the cylinder did not significantly change following any of the treatments, neither with L-DOPA nor through

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modulation of ChIs. (E-J) LIDs assessed through AIM scoring over time following L-DOPA treatment. (E, G and I) Animals injected with the activating DREADD (hM3Dq/rM3Ds) vector combination displayed increased overall AIMs at both mid dose (3mg/kg) (E) and high dose (9mg/kg) of (G) L-DOPA in combination with CNO treatment relative to LDOPA only baseline levels. CNO treatment in combination with mid dose L-DOPA treatment significantly increased the AIM score (I) in both the mid phase (yellow) (p=0.037) and the end phase (green) (p=0.043). High dose L-DOPA in combination with

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ACCEPTED MANUSCRIPT CNO significantly increased AIMs during the end phase (green) (p=0.007) but not in start or mid phases. (F, H and J) Animals injected with the KORD vector displayed no significant change in overall AIMs any dose of L-DOPA (E and H) with no change during either the start (red), mid (yellow) or end (green) phases (J) (See Supplemental File 2

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for detailed statistics).

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ACCEPTED MANUSCRIPT Figure 6 ChIs mediate their facilitation of AIMs through modulation of the indirect (D2) striatal output pathway. (A and C) Dyskinesia induced by activation of the direct pathway using a D1 specific

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agonist (SKF38393) in combination with CNO treatment over time (A). Animals receiving CNO treatment in combination with SKF38393 displayed a strong trend towards a reduction of SKF38393-induced dyskinesia (p=0.051) at the start phase but not at the mid- and end-phases (C). (B and D) Dyskinesia induced by inhibition of the

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indirect pathway using a D2 specific agonist (Quinpirole) in combination with CNO treatment over time (B). Animals receiving CNO treatment in combination with

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Quinpirole displayed a significant increase in D2-mediated dyskinesia in both the midphase (p=0.015) and end-phase (p=0.028) (See Supplemental File 2 for detailed

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statistics).

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ACCEPTED MANUSCRIPT Figure 7 Midbrain dopaminergic projections of TH-Cre/ChAT-Cre rats associate closely with ChIs within the striatum. (A and B) iDISCO tissue clearing of the entire right hemisphere (A) from TH-Cre/ChAT-

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Cre double transgenic rats, imaged using light sheet microscopy. The rats were injected with DIO-ChR2-IRES-YFP (green) in the SNpc and DIO-hM3Dq-HA (red) injected into the lateral striatum (B). The tissue clearing revealed strong YFP expression localized to the dopaminergic neurons of the SNpc together with strong HA expression localized only in

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the ChI of the striatum. (C-C’’) Maximum intensity overview of the lateral striatum of ChAT-Cre transgenic rats injected with hM3Dq-HA using confocal microcopy showed

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near perfect overlap between the phenotypic marker for ChIs, ChAT (C) and the HA-tag (C´). The overlap was quantified using on-line randomized confocal counting of 1000

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HA+ cells in 10 animals (graph inset in (C)). (D-D’’) Maximum intensity overview of the lateral striatum of TH-Cre/ChAT-Cre double transgenic rats using confocal microcopy

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revealed a dense fiber network of YFP-positive dopaminergic projections within the

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striatum, indicating that the majority of dopaminergic neurons of the SNpc were expressing ChR2 (D). ChIs transduced with the hM3Dq DREADD and stained using the

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HA-tag were shown to be scattered around the injection site within the lateral striatum (D’) and the close association between the striatal cholinergic and midbrain dopaminergic systems was clearly visualized in the overlay (D’’). (E-E’’) High magnification maximum projection of a representative ChI within striatum showing high degree of dopaminergic input (E) around the ChI (E’) with the close proximity of the midbrain dopaminergic and striatal cholinergic systems shown as an overlay (E’’). (FG’’) Stacks of representative ChI using confocal microscopy showing the where the

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ACCEPTED MANUSCRIPT projections originating from the midbrain dopaminergic neurons of the SNpc (F and G) are connecting with the ChI (F’ and G’). Dopaminergic projections made were shown to be in close proximity and appeared to make several connections with the ChI as shown

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in the overlays (F’’ and F’’).

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ACCEPTED MANUSCRIPT Figure 8 Proposed modulatory role of ChIs in the intact and DA-depleted striatum, controlling the balance between the two principal output pathways. (A) In the intact striatum, ChIs modulate the activity of both the D1 and D2 MSNs (i.e.

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dSPN and iSPN respectively) through muscarinic receptors as well as DA release from striatal terminals through nicotinic receptors (nAChR). In addition, they modulate striatal glutamate release by acting on M2 (inhibitory) receptors on glutamatergic terminals from the cortex and thalamus. ChIs are in turn modulated by DA acting

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primarily on inhibitory D2 receptors. (B) Activation of ChIs in the intact striatal circuitry stimulates DA release through activation of pre-synaptic nAChRs present on striatal DA

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terminals. This in turn drives the dSPNs through activation of the D1 receptor and reduces the activity of iSPNs by acting on D2 receptors. (C) In 6-OHDA lesioned rats, the

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loss of DA neurons of the SNpc leads to a decrease of dSPNs activity of the direct pathway while disinhibition of the iSPNs leads to increased activity of the indirect

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pathway. This change in the balance between the two output pathways is further

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amplified by an increase in striatal acetylcholine release from the ChIs (acting on both dSPNs and iSPNs) through reduced inhibition of ChIs resulting from the loss of DA

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afferents (Gerfen and Surmeier, 2011). (D) Potentiation of L-DOPA induced motor recovery in conjunction with chemogenetic activation of ChIs is proposed to be driven by acetylcholine inhibiting striatal glutamatergic terminals through the M2 receptor. This symmetrical inhibition enhances the effect of L-DOPA, creating an increased difference in activity between dSPNs and iSPNs. (E) Decreased dyskinesia seen after chemogenetic activation of ChIs combined with specific activation of dSPNs activated through the D1 receptor agonist is thought to be due to acetylcholine acting on pre-

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ACCEPTED MANUSCRIPT synaptic M2 receptors on glutamatergic terminals reducing the intrinsic drive of the dSPNs, thereby reducing dyskinesias. This may in turn be modulated by a direct interaction between the dSPNs and iSPNs through acetylcholine acting on the M4/M1 receptors. (F) Increased dyskinesia following chemogenetic stimulation of ChIs in conjunction with specific inhibition of iSPNs acting through the D2 receptor is proposed

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to be due to acetylcholine acting M1 receptors acting on the dSPNs as well as providing further silencing of the iSPNs through the activation of M2 receptors on the glutamatergic input.

Line thickness represents overall output activity of the different pathways and key

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pathways (based on literature support and behavior outcome) are represented in solid

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black colors while pathways that are known to exist but are thought to play a lesser role are colored in gray. Colored arrows have been filled with the same color as used to

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represent the peripheral drug administered (e.g., CNO in yellow).

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Zolotukhin, S., et al., 2002. Production and purification of serotype 1, 2, and 5

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recombinant adeno-associated viral vectors. Methods. 28, 158-67. Ztaou, S., et al., 2016. Involvement of Striatal Cholinergic Interneurons and M1 and M4

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Muscarinic Receptors in Motor Symptoms of Parkinson's Disease. J Neurosci. 36, 9161-72.

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ACCEPTED MANUSCRIPT Figure S1 Establishment of recording parameters for electrochemical detection of dopamine release induced by light. (A-K) Recordings at maximum laser intensity possible at the day of recording (8.48mW;

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validated before electrode/opticle fiber assembly) in the cortex (DV: -1.0mm). We incremented stimulation time with the laser between 10 to 160 seconds constant illumination as indicated. Note the background increase in signal that can be ascribed to the photovoltaic effect when light hits the electrode. (A’-I’)

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recordings using a high laser power (5mW) in the striatum (DV: -3.0mm) with increasing stimulus durations (constant stimulation). Dopamine release

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surpassing the background levels measured due to the photovoltaic effect could be detected from constant stimulation for 50 seconds and above (B’-I’). Maximum

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dopamine release at the site of recording was obtained after stimulation for longer than 160 seconds when release reached a peak plateau (see H’, I’). The

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maximum amplitude of light induced DA release (2µMol, H’) was close to the

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potassium induced release at the same location (3.4 µMol, L). Note that potassium (KCL) does depolarize all cells whereas Light does only induce

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Dopamine release in cells that express the channel rhodopsin. (A’’-H’’’) Control measurements to validate stimulation frequencies at maximum laser intensity. Pulses at a frequency of 25Hz were not able to induce significant levels of DA release at stimulation duration of 80 seconds (A’’) and 160 seconds (B’’), respectively. At constant stimulation, we could measure DA release exceeding the background photovoltaic effect when we used constant illumination for 20, 40, and 180 seconds (C’’-E’’). Using these stimulation parameters, we reached

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amplitude DA release at the site of recording (F’’’).

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Fig. 8

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ACCEPTED MANUSCRIPT Highlights Activation of striatal cholinergic interneurons increases motor function in rats.

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Cholinergic interneurons potentiate the effect of L-DOPA in Parkinsonian rats.

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L-DOPA induced dyskinesias are aggravated by the cholinergic interneurons.

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The potentiation of LIDs is driven through the indirect striatal output pathway.

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