Blockade of the dopaminergic neurotransmission with AMPT and reserpine induces a differential expression of genes of the dopaminergic phenotype in substantia nigra

Journal Pre-proof Blockade of the dopaminergic neurotransmission with AMPT and reserpine induces a differential expression of genes of the dopaminergic phenotype in substantia nigra Sergio Ortiz-Padilla, Elier Soto-Orduño, Marisa Escobar Barrios, Abril Armenta Manjarrez, Yadira Bastián, J. Alfredo Mendez PII:

S0028-3908(19)30491-5

DOI:

https://doi.org/10.1016/j.neuropharm.2019.107920

Reference:

NP 107920

To appear in:

Neuropharmacology

Received Date: 21 February 2019 Revised Date:

28 November 2019

Accepted Date: 20 December 2019

Please cite this article as: Ortiz-Padilla, S., Soto-Orduño, E., Barrios, M.E., Manjarrez, A.A., Bastián, Y., Mendez, J.A., Blockade of the dopaminergic neurotransmission with AMPT and reserpine induces a differential expression of genes of the dopaminergic phenotype in substantia nigra, Neuropharmacology (2020), doi: https://doi.org/10.1016/j.neuropharm.2019.107920. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Dr. J. Alfredo Mendez (Orcid ID : 0000-0002-2859-7086)

Blockade of the Dopaminergic neurotransmission with AMPT and reserpine induces a differential expression of genes of the Dopaminergic phenotype in substantia nigra

Sergio Ortiz-Padilla1, Elier Soto-Orduño1, Marisa Escobar Barrios1, Abril Armenta Manjarrez1, Yadira Bastián 2, J. Alfredo Mendez1*

1

Laboratory of Molecular Biophysics, Institute of Physics, Universidad Autónoma de San Luis Potosí. 2 Unidad de Investigación Biomédica, IMSS, Zacatecas. México

Address correspondence to: * J Alfredo Mendez. Universidad Autónoma de San Luis Potosí. Institute of Physics. Manuel Nava#6. Zona Universitaria. SLP. Mexico. CP 78290. Tel. +52(444)826 2300 X 5716. E-mail: [email protected]

Abbreviations used: SN, substantia nigra; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; AMPT, alpha-methyl-p-tyrosine; IPSCs, inhibitory postsynaptic currents, TH, tyrosine hydroxylase; VMAT2, vesicular monoamine transporter type 2; DA, Dopamine; DAT, Dopamine transporter, VGluT2, vesicular glutamate transporter type 2; DDC, DOPA descarboxylase; GAD67, Glutamate Descarboxylase type I; D1r, Dopamine receptor subtype 1, D2r, Dopamine receptor subtype 2; PD, Parkinson´s disease; ACSF, artificial cerebrospinal fluid; BSA, bovine serum albumin.

Abstract Dopaminergic neurons have the ability to release Dopamine from their axons as well as from their soma and dendrites. This somatodendritically-released Dopamine induces an autoinhibition of Dopaminergic neurons mediated by D2 autoreceptors, and the stimulation of neighbor GABAergic neurons mediated by D1 receptors (D1r). Here, our results suggest that the somatodendritic release of Dopamine in the substantia nigra (SN) may stimulate GABAergic neurons that project their axons into the hippocampus. Using semiquantitative multiplex RT-PCR we show that chronic blockade of the Dopaminergic neurotransmission with both AMPT and reserpine specifically decreases the expression levels of D1r, remarkably this may be the result of an antagonistic effect between AMPT and reserpine, as they induced the expression of a different set of genes when treated by separate. Furthermore, using anterograde and retrograde tracing techniques, we found that the GABAergic neurons that express D1r also project their axons in to the CA1 region of the hippocampus. Finally, we also found that the same treatment that decreases the expression levels of D1r in SN, also induces an impairment in the performance in an appetitive learning task that requires the coding of reward as well as navigational skills. Overall, our findings show the presence of a GABAergic interconnection between the SNr and the hippocampus mediated by D1r.

Key words: D1 receptors, AMPT, Reserpine, Dopaminergic neurons, Substantia nigra, GABAergic interconnection

1. Introduction Located in ventral mesencephalon, the substantia nigra (SN) plays important roles in the instrumentation of reward and in motor activity (Andersson et al., 2006; Chowdhury et al., 2013; Faure et al., 2005). It is anatomically divided into substantia nigra pars compacta (SNc) and substantia nigra pars reticulata (SNr), whereas the SNc is mainly comprised of Dopaminergic neurons, the SNr contains GABAergic neurons. Intermingled with these neurons, a population of purely Glutamatergic neurons can be found (Mendez et al., 2008; Yamaguchi et al., 2013). In addition, subpopulations of these neurons have the ability to express more than one neurotransmitter phenotype (Chuhma et al., 2004; Mendez et al., 2008; Tritsch et al., 2012). Information coded by Dopaminergic neurons of the SNc is sent into the striatum through the Dopaminergic nigrostriatal axonal projection (Anden et al., 1964) while the SNr conveys the coded information by the basal ganglia into the thalamus and superior colliculus through GABAergic axonal projections (Azdad et al., 2009; Kase et al., 2015; Zhou and Lee, 2011). Dopaminergic neurons release dopamine (DA) from their axons as well as from their soma and dendrites (Bjorklund and Lindvall, 1975; Cheramy et al., 1981). The DA somatodendritically released within the SN acts on Dopaminergic neurons through D2 subtype receptors (D2r) inducing a self-regulatory inhibition mediated by inhibitory postsynaptic currents (IPSCs) (Beckstead et al., 2004); and on GABAergic neurons as well as on presynaptic axons in the SNr stimulating the release of GABA, which modulates motor activity and muscle tone (Bergquist et al., 2003; Double and Crocker, 1995; Hemsley and Crocker, 2001). The ability of Dopaminergic neurons to use DA as neurotransmitter depends basically on the enzymatic activity of tyrosine hydroxylase (TH) to start the DA biosynthetic pathway, and on the activity of the vesicular monoamine transporter type 2 (VMAT2), which loads

both synaptic (Nirenberg et al., 1995) and tubulo vesicles (Beckstead et al., 2004; Nirenberg et al., 1996; Rice et al., 1994) to allows its release during neurotransmission. TH and VMAT2 can be inhibited with Alpha-methyl-p-tyrosine (AMPT) and with reserpine, respectively. Whereas AMPT is currently used for the treatment of pheochromocytoma, reserpine is used for the treatment of high blood pressure, and in the past, it was used as an antipsychotic drug (Preskorn, 2007). Also, reserpine has been used as a model for Parkinson´s disease (PD) (Fernandes et al., 2012) and of tardive diskinesia (Naidu et al., 2004) while AMPT has been used to induce a model of depression (Caldecott-Hazard et al., 1988) or to study behavioral responses (van Enkhuizen et al., 2014). Furthermore, these compounds have also been used together to induce the depletion of DA to study striatal neuronal excitability (Azdad et al., 2009), locomotor activity (Finn et al., 1990) and as a model of PD (Stauch Slusher et al., 1994). Considering that blockade of both TH and VMAT2 should lead to phenotypic adaptations in response to low levels of DA, we used AMPT and reserpine to pharmacologically inhibit the DA phenotype and evaluate whether blockade of DA neurotransmission induces changes in the expression levels of genes involved in DA neurotransmission in the adult SN. 2. Experimental procedures 2.1 Animals, treatment and tissue processing. All experiments were performed using adult male mice of 90-105 days old of the BALB/cJ strain obtained from the UASLPBioscience Center. Animals were kept under 12h/12h cycle under a scheme of 2/3 mice per cage at 25°C with water and food at libitum. Welfare assessments and monitoring of mice were carried out by the staff of the UASLP-Bioscience Center on a regular basis. Both alpha-methyl-p-tyrosine (AMPT, #M8131, Sigma-Aldrich, St. Louis, MO, USA) and reserpine (#83580, Sigma-Aldrich) were diluted in sterile saline solution (NaCl 150 mM)

and intraperitoneally injected at a dose of 537.245 mg/kg and 0.66 mg/kg, respectively. Doses used for AMPT and reserpine correspond to the natural logarithm of their effective dose after 24h as this should compensate for drug elimination and assure a greater pharmacological effect. The effective dose was calculated using their respective half life and IC50 values assuming a first order kinetic rate of elimination and a 24 h period as dosing interval. Control mice were injected with the same amount of vehicle (saline solution). All mice were randomly allocated into the necessary groups according to each experiment and received no specific order in treatment unless they were used for the appetitive learning task, in that case, all animals were injected 3 h before their respective first training/test. All handling animal procedures were approved by the UASLP bioethics committee (UASLP-Biociencias CB-2016-005). To obtain the brains, mice were deeply anaesthetized with isoflurane and decapitated. The encephalon was immediately collected in ice-cold artificial cerebrospinal fluid (ACSF, in mM: 125 NaCl, 25 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 2 MgCl2, 23 glucose) saturated with 95% O2-5% CO2 (Infra, San Luis Potosí, SLP, Mexico). Brains were then sliced using a vibrating microtome (VT1000S, Leica Microsystems, Wetzlar, Germany) in ice-cold oxygenating ACSF and slices were left to recuperate in oxygenating ACSF at room temperature for 1h. The SN tissue was manually dissected from 250 µm thick horizontal slices using the mouse brain atlas of Paxinos as a guide (Paxinos and Franklin, 2007). 2.2 Anterograde and retrograde tracers injections. On the day of surgery, animals were anesthetized with an intramuscular injection of a ketamine/xylazine/acepromazine mix (100/10/5 mg/kg) and stabilized in a stereotaxic apparatus for mice (#51725, Stoelting, Wood Dale, IL, USA). After a minimal trepanation, 400 nl of the anterograde tracer Dextran Alexa Fluor-546 10,000 MW (DA546, #D22911, Thermo Fisher Scientific,

Waltham, MA, USA) or retrograde tracer Fluorogold (Fluorochrome, Denver, CO, USA), were injected at a speed of 40 nl/min using a Hamilton syringe (#7000.5, Hamilton, Reno, Nv, USA). Both tracers were prepared at 4% in sterile saline solution (150 mM NaCl). After injection, the needle was left for 10 more min, and then slowly removed on a period of 5 min. After surgery, animals were left to recover on a 37 °C thermal surface and returned to their cages after a full recovery of locomotor activity. During and after the surgery all animals received ciprofloxacine treatment for their scalp wound. Coordinates used for the injection of DA546 into the SN were: AP -3.16, ML 0.5 and DV 4.3 from bregma, whereas coordinates for the injection of Fluorogold into CA1 were: AP -1.7, ML 1.75 and DV 1, and for CA3: AP 1.7, ML -2 and DV 2, all from bregma (Paxinos and Franklin, 2007). 10 days later, mice were sacrificed, brain slices of 200 µm obtained and axonal projections analyzed using a CKX40 inverted microscope (Olympus, Shinjuku, Tokyo, Japan) equipped with epifluorescence and a C9100-14 EM-CCD camera (Hamamatsu Photonics, Shizuoka, Japan). All images were processed using the MetaMorph 7.6 image software (MDS Analytical Technologies, USA) and identification of the different brain regions was based upon the mouse brain atlas of Paxinos (Paxinos and Franklin, 2007). 2.3 Fluorescent immunolabeling. Slices from mice injected with Fluorogold were immediately fixed using 4% paraformaldehyde (PFA) in Phosphate-buffered saline (PBS) (in mM: 10 N Na2HPO4, 2.6 KH2PO4, 133 NaCl, 2.7 KCl). After fixation, slices were permeabilized with 0.3% TritonX-100 in PBS and non-specific binding sites blocked with PBS-BSA 5% (in 0.3% Triton X-100 and 5% goat serum). Slices were incubated overnight with the anti-GABA (#A-2052, Sigma-Aldrich) and anti-TH (#T-2928, Sigma-Aldrich)

antibodies at room temperature in incubating solution (0.3% Triton X-100, 0.5% BSA, 5% goat serum in PBS). After three washes, slices were incubated for 1h at 37°C with the appropriated Alexa-labeled secondary antibodies (Molecular Probes) in incubating solution. Documentation was performed using a CKX40 inverted microscope (Olympus) equipped with epifluorescence and a C9100-14 EM-CCD camera (Hamamatsu). All images were processed using the MetaMorph 7.6 image software (MDS Analytical Technologies) and identification of the different brain regions was based on the mouse brain atlas of Paxinos (Paxinos and Franklin, 2007). 2.4 Appetitive learning task using a multiple choice maze. Although mice were housed together, they were separated in individual cages at the beginning of the behavioral experiment. First, mice were allowed to freely explore the maze for 3 days, 2 sessions of 20 min each day. On the second day of habituation, food was removed but water supply was kept ad libitum. After 6 habituation sessions, on day 4 and 5 (2 sessions per day), mice were allowed to explore the maze to find a food pellet previously placed and the end of one of the arms. When mice found the food pellet, they were permitted to eat for 5 seconds and then retired from the maze. This training was repeated 20 min later. At the end of the training, a restricted amount of food (1.2 g) was given to each mouse in their individual cage. Mice that failed to explore or took more than 20 min to find the food pellet were considered as nonperformers and thus discarded. Also, animals that ignored the food pellet after they found it were considered as nonperformers as this was considered as a nonreward related event. After 4 training sessions, on day 6 mice were allowed to search for the food pellet and the time spent to reach the target recorded. Animals treated with AMPT and reserpine received the drugs 3 h before the first training session on day 4 and 5 (2

intraperitoneal injections 24 h apart), and on evaluation day, no drugs were applied. Control mice received saline solution without drugs. For the chronic treatment of AMPT together with reserpine, mice were started with the treatment, and on day 6 they were started with the habituation sessions for 3 days (2 sessions of 20 min each day). As in the acute treatment, training lasted for 2 days (2 sessions of 20 min each day) and on test day (day 11), the time they spent to reach and eat the food pellet recorded. As in the acute treatment, from the second day of habituation, food was removed but mice were allowed to eat for 5 s when the pellet was found in the maze and the food restriction continued when mice were individually housed. Mice were weighed every day to calculate the dose and not significant weight loss was found with respect to the control group. The ability of mice to learn the location of an appetitive reward requires of spatial learning (navigation) and the memorization of the route (path integration) to reach the target. To facilitate these processes, each bifurcation in the maze was labeled with a different visual cue, which mice must use to navigate through this slightly intricate maze and to learn the path. The wide of the path along the maze is of 10 cm. The end of all arms are equidistant to first bifurcation site, thus the time each mice takes to reach the food pellet is independent of the location of the food reward. To avoid any negative associations during these experiments, the person that intraperitoneally applied the drugs was never the observer in the appetitive learning task. 2.5 Evaluation of locomotor activity. When locomotor performance was evaluated using the rotarod, mice were placed on the middle of a rotating rod at a constant speed of 15 rpm. Two trials per day, 5 min apart. Time that mice took to fall off of the rod (latency) was recorded. When the locomotor activity was evaluated in the open field maze, individual

mice were place in the center of a clear maze free of obstacles (50 X 50 X 35 cm) under uniform bright light to avoid dark spots. Ambulatory activity was recorded using 30 fps at 1080p of resolution. Videos were analyzed off line using the ToxId algorithm (Rodriguez et al., 2017) under the ToxTrac software. Data was exported to excel sheets for further analysis. 2.6 RNA extraction and multiplex RT-PCR. Total RNA was extracted from manually dissected tissue using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer´s instructions. RNA was quantified by spectrophotometry at 260/280 nm and subjected to cDNA synthesis for 1h using 0.5 mM dNTPs mix, 2.5 µM random hexamers, 2 µg of total RNA, 10 mM DTT, 40 units RNaseOUT and 200 units MMLV RT under RT buffer (50 mM Tris-HCl, 75 mM KCl and 3 mM MgCl2, pH 8.3). After RT enzyme was denatured and cDNAs stored at -20°C until use. Multiplex PCR was performed using 100 ng of cDNA, 1.8 mM MgCl2, 0.5 mM dNTPs mix, 10-200 pmol of each primer, 5 units of Platinum Taq-DNA polymerase under PCR buffer (20 mM TrisHCl, 50 mM KCl, pH 8.3) with 55°C of annealing temperature for 1 min. All reagents used in this section were purchased from Thermo Fisher Scientific (San Jose, CA, USA). Relative levels of all genes were evaluated through semiquantitative (r2>0.95) PCR reactions normalized with the constitutive levels of β-actin. Custom made primers (Oligo T4, Gto, Mexico), were tested using the appropriate positive and negative controls. Upper (right) primers are followed by lower (left) primers: TH, 5´-gtacaaaaccctcctcactgtctc-3´ and 5´-cttgtattggaaggcaatctctg-3´; gatccacacagatgcctcac-3´; gatagtgctgttgttgaccatgt-3´;

DAT

5´-

gtattttgagcgtggtgtgct-3´

and

5´-

VGluT2

5´-

atctacagggtgctggagaagaa-3´

and

5´-

GAD

5´-

tctttgtcatctctttagctgtgtc-3´

and

5´-

gtgactgtgttctgaggtgaagag-3´;

VMAT2

5´-

gctgatcctgttcatcgtgtt-3´

and

5´-

tagaagtcctatgaatgggttggt-3´; D1r 5´-tctttgtcatctctttagctgtgtc-3´ and 5´-ttcggagtcatcttcctctca3´;

D2r

5´-tactcctccatcgtctcgttcta-3´

and

5´-atgcccattcttttctggttt-3´;

β-actin

5´-

tggagaagagctatgagctgc-3´ and 5´-tgttggcatagaggtctttacg-3´. All primers were designed with a similar TM and to not interact with the other primers in the multiplex PCR. Also, all primers were designed to bind within different exons of the same target, thus the signal we obtained could not come from genomic DNA. D2r primers were designed to recognize both short (D2rs) and long (D2rl) isoforms of the D2 receptor. In addition, β-actin primers were designed to distinguish between mRNA and genomic DNA. Samples with a genomic βactin PCR product were discarded. PCR products were resolved in 2 % agarose gels and the identity of all PCR products was confirmed by sequencing. 2.7 Multiplex single cell RT-PCR. Manually dissected SN tissue was treated with Trypsin (2 µg/ml) for 30 min at 30°C in oxygenating (100% O2, Infra) PIPES-buffer (in mM: 115 NaCl, 5 KCl, 1 CaCl2, 4 MgCl2, 25 Glucose, 20 PIPES, pH 7.0) (Mendez et al., 2008). Trypsin was then neutralized by 2 rinses with 10% fetal bovine serum (FBS) in oxygenated PIPES-buffer and the tissue was left to stabilize for 1.25 h at room temperature in oxygenating PIPES-buffer. Tissue was then triturated using trituration solution (0.1 % BSA, 1% FBS in PIPEs-buffer) by several passages through glass pipettes of decreasing diameter to obtain a cell suspension. Death cells and debris were discarded through a differential centrifugation (Mendez et al., 2008). The recovered cell suspension was left to adhere on poly-L-lysine-coated coverslips for 15 min and washed with oxygenated KrebsRinger buffer (in mM: 140 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, 10 glucose, 6 sucrose, pH 7.35, and 305 mOsm) to remove non-attached cells. Cells were individually

collected using sterile borosilicate patch pipettes by applying light negative pressure to the pipette. Collected cells were immediately transferred into iced-cold PCR tubes containing 6 µl of a freshly prepared preservation solution (RNaseOUT 20 U and DTT 8.3 mM) and frozen until use. Individual frozen cells were then thawed on ice and subjected directly to cDNA synthesis and two rounds of PCR reactions. cDNA synthesis was performed using 1.25 µM of random hexamers, 100 units of M-MLV RT and 20 additional units of RNaseOUT. The first round of PCR was performed as describe above by using the entire RT reaction and 23 cycles. The second round of PCR was performed using 20% of the first PCR reaction and 38 cycles. PCR reactions were resolved in 1.5 % agarose gels and the identity of all PCR products was confirmed by sequencing. All reagents for molecular biology were purchased from Thermo Fisher Scientific whereas all other reagents were from Sigma-Aldrich. 2.8 Statistical analysis. All analysis and observations were blindly performed to avoid any observer bias. For the appetitive learning task and the evaluation of motor activity, the person who injected the compounds was different from the persons that performed the behavior experiments. For the evaluation of the gene expression levels, the experimenter only knew the identity of the mice that were injected, but once the tissues were collected, the whole process was blind until the loading of the gels. For the single cell RT-PCR experiments, the whole process was blind and the phenotypes were established until the gels were revealed. All data shown here were analyzed using the Prism v5.03 software (GraphPad, San Diego, CA, USA). The experimental unit was the individual mice, thus for the evaluation of gene expression levels, each experiment involved the use of 3 slices per animal that were pooled together for the RNA extraction. The primary outcomes variables

were the gene expression levels and the time that mice took to reach the food pellet. Gene expression data plots shown are mean ± S.D from 4 mice. Quantifications of the time that mice expend to reach the food pellet were plotted using box-and-whisker diagrams. Statistically-significant differences among conditions were analyzed using the student´s ttest. p values <0.05 were considered significant and were not adjusted for multiple comparisons. Normality of data distribution was tested using the Shapiro-Wilk test (Prism v5.03). Identification of outliers was performed using the iterative Grubbs´ tool contained within the column data analysis of Prism v7, however, none was identified. No specific statistical analysis to control for confounding factors was applied as that was controlled through randomization of both mice and slices as well as age-matching and sex/gender restriction. 3 Results 3.1 Blockade of Dopaminergic phenotype decreases the expression of D1r in substantia nigra In addition to the synthesis of DA through the enzymatic activity of TH and DOPA descarboxylase (DDC), Dopaminergic cells have the ability to obtain DA through the activity of the DA transporter (DAT) that re-uptakes the extracellular DA. In either case, to be released during neurotransmission, cytosolic accumulated DA has to be loaded into vesicles through the activity of the vesicular monoamine transporter type 2 (VMAT2). Considering that AMPT blocks the synthesis of DA by inhibiting TH, and that reserpine blocks the vesicular transport of DA by inhibiting VMAT2, we used both compounds to produce an effective blockade of DA phenotype and evaluated whether this blockade induces changes in the expression levels of genes involved in DA neurotransmission in the adult SN. To do so, we first established multiplex RT-PCR assays to detect the expression

of genes of the DA system (TH, VMAT2, DAT, D1r and D2r) as well as of VGluT2 and GAD67, as representatives of the ability of DA cells to use glutamate and GABA as neurotransmitters, respectively (Supplementary Fig 1). Once the expression of these genes was readily detected, we established the experimental conditions to perform semiquantitative RT-PCR assays in which RT-PCR measurements were able to give an error of less than 5% (r2>0.95) when mesencephalic total RNA was serially increased by 10%. An example of this can be found in Supplementary Fig. 1e and f, in which the results for VGluT2 and TH are shown. Once the conditions to detect differences of at least 10% in the expression levels were established, we then evaluated the effect of the blockade of DA neurotransmission in the expression of TH, VMAT2, DAT, D1r, D2r as well as of VGluT2 and GAD67. As can be seen in Fig. 1, only D1r changed its expression levels in SN when mice were treated for 10 days with both compounds (Fig. 1H). On the contrary, when mice were treated for only one day, no changes in the expression could be detected (Fig.1m and n). Thus the decrease on the expression of D1r was the result of the chronic treatment with both AMPT and reserpine and not the response after a single (the last) dose. Since inhibition of VMAT2 with reserpine downregulates the activity of DAT (Mendz et al., 1988) leading to a severe reduction in the storage and release of DA, and TH inhibition with AMPT leads to significant decrease in the ability of Dopaminergic neurons to release DA (Watanabe et al., 2005), our results were somehow unexpected as the complete pharmacological blockade of DA neurotransmission should have resulted in more phenotypic adaptations in response to the low levels of DA. We thus evaluated the effect of each compound treated by separate. As can be seen in Fig. 2H and Fig. 3H, neither AMPT or reserpine were able to decrease the expression levels of D1r when mice were separately

treated for 10 days with AMPT or reserpine. Thus the effect on D1r expression in SN is specific of the treatment with both compounds. Furthermore, when treated alone for 10 days, AMPT induced the expression of VMAT2 and of the short isoform of D2r as well as of GAD67 (Fig. 2), whereas reserpine induced the expression of TH, VGluT2 and of both isoforms of D2r (Fig. 3). Thus when treated alone, AMPT and reserpine differentially induced phenotypic adaptations in the SN. 3.2 GABAergic and Glutamatergic neurons of the substantia nigra express D1r Considering that chronic treatment with both AMPT and reserpine modulates only the expression of D1r, we decided to evaluate which cells of the SN express this receptor. In addition to Dopaminergic and GABAergic cells, within the SN we can also find Glutamatergic cells as well was neurons that corelease these neurotransmitters (Chuhma et al., 2004; Mendez et al., 2008; Tritsch et al., 2012; Yamaguchi et al., 2013). We thus isolated freshly-dissociated individual cells from the SN and searched for the expression of D1r by single cell RT-PCR, after this, the identity of the D1r expressing cells was identified by the expression of TH, VGluT2 or GAD67. Of 33 cells collected, D1r was expressed in 15 cells (45 %) (Fig. 4a), of which, 11 were GABAergic (GAD67 positive) and 3 were Glutamatergic (VGluT2 positive). Whereas none of the D1r expressing cells were Dopaminergic (TH positive), the identity of one cell could not be established. To control for our ability to detect Dopaminergic cells, the D1r negative cells (18 cells) were subjected to TH detection, of these cells, 10 were positive to TH, thus 30% (10/33) of cells individually collected were Dopaminergic, but none of them expressed the mRNA for D1r (Fig. 4a and b). 3.3 GABAergic neurons expressing D1r project axons to the hippocampus

Besides their well established role in regulating the Dopaminergic neurons within the SN, GABAergic neurons also send axons to several nuclei, primary to the thalamus (Kase et al., 2015; Zhou and Lee, 2011), which serves as the main output of the basal ganglia; and among others, to the superior colliculus and the mesencephalic reticular formation (Hopkins and Niessen, 1976). On the other side, Glutamatergic neurons, which are mainly located within the SNc, send axonal projections to the posterior thalamic nuclei as well as to the nucleus reticularis of the thalamus (Antal et al., 2014). Thus, with the goal of determining where the D1r expressing cells project their axons, we first injected the anterograde tracer Dextran Alexa-Fluor 546 (DA546) into the SN (Supplementary Fig.2A and B) and search for sites of possible axonal projection throughout all the brain using coronal slices. As expected, DA546 signal was identified within the striatum (Caudate Putamen, CPu) (Dopaminergic nigrostriatal projection) (Fig. 4D) (Anden et al., 1964), the thalamic ventral medial nucleus, the superior colliculus (Fig. 4E) and the mesencephalic reticular formation (GABAergic projections) (Hopkins and Niessen, 1976; Kase et al., 2015; Zhou and Lee, 2011) as well as in the posterior thalamic nuclei and the nucleus reticularis of the thalamus (Glutamatergic projections) (Antal et al., 2014). In addition, we also found DA546 signal in the hippocampus (CA1 and CA3) (Fig.4C), the dorsal groups of thalamus (anterior pretectal nucleus, lateral posterior thalamic nucleus, dorsal lateral geniculate) and the upper region of the posterior thalamic nucleus (Fig. 4C), among others. Because of the importance of the hippocampus in memory formation and consolidation, we decided to confirm this projection by the use of the retrograde tracer Fluorogold. Injection of Fluorogold into the CA3 region did not produce any fluorescent signal of the retrograde tracer within the SN (Data not shown). However, when Fluorogold was injected into the CA1 (Supplementary Fig.2C), its fluorescent signal was strongly recovered in the caudal

tier of the dorsolateral SNr (Fig. 4G) and scarcely weak in the medial tier of the ventral SNr (Supplementary Fig. 2D). As expected, injection of Fluorogold into de CA1 also produced signal within the ventral tegmental area (Fig. 4F) (Swanson, 1982). Then, with the goal of identifying the phenotype of the Fluorogold-labeled cells, fluorescent individual cells were individually collected using cell suspensions prepared from SN tissue dissected from mice injected with Fluorogold. Individual cells were then subjected to single cell RT-PCR. Of 29 Fluorogold-labeled cells we collected, 18 (62 %) were GABAergic whereas 2 cells (7 %) were Glutamatergic. Surprisingly, 9 cells (31 %) could not be identified (they were also TH negative) (Fig. 4H). To confirm that the majority of SN projecting cells were GABAergic, we performed double immunolabeling against GABA and TH on slices from mice injected with Fluorogold into the CA1 (Fig. 4J-M). All GABAergic neurons projecting into de hippocampal CA1 region (positive to both Fluorogold and GABA) were located within the caudal tier of the SNr (Fig. 4J, K and M). When quantified, we found that 82.2% (212 of 258) of Fluorogold- labeled cells were GABAergic (Fig. 4N) (6 slices from 3 mice, 2 slices each). Conversely, 13.5% of GABAergic neurons (212 of 1559) were positive to Fluorogold (Fig. 4N). Hence, the GABAergic nigrocamppal projection into the hippocampus is an important component of the SNr. In addition, as expected from our single cell RT-PCR assays (Fig. 4H), some of the Fluorogold positive cells were negative to either GABA or TH (17.8%) (Fig. 4J, M and N). These cells could be either Glutamatergic or of a not-yet identified phenotype (Fig 4H). In this regard, it is important to note that VGluT2, the phenotypic marker of SN Glutamatergic neurons is an axonal marker not expressed within the cell body, we therefore could not find any VGluT2 immunolabeling in our experiments (Data not shown). Interestingly, despite the presence of TH positive cells within the SNr, none of these Dopaminergic cells projected to the CA1 as

they were negative to Fluorogold (Fig. 4N). Remarkably, some of the TH positive cells were also positive to GABA (Fig. 4L and N). We next evaluated the expression of D1r in 10 cells from our single cell RT-PCRs shown in Fig. 4H (7 GABAergic, 2 Glutamatergic and 1 no-identified). We found that 5 of the 7 GABAergic cells (71%) expressed the mRNA for D1r whereas none of the Glutamatergic cells expressed D1r, and the only unidentified cell also expressed the mRNA for D1r (Fig. 4I). Thus the majority of the GABAergic cells projecting their axons to the hippocampus express D1r. 3.4 Blockade of dopaminergic phenotype impairs memory formation At this point, we have shown that combined treatment with AMPT and reserpine reduces the expression of D1r in the SN, which is mainly expressed by GABAergic neurons that send their axons into the CA1 region of the hippocampus. We thus decided to test whether the blockade of the DA phenotype with both AMPT and reserpine might impair the memory formation in a appetitive learning task using a multiple choice eight arm maze (Fig.5A). In this task mice search for a food pellet placed at the end of one of the arms. Each bifurcation in the maze was labeled with a different visual cue, which mice must use to navigate through the maze. After six habituation sessions of 20 min each during three days, on day 4 and 5, mice had 4 training sessions 20 min apart in which they were allowed to freely explore the maze until they found the food pellet and bit it (Fig. 5B). On day 6, mice were allowed to search for the food pellet and the time spent to reach the target recorded. On average, it took 395 ±148 s to naive mice to find the food (Fig. 5D), however on the test day (fifth trial), control mice found the food pellet in only 16.8 ± 7.1 s (Fig. 5D), basically, mice went straight through directly to the food. When mice treated with both AMPT and reserpine for two days (on day 4 and 5) were evaluated on the same task, it took 13.5 ± 2.5 s for them to find the food pellet. Thus no significant differences were found

(Fig. 5D and E). However, because the change in the expression levels of D1r is readily detected after 10 days of treatment, we thus decided to evaluate the same appetitive learning task after 10 days of combined treatment. For this experiment, on day 6-8 of treatment, mice were allowed to habituate, and on day 9 and 10, they received the four training sessions of 20 min (Fig. 5C), just as in the previous protocol. On test day (day 11), control mice found the food pellet in just 13.8 ± 3.1 s whereas mice that chronically received both AMPT and reserpine required 377 ± 146 s to do so (Fig. 5F). Finally, because both AMPT and reserpine are compounds known to decrease locomotor activity, we decided to evaluate the motor performance of mice chronically treated with both AMPT and reserpine. As can be seen in Fig. 5G, when the latency to fall off of a rotating rod was measured, mice treated with both AMPT and reserpine for one day showed an important decrease in their ability to stay on the rod, however, this motor impairment gradually recovered over time, and by day 9, when the training of mice was initiated, no statistical differences versus control were found. Moreover, when the total locomotor activity was evaluated in a open field maze, the total distance traveled by mice treated with both compounds had a similar pattern than in the rotarod test: it was decreased after one day of treatment, and was not different from control mice by day 9 (Fig. 5H). We thus conclude that in addition to the decrease in the expression of D1r, the combined chronic treatment with AMPT and reserpine induces an impairment in memory formation not related to a decrease in locomotor activity. 4. Discussion 4.1 GABAergic interconnection to the hippocampus In addition to the classical axonal release of neurotransmitters, Dopaminergic neurons have the ability to release DA from their soma and dendrites (Bjorklund and Lindvall,

1975; Cheramy et al., 1981). This form of neurotransmission downregulates the electrical activity of Dopaminergic neurons through IPSCs mediated by D2 autoreceptors (Beckstead et al., 2004). In SN, the somatodendritic (STD) release of DA have been implicated in the regulation of motor activity (Andersson et al., 2006; Bergquist et al., 2003) and of muscle tone (Double and Crocker, 1995) through activation of D1r located within SNr (Bergquist et al., 2003; Double and Crocker, 1995; Hemsley and Crocker, 2001). Here we show that SNr GABAergic neurons that project to the hippocampus express D1r, thus suggesting that STD release of DA might be involved in memory formation as DA released from soma and dendrites is responsible for their activation (Zhou et al., 2009). In this regard, it is promising that blockade of the DA phenotype with AMPT and reserpine induces both a downregulation of D1r expression in the SN and an impairment in the performance in an appetitive learning task that requires the coding of reward as well as navigational skills, which depend on Dopaminergic neurons and on the activity of the hippocampus, respectively. Further work is necessary to strength the link between STD release of DA and the hippocampus via a GABAergic interconnection. It is important to note that although a Dopaminergic nigrocampal projection has already been shown in the rat (Swanson, 1982; Wyss et al., 1979), this is the first report of a GABAergic nigrocampal projection (Fig.4). Moreover, although the expression of D1r in GABAergic neurons has also been previously reported (Zhou et al., 2009), the novelty of our work relies in the finding that GABAergic neurons of the SNr, who have the ability to respond to somatodendritically released DA because of the expression of D1r, project axons into the hippocampus. Indeed, the role of GABA in the activity of the hippocampus has been already explored: locally released GABA has been implicated in schizophrenia (Gilani et al., 2014; Konradi et al., 2011), chronic stress (Czeh et al., 2015), anxiety (Crestani et al., 1999; Muller et

al., 2015) and the learning of aversive events (Crestani et al., 2002) as well as mnemonic control (Schmitz et al., 2017); whereas GABAergic inputs to the hippocampus from the septum and VTA modulate memory (Krebs-Kraft et al., 2007; Ntamati and Luscher, 2016), thus our findings may shed some light on how SNr is linked to the hippocampal activity. 4.2 AMPT and reserpine antagonize each other AMPT is used for treatment of pheochromocytoma and induces some level of sedation that decreases quite a few days later. On its side, reserpine, which is used for the treatment of hypertension, and in the past for the relief of psychotic symptoms, induces some level of hypoactivity, thus both compounds have the ability to reduce the overall locomotor activity. In our experiments, when AMPT and reserpine were injected together, the locomotor activity was initially decreased (Fig. 5G and H), and by day 9 it was fully recovered, therefore, at the level of locomotor activity, some sort of functional rescue was found, and thus the reduction in the time that mice took to reach the food pellet cannot be attributed to a decrease in the overall locomotor activity induced by both compounds. Remarkably, at the gene expression level, the effects of both compounds were dissimilar, besides the short isoform of D2r, they induced the expression of different genes (VMAT2 and GAD67 for AMPT; TH, VGluT2 and the long isoform of D2r for reserpine). Remarkably, the expression of D1r was the only gene that was modified when both compounds were administered together, and it was a decrease. Therefore, as in the locomotor activity, some sort of reciprocal rescue was found, suggesting an antagonistic activity between them at the transcriptional level. Because it has not been reported that AMPT or reserpine induce cell death, and considering that the combined treatment of AMPT and reserpine blocks the DA phenotype, we expected the induction of genes of the Dopaminergic phenotype or the overexpression of the

GABAergic/Glutamatergic phenotype as a compensatory adaptation, which was something we observed when either AMPT or reserpine were injected alone. However that was not the case for the double treatment. Thus our results are in line with a recent meta-analysis of the literature of gene expression in Parkinson´s disease (Lewis and Cookson, 2012), in which no increase of any of the genes we searched for was reported. Furthermore, our results showing a decrease in the expression of D1r are also in line with previous reports showing that as a compensatory response, DA depletion induces a functional supersensitivity of D1r (Corvol et al., 2004; Ding et al., 2015), instead of a increase in its expression (Hurley et al., 2001; Shinotoh et al., 1993). We hypothesize that this compensatory D1r supersensitivity in response to DA depletion underlies the reciprocal rescue in locomotor activity we found. Likely, this D1r supersensitivity is reflected as a locomotor sensitization, and could be the reflect of the reciprocal antagonistic effect of AMPT and reserpine at the transcriptional level, in which only the expression of D1r is modified when both compounds are chronically applied (Fig.1-3). 4.3 Phenotype of D1r expressing cells In our scRT-PCR assays we found that glutamatergic neurons of the SN also express D1r, though they do not project axons into the hippocampus. In view of the report of Tritsch et al., 2012, it seems likely that these cells project their axons into the striatum. Currrent work in our lab is devoted to determine other sites of axonal projection for these type of neurons. Interestingly, we also found cells that project to the hippocampus whose phenotype could not be identified (31%). Although the expression of D1r has been reported in astrocytes in the adult SNr (Nagatomo et al., 2017), the Fluorogold-labeled cells could only be neurons as astrocytes cannot send projections to other nuclei. However, unidentified cells expressing D1r in experiments no involving the retrograde tracer have the possibility of

being astrocytes since we did not use any specific labeling, like sulforhodamine 101 (Nimmerjahn et al., 2004), to negatively select glial cells. Although these unidentified cells did not express TH, they can still be Dopaminergic of the alternative phenotype, this is, unable to produce DA but still able to up-take it using DAT and releasing it with the help of VMAT2. Interestingly, despite the ability of Dopaminergic neurons to co-express other neurotransmitter phenotypes, none of the adult SN cells we identified in our scRT-PCR assays have any double phenotype. Taking into account that all experiments here were performed using adult mice, and that a developmental downregulation of the expression of VGluT2 has been previously suggested (Mendez et al., 2008), our results further support this scenario. Taken together, our findings suggest that Dopaminergic neurons of the substantia nigra may participate in memory formation through a GABAergic interconnection to the hippocampus mediated by D1r.

Acknowledgements This study was supported by grants from Conacyt-Mexico (CB-154645) and PROMEP/SEP (103.5/107324) to JAM. In addition, this study benefitted from an installation grant from UASLP to JAM. SO-P and ES-O were supported by a ConacytMexico Fellowship.

Conflict of interest: We have no conflicts of interest to declare.

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Figure Legends Figure 1. Combined chronic treatment of AMPT with reserpine decreases the expression levels of D1r. The SN of mice treated with both AMPT and reserpine for 10 days was dissected and subjected to semiquantitative multiplex RT-PCR. Representative agarose gels showing the expression levels of TH and VGluT2 (A) as well as of VMAT2 and DAT (B), D2r and GAD67 (C), and of D1r (D). Their relative expression levels normalized to β-actin are shown in E-L. The expression levels of D1r after 1 day of treatment with AMPT and reserpine is shown in M whereas its respective quantification is shown in N. Bars represent mean ± SD of the expression from 4 animals (3 pooled slices per animal). Statistical differences were analyzed using student´s t-test. *, p=0.0396. Ctl, vehicle-injected mice; AMPT/res, mice daily treated with 537.245 mg/kg of AMPT combined with 0.66 mg/kg of reserpine. C-, RT-PCR reaction with a sample in which water was used instead of total RNA for the RT reaction; C+, mesencephalon plus striatum used as positive control.

Figure 2. Chronic AMPT treatment induces the expression of VMAT2, D2rs and GAD67. The SN of mice treated with AMPT for 10 days was dissected and subjected to semiquantitative multiplex RT-PCR. Representative agarose gels showing the expression levels of TH and VGluT2 (A) as well as of VMAT2 and DAT (B), D2r and GAD67 (C), and of D1r (D). Their relative expression levels normalized to β-actin are shown in E-L. Bars represent mean ± SD of the expression from 4 animals (3 pooled slices per animal). Statistical differences were analyzed using student´s t-test. * in F, p=0.0313; * in I, p=0.0485; * in K, p=0.0412. Ctl, vehicle-injected mice; AMPT, mice daily treated with 537.245 mg/kg of AMPT. C-, water instead of total RNA for the RT reaction; C+, mesencephalon plus striatum used as positive control. Figure 3. Chronic reserpine treatment induces the expression of TH, D2rs, D2rl and VGluT2. The SN of mice treated with AMPT for 10 days was dissected and subjected to semiquantitative multiplex RT-PCR. Representative agarose gels showing the expression levels of TH and VGluT2 (A) as well as of VMAT2 and DAT (B), D2r and GAD67 (C), and of D1r (D). Their relative expression levels normalized to β-actin are shown in E-L. Bars represent the mean ± SD of the expression from 4 animals (3 pooled slices per animal). Statistical differences were analyzed using student´s t-test. ** in E, p=0.0012; ** in I, p=0.0011; ** in J, p=0.0012. * in L, p=0.0494. Ctl, vehicle-injected mice; res, mice treated with 0.66 mg/kg of reserpine. C-, water instead of total RNA for the RT reaction; C+, mesencephalon plus striatum used as positive control. Figure 4. D1r is expressed in GABAergic neurons that project their axons into the hippocampus. Freshly-dissociated mesencephalic cells were individually collected and subjected to multiplex single cell RT-PCR. A. representative gel showing the expression

profiles of selected individual cells. Cells were first evaluated against the expression of D1r (upper gel) and the expression of the phenotypic markers for DA (TH), GABA (GAD67) and Glutamate (VGluT2) was subsequently evaluated (lower gel). The expression of β-actin was used to confirm the presence of intact RNA and to evaluate any genomic DNA contamination. Cell 3 (C3) is a GABAergic neurons expressing D1r, whereas Cell 4 (C4) is a glutamatergic cell that expresses D1r. Cell 1 (C1) is a D1r expressing cell of unidentified (NI) phenotype (negative to TH, VGluT2 and GAD67). B. Pie chart representing the proportion of neurons expressing D1r. The percent values outside circular graph represent the proportion with respect to whole sample (n=33 cells from 3 experiments, two mice in each experiment). The numbers inside the graph represent the proportion of the respective gene in terms of the D1r expressing cells (15 cells). TH expressing cells (TH, 10 cells) represent 30% of the whole sample, but none of them were D1r positive. ND, cells negative to both D1r and TH whose phenotype was not further determined (8 cells). n= 3 experiments, 2 mice in each experiment. To determine sites of axonal projection of D1r cells, mice were injected with the anterograde tracer Dextran Alexa-546 (DA546) into the SN. 10 days later brains were coronally sliced and serially analyzed (n=5 mice). The signal of DA546 was recovered within the CA1 and CA3 regions of the hippocampus (C), the Caudate-Putamen (CPu, striatum) (D), the periacueductal gray (PAG) (E), the superior colliculus (SC) and the dorsal part of the Thalamus (APD, PTN, DLG) (C). For illustrative landmarking purposes, C´, D´ and E´ are schematic representations of the slices from which the images were respectively taken. The signal reported here was found in all 5 injected animals. Ctx, brain cortex; DG, Dentate gyrus; APD, Dorsal tier of the Anterior Pretectal Nucleus; PTN, Posterior thalamic nuclei (both mediorostral and laterorostral part

of the Lateral Posterior Thalamic nucleus); DLG, Dorsolateral geniculate nucleus; SGSC, dorsal part of the superior colliculus; SC, internal part of the superior colliculus; V1, primary visual cortex; S1, primary somatosensory cortex; Au1, primary auditory cortex; PF, parafascicular thalamic nucleus; Pir, piriform cortex; M1, primary motor cortex; M2, secondary motor cortex, S1Ulp, upper lip of the primary somatosensory cortex; S2, secondary somatosensory cortex; ACA, anterior commissure; Tu, olfactory tubercle; V1M, monocular area of the primary visual cortex; V2L, lateral part of the secondary visual cortex; mRT, mesencephalic reticular formation. 10 days after the injection of the retrograde tracer Fluorogold into the CA1 region of the hippocampus (n=4 mice), the fluorescent signal was recovered within the rostral tier of the ventral tegmental area (VTA) (F). Image in G shows the Fluorogold signal located within the caudal tier of the SNr. The signal was intense within the dorsolateral part of the caudal tier and weak within the medial tier of the ventral SNr (Supplementary Fig. 2D). Identification of . H, Identification of the phenotype of the SNr projecting cells into the hippocampus was determined by multiplex single cell RT-PCR of individual Fluorogold-labeled cells. 62% of cells (18/29) were GAD67 positive, 7% (2/29) were VGluT2 positive and none of the remaining 9 cells were TH positive. I, representative gel showing the expression profiles of selected individual cells. n= 29 cells from 4 individual experiments, one mice in each experiment. The expression of D1r is shown in the upper gel whereas the expression of the phenotypic markers for GABA (GAD67) and glutamate (VGluT2) is shown in the lower gel. Cell 1 and 2 (C1 and C2) are GABAergic neurons expressing D1r, whereas Cell 3 (C3) is a GABAergic cell D1r negative and cell 4 (C4) is D1r expressing cell of unidentified phenotype. J, slices from mice injected with Fluorogold into the CA1 region of hippocampus were subjected to immunolabeling against GABA (red) and TH (blue).

GABAergic neurons (red) containing Fluorogold (green) were located only in the caudal tier of the SNr (yellow-merged signal). Image in K shows in detail the presence of GABAergic neurons (GABA positive cells in red) containing Fluorogold (green) in the SNr. Note the presence of Dopaminergic neurons (TH positive in blue) lacking Fluorogold. L, amplification of the SNr-SNc boundary, note the presence of neurons of double GABAergic-Dopaminergic phenotype (arrows). M, amplification of the immunolabeling signal within the dorsolateral part of SNr, note the presence of Fluorogold containing neurons (green) negative to GABA (arrows) in addition to the projecting GABAergic neurons (yellow-merged cells). Images shown in K, L and M are amplifications from slices different from the one shown in J. Quantifications of cell populations is shown in N. Numbers outside the graph represent the total number of cells for its corresponding population; numbers inside each diagram represent cells of pure phenotype (GABA, TH or Fluorogold), whereas numbers within the merged areas represent cells of double phenotype. Percentages within the merged areas are with respect to GABA positive cells. n= 6 slices from 3 mice, 2 slices from each mouse. Identification of all brain structures was based upon the mouse brain atlas of Paxinos (Paxinos and Franklin, 2007). Figure 5. Chronic blockade of the dopaminergic phenotype with both AMPT and reserpine impairs memory formation. Mice were subjected to a learning paradigm in which they have to learn to locate a food reward (0.6 g food pellet) placed at the end of one of the 8 arms of the maze shown in A. Animals were allowed to habituate to the maze for 3 days (2 daily sessions of 20 min) followed by 2 days of training (2 daily sessions of 20 min), and on next day, the time that mice took to reach the food pellet was recorded (test day). B, Timeline diagram of the learning protocol under a 2-day treatment with AMPT and reserpine. C,

Timeline diagram of the learning protocol under a 10-day treatment. Both compounds and vehicle were intraperitoneally applied. The time that mice took to reach the food pellet in their first training session (naive) is shown in D, as well as the time they took on the third training session (Training) and on test day under 2 days of treatment. To better illustrate the time mice spent under 2 days of treatment, this part of the graph is expanded in E. The time mice spent to reach the food pellet under 10 days of treatment is shown in F. Graphs represent the mean of the time used to reach the target from the origin of the maze. The error bars indicate SD. n= 6 mice for both experiments. ***, p=0.0001. G, locomotor performance of mice under 10 days of treatment with AMPT and reserpine was evaluated using a rotarod at a constant rotating speed of 15 rpm. Latency to fall (in seconds) was evaluated one day before treatment (D-1) to establish basal motor performance, and then every second day up to day 11 (D1-D11). Graph represents the time (average of 2 measurements per day) mice stayed on the rotarod before falling off. The error bars indicate SD. n= 4 mice in each group. **, p=0.0086; *, p=0.0212. F, locomotor activity of mice under 10 days of treatment with AMPT and reserpine was daily evaluated by measuring the total distance (in cm) traveled by mice in a bare open field maze. Basal locomotor activity was measured one day before treatment (D-1). Graph represents the ambulatory activity developed during a 5 min period. The error bars indicate SD. n= 4 mice in each group. ***, p=0.0001. Supplementary Figure 1. Standardization of multiplex RT-PCR. Representative agarose gels showing the expression of TH and VGluT2 (A) as well as of VMAT2 and DAT (B), D2r and GAD67 (C), and of D1r (D). The expression of all these genes is accompanied with the expression of β-actin. Mesencephalon plus striatum was used as the source of transcripts

(C+). E, representative gel of a relative standard curve for TH and VGluT2, in which the cDNA was serially increased by 10 %. The numbers on top of the gel indicate the amount of cDNA used for the PCR reaction. F, linear regression of the band intensity data of gels from 3 relative standard curves for TH and VGluT2. The gel (E) and the linear regression plot (F) represent our ability to detect differences of at least 10% with a error of less than 5 % (r2 < 0.05). Supplementary Figure 2. Anterograde and retrograde tracer injections. A, Representative image of the injection of the anterograde tracer Dextran Alexa-546 (DA546) into the SNc (n=2 mice). Slices were subjected to immunolabeling against TH (yellow) to delineate the boundaries of SN and VTA (dashed lines) by means of the presence of Dopaminergic neurons, note that the tip of the injection sites is located within the SNc. B, Representative image of the injection of the anterograde tracer DA546 into the SNr (n=3 mice). Dashed lines delineate SN and VTA using the expression of TH (yellow). Note that the tip of the injection sites is located within the SNr. 10 days after the injection of DA546, mice were sacrificed and their brains processed. C, Representative image of the injection of the retrograde tracer Fluorogold into the CA1 region of the hippocampus (n=4 mice). 10 days later, mice were sacrificed and their brains processed to identify fluorescent signal within the SN. D, Magnification image of the SNr to show the weak puncta-like signal Fluorogold (red) signal located within the medial tier of the ventral SNr. Slices were subjected to immunolabeling against GABA (green) and TH (blue), note that puncta signal does not colocalize with the GABA or the TH signal. SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; ATV, ventral tegmental area; LHip, Lateral hippocampus; SC, superior colliculus; PAG, periacueductal gray; MG, medial geniculate; GD, dentate

gyrus; APD, Dorsal tier of the Anterior Pretectal Nucleus; PTN, Posterior thalamic nuclei (both mediorostral and laterorostral part of the Lateral Posterior Thalamic nucleus); DLG, Dorsolateral geniculate nucleus. Ctx, cortex.

Dr. J. Alfredo Mendez (Orcid ID : 0000-0002-2859-7086)

Blockade of the Dopaminergic neurotransmission with AMPT and reserpine induces a differential expression of genes of the Dopaminergic phenotype in substantia nigra

Sergio Ortiz-Padilla1, Elier Soto-Orduño1, Marisa Escobar Barrios1, Abril Armenta Manjarrez1, Yadira Bastián 2, J. Alfredo Mendez1*

Highlights •

Blockade of the Dopaminergic phenotype reduces the expression of the D1 receptor

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GABAergic neurons expressing the D1 receptor interconnect SN with the hippocampus

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AMPT and Reserpine have antagonistic effects at the transcriptional level

Sergio Ortiz-Padilla: Investigation, Formal analysis, Data curation. Elier Soto-Orduño: Investigation, Formal analysis, Data curation, Validation. Marisa Escobar Barrios: Investigation, Validation. Abril Armenta Manjarrez: Investigation, Validation. Yadira Bastián: Investigation, Validation, Formal analysis. J. Alfredo Mendez: Conceptualization, Investigation, Data curation, Supervision, Formal analysis, Visualization, Writing- Original draft preparation, Writing- Reviewing and Editing, Funding acquisition.