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• W2912884060 abstract "•DR serotonin neurons make symmetric or asymmetric synapses on VTA dopamine neurons•DR serotonin-VGluT3 axon terminals make asymmetric synapses on VTA dopamine neurons•VTA activation of DR serotonin terminals elicits excitation of dopamine neurons•VTA photoactivation of SERT fibers promotes CPP and evokes dopamine release in nAcc Dorsal raphe (DR) serotonin neurons provide a major input to the ventral tegmental area (VTA). Here, we show that DR serotonin transporter (SERT) neurons establish both asymmetric and symmetric synapses on VTA dopamine neurons, but most of these synapses are asymmetric. Moreover, the DR-SERT terminals making asymmetric synapses on VTA dopamine neurons coexpress vesicular glutamate transporter 3 (VGluT3; transporter for accumulation of glutamate for its synaptic release), suggesting the excitatory nature of these synapses. VTA photoactivation of DR-SERT fibers promotes conditioned place preference, elicits excitatory currents on mesoaccumbens dopamine neurons, increases their firing, and evokes dopamine release in nucleus accumbens. These effects are blocked by VTA inactivation of glutamate and serotonin receptors, supporting the idea of glutamate release in VTA from dual DR SERT-VGluT3 inputs. Our findings suggest a path-specific input from DR serotonergic neurons to VTA that promotes reward by the release of glutamate and activation of mesoaccumbens dopamine neurons. Dorsal raphe (DR) serotonin neurons provide a major input to the ventral tegmental area (VTA). Here, we show that DR serotonin transporter (SERT) neurons establish both asymmetric and symmetric synapses on VTA dopamine neurons, but most of these synapses are asymmetric. Moreover, the DR-SERT terminals making asymmetric synapses on VTA dopamine neurons coexpress vesicular glutamate transporter 3 (VGluT3; transporter for accumulation of glutamate for its synaptic release), suggesting the excitatory nature of these synapses. VTA photoactivation of DR-SERT fibers promotes conditioned place preference, elicits excitatory currents on mesoaccumbens dopamine neurons, increases their firing, and evokes dopamine release in nucleus accumbens. These effects are blocked by VTA inactivation of glutamate and serotonin receptors, supporting the idea of glutamate release in VTA from dual DR SERT-VGluT3 inputs. Our findings suggest a path-specific input from DR serotonergic neurons to VTA that promotes reward by the release of glutamate and activation of mesoaccumbens dopamine neurons. Although selective serotonin uptake inhibitors comprise the major class of modern antidepressants, the role of serotonin in reward function remains poorly understood. Lesion and pharmacological studies implicate serotonin in a wide array of cognitive and behavioral functions, including mood and reward (Lucki, 1998Lucki I. The spectrum of behaviors influenced by serotonin.Biol. Psychiatry. 1998; 44: 151-162Abstract Full Text Full Text PDF PubMed Scopus (948) Google Scholar). Alterations in serotonergic function and reward-related processing have been hypothesized in several psychiatric disorders, including schizophrenia (Kapur and Remington, 1996Kapur S. Remington G. Serotonin-dopamine interaction and its relevance to schizophrenia.Am. J. Psychiatry. 1996; 153: 466-476Crossref PubMed Scopus (755) Google Scholar, Ziauddeen and Murray, 2010Ziauddeen H. Murray G.K. The relevance of reward pathways for schizophrenia.Curr. Opin. Psychiatry. 2010; 23: 91-96Crossref PubMed Scopus (68) Google Scholar), depression (McCabe et al., 2012McCabe C. Woffindale C. Harmer C.J. Cowen P.J. Neural processing of reward and punishment in young people at increased familial risk of depression.Biol. Psychiatry. 2012; 72: 588-594Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, Nestler and Carlezon, 2006Nestler E.J. Carlezon Jr., W.A. The mesolimbic dopamine reward circuit in depression.Biol. Psychiatry. 2006; 59: 1151-1159Abstract Full Text Full Text PDF PubMed Scopus (1516) Google Scholar, Ruf and Bhagwagar, 2009Ruf B.M. Bhagwagar Z. The 5-HT1B receptor: a novel target for the pathophysiology of depression.Curr. Drug Targets. 2009; 10: 1118-1138Crossref PubMed Scopus (69) Google Scholar, Watson and Dawson, 2007Watson J.M. Dawson L.A. Characterization of the potent 5-HT1A/B receptor antagonist and serotonin reuptake inhibitor SB-649915: preclinical evidence for hastened onset of antidepressant/anxiolytic efficacy.CNS Drug Rev. 2007; 13: 206-223Crossref PubMed Scopus (22) Google Scholar), and drug abuse (Higgins and Fletcher, 2003Higgins G.A. Fletcher P.J. Serotonin and drug reward: focus on 5-HT2C receptors.Eur. J. Pharmacol. 2003; 480: 151-162Crossref PubMed Scopus (140) Google Scholar, Kirby et al., 2011Kirby L.G. Zeeb F.D. Winstanley C.A. Contributions of serotonin in addiction vulnerability.Neuropharmacology. 2011; 61: 421-432Crossref PubMed Scopus (108) Google Scholar, Müller et al., 2007Müller C.P. Carey R.J. Huston J.P. De Souza Silva M.A. Serotonin and psychostimulant addiction: focus on 5-HT1A-receptors.Prog. Neurobiol. 2007; 81: 133-178Crossref PubMed Scopus (249) Google Scholar, Vengeliene et al., 2008Vengeliene V. Bilbao A. Molander A. Spanagel R. Neuropharmacology of alcohol addiction.Br. J. Pharmacol. 2008; 154: 299-315Crossref PubMed Scopus (423) Google Scholar). The role of serotonin in reward-related processing has been investigated for several decades, and although some have previously suggested that serotonin is antagonistic to reward function, the literature describing the role of serotonin in reward is equivocal (Boureau and Dayan, 2011Boureau Y.L. Dayan P. Opponency revisited: competition and cooperation between dopamine and serotonin.Neuropsychopharmacology. 2011; 36: 74-97Crossref PubMed Scopus (322) Google Scholar, Cools et al., 2011Cools R. Nakamura K. Daw N.D. Serotonin and dopamine: unifying affective, activational, and decision functions.Neuropsychopharmacology. 2011; 36: 98-113Crossref PubMed Scopus (294) Google Scholar, Hayes and Greenshaw, 2011Hayes D.J. Greenshaw A.J. 5-HT receptors and reward-related behaviour: a review.Neurosci. Biobehav. Rev. 2011; 35: 1419-1449Crossref PubMed Scopus (115) Google Scholar, Kranz et al., 2010Kranz G.S. Kasper S. Lanzenberger R. Reward and the serotonergic system.Neuroscience. 2010; 166: 1023-1035Crossref PubMed Scopus (197) Google Scholar). The inhibitory role of serotonin in reward function was initially suggested from studies showing that depletion of serotonin increased responding for brain-stimulation reward (Phillips et al., 1976Phillips A.G. Carter D.A. Fibiger H.C. Differential effects of para-chlorophenylalanine on self-stimulation in caudate-putamen and lateral hypothalamus.Psychopharmacology (Berl.). 1976; 49: 23-27PubMed Google Scholar, Poschel and Ninteman, 1971Poschel B.P. Ninteman F.W. Intracranial reward and the forebrain’s serotonergic mechanism: studies employing para-chlorophenylalanine and para-chloroamphetamine.Physiol. Behav. 1971; 7: 39-46Crossref PubMed Scopus (67) Google Scholar, Poschel et al., 1974Poschel B.P. Ninteman F.W. McLean J.R. Potoczak D. Intracranial reward after 5,6-dihydroxytryptamine: further evidence for serotonin’s inhibitory role.Life Sci. 1974; 15: 1515-1522Crossref PubMed Scopus (30) Google Scholar) and from studies in which local inhibition of serotonin neurons facilitated brain stimulation reward (Fletcher et al., 1995Fletcher P.J. Tampakeras M. Yeomans J.S. Median raphe injections of 8-OH-DPAT lower frequency thresholds for lateral hypothalamic self-stimulation.Pharmacol. Biochem. Behav. 1995; 52: 65-71Crossref PubMed Scopus (55) Google Scholar) or established conditioned place preference (Fletcher et al., 1993Fletcher P.J. Ming Z.H. Higgins G.A. Conditioned place preference induced by microinjection of 8-OH-DPAT into the dorsal or median raphe nucleus.Psychopharmacology (Berl.). 1993; 113: 31-36Crossref PubMed Scopus (64) Google Scholar). Place preference was also observed after pharmacological manipulations aimed to inhibit serotonergic function (Liu and Ikemoto, 2007Liu Z.H. Ikemoto S. The midbrain raphe nuclei mediate primary reinforcement via GABA(A) receptors.Eur. J. Neurosci. 2007; 25: 735-743Crossref PubMed Scopus (34) Google Scholar, Shin and Ikemoto, 2010Shin R. Ikemoto S. The GABAB receptor agonist baclofen administered into the median and dorsal raphe nuclei is rewarding as shown by intracranial self-administration and conditioned place preference in rats.Psychopharmacology (Berl.). 2010; 208: 545-554Crossref PubMed Scopus (19) Google Scholar). In contrast, direct electrical stimulation of the dorsal raphe (DR) is rewarding (Deakin, 1980Deakin J.F. On the neurochemical basis of self-stimulation with midbrain raphe electrode placements.Pharmacol. Biochem. Behav. 1980; 13: 525-530Crossref PubMed Scopus (13) Google Scholar, Miliaressis et al., 1975Miliaressis E. Bouchard A. Jacobowitz D.M. Strong positive reward in median raphe: specific inhibition by para-chlorophenylalanine.Brain Res. 1975; 98: 194-201Crossref PubMed Scopus (50) Google Scholar, Rompre and Miliaressis, 1985Rompre P.P. Miliaressis E. Pontine and mesencephalic substrates of self-stimulation.Brain Res. 1985; 359: 246-259Crossref PubMed Scopus (81) Google Scholar, Simon et al., 1976Simon H. Le Moal M. Cardo B. Intracranial self-stimulation from the dorsal raphe nucleus of the rat: effects of the injection of para-chlorophenylalanine and of alpha-methylparatyrosine.Behav. Biol. 1976; 16: 353-364Crossref PubMed Scopus (38) Google Scholar, Van Der Kooy et al., 1978Van Der Kooy D. Fibiger H.C. Phillips A.G. An analysis of dorsal and median raphe self-stimulation: effects of parachlorophenylalanine.Pharmacol. Biochem. Behav. 1978; 8: 441-445Crossref PubMed Scopus (31) Google Scholar), and DR in vivo recordings in monkeys (Bromberg-Martin et al., 2010Bromberg-Martin E.S. Hikosaka O. Nakamura K. Coding of task reward value in the dorsal raphe nucleus.J. Neurosci. 2010; 30: 6262-6272Crossref PubMed Scopus (111) Google Scholar, Nakamura et al., 2008Nakamura K. Matsumoto M. Hikosaka O. Reward-dependent modulation of neuronal activity in the primate dorsal raphe nucleus.J. Neurosci. 2008; 28: 5331-5343Crossref PubMed Scopus (186) Google Scholar) and rats (Miyazaki et al., 2011Miyazaki K.W. Miyazaki K. Doya K. Activation of the central serotonergic system in response to delayed but not omitted rewards.Eur. J. Neurosci. 2011; 33: 153-160Crossref PubMed Scopus (49) Google Scholar, Ranade and Mainen, 2009Ranade S.P. Mainen Z.F. Transient firing of dorsal raphe neurons encodes diverse and specific sensory, motor, and reward events.J. Neurophysiol. 2009; 102: 3026-3037Crossref PubMed Scopus (131) Google Scholar) suggest that DR neurons are activated by reward and contribute to reward function. Recent recordings from genetically identified serotonin neurons have provided evidence for neuronal activation of some DR serotonin neurons in response to cues predicting reward (Cohen et al., 2015Cohen J.Y. Amoroso M.W. Uchida N. Serotonergic neurons signal reward and punishment on multiple timescales.eLife. 2015; 4: 4Crossref Scopus (205) Google Scholar, Liu et al., 2014Liu Z. Zhou J. Li Y. Hu F. Lu Y. Ma M. Feng Q. Zhang J.E. Wang D. Zeng J. et al.Dorsal raphe neurons signal reward through 5-HT and glutamate.Neuron. 2014; 81: 1360-1374Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar) or reward consumption (Li et al., 2016Li Y. Zhong W. Wang D. Feng Q. Liu Z. Zhou J. Jia C. Hu F. Zeng J. Guo Q. et al.Serotonin neurons in the dorsal raphe nucleus encode reward signals.Nat. Commun. 2016; 7: 10503Crossref PubMed Scopus (222) Google Scholar). In addition, behavioral studies have shown that photoactivation of DR serotonin neurons reinforces instrumental behavior (Li et al., 2016Li Y. Zhong W. Wang D. Feng Q. Liu Z. Zhou J. Jia C. Hu F. Zeng J. Guo Q. et al.Serotonin neurons in the dorsal raphe nucleus encode reward signals.Nat. Commun. 2016; 7: 10503Crossref PubMed Scopus (222) Google Scholar, Liu et al., 2014Liu Z. Zhou J. Li Y. Hu F. Lu Y. Ma M. Feng Q. Zhang J.E. Wang D. Zeng J. et al.Dorsal raphe neurons signal reward through 5-HT and glutamate.Neuron. 2014; 81: 1360-1374Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). However, other studies have reported that photoactivation of DR serotonin neurons do not reinforce behavior (Fonseca et al., 2015Fonseca M.S. Murakami M. Mainen Z.F. Activation of dorsal raphe serotonergic neurons promotes waiting but is not reinforcing.Curr. Biol. 2015; 25: 306-315Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, McDevitt et al., 2014McDevitt R.A. Tiran-Cappello A. Shen H. Balderas I. Britt J.P. Marino R.A.M. Chung S.L. Richie C.T. Harvey B.K. Bonci A. Serotonergic versus nonserotonergic dorsal raphe projection neurons: differential participation in reward circuitry.Cell Rep. 2014; 8: 1857-1869Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Because global serotonin brain manipulations can alter multiple serotonergic projections and transduction pathways as well as multiple components of a given aspect of reinforcement, the role of serotonin on reward function may be better understood by studying the contribution of specific serotonergic pathways. In this regard, DR serotonin neurons heavily innervate the ventral tegmental area (VTA) (Bobillier et al., 1976Bobillier P. Seguin S. Petitjean F. Salvert D. Touret M. Jouvet M. The raphe nuclei of the cat brain stem: a topographical atlas of their efferent projections as revealed by autoradiography.Brain Res. 1976; 113: 449-486Crossref PubMed Scopus (572) Google Scholar, Pierce et al., 1976Pierce E.T. Foote W.E. Hobson J.A. The efferent connection of the nucleus raphe dorsalis.Brain Res. 1976; 107: 137-144Crossref PubMed Scopus (207) Google Scholar), the origin of the mesolimbic dopamine system, a network of known importance for reward and motivational function (Wise, 2004Wise R.A. Dopamine, learning and motivation.Nat. Rev. Neurosci. 2004; 5: 483-494Crossref PubMed Scopus (2332) Google Scholar). Immuno ultrastructural studies have demonstrated that DR serotonin neurons establish synaptic contacts on VTA dopamine neurons (Hervé et al., 1987Hervé D. Pickel V.M. Joh T.H. Beaudet A. Serotonin axon terminals in the ventral tegmental area of the rat: fine structure and synaptic input to dopaminergic neurons.Brain Res. 1987; 435: 71-83Crossref PubMed Scopus (343) Google Scholar, Van Bockstaele et al., 1994Van Bockstaele E.J. Cestari D.M. Pickel V.M. Synaptic structure and connectivity of serotonin terminals in the ventral tegmental area: potential sites for modulation of mesolimbic dopamine neurons.Brain Res. 1994; 647: 307-322Crossref PubMed Scopus (112) Google Scholar), and pharmacological and electrophysiological studies have shown that serotonin is capable of inhibiting or exciting VTA dopamine neurons. The mixed effects of serotonin on VTA dopamine neurons is likely to reflect activation of multiple serotonin receptor subtypes, some of which excite or inhibit dopamine neurons (Alex and Pehek, 2007Alex K.D. Pehek E.A. Pharmacologic mechanisms of serotonergic regulation of dopamine neurotransmission.Pharmacol. Ther. 2007; 113: 296-320Crossref PubMed Scopus (477) Google Scholar, Cameron et al., 1997Cameron D.L. Wessendorf M.W. Williams J.T. A subset of ventral tegmental area neurons is inhibited by dopamine, 5-hydroxytryptamine and opioids.Neuroscience. 1997; 77: 155-166Crossref PubMed Scopus (145) Google Scholar, Di Giovanni et al., 2008Di Giovanni G. Di Matteo V. Pierucci M. Esposito E. Serotonin-dopamine interaction: electrophysiological evidence.Prog. Brain Res. 2008; 172: 45-71Crossref PubMed Scopus (103) Google Scholar, Pessia et al., 1994Pessia M. Jiang Z.G. North R.A. Johnson S.W. Actions of 5-hydroxytryptamine on ventral tegmental area neurons of the rat in vitro.Brain Res. 1994; 654: 324-330Crossref PubMed Scopus (131) Google Scholar). Nevertheless, a role of VTA in serotonin-mediated reward was initially proposed from studies showing that infusion of serotonin into the VTA potentiates medial forebrain bundle electrical self-stimulation (Redgrave and Horrell, 1976Redgrave P. Horrell R.I. Potentiation of central reward by localised perfusion of acetylcholine and 5-hydroxytryptamine.Nature. 1976; 262: 305-307Crossref PubMed Scopus (50) Google Scholar). In the present study, we examined the ultrastructural and molecular characteristics of the synaptic connectivity between DR serotonin neurons and VTA dopamine neurons and determined the role of these connections in behavior. Our results demonstrate that axon terminals from DR serotonin neurons establish symmetric or asymmetric synapses on VTA dopamine neurons. We found that the axon terminals from DR serotonin neurons making asymmetric (putative excitatory) synapses on VTA dopamine neurons coexpress vesicular glutamate transporter 3 (VGluT3). VTA activation of DR serotonin terminals elicits excitation of dopamine neurons, and the release of serotonin and glutamate from these terminals activates serotonin and glutamate receptors, induces release of dopamine in nucleus accumbens (nAcc), and promotes conditioned place preference (CPP). Tracing studies in the rat have shown that two types of DR serotonergic neurons innervate the VTA; serotonin-only and dual serotonin-VGluT3 neurons (Geisler et al., 2007Geisler S. Derst C. Veh R.W. Zahm D.S. Glutamatergic afferents of the ventral tegmental area in the rat.J. Neurosci. 2007; 27: 5730-5743Crossref PubMed Scopus (365) Google Scholar, Hioki et al., 2010Hioki H. Nakamura H. Ma Y.F. Konno M. Hayakawa T. Nakamura K.C. Fujiyama F. Kaneko T. Vesicular glutamate transporter 3-expressing nonserotonergic projection neurons constitute a subregion in the rat midbrain raphe nuclei.J. Comp. Neurol. 2010; 518: 668-686Crossref PubMed Scopus (165) Google Scholar). Here, we examined, in mouse VTA, the frequency of axon terminals (punctate structures containing synaptophysin) expressing SERT alone (SERT-only terminals) or together with VGluT3 (SERT-VGluT3 terminals) (Figure 1A). We determined that, from a population of 14,342 counted SERT terminals in the VTA, 67.66% ± 1.43% were SERT-only and 32.34% ± 1.43% were dual SERT-VGluT3 terminals (Figure 1B; t(3) = 12.33, p = 0.0011). By triple-immunolabeling and electron microscopy, we determined the extent to which SERT-only or SERT-VGluT3 terminals synapse on VTA dopamine neurons (expressing tyrosine hydroxylase [TH]). Given that VGluT3 and SERT are each confined to presynaptic compartments in VTA and TH has a postsynaptic distribution in dendrites and cell bodies (Bayer and Pickel, 1991Bayer V.E. Pickel V.M. GABA-labeled terminals form proportionally more synapses with dopaminergic neurons containing low densities of tyrosine hydroxylase-immunoreactivity in rat ventral tegmental area.Brain Res. 1991; 559: 44-55Crossref PubMed Scopus (108) Google Scholar, Carr and Sesack, 2000Carr D.B. Sesack S.R. Projections from the rat prefrontal cortex to the ventral tegmental area: target specificity in the synaptic associations with mesoaccumbens and mesocortical neurons.J. Neurosci. 2000; 20: 3864-3873Crossref PubMed Google Scholar, Omelchenko and Sesack, 2005Omelchenko N. Sesack S.R. Laterodorsal tegmental projections to identified cell populations in the rat ventral tegmental area.J. Comp. Neurol. 2005; 483: 217-235Crossref PubMed Scopus (185) Google Scholar), this segregation allowed us the simultaneous detection of presynaptic VGluT3 or SERT and postsynaptic TH. By electron microscopy, we found that terminals from both SERT-only and SERT-VGluT3 neurons established synapses on VTA TH- neurons, but 61.17% ± 2.59% were from SERT-VGluT3 terminals (t(3) = 4.313, p = 0.0230; 150 of 251 SERT-VGluT3 terminals) and 36.99% ± 3.72% from SERT-only terminals (36.99% ± 3.72%, t(3) = 3.498, p = 0.0395; 180 of 535 SERT-only terminals) (Figures 1C, 1D, and S1). Furthermore, we found that SERT-positive terminals establishing asymmetric synapses on VTA TH-positive neurons coexpressed VGluT3, suggesting that serotonergic terminals establishing asymmetric synapses are excitatory because of their capability to co-release glutamate. These findings indicate that, within the VTA, TH-positive neurons are the major target of dual SERT-VGluT3 neurons and that most DR serotonin neurons synapsing on TH-positive neurons are from dual SERT-VGluT3 neurons. We next determined whether DR-SERT-VGluT3 neurons synapse on TH neurons that innervate nAcc (mesoaccumbens neurons). For these studies, we injected an adeno-associated virus (AAV, encoding Cre-dependent channelrhodopsin-2 [ChR2], tethered to mCherry or enhanced yellow fluorescent protein [eYFP]) in DR of SERT::Cre mice (referred to here as SERT-ChR2-mCherry or SERT-ChR2-eYFP mice) to drive expression of ChR2-mCherry or ChR2-eYFP in DR-SERT neurons (Figure 2). Next, in the nAcc of these mice, we injected the retrograde tract tracer Fluoro-Gold (FG) to tag mesoaccumbens neurons (Figures 2A, 2A’, and S2A). We observed that DR inputs mostly innervated the lateral VTA, where most FG-positive neurons were detected, FG-neurons that were mostly TH-positive (81.02%; 397 of 490 FG neurons). Then, by quadruple immunofluorescence, we found that SERT-VGluT3 axon terminals targeted dual FG-TH dendrites, which were confined to lateral VTA (Figures 2B–2D). By VTA electron microscopy, we confirmed that dual SERT-VGluT3 axon terminals made asymmetric synapses on dendrites coexpressing TH and FG (Figures 2E–2G). Together these fluorescence and ultrastructural findings indicate that dopamine mesoaccumbens neurons are a major target of dual DR serotonergic-VGluT3 neurons. These anatomical findings provide evidence suggesting an excitatory control of mesoaccumbens VTA dopamine neurons by dual SERT-VGluT3 afferents from DR neurons. Published studies using whole-cell patch-clamp recordings have shown that optical stimulation of inputs from Pet-1 neurons (express mostly in serotonergic neurons) releases glutamate and serotonin within the VTA (Liu et al., 2014Liu Z. Zhou J. Li Y. Hu F. Lu Y. Ma M. Feng Q. Zhang J.E. Wang D. Zeng J. et al.Dorsal raphe neurons signal reward through 5-HT and glutamate.Neuron. 2014; 81: 1360-1374Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). To directly test glutamate release from serotonergic neurons on VTA dopamine neurons innervating nAcc, we prepared VTA slices from SERT-ChR2-eYFP mice that were also injected in nAcc with the retrograde tracer cholera toxin subunit B (CTb) (n = 6 mice; Figures 2H and S2B). We performed patch-clamp recordings from 24 CTb-labeled (mesoaccumbens) neurons distributed in the lateral VTA and found that VTA optical stimulation of SERT-fibers evoked excitatory postsynaptic currents (EPSCs) in seven of the CTb recorded neurons, whose currents were abolished by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (Figure 2J). We filled the recorded neurons with biocytin and found that five CTb-recorded neurons showing optical-evoked EPSCs were TH positive (Figure 2I). To determine whether DR-SERT inputs are capable of exciting TH mesoaccumbens neurons, we performed VTA cell-attached and current-clamp recordings in response to optical stimulation and found increased firing of three CTb-TH neurons (Figure 2K). The optical-evoked EPSCs were eliminated by bath application of tetrodotoxin (TTX; 500 nM) and were restored by application of 4-aminopyridine (4-AP; 200 μM) (Figure S3), suggesting that optical-evoked EPSCs are mediated by monosynaptic glutamatergic transmission. These findings indicate that VTA glutamate release from DR dual SERT-VGluT3 terminals is capable of evoking the firing of mesoaccumbens dopamine neurons. In addition, by whole-cell voltage-clamp recordings, we found that either 0.5-ms pulses of VTA electrical stimulation of DR fibers or 2-ms pulses of VTA optical stimulation of DR-SERT fibers evoked EPSCs on VTA dopamine neurons, clamped at −60 mV and found that those currents decreased after VTA blockade of 5-HT3 receptor antagonist Y-25130. As a result of 5-TH3 receptor blockade, the current evoked by optical stimulation decreased from 63.78 ± 20.8 pA to 53.07 ± 19.22 pA (Figures 3A and 3B ), and that evoked by electrical stimulation decreased from −1,686 ± 322.7 pA to −1,511 ± 262.6 pA (Figures 3D and 3E). The 5-HT3 receptor-mediated current was calculated by subtracting the total current from the current remaining after 5-HT3 receptor antagonism (optical 5-HT3-mediated current = 13.14 ± 3.3 pA; electrical 5-HT3-mediated current = 80.3 ± 46.33 pA). Additionally, the rise time of the optically evoked, 5-HT3-mediated current was faster (0.47 ± 0.094 ms) than the remaining current (0.84 ± 0.11 ms; Figure 3C). This kinetic characteristic was also similar for electrical stimulation, faster (0.60 ± 0.094 ms) than the remaining current (0.79 ± 0.23 ms), although that was not statistically significant (paired t test, p > 0.05, n = 3). We found that blockade of 5-HT3 receptors increased the optically induced, paired pulse ratio (from 0.34 ± 0.1 to 0.62 ± 0.12; Figure 3F), suggesting that 5-HT3 receptors can also affect the probability of neurotransmitter release. These findings indicate that VTA release of serotonin from DR inputs excites dopamine neurons via activation of 5-HT3 receptors. By in vivo VTA recordings in urethane-anesthetized mice, we found that VTA optical stimulation of SERT fibers evoked either neuronal excitation or inhibition (Figure 3G). From a total of 80 VTA-recorded neurons in response to photoactivation of the DR to VTA pathway, 20% (16 of 80) of the neurons were excited and 28.8% (23 of 80) were inhibited (Figure 3H); the excitatory and the inhibitory responses had similar onset (reaching significance at 1.5 s after stimulus) and duration (6.5 s from prestimulation firing rates to onset; Figures 3I and 3J). Among the neurons that showed excitation, 50% (8 of 16) remained excited during the 6 s immediately after the stimulus, and in only one of those neurons was the excitation followed by inhibition (Figure 3I, individual traces). Among the neurons that were inhibited, 44% (10 of 23) remained inhibited during the 6 s immediately after the stimulus, and in 21.8% (5 of 23) of those neurons, the inhibition was followed by excitation (Figure 3J, individual traces). These findings from in vitro and in vivo VTA recordings, together with those from immuno ultrastructural studies (Figures 1C and 1D), indicate that (1) some SERT inputs from DR to VTA release glutamate, and (2) DR dual SERT-VGluT3 terminals establish asymmetric synapses, mostly on TH neurons, and have an excitatory effect on some dopamine neurons that innervate the nAcc. We next determined whether stimulation of DR neurons or their VTA inputs produce release of dopamine in nAcc. We first tested the effects of DR electrical stimulation on nAcc dopamine release (Figures S4A–S4D) because previous studies had reported that electrical stimulation of DR inhibits dopamine neurons (Dray et al., 1976Dray A. Gonye T.J. Oakley N.R. Tanner T. Evidence for the existence of a raphe projection to the substantia nigra in rat.Brain Res. 1976; 113: 45-57Crossref PubMed Scopus (243) Google Scholar, Trent and Tepper, 1991Trent F. Tepper J.M. Dorsal raphé stimulation modifies striatal-evoked antidromic invasion of nigral dopaminergic neurons in vivo.Exp. Brain Res. 1991; 84: 620-630Crossref PubMed Scopus (59) Google Scholar, Tsai, 1989Tsai C.T. Involvement of serotonin in mediation of inhibition of substantia nigra neurons by noxious stimuli.Brain Res. Bull. 1989; 23: 121-127Crossref PubMed Scopus (11) Google Scholar). In contrast with previous, published findings, we found that DR electrical stimulation evoked rapid and robust release of dopamine in nAcc core, as measured by fast-scan cyclic voltammetry (Figure S4). We administered the D2-receptor antagonist eticlopride (2 mg/kg, intraperitoneally [i.p.]) to confirm that the principal electrochemical contribution to the signal was from dopamine. Eticlopride treatment significantly increased the signal due to autoreceptor blockade (Figures S4B and S4C), suggesting that the recorded electrochemical analyte was dopamine. Additionally, we did not detect changes in the rate of decay of the signal in the nAcc of animals that received systemic administration of the serotonin reuptake inhibitor fluoxetine (10 mg/kg, i.p.), suggesting that serotonin was not released in a detectable fashion in the nAcc core after DR stimulation (Figure S4D). Because previous in vivo microdialysis studies have shown that 5-HT3 receptors affect dopamine release within the mesoaccumbens system (Campbell et al., 1996Campbell A.D. Kohl R.R. McBride W.J. Serotonin-3 receptor and ethanol-stimulated somatodendritic dopamine release.Alcohol. 1996; 13: 569-574Crossref PubMed Scopus (106) Google Scholar, Imperato and Angelucci, 1989Imperato A. Angelucci L. 5-HT3 receptors control dopamine release in the nucleus accumbens of freely moving rats.Neurosci. Lett. 1989; 101: 214-217Crossref PubMed Scopus (152) Google Scholar, McBride et al., 2004McBride W.J. Lovinger D.M. Machu T. Thielen R.J. Rodd Z.A. Murphy J.M. Roache J.D. Johnson B.A. Serotonin-3 receptors in the actions of alcohol, alcohol reinforcement, and alcoholism.Alcohol. Clin. Exp. Res. 2004; 28: 257-267Crossref PubMed Scopus (66) Google Scholar), we tested the effect of ondansetron on evoked accumbal dopamine release driven by DR electrical stimulation. We found that the baseline (predrug) level of dopamine release in nAcc core evoked by DR electrical stimulation (Figures S4E–S4G; 141.9 ± 8.8 nM, n = 4) was significantly reduced by systemic application of ondansetron (2 mg/kg i.p.) (Figure S4E; 89.4 ± 5.0 nM, p < 0.01, n = 4)." @default.
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• W2912884060 date "2019-01-01" @default.
• W2912884060 modified "2023-10-18" @default.
• W2912884060 title "Dorsal Raphe Dual Serotonin-Glutamate Neurons Drive Reward by Establishing Excitatory Synapses on VTA Mesoaccumbens Dopamine Neurons" @default.
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