Serotonin-producing neurons profusely innervate brain regions via long-range projections. However, it remains unclear whether and how endogenous serotonergic transmission specifically influences regional or global functional activity. We combined designed receptors exclusively activated by designed drugs (DREADD)-based chemogenetics and functional magnetic resonance imaging (fMRI), an approach we term “chemo-fMRI,” to causally probe the brain-wide substrates modulated by endogenous serotonergic activity. We describe the generation of a conditional knockin mouse line that, crossed with serotonin-specific Cre-recombinase mice, allowed us to remotely stimulate serotonergic neurons during fMRI scans. We show that endogenous stimulation of serotonin-producing neurons does not affect global brain activity but results in region-specific activation of a set of primary target regions encompassing corticohippocampal and ventrostriatal areas. By contrast, pharmacological boosting of serotonin levels produced widespread fMRI deactivation, plausibly reflecting the mixed contribution of central and perivascular constrictive effects. Our results identify the primary functional targets of endogenous serotonergic stimulation and establish causation between activation of serotonergic neurons and regional fMRI signals.

To allow for stable and reproducible endogenous serotonin stimulation across animals, we generated a conditional knockin mouse that we crossed with Pet1-Cre transgenic mice (), endowing serotonin specificity (). This approach allowed us to remotely stimulate serotonin-producing neurons during fMRI scans, with reduced inter-subject variability. Our results show that chemogenetic serotonin stimulations do not affect global brain activity but result in region-specific activation of a set of primary target regions encompassing corticohippocampal and midbrain structures, as well as components of the mesolimbic reward systems. Notably, pharmacological boosting of serotonin level via systemic administration of the serotonin reuptake inhibitor citalopram produced widespread fMRI deactivation, plausibly reflecting the mixed contribution of central and perivascular constrictive effects. Collectively, our results reveal a set of regional substrates that act as primary functional targets of endogenous serotonergic stimulation and establish causation between endogenous activation of serotonin neurons and regional fMRI signals. They also provide a framework for understanding serotonin-dependent functions and interpreting data obtained from human fMRI studies of serotonin modulating agents.

Light-based or chemically mediated cell manipulations have made it possible to causally link the activity of focal neuronal populations with specific circuital and behavioral outputs (). When combined with non-invasive neuroimaging methods, cell-type-specific manipulations can be used to map the functional substrates of endogenous modulatory transmission without the confounding contribution of peripherally elicited vasoactive or pharmacological effects. Toward this goal, we describe the combined use of mouse cerebral-blood-volume-based fMRI (rCBV;) and cell-type-specific DREADD (designed receptors exclusively activated by designed drugs;) chemogenetics, an approach we term “chemo-fMRI,” to map the brain-wide functional targets of sustained serotonin stimulation.

Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand.

Neuroimaging studies have employed pharmacological manipulations to probe the brain-wide targets of serotonin. The vast majority of these studies have primarily focused on the use of functional magnetic resonance imaging (fMRI) as a proxy for the neuronal activity directly elicited by pharmacological agents or by mapping second-order modulatory effects on task-elicited fMRI responses (). This approach, termed pharmacological fMRI, has also been back-translated to animal studies (). However, while useful in identifying possible brain-wide substrates of neuromodulatory action, drug-based approaches are often contaminated by off-target receptor contributions () and often contain peripheral vasotonic or cerebrovascular contributions that cannot be easily disentangled from the drug’s primary neural effects (). This aspect is especially relevant when hemodynamic measures of brain function are used, given that serotonin can perivascularly modulate blood flow, independent of its central effects (). As a result, it remains unclear where and how endogenous serotonergic activity specifically influences regional or global brain functional activity and the corresponding fMRI responses.

The use of pharmacological-challenge fMRI in pre-clinical research: application to the 5-HT system.

Serotonin (5-HT) is a modulatory transmitter produced by a small set of midbrain and brainstem nuclei that richly innervate forebrain target regions via long-range projections (). Such profuse innervation, along with the combined release of transmitters via pointwise synaptic signaling as well as non-synaptic “volume transmission” (), can alter neuronal functions to define internal states that affect behavioral outputs in response to external stimuli. Recent research has shed light on the basic cellular and molecular machinery engaged by serotonin (). However, many questions regarding the systems-level substrates modulated by serotonin transmission remain unanswered. For one, it is unclear whether serotonin’s modulatory action entails a global regulation of neural activity or is relayed and encoded by a set of primary functional targets.

An underlying assumption of our work is that the signals elicited by CNO in hM3Dq/Pet1-Cre mice primarily reflect central serotonin neuronal stimulation, which in turn elicits rCBV changes via neurovascular and neurometabolic coupling. By contrast, fMRI mapping of systemically administered serotonin ligands may be contaminated or masked by direct stimulation of endothelial serotonin receptors, which produces vasoconstrictive responses (). To compare the effect of endogenous versus systemic pharmacological serotonin stimulation, we carried out fMRI mapping upon administration of the selective serotonin reuptake inhibitor citalopram (5–10 mg/kg), a drug leading to increased central and circulating serotonin levels (). Interestingly, i.v. citalopram (10 mg/kg) produced widespread foci of rCBV signal decrease in a widespread set of cortical and subcortical regions, as seen with voxelwise mapping ( Figure S7 ; Z > 1.8, cluster corrected p = 0.05). Although the effect was widely distributed, the deactivation was particularly prominent in the prefrontal cortex ( Figures 4 B and S7 ). Citalopram administration was not associated with appreciable blood pressure alteration at any of the doses tested ( Figure S2 ).

(B) Illustrative fMRI time course in the prefrontal cortex (mean ± SEM); citalopram was administered at time 0.

To probe a neural origin of hM3Dq-mediated fMRI response, we performed local field potential (LFP) measurements in the dorsal hippocampus of halothane-anesthetized animals (). Intravenous (i.v.) CNO administration (0.5 mg/kg) produced significant LFP power increases in the theta (F= 110.2, p < 0.001) and gamma bands (F= 239.4, p < 0.001) in hM3Dq/Pet1-Cre, but not in DIO-hM3Dq control animals ( Figure S4 ). To rule out an interaction between the anesthesia used for fMRI and serotonin chemogenetic manipulations, we carried out c-Fos immunofluorescence mapping in unanesthetized mice upon intraperitoneal administration of 2 mg/kg CNO (see dose selection in Supplemental Experimental Procedures ). This dosing strategy does not elicit substantial fMRI signal alterations ( Figure S5 ; p > 0.26, all regions). We observed robust and recognizable increases in c-Fos-positive neurons in many regional substrates identified with fMRI, including the nucleus accumbens, hippocampus, anterior thalamus, hypothalamic nuclei, and raphe nuclei ( Figure S6 A–S6E; p < 0.05, all regions, q = 0.05). No inter-group differences were observed in somatosensory cortex ( Figures S6 A–S6E), and no detectable c-Fos reactivity was observed in the cerebellum in either CNO-treated group (data not shown). The same dose of CNO (2 mg/kg i.p.) elicited significant release of extracellular serotonin in hM3Dq/Pet1-Cre animals, but not in DIO-hM3Dq control animals, as assessed with hippocampal microdialysis in unanesthetized animals ( Figure S6 F; F= 7.76, p = 0.02).

Chemogenetic activation of serotonin-producing neurons elicited robust rCBV increases in several cortical and non-cortical substrates in hM3Dq/Pet1-Cre mice ( Figure 3 ; Z > 2, cluster corrected p = 0.05; Figure S3 ). The elicited chemo-fMRI response encompassed parietal cortical and motor cortices, as well as posterior insular and temporal association regions, plus several subcortical substrates, including hippocampal areas, the hypothalamus, the dorsal raphe, and the cerebellum. A prominent involvement of ventral tegmental area and its mesolimbic terminals in the nucleus accumbens was also apparent.

(B) Illustrative fMRI time course in the hippocampus; CNO was administered at time 0 (mean ± SEM).

To control for possible off-target effects of CNO and its metabolites, we first performed a dose-response titration of the fMRI response produced by CNO in wild-type mice ( Figure S1 ). Intravenous CNO administration at doses of 1 and 2 mg/kg produced significant rCBV increases in most of the examined regions ( Figure S1 ; p < 0.05, corrected at q = 0.05). Upon intravenous dosing of CNO at 0.5 mg/kg, the rCBV signal was qualitatively distinguishable from reference baseline signal only in hypothalamic and hippocampal areas, and no statistically significant alterations were detected in regional or voxelwise quantifications with respect to vehicle (p > 0.42, Z > 1.6 in voxelwise maps). Pharmacokinetic profiling of this dose ( Figure S1 C) revealed sustained plasma CNO exposure throughout the imaging time window, as well as the presence of low but detectable levels of clozapine. The observed peak exposure of clozapine (8 ± 6 ng/mL) is ∼25-fold lower than behaviorally relevant plasma levels of this compound in rodents () and below the range of values previously associated with detectable brain exposure (). Based on these assessments, we chose to perform subsequent chemogenetic-fMRI mapping using an i.v. dose of 0.5 mg/kg. As a further control for possible CNO/clozapine off-target effects, fMRI mapping and all the supporting experiments were performed and quantified by administering CNO to both hM3Dq/Pet1-Cre and control DIO-hM3Dq reference mice. CNO administration did not alter peripheral blood pressure at any of the doses tested ( Figure S2 ).

We next recorded the electrophysiological response induced by clozapine-N-oxide (CNO) administration in hM3Dq-expressing serotonin neurons via whole-cell patch-clamp recordings in the dorsal raphe nucleus (DRN). mCherry-positive neurons in hM3Dq/Pet1-Cre mice were robustly activated by CNO, resulting in sustained firing rates ( Figure 2 E). We did not observe any CNO-induced firing rate alterations in any of the neighboring mCherry-negative neurons probed or in serotonin neurons in DIO-hM3Dq control mice ( Figures 2 F and 2G ).

(E) CNO application (5 μM, purple bar) in DRN brain slices from hM3Dq/Pet1-Cre mice triggered firing activity in mCherry(+)serotonin neurons (discharge rate, 0.19 ± 0.03 Hz; p = 0.003), identified by their electrophysiological properties (inset). CNO did not elicit activity in mCherry(−) cells (F) or DIO-hM3Dq control cells (G). (E–G) Left, image of recorded cells; middle, sample traces of recorded cell membrane potential; right, boxplot summary of changes in discharge rate in response to CNO application.

(A′–C″) High-power magnification highlighting Tph2 (A′) and mCherry (B′) co-localization (C′) in the DRN lateral wings, and Tph2 (A″) and mCherry (B″) co-localization (C″) in MRN.

(A–C) Double immunohistochemical assay showing Tph2 (A) and mCherry expression (B) and merged channels (C) in medial and dorsal raphe nucleus (MRN and DRN, respectively) of hM3Dq/Pet1-Cre mice (n = 4).

To enable stable pan-neuronal stimulation of serotonin-producing cells, we first generated an hM3Dq conditional knockin mouse encompassing the integration of hM3Dq-mCherry double-floxed inverse open reading frame (DIO-hM3Dq) within the ROSA26 genomic locus ( Figure 1 ). In the designed mouse line, stable Cre-mediated somatic recombination is required for conditional transcriptional activation (CAG-promoter driven) of the hM3Dq gene, whose expression could be probed by the presence of in-frame-fused mCherry reporter ( Figure 1 ). To enable hM3Dq expression in serotonin-producing neurons, DIO-hM3Dq mice were crossed with the Pet1-Cre transgenic mouse line (). Immunofluorescence analysis on double trans-heterozygous hM3Dq/Pet1-Cre mice revealed that hM3Dq is expressed in the vast majority (95.9%) of serotonin-producing neurons ( Figures 2 A–2D′).

(C) PCR amplification of the ROSA26 locus from DNA extracted from a wild-type mouse (3), two mutant mice (1 and 4), and two heterozygous DIO-hM3Dq mice (2 and 5).

(A) Top to bottom: wild-type organization of ROSA26 mouse genomic locus; hM3Dq targeting vector (green, loxP; blue, Lox2722; arrowheads indicate the location of Lox sites, and yellow circles indicate FRT sites); transcriptionally inactive DIO-hM3Dq allele; and transcriptionally active hM3Dq allele.

Discussion

Serotonin is an archetypical neuromodulatory monoamine characterized by broad and far-ranging modulatory properties. Despite extensive research, the macroscale functional substrates endogenously modulated by serotonin in the intact brain remain elusive. By using chemo-fMRI, we describe the primary brain-wide targets of sustained serotonergic stimulation and establish causation between endogenous activation of serotonin neurons and regional fMRI signals. Our results provide a framework for understanding serotonin-dependent function and interpreting data obtained from human fMRI studies of serotonin modulatory agents.

Urban et al., 2016 Urban D.J.

Zhu H.

Marcinkiewcz C.A.

Michaelides M.

Oshibuchi H.

Rhea D.

Aryal D.K.

Farrell M.S.

Lowery-Gionta E.

Olsen R.H.

et al. Elucidation of the behavioral program and neuronal network encoded by dorsal raphe serotonergic neurons. Gomez et al., 2017 Gomez J.L.

Bonaventura J.

Lesniak W.

Mathews W.B.

Sysa-Shah P.

Rodriguez L.A.

Ellis R.J.

Richie C.T.

Harvey B.K.

Dannals R.F.

et al. Chemogenetics revealed: DREADD occupancy and activation via converted clozapine. Gomez et al., 2017 Gomez J.L.

Bonaventura J.

Lesniak W.

Mathews W.B.

Sysa-Shah P.

Rodriguez L.A.

Ellis R.J.

Richie C.T.

Harvey B.K.

Dannals R.F.

et al. Chemogenetics revealed: DREADD occupancy and activation via converted clozapine. Olsen et al., 2008 Olsen C.K.

Brennum L.T.

Kreilgaard M. Using pharmacokinetic-pharmacodynamic modelling as a tool for prediction of therapeutic effective plasma levels of antipsychotics. Baldessarini et al., 1993 Baldessarini R.J.

Centorrino F.

Flood J.G.

Volpicelli S.A.

Huston-Lyons D.

Cohen B.M. Tissue concentrations of clozapine and its metabolites in the rat. Interestingly, recent mapping of chemogenetically stimulated neurons via 2-fluodeoxygucose positron emission tomography (PET) did not highlight corticohippocampal modulation of brain activity, but revealed small foci of reduced metabolism in thalamic areas (). Several experimental factors could account for these discrepant results, including the use of deep anesthesia versus light sedation, and the possible contribution of unspecific effects of CNO, given that Urban et al. did not employ a CNO-treated group as a baseline reference for brain mapping. This aspect is of especial importance in the light of a recent report pointing at a possible role of converted clozapine as primary effector of DREADD-induced manipulations (). Our pharmacokinetic data are consistent with this view, showing that CNO can be converted to clozapine in vivo. However, several lines of evidence argue against a significant off-target contribution of converted clozapine to our mapping. First, our chemo-fMRI mapping was performed at CNO doses that negligibly affect fMRI signals. Second, we have quantified and described all chemogenetic responses with respect to a CNO-treated DIO-hMD3q control group of animals. Third, in vivo LFP recordings and microdialysis studies provide evidence of neural responses and extracellular serotonin release in hM3Dq/Pet1-Cre animals, but not in DIO-hM3Dq control animals. Finally, consistent with the notion of “subthreshold clozapine exposure” (), the measured peak of clozapine concentration is ∼25-fold lower the exposure required to obtain behaviorally relevant responses in rodents () and below the range of values previously associated with detectable brain exposure in pharmacokinetic studies (). Taken together, these findings strongly argue against a contribution of unspecific clozapine-related effects to the signals mapped with our approach.

1A , serotonin 1B and serotonin 2A receptors located in brain and peripheral vascular cells (reviewed by Cohen et al., 1996 Cohen Z.

Bonvento G.

Lacombe P.

Hamel E. Serotonin in the regulation of brain microcirculation. Blardi et al., 2005 Blardi P.

de Lalla A.

Urso R.

Auteri A.

Dell’Erba A.

Bossini L.

Castrogiovanni P. Activity of citalopram on adenosine and serotonin circulating levels in depressed patients. Zolkowska et al., 2008 Zolkowska D.

Baumann M.H.

Rothman R.B. Chronic fenfluramine administration increases plasma serotonin (5-hydroxytryptamine) to nontoxic levels. Toda and Fujita, 1973 Toda N.

Fujita Y. Responsiveness of isolated cerebral and peripheral arteries to serotonin, norepinephrine, and transmural electrical stimulation. Cohen et al., 1996 Cohen Z.

Bonvento G.

Lacombe P.

Hamel E. Serotonin in the regulation of brain microcirculation. Lesch and Waider, 2012 Lesch K.-P.

Waider J. Serotonin in the modulation of neural plasticity and networks: implications for neurodevelopmental disorders. Our results provide a reference framework for the interpretation of fMRI imaging studies employing serotonin -targeting agents. In this respect, the divergent functional effects observed with chemogenetic stimulation and serotonin reuptake inhibition are especially relevant, as they suggest that endogenous versus systemic serotonin level boosting can lead to opposing hemodynamic responses, a factor that needs to be taken into account when hemodynamic readouts are used as surrogates for serotonin induced neural manipulations. Serotonin is a key mediator of central and peripheral vasoactivity, with predominant evidence of a vasoconstrictive action via serotonin, serotoninand serotoninreceptors located in brain and peripheral vascular cells (reviewed by). Because serotonin reuptake inhibition rapidly increases circulating levels of this neurotransmitter () and systemic serotonin administration produces endothelial vasoconstriction (), citalopram-induced rCBV decreases could reflect a contribution of direct vasoactive effect of circulating serotonin on cerebral vasculature. Nonvascular mechanisms could also be involved in this effect, such as a possible engagement of different receptor populations as a function of serotonin release site, a hypothesis consistent with the wide variety in the anatomical and cellular distribution patterns exhibited by the serotonin receptor subtypes identified thus far ().

McBean et al., 1999 McBean D.E.

Ritchie I.M.

Olverman H.J.

Kelly P.A.T. Effects of the specific serotonin reuptake inhibitor, citalopram, upon local cerebral blood flow and glucose utilisation in the rat. Geday et al., 2005 Geday J.

Hermansen F.

Rosenberg R.

Smith D.F. Serotonin modulation of cerebral blood flow measured with positron emission tomography (PET) in humans. Sekar et al., 2011 Sekar S.

Verhoye M.

Van Audekerke J.

Vanhoutte G.

Lowe A.S.

Blamire A.M.

Steckler T.

Van der Linden A.

Shoaib M. Neuroadaptive responses to citalopram in rats using pharmacological magnetic resonance imaging. Schridde et al., 2008 Schridde U.

Khubchandani M.

Motelow J.E.

Sanganahalli B.G.

Hyder F.

Blumenfeld H. Negative BOLD with large increases in neuronal activity. While reduced cerebral blood flow in humans and rats has been reported upon citalopram administration (), prior blood-oxygen-level-dependent (BOLD) fMRI mapping of citalopram in rats revealed increased cortical responses (). A number of methodological discrepancies could account for this result, the two most notable being the use of deeper anesthesia levels by Sekar et al., as well as the use of BOLD fMRI, as opposed to rCBV (like in the present work). In this respect, it should be emphasized that CBV is a direct and reliable indicator of microvascular activity that appears to be more directly related to underlying neuronal activity than BOLD fMRI (). Further opto- or chemogenetic fMRI mapping of serotonin function employing BOLD fMRI could help conciliate these experimental discrepancies.

Stark et al., 2006 Stark J.A.

Davies K.E.

Williams S.R.

Luckman S.M. Functional magnetic resonance imaging and c-Fos mapping in rats following an anorectic dose of m-chlorophenylpiperazine. Gozzi et al., 2012 Gozzi A.

Colavito V.

Seke Etet P.F.

Montanari D.

Fiorini S.

Tambalo S.

Bifone A.

Zucconi G.G.

Bentivoglio M. Modulation of fronto-cortical activity by modafinil: a functional imaging and fos study in the rat. Gass et al., 1997 Gass P.

Bruehl C.

Herdegen T.

Kiessling M.

Lutzenburg M.

Witte O.W. Induction of FOS and JUN proteins during focal epilepsy: congruences with and differences to [14C]deoxyglucose metabolism. Logothetis et al., 2001 Logothetis N.K.

Pauls J.

Augath M.

Trinath T.

Oeltermann A. Neurophysiological investigation of the basis of the fMRI signal. Cirelli and Tononi, 2000 Cirelli C.

Tononi G. On the functional significance of c-fos induction during the sleep-waking cycle. The results of our microdialysis and LFP recordings, plus the observation of c-Fos induction in many regions exhibiting significant chemo-fMRI activation argue against a significant interference of anesthesia on our functional mapping, and support a neural origin of the mapped signals. Not surprisingly, discrepancies between imaging and c-Fos mapping were noted (e.g., in cortical regions or the cerebellum). Such differences are not surprising given the diverse neurophysiological mechanisms underlying these two experimental readouts and previous evidence showing that c-Fos induction does not always correspond to activation shown by metabolic or hemodynamic-based brain mapping (). Indeed, hemodynamic responses are generally thought to reflect local synaptic input (), whereas the c-Fos protein induction is an indirect marker of cellular activation that is not elicited in all CNS cells and can be affected by the behavioral state of the animal ().

Urban et al., 2016 Urban D.J.

Zhu H.

Marcinkiewcz C.A.

Michaelides M.

Oshibuchi H.

Rhea D.

Aryal D.K.

Farrell M.S.

Lowery-Gionta E.

Olsen R.H.

et al. Elucidation of the behavioral program and neuronal network encoded by dorsal raphe serotonergic neurons. Lee et al., 2010 Lee J.H.

Durand R.

Gradinaru V.

Zhang F.

Goshen I.

Kim D.S.

Fenno L.E.

Ramakrishnan C.

Deisseroth K. Global and local fMRI signals driven by neurons defined optogenetically by type and wiring. Rungta et al., 2017 Rungta R.L.

Osmanski B.-F.

Boido D.

Tanter M.

Charpak S. Light controls cerebral blood flow in naive animals. Gozzi and Schwarz, 2016 Gozzi A.

Schwarz A.J. Large-scale functional connectivity networks in the rodent brain. From a methodological standpoint, this work paves the way to the combined use of chemogenetics and fMRI to unravel the large-scale substrates modulated by pan-neuronal populations in the living mouse brain. Our approach follows analogous attempts to combine chemogenetics with noninvasive mouse brain imaging (). Our data demonstrate that properly controlled chemogenetic-fMRI studies can elicit large and sustained functional responses that exhibit neuronal specificity and can be employed to noninvasively map the brain-wide effect of regional neural manipulations. This approach crucially complements current opto-fMRI stimulations () by permitting brain-wide functional mapping without the need for invasive cranial probes, and it is devoid of the confounding contribution of heat-induced vasodilation in anesthetized animals (). As such, chemo-fMRI appears to be optimally suited to the investigation of drug-like sustained modulatory stimulation/inhibition and for the deconstruction or manipulation of steady-state network activity via fMRI connectivity mapping (). While here employed to study pan-neuronal serotonin modulation, this approach can be straightforwardly expanded to manipulate focal circuits and neuronal ensembles via intersectional genetics or retrograde viral vectors.

In conclusion, we describe the use of chemo-fMRI to map the brain-wide substrates of modulatory transmission in the intact mammalian brain. We show that chemogenetic stimulation of endogenous serotonin neurons results in regionally specific activation of a set of cortical and subcortical targets that serve as primary functional mediators of sustained endogenous serotonin transmission. We also show that endogenous activation of serotonergic neurons and systemic serotonin reuptake inhibition can produce opposing hemodynamic effects. Our findings provide a framework for understanding serotonin-dependent functions and interpreting data obtained from human fMRI studies of serotonin modulating agents.