# Dopaminergic neuromodulation of prefrontal cortex activity requires the NMDA receptor co-agonist D-serine

Glenn Dallérac, Xia Li, Pierre Lecouflet, Nadège Morisot, Silvia Sacchi, Rachel Asselot, Thu Ha Pham, B Potier, David JG Watson, Staffan Schmidt, et al.

► To cite this version:

Glenn Dallérac, Xia Li, Pierre Lecouflet, Nadège Morisot, Silvia Sacchi, et al. Dopaminergic neuro-
modulation of prefrontal cortex activity requires the NMDA receptor co-agonist D-serine. Proceedings
of the National Academy of Sciences of the United States of America, 2021, 118 (23), pp.e2023750118.
10.1073/pnas.2023750118. hal-03331836

HAL Id: hal-03331836

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Distributed under a Creative Commons Attribution 4.0 International License
Dopaminergic neuromodulation of prefrontal cortex activity requires the NMDA receptor co-agonist D-serine

Glenn Dallérac<fnref n="1" />,<fnref n="2" />, Xia Li<fnref n="2" />,*, Pierre Lecouflet<fnref n="3" />,*, Nadège Morisot<fnref n="4" />#, Silvia Sacchi<fnref n="5" />#, Rachel Asselot<fnref n="6" />#,

Thu Ha Pham<fnref n="7" />#, Brigitte Potier<fnref n="3" />, David JG Watson<fnref n="8" />, Staffan Schmidt<fnref n="9" />, Grégoire Levasseur<fnref n="1" />, Pascal

Fossat<fnref n="10" />, Andrey Besedin<fnref n="6" />, Jean-Michel Rivet<fnref n="11" />, Joseph T Coyle<fnref n="12" />, Ginetta Collo<fnref n="13" />, Loredano

Pollegioni<fnref n="5" />, Jan Kehr<fnref n="9" />, Micaela Galante<fnref n="2" />, Kevin CF Fone<fnref n="8" />, Alain M Gardier<fnref n="7" />, Thomas Freret<fnref n="6" />, Angelo

Contarino<fnref n="4" />,§, Mark J. Millan<fnref n="11" />,§#, Jean-Pierre Mothet<fnref n="1" />,<fnref n="3" />

**Correspondance to:**

Dr Jean-Pierre Mothet

Laboratoire LuMin, UMR 2036

Université Paris-Saclay, CNRS, ENS Paris Saclay

91190 Gif-sur-Yvette

France

e-mail: jean-pierre.mothet@universite-paris-saclay.fr

Dr Glenn Dallerac

Institut des Neurosciences Paris-Saclay (Neuro-PSI), UMR 9197

Université Paris Saclay, CNRS

Orsay F-91405

France

e-mail: glenn.dallerac@universite-paris-saclay.fr

**Keywords:** D-serine; NMDA receptor; cortex; schizophrenia; D₁- and D₂-type receptors; serine

racemase knock-out mice

**Short title:** Dopamine tunes D-serine modulation of PFC functions

<footnote n="1">¹ Aix-Marseille Université, Centre de Recherche en Neurobiologie et Neurophysiologie de Marseille, UMR7286 CNRS 13344 Marseille Cedex 15, France</footnote>

<footnote n="2">² Institut des Neurosciences Paris-Saclay (Neuro-PSI), UMR 9197, CNRS, Université Paris Saclay, Orsay F-91405, France</footnote>

<footnote n="3">³ LuMin UMR2036, CNRS Université Paris-Saclay, ENS Paris-Saclay, CentraleSupélec, 91190, Gif-sur-Yvette, France.</footnote>

<footnote n="4">⁴ University of Bordeaux, INCIA, UMR 5287, Bordeaux, France</footnote>

<footnote n="5">⁵ Dipartimento di Biotecnologie e Scienze della Vita, Università degli Studi dell'Insubria, Varese, Italy</footnote>

<footnote n="6">⁶ Normandie Université, UNICAEN, INSERM, COMETE, 14000 Caen, France</footnote>

<footnote n="7">⁷ Laboratoire de Neuropharmacologie, CESP/UMR-S 1178, Faculté Pharmacie, Université Paris-Saclay, F-92296 Chatenay-Malabry cedex, France</footnote>

<footnote n="8">⁸ School of Life Sciences, Queen's Medical Centre, The University of Nottingham, Nottingham, NG7 2UH UK</footnote>

<footnote n="9">⁹ Pronexus Analytical AB, Grindstuvägen 44, 167 33 Bromma, Sweden</footnote>

<footnote n="10">¹⁰ INSERM U.862, Neurocentre Magendie 33077 Bordeaux, France</footnote>

<footnote n="11">¹¹ Centre for Thérapeutique Innovation in Neuropsychiatry, Institut de Recherche Servier, 125 Chemin de Ronde, 78290 Croissy-sur-Seine, Paris, France</footnote>

<footnote n="12">¹² Department of Psychiatry, Harvard Medical School, Boston, MA 02115, USA; Laboratory for Psychiatric and Molecular Neuroscience, McLean Hospital, Belmont, MA 02478, USA.</footnote>

<footnote n="13">¹³ Division of Pharmacology, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy</footnote>

<footnote>* Equal contributing authors</footnote>

<footnote># Equal contributing authors</footnote>

<footnote>§ Equal senior contribution</footnote>

<footnote>\#Current coordinates: Institute of Neuroscience and Psychology, College of Medicine, Life and Vet Sciences Glasgow University, Scotland, UK</footnote>
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## Significance

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Dopamine and glutamate in the prefrontal cortex are important substrates of higher cognitive functions, which are impaired in neuropsychiatric disorders. As regards glutamatergic pathways, a role for the NMDA receptor co-agonist D-serine has been highlighted yet its relationship to dopaminergic transmission remains unclear. In this study we reveal that D-serine plays a pivotal role in the modulation by dopamine of NMDA receptor activity and cognitive performance in the prefrontal cortex. Comprehensive evidence for this interaction is provided at the synaptic, neuronal, network, and behavioral level. These observations are of relevance to the pathophysiology and treatment of cognitive impairment in numerous disorders involving disruption of the frontocortical dialogue between dopamine and glutamate.

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## Abstract

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Prefrontal control of cognitive functions critically depends upon glutamatergic transmission and NMDA receptors (NMDARs), the activity of which is regulated by dopamine. Yet, whether the NMDA receptor co-agonist D-serine is implicated in the dopamine-glutamate dialogue in prefrontal cortex (PFC) and other brain areas remains unexplored. Here, using electrophysiological recordings, we show that D-serine is required for the fine-tuning of glutamatergic neurotransmission, neuronal excitability and synaptic plasticity in the PFC through the actions of dopamine at D₁- and D₃- receptors. Using *in vivo* microdialysis, we show that D₁- and D₃- receptors exert a respective facilitatory and inhibitory influence on extracellular levels and activity of D-serine in the PFC, actions expressed primarily *via* the cAMP/PKA signalling cascade. Further, using fMRI imaging and behavioral assessment, we show that D-serine is required for the potentiation of cognition by D₃R blockade as revealed in a test of novel object recognition memory. Collectively, these results unveil a key role for D-serine in the dopaminergic neuromodulation of glutamatergic transmission and PFC activity, findings with clear relevance to the pathogenesis and treatment of diverse brain disorders involving alterations in dopamine-glutamate cross-talk.

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Introduction

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The prefrontal cortex (PFC) supports the higher-order and top-down coordination of complex behaviors, including executive function, working memory and social interactions (1). Dopaminergic (DAergic) inputs exert a marked influence on the activity of frontocortical circuits and the dynamic regulation of background dopamine (DA) levels allows for the optimization of PFC cognitive performances (2). Interestingly, DA exerts opposite effects on glutamatergic activity through activation of D₁-type (D₁- and D₅-) and D₂-type (D₂-, D₃-, and D₄-) receptors, respectively (3, 4). Specifically, D₁ receptor (D₁R) activation increases neuronal excitability and synaptic efficacy through modulation of *N*-methyl D-aspartate receptors (NMDARs), whilst D₂ receptor (D₂R) signaling produces opposite effects (4). Furthermore, activation of D₁R promotes Long-Term Potentiation (LTP) in the PFC (5, 6) whereas D₂R stimulation is required for inducing Long-Term Depression (LTD) (6). These observations support the current view that distinct cortical DA receptors differentially regulate cognitive functions through the modulation of NMDARs. However, the underlying mechanisms still await clarification. While D₁R activation in the PFC consistently facilitates working memory and executive function (7), the significance of PFC-localized D₂-type receptors is more complex inasmuch as D₂R and D₃R subtypes enact contrasting roles: activation of D₂R favors cognitive processes whereas D₃R stimulation exerts a negative impact on cognition (8). Accordingly, blockade of D₃Rs results in a broad-based positive influence upon cognition, including improvements in social novelty discrimination and novel object recognition (NOR) and a reduction in the cognitive deficits associated with a developmental model for schizophrenia (SCZ) (9, 10). Moreover, it has been proposed that DAR and NMDARs can form clusters at the surface of neurons (11) and that their reciprocal dialogue relies on intracellular signaling cascades involving protein kinases (12). Whether functional DA-NMDAR cross-talk involves others mechanisms is not known.

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Activation of the canonical GluN1-GluN2 containing NMDARs requires binding of both glutamate and a co-agonist, either glycine or D-serine (13). We have previously shown that D-serine rather than glycine is the primary endogenous co-agonist of synaptic NMDARs at glutamatergic synapses connecting pyramidal neurons in the PFC of adult rats (14). This is important since genetic linkage and association studies show that the genes encoding the D-serine producing and degrading enzymes serine racemase (SR) and D-amino acid oxidase (DAAO), respectively, are risk genes for SCZ (15, 16). Accordingly, serum and cerebrospinal fluid levels of D-serine are reduced in SCZ patients (16-18). Moreover, clinical trials of D-serine supplementation or the administration of DAAO inhibitors have yielded encouraging results
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(17, 18). An involvement of D-serine in SCZ is further supported by animal studies showing that multiple risk pathways for SCZ converge in SR deficient mice (19). Yet, our understanding of the significance of D-serine to the pathology and symptoms of SCZ remains limited, and the question of how it interacts with DA signaling remains to be resolved. This issue is, moreover, of broader pertinence in view of accumulating evidence that a malfunction of NMDAR is central to the pathophysiology of multiple brain disorders (20).

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In the current study, we sought to determine whether and how D-serine and DA interact in the PFC. We show that D-serine is indeed required for the dopaminergic modulation of NMDARs in the PFC, as expressed in measures of neuronal excitability, synaptic plasticity and cognitive function, providing a novel framework for understanding PFC integrated DA-glutamate crosstalk under physiological and pathological conditions.

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## Results

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### Modulation of NMDAR and neuronal excitability by DA involves D-serine

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To test the hypothesis that D-serine may be implicated in DA modulation of NMDAR functions, we first investigated whether DA modulations of isolated synaptic NMDAR-mediated excitatory postsynaptic currents (NMDA-EPSCs) recorded at +40 mV and -70 mV in adult rat prelimbic cortex (PrL) layer 5 pyramidal neurons (Fig.1 A-C) require D-serine. Low concentrations of DA (1 µM) potentiated NMDA-EPSCs by 73.9 ± 14.1% (Fig.1D, I, *SI appendix Fig.S8*) while higher concentrations (10 µM) decreased NMDA-EPSCs by 44.7 ± 6.7% (Fig.1D, I, *SI appendix Fig.S8*) confirming previous studies (21, 22). To determine if these bidirectional modulations of NMDA-EPSCs by DA imply changes in the occupancy of NMDARs by a co-agonist, we monitored the effects of DA after pre-saturation of the co-agonist binding site by exogenous D-serine (100 µM) (Fig.1E, I). This resulted in the occlusion of DA-effected NMDA-EPSCs modulation. We then directly addressed the implication of D-serine in the DA modulation of NMDA-EPSCs by treating the slices with *Rhodotorula gracilis* D-amino acid oxidase (*RgDAAO*) (0.2 U/mL) to specifically deplete endogenous D-serine (14, 23) (Fig.1F, I). Akin to the effect of saturating the D-serine binding site, removal of D-serine with *RgDAAO* markedly reduced the potentiating and inhibitory effect of 1 µM and 10 µM DA, respectively. As an important control we also confirmed, using enzymatic assays, that DA is not a substrate or an inhibitor for *RgDAAO* (*SI appendix Fig.S1*). These data therefore indicate that positive and negative modulations of NMDAR by exogenous DA requires D-serine.
We next assessed whether D-serine is involved in the regulation of NMDA-EPSCs by endogenous DA. To this end, we first studied the effects of the DA reuptake inhibitor GBR12909 on NMDA-EPSCs. Bath applied GBR12909 (0.5 µM) potentiated NMDA-EPSCs (Fig. 1G, I) similarly to exogenous low DA (1 µM). Remarkably, performing this experiment under D₁R blockade by addition of the antagonist SCH39166 (1 µM) not only abolished the potentiating effect of GBR12909, but also unmasked a depressant action on NMDA-EPSCs (Fig. 1G, I). To directly address whether the potentiating effect of GBR12909 requires D-serine acting on NMDARs, we next recorded NMDAR currents on PFC slices isolated from mice lacking the D-serine producing enzyme serine racemase (SR⁻/⁻), in which D-serine content is ~10% of wild-type (SR⁺/⁺) mice (24). Bath application of GBR12909 (0.5 µM) potentiated NMDA-EPSCs in SR⁺/⁺ control slices and depressed these currents upon blockade of D₁Rs. In contrast, in slices from SR⁻/⁻ mice, GBR12909 failed to induce any changes in NMDA-EPSCs (Fig. 1H, I). Together, these results indicate that D-serine is required for appropriate DA modulation of NMDA-EPSCs.

As DA is known to regulate PFC activity through modulation of pyramidal neurons excitability (4) and modulation of NMDARs also influences PFC neuronal firing activity (25, 26), we next investigated whether D-serine is also involved in the DA modulation of layer 5 pyramidal neurons excitability. To this end, cell firing was induced by incremental square depolarizing current pulses during whole-cell current-clamp recordings. DA concentrations of 1 µM and 10 µM applied to rat PFC slices respectively increased and decreased neuronal excitability (SI appendix Fig.S2A,B) without altering membrane properties (SI appendix Table 1). Depletion of D-serine with RgDAAO prevented these changes in excitability (SI appendix Fig.S2C,D). We also undertook these experiments in PFC slices from SR⁻/⁻ mice and SR⁺/⁺ mice. DA at 1 µM and 10 µM induced changes in SR⁺/⁺ mice cell excitability similar to those observed in rats, without altering membrane properties (SI appendix Table 2; Fig.S2E,F). Absence of D-serine in SR⁻/⁻ mice resulted in a complete loss of these DA modulations, mirroring the effect observed with RgDAAO (SI appendix Fig.S2G,H). These results thus indicate that D-serine is required for DA-induced changes in NMDAR-dependent neurotransmission and excitability at PFC pyramidal neurons.

## Opposite influence of D₁R and D₃R activation on NMDAR

Positive DA modulation on PFC pyramidal neurons is classically attributed to D₁-type receptor activation whereas negative modulation is thought to be mediated by the D₂-type receptor (3). We then tested whether modulation of NMDA-EPSCs by selective D₁-type and D₂-type agonists required D-serine. As D₃Rs were recently found to play an important
regulatory role in the PFC and in SCZ (27), we ensured that we could detect these receptors in patched PFC neurons using single cell RT-PCR (Fig.2A) and particularly focused on this subtype of receptor. Bath application of the preferential D₃R agonist PD128907 (1 µM) reduced NMDA-EPSCs whereas the D₁R agonist SKF81297 (10 µM) induced a reliable potentiation of NMDA-EPSCs (Fig.2C,E). As with DA, actions of these two agonists were strongly impaired by saturating the NMDARs co-agonist binding site with exogenous D-serine (100 µM, Fig.2D,E). Furthermore, application of S33084 (0.1 µM), a selective D₃R antagonist (10), fully abolished the inhibitory effect of PD128907 on NMDA-EPSCs but did not alter the potentiating effect of the D₁R agonist SKF81297 (Sl appendix Fig.S3).

We next analyzed the effects of D₁- and D₃R agonists on NMDA-EPSCs in SR⁻/⁻ mice. Mirroring rat PFC slices, application of SKF81297 and PD128907 respectively increased and decreased NMDAR-EPSCs in SR⁺/⁺ slices (Fig.2F,H) and these effects were markedly compromised in SR⁻/⁻ slices (Fig.2G,H). To further confirm that synaptic deficits in SR⁻/⁻ mice were specifically attributable to a lack of D-serine and not to other putative modulators such as glycine or D-aspartate, we restored its levels in SR⁻/⁻ mice by chronic subcutaneous administration ([D-serine]=150 mg/kg) for 21 days, as previously described (19). While levels of D-serine, D-aspartate and glycine in the medial PFC (mPFC) were respectively decreased by ~89%, ~35%, and ~69% in saline-treated SR⁻/⁻ mice, treatment with D-serine selectively re-established levels of D-serine alone to near control values in mutant mice (Sl appendix Fig.S4). Importantly, this resulted in the normalization of the modulatory actions of D₁R and D₃R agonists on NMDA-EPSCs in D-serine-treated but not in saline-treated SR⁻/⁻ mice (Fig.2I-K). Thus, these rescue experiments confirm that in SR⁻/⁻ mice, D-serine is the missing element affecting DAergic modulation of NMDARs. Collectively, these experiments demonstrate that D-serine is a critical factor for DA modulation of NMDARs via D₁- and D₃-Rs.

## D-serine mediates DA modulations of synaptic plasticity

Activity-dependent changes of synaptic strength in the PFC rely on NMDARs (6, 14) and are strongly regulated by DA (3, 5, 6). In particular, D₁-type receptor stimulation enhances LTP whilst LTD requires the activation of both D₁- and D₂-type receptors (5, 6). To specifically determine the contribution of D-serine to DA modulation of NMDAR-dependent long-term changes in synaptic plasticity, we investigated the effect of DA receptor stimulation on excitatory post-synaptic field potential (fEPSP) plasticity in the PrL of SR⁺/⁺ and SR⁻/⁻ mice. Analyses of SR⁻/⁻ mice synaptic function revealed that basal synaptic transmission was reduced by ~26% (Fig.3A), a decrease most likely attributable to post-synaptic mechanisms as paired-pulse experiments did not show any changes in release probability (Fig.3B). These
results are reminiscent of previously reported NMDAR hypofunction in SR⁻/⁻ mice (19, 24).
Although no difference in LTP magnitude between SR⁺/⁺ and SR⁻/⁻ mice was detectable
(Fig.3C), D₁-type receptor activation by SKF81297 (10 µM) enhanced LTP in SR⁺/⁺ (Fig.3D),
consistent with previous investigations (5, 6), but not SR⁻/⁻ mice (Fig.3E), thereby indicating
that D-serine is required for DAergic modulation of PrL LTP.

We then investigated LTD. As previously reported (6), low-frequency stimulation (LFS) could only induce LTD when performed in conjunction with D₁- and D₂-type receptor activation (SI appendix Fig.S5, Fig.3F). Indeed, combining SKF81297 (10 µM) and quinpirole (1 µM) with LFS elicited LTD (SI appendix Fig.S5D). Strikingly the latter effect was abolished in D₃R knock-out (D₃R⁻/⁻) mice (SI appendix Fig.S5D), indicating that D₃Rs play a critical role in the DA-dependent generation of LTD in the PrL. Accordingly, co-stimulation of D₁R and D₃R with SKF81297 and PD128907 combined with LFS reliably induced LTD in SR⁺/⁺ mice (Fig.3F). Importantly, this DA-dependent LTD was markedly reduced by ~ 62% in SR⁻/⁻ mice (Fig.3F). Collectively, these data indicate that D-serine is critical for the dopaminergic modulation of long-term synaptic plasticity in the PrL *via* both D₁R and D₃R.

## Activation of D₁R and D₃R modulates PFC D-serine levels *in vivo*

To gain insights into how D-serine participates in the DAergic modulations of NMDAR function, we tested whether activation of D₁- and D₃Rs could regulate extracellular levels of this NMDAR co-agonist. To this end, we performed bilateral *in vivo* microdialysis in the mPFC of freely moving mice measuring basal extracellular levels of D- and L-serine for 1 h in response to perfusion of artificial cerebrospinal fluid (aCSF) as a control, the D₁R agonist SKF81297 (10 µM), the D₃R agonist PD128907 (1 µM) or a combination of both (Fig.4A-C). The basal levels of D-serine and L-serine in the mPFC were 812.80 ± 22.97 nM and 1514.00 ± 126.30 nM respectively, in the range of previous studies (28, 29). SKF81297 increased extracellular levels of D-serine level by ~17%, providing a mechanism for the D-serine dependent activation of NMDARs by D₁Rs reported herein. Although activation of D₃Rs with PD128907 alone did not induce any change in extracellular D-serine levels, it blunted the increase in D-serine levels elicited by D₁R activation (Fig.4C, left) indicating functional cross-talk between D₁R and D₃R. Interestingly, local infusion of SKF81297 also increased levels of L-serine, the precursor of D-serine, and this effect was also blunted by D₃Rs activation (Fig.4C, right). These data therefore indicate that D₁R and D₃R activation modulate extracellular levels of D-serine *via* direct release as well as increased biosynthesis by SR and/or decreased degradation by DAAO. Since we found that chronic supplementation of D-serine was sufficient to restore D₁R and D₃R
modulations of NMDARs in SR$^{-/}$ mice, we conclude that release of D-serine is the primary
process at play.

To ascertain whether the inhibitory influence of D₃R on D₁R is indeed functionally relevant at NMDARs, we recorded NMDA-EPSCs in the presence of PD128907 (1 µM) and under these conditions applied SKF81297 (10 µM). Instead of potentiating NMDA-EPSCs as occurs when applied alone (Fig.2F), SKF81297 in the presence of PD128907 induced no change (Fig.4D), thus corroborating the inhibitory action that D₃R activation exerts on D₁R function. These data suggest that D-serine function at synaptic NMDARs can be fine-tuned by DAergic input via the balanced activation of D₁- and D₃Rs (Fig.4D).

The balance of D₁ vs D₃R activation regulates D-serine functions via the cAMP/PKA pathway

We next sought to determine the signaling cascade underlying the D₁/D₃R cross-talk regulating D-serine function. To this end, we focused on the adenylyl cyclase (AC) – cAMP – protein kinase A (PKA) pathway because AC activity is differentially influenced by both D₁- and D₃R (3) and PKA is known to regulate exocytosis (30, 31). We posited that D₁R activation leads to an upregulation of AC activity to induce an increase in D-serine release whereas D₃Rs activation inhibits this process by suppression of AC. To test this hypothesis, we inhibited AC with SQ22536 (20 µM) (Fig.5A) or PKA with H89 (10 µM) (Fig.5B) in SR⁺/⁺ PFC slices and under these conditions activated D₁Rs with SKF28197 (10 µM). Blocking AC or PKA yielded a similar blunting effect on D₁R-induced facilitation of NMDA-EPSCs (Fig.5A-B) as activation of D₃Rs (Fig.4D), thus corroborating our hypothesis. Accordingly, blockade of PKA also occluded the effect of D₃R activation (Fig.5C). To directly test whether this signaling pathway is indeed underlying DAergic modulation of D-serine functions, we assessed the effect of AC activation with forskolin (50µM) on SR⁺/⁺ and SR⁻/⁻ NMDA-EPSCs. While, akin to D₁R stimulation, AC activation induced a robust increase in NMDA-EPSCs amplitude in SR⁺/⁺ mice (+57.60 ± 10.94%), this effect was abolished in SR⁻/⁻ mice (Fig.5D). Together these data identify the AC-cAMP-PKA signaling cascade as a key intracellular pathway transducing the opposing actions of D₁R⁻ and D₃R on D-serine function.

Dopaminergic modulation of PFC cognitive functions involves D-serine

To evaluate the implication of this newly identified neuromodulatory interplay on
cognition, we next investigated the role of D-serine in D₃R modulation of PFC-dependent
cognitive processes. We previously showed that antagonizing D₃Rs with systemic
administration of S33084 (0.63 mg/kg) enhances rat short-term memory in the novel object
284 recognition (NOR) task (10). Here, we further established that this effect was attributable to
285 changes in PFC function by testing NOR memory in rats after intra-PFC administration of
286 S33084 5 min prior to the acquisition phase (T1, *SI appendix* Fig.S6A). As with systemic
287 administration, treatment resulted in the retention of the novel object 4h after T1, while in
288 control vehicle-treated rats NOR memory was lost at this inter-trial interval (ITI) (10), *SI
289 appendix* Fig.S6B), thus confirming the PFC origin of the pro-cognitive effect of D₃R blockade
290 on recognition memory.

291 To test the involvement of NMDARs in this pro-cognitive effect, the non-competitive
292 NMDAR channel blocker MK801 (0.05 mg/kg, i.p.) or the selective and competitive NMDAR
293 antagonist CPP (10 mg/kg, i.p) were administered 15 min prior to S33084 (0.63 mg/kg, s.c).
294 Both NMDAR blockers prevented the pro-cognitive effect of D₃R antagonism on NOR memory
295 (Fig.6A). We also studied the contribution of the co-agonist modulatory binding site using the
296 selective D-serine/glycine binding site antagonist, L701,324 (5 mg/kg, i.p.) (32), which also
297 abolished the reversal of delay-induced impairment in NOR memory by S33084 (Fig.6A).
298 These results indicate that the pro-cognitive action of S33084 depends on NMDAR function
299 and activation of the co-agonist site, possibly *via* D-serine.

300 To further explore this possibility, we assessed NOR memory in SR<sup>+/+</sup> and SR<sup>-/-</sup> mice.
301 The two genotypes displayed similar baseline NOR performances, being intact at 2 min and
302 1 h after the acquisition trial but lost after a 2 h ITI (*SI appendix* Fig.S6C). Thus, a deficit in D-
303 serine does not affect NOR performance *per se*. However, S33084 applied systemically
304 (0.63 mg/kg, s.c) 30 min before T1 or by local intra-PFC injection (2.5 µg, 0.5 µL) 5 min prior
305 to T1 reversed the 2 h delay-induced NOR impairment in SR<sup>+/+</sup> mice (Fig.6B, *SI appendix*
306 Fig.S6D). This result indicates that, in mice, D<sub>3</sub>R antagonism also yields a pro-cognitive action
307 that relies on PFC neuromodulation. Strikingly, S33084 (0.63 mg/kg, s.c.) was ineffective in
308 SR<sup>-/-</sup> mice (Fig.6B), thus demonstrating the pivotal role of D-serine in the DA modulation of
309 PFC-dependent cognitive functions. We here further ensured that this impairment was indeed
310 attributable to the lack of D-serine in SR<sup>-/-</sup> mice by performing rescue experiments in which D-
311 serine supplementation of SR<sup>-/-</sup> mice reversed this deficit (Fig. 6B). Taken together, these data
312 demonstrate that, in the PFC, modulation of D-serine function *via* D<sub>3</sub>Rs impacts cognition.

313 Dopaminergic regulation of PFC global activity is impaired in SR knock-out mice.

314 We finally tested *in vivo* whether D₃R modulation of PFC activity is impaired in SR<sup>-/-</sup>
315 mice by pharmacological magnetic resonance imaging (phMRI). Since antagonizing D₃Rs
316 potentiates the cerebral blood volume response (rCBV) to the DA-releasing compound D-
317 amphetamine in cingulate cortex (33), we reasoned that if D-serine is required for optimal
318 DAergic modulation of PFC activity, the rCBV change in response to D-amphetamine in the
319 presence of S33084 (0.63 mg/kg, s.c.) should be impaired in SR<sup>-/-</sup> mice. As predicted, SR<sup>-/-</sup>
320 mice displayed a blunted rCBV response to D-amphetamine (1 mg/kg, i.p.), as compared to
321 control SR<sup>+/+</sup> mice (SI appendix Fig.S7A-C). These data therefore confirm, *in vivo*, that DA
322 modulation of PFC activity occurs through regulation of D-serine.
323

324

Discussion
=========

325 The present study reveals a novel role for D-serine in driving PFC dopamine-glutamate
326 interactions at the cellular, network, and behavioral level. The underlying signaling pathway
327 involves differential regulation of AC activity by D₁- and D₃-R, most likely resulting in PKA-
328 mediated changes in D-serine release. The pharmacological tools employed in this study were
329 in general used at a single dose which may perhaps be a limitation. Yet we selected doses
330 selective for their respective targets and the use of several agents, together with other
331 complementary approaches, reinforces our conclusions.
332

332 Activation of D₁R enhances NMDAR-dependent functions to influence
333 neurotransmission, synaptic plasticity and cell excitability through D-serine. Conversely,
334 activation of D₃R exerts an opposite and inhibitory action. Although contrasting effects of D₁-
335 and D₂-type receptors on NMDAR currents have been reported to partly act *via* distinct
336 excitatory and inhibitory microcircuits (34), such a regulatory mode can be ruled-out since in
337 this study all electrophysiological recordings were performed in the presence of the GABA<sub>A</sub>
338 receptor antagonist picrotoxin to block GABAergic inhibitory transmission. Still, the question
339 remains of whether the DA modulations of D-serine functions unveiled herein are cell-
340 autonomous. We found that activation of D₁Rs increased extracellular levels of D-serine and
341 in consequence NMDAR-dependent functions in the PFC, an effect counteracted by activation
342 of D₃Rs, indicating functional interactions between the two receptors. Our data further indicate
343 that the underlying mechanism involves differential regulation of the AC-PKA pathway,
344 consistent with a cell-autonomous effect. As previous studies showed that D₁- and D₃R
345 positive pyramidal neurons are particularly abundant in L5 of mPFC and that the two receptors
346 may reside on the same neurons (35), such a scenario is indeed conceivable.
347

347 Intriguingly, we found that modulation of L-serine levels by D₁- and D₃Rs mirrored their
348 influence on D-serine. Although these observations might suggest intensification of the D-
349 serine biosynthesis pathway through activation of the glia-to-neuron serine shuttle (36), the
fact that we could rescue DAergic control of synaptic and cognitive functions by D-serine supplementation in SR⁻/⁻ mice indicates that modulation of D-serine biosynthesis is not the limiting step in DA regulation. Rather, DA-induced changes in neuronal D-serine release are most likely central to these processes, while L-serine shuttling and D-serine synthesis by SR would be secondarily upregulated as a homeostatic loop to support the metabolic demands imposed on neuronal circuitry.

Modulation of D-serine levels upon activation of D₁- or D₃R offers a multifaceted signaling system for DA fine-tuning of NMDAR function, thereby optimizing synaptic and network activity. Tonic, spontaneous DA release may increase NMDAR function through D₁-type receptors whilst the higher extracellular level of DA attained during phasic firing of VTA-derived mesocortical pathways would recruit D₃Rs. By inhibiting the AC-PKA pathway, D₃R would counteract D₁R activation to lower D-serine levels and as a consequence the operation of NMDAR. In line with this notion, enhancement and reduction of NMDAR activity by respectively low and high concentrations of DA has previously been reported (21, 22) and is coherent with the U-shaped function of the influence of DA receptors activation upon cognition (37, 38). In the case of PFC, such effects have been attributed to the contrasting level of expression of D₁- and D₂-type receptors (39) as well as the differential localization of these receptors (3, 39). In addition, there is compelling evidence for a differential facilitatory and suppressive influence upon cognitive function of D₁ and D₃R respectively (27, 38).

The striking defects in D₁- and D₃R-dependent modulation of neurotransmission and PFC function in SR⁻/⁻ mice suggest that DA modulation specifically involves NMDAR co-agonism by D-serine but not glycine. It might then be enquired why SR⁻/⁻ mice did not exhibit baseline deficits in the NOR procedure which requires functionally-intact NMDARs (40). We propose that, under conditions of a persistent lack of D-serine in the mPFC, glycine can, to some extent, assume a compensatory role at NMDARs. Possible reasons for the specific role of D-serine rather than glycine as a mediator of DA modulation of PFC functions include differential actions related to their spatial segregation. Indeed, we previously found that under physiological conditions synaptic pools of D-serine but not glycine gate synaptic NMDARs in the mPFC (14), whereas glycine preferentially activates extrasynaptic NMDARs (41).

As for potential pathophysiological relevance, by identifying D-serine as a novel substrate for the influence of DA upon glutamatergic transmission in PFC, the current observations are relevant to several neuropsychiatric disorders, such as SCZ where there is a well-established link between a dysfunction of D-serine-mediated NMDAR signaling and both cognitive and positive symptoms (19). Our findings are also consistent with a previous report
by Nomura and colleagues showing that neonatal D-serine supplementation of mice displaying
a knockout for the SCZ susceptibility gene *pick1* rescues DA modulation of NMDAR-mediated
control of pyramidal cell excitability (42). Interestingly, the atypical antipsychotic clozapine
enhances D-serine release in rat frontal cortex (43) and elevates plasma levels of D-serine and
L-serine in patients with SCZ (44). As clozapine notably acts on DA receptors, in light of our
findings, one interpretation might be that clozapine actually changes the balance between D1-
and D2-type receptors activation, thereby leading to an increase in D-serine levels. The
present study may also offer a cellular explanation for previous studies showing that D-serine
plays a major role in cocaine-induced sensitization (45, 46) and to studies suggesting that the
novel antipsychotics cariprazine and blonaneserin strengthen cognitive functions by virtue of
their attenuation of D₃R activity in the PFC (47,48). In addition, there is increasing interest in
the potential utility for ligands at NMDAR in the management of other disorders from autism to
Alzheimer's disease (49,50).

Finally, gender is clearly a factor related to the prevalence, clinical picture and treatment
of many psychiatric disorders, so it should be pointed out that only males were used in the
present study, representing a potential limitation to any clinical extrapolation. Nonetheless the
present study provides a novel framework for an improved understanding and, ultimately,
management of the diversity of brain disorders involving disruption of the frontocortical DA-
glutamate interface (51,52).

403

404 Methods and Materials

405

406 A detailed description of all methods and materials can be found in the SI Appendix.

All experiments were conducted in accordance with European and French directives on animal
experimentation and with local ethical committee approval. Electrophysiological recordings
and behavioral analyses were performed on adult Wistar and Lister hooded rats as well as
serine racemase knock-out mice (SR<sup>-/-</sup>) and control littermates (SR<sup>+/+</sup>) while *in vivo*
microdialysis and phMRI experiments were performed only on mice. Only males were used.
All quantitative data are expressed as mean ± SEM. Statistical analyses performed are
detailed in the *SI Appendix* and in figure legends.

414

415 Acknowledgments
The authors thank Dr Philippe Fossier for sharing electrophysiological equipment. We are
416 grateful to Gérard Sadoc for his help using Elphy™. Thanks to Julien Moulard for his help in
417 analyzing cell excitability. This work was supported by operating grants from Centre National
418 de la Recherche Scientifique and INSERM (to J-P.M.), Université Aix-Marseille (to J-P.M.),
419 Agence Nationale de la Recherche (ANR-09-MNPS-022-01 to J-P.M.), Institut de Recherche
420 Servier (to J-P.M and KCFF), and the Fondation pour la Recherche Médicale (Equipe FRM
421 DEQ20150331734 to J-P.M.). NM and AC were supported by the Institut de Recherche
422 Servier, the Centre National de la Recherche Scientifique (CNRS) and the University of
423 Bordeaux. LP and SS were supported by a grant from Fondo di Ateneo per la Ricerca
424

425
Financial Disclosures

GD, XL, PL, NM, SS, RA, THA, BP, DJGW, GL, PF, AB, JTC, GC, LP, MG, KCFF,
426 AMG, TF, AC, and JPM declare no conflict of interest. MJ Millan and JM Rivet respectively
427 were and are full time employees of Institut de Recherche Servier and have no other interests
428 to declare. S Schmidt and J Kehr are employees of Pronexus Analytical AB but declare no
429 conflicts of interests.
430

431
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Figures legends

**Fig.1.** D-serine is required for dopamine modulations of NMDA-EPSCs. A: Diagram showing the slice and electrodes placements for recording NMDA-EPSCs at pyramidal neurons in layer 5 of the prelimbic (PrL) cortex. B-C: Pharmacologically isolated NMDA-EPSCs can be recorded in PrL neurons from rat slices at +40mV and at -70 mV in the presence of GABA-receptor blocker PTX (50 µM) and AMPA-receptor blocker NBQX (10 µM) and are blocked by D-AP5 (50 µM). D: Bath application of low (light green, 1 µM, n=6) or high (dark green, 10 µM, n = 7) concentrations of DA respectively up- (+73.9 ± 14.1%) or downregulate (-44.7 ± 6.7%) NMDA-EPSCs. E-F: Pre-incubation with 100 µM D-serine (triangle symbols) or treatment with RgDAAO (square symbols) opposes occluding action on the positive (DA₁µM+D-Ser: +22.5 ± 4.6% n=7; DA₁µM+RgDAAO: +5.4 ± 5.93%, n=7) and negative (DA₁₀µM+D-Ser: -18.7 ± 6.7%, n=6; DA₁₀µM+RgDAAO: -16.8 ± 6.2%, n=5) regulations of NMDA-EPSCs by exogenous DA. G: Accumulation of endogenous DA by application of the DA reuptake inhibitor GBR12909 (0.5 µM) increases NMDA-EPSCs in control rat slices (filled light green circles, +73.78 ± 21.35%, n=11) and decreases NMDA-EPSCs in the presence of the D1R antagonist SCH39166 (1 µM) (filled dark green circles, -35.68 ± 1.45%, n=13). H: GBR12909 (0.5 µM) increases NMDA-EPSCs in mouse SR⁺/⁺ control slices (filled light green circles, +41.73 ±
12.12%, n=5) and decreases NMDA-EPSCs in the presence of the D1R antagonist SCH39166 (1 µM) (filled dark green circles, -22.84 ± 1.79%, n=5). GBR12909 (0.5 µM) induces no change in slices from SR⁻/⁻ mice (empty green circles, +0.93 ± 4.22%, n=6). Top traces scale bars: 50 pA, 100 ms. Bottom traces scale bars: 50 pA, 500 ms. H: Histograms summarizing the DA modulations of NMDA-EPSCs and the opposing action of D-serine binding site occlusion, RgDAAO treatment and serine racemase deletion. * p<0.05, ** p<0.01, ***p<0.001.

**Fig.2. Modulations of NMDA-EPSCs by D₁- and D₃-receptors involves D-serine.** A-B: Single-Cell multiplex RT-PCR showing the expression of D1 and D2 receptors subtypes in L5 excitatory neurons of the PrL area. C: Bath application of D₁R agonist SKF81297 (10 µM) increases NMDA-EPSCs (93.93 ± 14.05%, n=11) while the D₃R specific agonist PD128907 (1 µM) has an opposing action (-39.67 ± 8.1% n=8). D: Occluding the co-agonist binding site of NMDAR with 100 µM D-serine markedly impairs NMDAR-EPSCs modulations by DA receptors activation (SKF28197+D-ser: +17.04 ± 2.48%, n=6; PD128907+D-ser: -21.7 ± 5.6%, n=6). E: Histograms summarizing the effects of D₁R and D₃R agonists in the absence and presence of occluding D-serine. F: Modulations of NMDAR-EPSCs by D₁Rs and D₃Rs activation in SR⁺/⁺ mice (SKF81297: +92.66 ± 23.5%, n=8; PD128907: -34.92 ± 4.95%, n=7). G: Modulations of NMDAR-EPSCs by D₁Rs and D₃Rs activation in SR⁻/⁻ mice (SKF81297; +51.03 ± 10.91%, n=7; PD128907: -10.36 ± 11.75%, n=7). H: Histograms showing that modulations of NMDAR-EPSCs by D₁Rs and D₃Rs activation is impaired in SR⁻/⁻ mice. I: Modulations of NMDAR-EPSCs by D₁Rs and D₃Rs activation in SR⁻/⁻ mice chronically supplemented with D-serine (SKF81297; +86.86 ± 30.01%, n=7; PD128907: -26.46 ± 4.19%, n=14). J: Modulations of NMDAR-EPSCs by D₁Rs and D₃Rs activation in SR⁻/⁻ mice chronically supplemented with control saline (SKF81297; +11.98 ± 10.32%, n=6; PD128907: -3.70 ± 4.56%, n=14). H: Histograms showing that chronic D-serine supplementation rescues modulations of NMDAR-EPSCs by D₁Rs and D₃Rs activation in SR⁻/⁻ mice. * p<0.05; ** p<0.01. Top traces scale bars: 50 pA, 100 ms. Bottom traces scale bars: 50 pA, 500 ms.

**Fig.3. D-serine mediates DA regulations of synaptic plasticity.** A: Input/output relationship in wild-type vs SR deficient mice (SR⁺/⁺ n=44; SR⁻/⁻ n=21). B: Paired-pulse ratio profiles in wild-type vs SR deficient mice (SR⁺/⁺ n=44; SR⁻/⁻ n=21). C: LTP of the field potential in wild-type vs SR deficient mice (SR⁺/⁺: +48.88 ± 9.20%, n=9; SR⁻/⁻: +48.13 ± 14.6%, n=8). D: Effect of the D₁R-type agonist SKF81297 (10 µM) on LTP in SR⁺/⁺ mice (+83.66 ± 10.72%, n=10). E: Effect of SKF81297 (10 µM) on LTP in SR⁻/⁻ mice (+61.64 ± 14.66%, n=9). F: LTD induced by concomitant application of SKF81297, PD128907 and LFS in wild-type vs SR deficient mice (SR⁺/⁺: -32.39 ± 7.58%, n=6; SR⁻/⁻: -12.20 ± 5.44%, n=6). * p<0.05. Scale bars: 0.1 mV, 2 ms.
**Fig.4.** DA receptors activation regulates extracellular mPFC D-serine levels in freely moving mice. A: Mice were implanted with two microdialysis probes placed in the left and right mPFC. Microdialysates were collected every 20 min and D- and L-serine were quantified using UHPLC-MS/MS. B: Left chromatogram shows separation of a standard mixture of D-serine at LLOQ (20 nM) and L-serine in AAS18 in a CSF (black line) and D,L-serine-d3 (230 nM, internal standard, gray line). Right chromatogram shows a typical microdialysis sample in basal conditions (vehicle, black line) and D,L-serine-d3 (gray line). C: Changes in extracellular D-serine and L-serine levels from 20 to 60 min during drug infusion in the mPFC. Histograms represent values for area under the curve (AUC). Left, the D₁R agonist SKF81297 increased the extracellular levels of D-serine by 17.40 ± 4.73% (n=8 samples, 6 mice) while D₃R agonist PD128907 on its own had no effect (vehicle: -0.25 ± 0.49% change, n=10 samples from 6 mice vs PD128907: -1.90 ± 2.4% change, n=10 samples from 6 mice) but blocked the potentiating effect of SKF81297 (-0.08 ± 3.13% change, n=13 samples from 7 mice). Right, SKF81297 also increased the extracellular levels of L-serine by 33.10 ± 15.05% while D₃R agonist PD128907 had no effect on its own (vehicle: -1.31 ± 1.15% change vs PD128907: 0.00 ± 8.90% change) but blocked the potentiating effect of SKF81297 (-9.15 ± 6.32% change). \* p<0.05, \*\*p<0.01, \*\*\*p<0.001. D: Recordings of NMDA-EPSCs in the mPFC of mice showing that in the presence of PD128907, SKF81297 fails to potentiate NMDA responses (-3.15 ± 6.16% change, n=6).

**Fig.5:** DA modulations of D-serine NMDAR co-agonist functions occurs through cAMP/PKA signaling. A: The facilitating effect of D₁Rs activation on NMDA-EPSCs is abolished by blocking production of cAMP by the adenylate cyclase enzyme (AC) (-4.52±3.21%, n=12). B: Similarly, blocking downstream the PKA enzyme prevents the facilitating effect of D₁Rs activation on NMDA-EPSC (+6.18 ± 3.27%, n=12). C: Downregulation of NMDAR-EPSCs by D₃Rs activation is also abolished by H89 blockade, which is indicative of an occlusion. D: Activating AC increased NMDA-EPSCs in SR<sup>+/+</sup> mice (+51.61 ± 11.62%, n=10) but not in SR<sup>-/-</sup> mice (-0.63 ± 2.01%, n=9; p<0.001). Scale bars: 20 pA, 100 ms.

**Fig.6.** Pro-cognitive action of D₃R antagonist S33084 relies on D-serine modulation of NMDARs. A: Administration of the D₃R antagonist S33084 (0.63 mg/kg, s.c.) reverses the impairment in NOR memory induced by a 4 hours inter-trial interval (ITI) in rats (n=12). Systemic administration of the NMDAR antagonists, MK801 (B, n=12) and CPP (C, n=12) as well as the glycine/D-serine binding site blocker L701,324 (C, n=12) all prevented the enhancement of NOR memory induced by S33084. B: Administration of the D₃R antagonist S33084 (0.63 mg/kg, s.c.) reversed the impairment in novel object recognition (NOR) memory
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663

664

induced by a 2 hours ITI in SR<sup>+/+</sup> mice (vehicle, n=14; S33084, n=18) but not in SR<sup>-/-</sup> mice (vehicle: n=9; S33084, n=10). The pro-cognitive effect of S33084 is rescued in SR<sup>-/-</sup> mice by chronic supplementation of D-serine (SR<sup>+/+</sup>+saline, n=7; SR<sup>-/-</sup>+D-ser, n=8) performed by s.c. administration with 300 mg/kg D-serine (or control saline) on day 1 and then 150 mg/kg D-serine (or control saline) daily for 20 days. **p<0.01, ***p<0.001 compared to control SR<sup>+/+</sup> mice treated with vehicle. ++p<0.01, +++p<0.001 compared to SR<sup>+/+</sup> mice treated with S33084.
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

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<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>
<figure><img src="image_11.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /><figcaption>J</figcaption></figure>

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /><figcaption>K</figcaption></figure>

<figure><img src="image_27.png" /></figure>
<figure><img src="image_28.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_29.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_32.png" /><figcaption>F</figcaption></figure>

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<figure><img src="image_38.png" /></figure>
A

(1)

<figure><img src="image_39.png" /></figure>

(2)

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /></figure>

<figure><img src="image_43.png" /></figure>

B

(1)

<figure><img src="image_44.png" /></figure>

(2)

<figure><img src="image_45.png" /></figure>

<figure><img src="image_46.png" /></figure>

<figure><img src="image_47.png" /></figure>

<figure><img src="image_48.png" /></figure>

<figure><img src="image_49.png" /></figure>

<figure><img src="image_50.png" /></figure>

C

(1)

<figure><img src="image_51.png" /></figure>

(2)

<figure><img src="image_52.png" /></figure>

<figure><img src="image_53.png" /></figure>

<figure><img src="image_54.png" /></figure>

<figure><img src="image_55.png" /></figure>

D

(1)

<figure><img src="image_56.png" /></figure>

(2)

<figure><img src="image_57.png" /></figure>

<figure><img src="image_58.png" /></figure>
<figure><img src="image_59.png" /></figure>

<figure><img src="image_60.png" /></figure>

<figure><img src="image_61.png" /></figure>

<figure><img src="image_62.png" /></figure>