Shinobu Nomura
email: shinobu.nomura@oist.jp

Ludovic Tricoire

Ivan Cohen

Bernd Kuhn

Bertrand Lambolez
email: bertrand.lambolez@upmc.fr

Régine Hepp
email: regine.hepp@upmc.fr

Katia Boutourlinski

Bruno Giros

Hirokazu Tanimoto

Combined optogenetic approaches reveal quantitative dynamics of endogenous noradrenergic transmission in the brain

Keywords: noradrenaline, dopamine, locus coeruleus, cerebral cortex, pyramidal neuron, cAMP/PKA signaling, optogenetics, channelrhodopsin, fluorescent biosensor, 2-photon imaging

Despite the importance of catecholamines in brain functions, little is known about the real-time cellular dynamics triggered by the release of endogenous catecholamines. To address this issue, we expressed channelrhodopsin in locus coeruleus neurons and protein kinase A activity biosensors in cortical pyramidal neurons, and combined 2-photon imaging of biosensors with 1-photon stimulation of locus coeruleus axons in the neocortex, in acute slices and in vivo. We found that burst photostimulation of these axons for 5-10 s elicited robust, minutes-lasting increases of protein kinase A activity in individual neurons. Selected burst stimulation patterns yielded stable response amplitudes upon repetitive stimulation. Protein kinase A activation was mediated by β1 adrenoceptors, dampened by co-activation of α2 adrenoceptors, and dramatically increased upon inhibition of noradrenaline reuptake transporter. Conversely, dopamine receptors were not involved, showing that protein kinase A activation triggered by burst photostimulation was primarily due to noradrenaline release. These results indicate that a single episode of synchronized burst firing in locus coeruleus neurons has rapid and long lasting effects on the cortical network. Our study shows that noradrenergic neurotransmission events can be characterized with high spatiotemporal resolution in brain slices and in vivo using optogenetic tools.

INTRODUCTION (448 words)

The catecholamines dopamine (DA) and noradrenaline (NA) are neurotransmitters that widely modulate brain circuits and behaviors. Their dysfunctions are associated with cognitive, emotional, and motor disorders, and many drugs target catecholaminergic transmission in therapy of human brain diseases. Little is known about the cellular dynamics of endogenous catecholaminergic transmission, because of the difficulty of selectively stimulating catecholaminergic fibers, and because catecholamines modulate neuronal excitability but do not elicit fast and prominent membrane currents, thereby limiting the sensitivity of electrophysiological readout. However, recent reports demonstrate that optogenetic tools allow the detection of catecholamine release and of their effects at the cellular level with physiologically relevant temporal resolution in brain slices and in vivo [START_REF] Goto | Circuit-dependent striatal PKA and ERK signaling underlies rapid behavioral shift in mating reaction of male mice[END_REF][START_REF] Ma | A Highly Sensitive A-Kinase Activity Reporter for Imaging Neuromodulatory Events in Awake Mice[END_REF][START_REF] Sun | A Genetically Encoded Fluorescent Sensor Enables Rapid and Specific Detection of Dopamine in Flies, Fish, and Mice[END_REF][START_REF] Feng | A Genetically Encoded Fluorescent Sensor for Rapid and Specific In Vivo Detection of Norepinephrine[END_REF][START_REF] Oe | Distinct temporal integration of noradrenaline signaling by astrocytic second messengers during vigilance[END_REF].

NA is involved in arousal, attention, memory and stress [START_REF] Sara | The locus coeruleus and noradrenergic modulation of cognition[END_REF][START_REF] Benarroch | The locus ceruleus norepinephrine system: functional organization and potential clinical significance[END_REF]. It is synthetized in discrete brainstem nuclei from the aminoacid tyrosine, transformed by tyrosine hydroxylase (TH) into DA, which is in turn converted into NA by dopamine beta hydroxylase (DBH). NA effects are mediated by G protein-coupled receptors, among which Gs-coupled β1-3 and Gi-coupled α2 receptors activate and inhibit adenylate cyclase, respectively, whereas Gqcoupled α1 receptors activate phospholipase C [START_REF] Bylund | Subtypes of alpha 1-and alpha 2-adrenergic receptors[END_REF][START_REF] Cotecchia | The alpha1-adrenergic receptors: diversity of signaling networks and regulation[END_REF][START_REF] Evans | Ligand-directed signalling at betaadrenoceptors[END_REF]. NA-releasing fibers are widely distributed in the brain and, in the cerebral cortex, stem from neurons of the locus coeruleus (LC), whose axons innervate all layers of the entire cortical mantle [START_REF] Morrison | The distribution and orientation of noradrenergic fibers in neocortex of the rat: an immunofluorescence study[END_REF][START_REF] Morrison | Noradrenergic innervation of cerebral cortex: widespread effects of local cortical lesions[END_REF][START_REF] Latsari | Noradrenergic innervation of the developing and mature visual and motor cortex of the rat brain: a light and electron microscopic immunocytochemical analysis[END_REF][START_REF] Nomura | Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex[END_REF]. In pyramidal cells, the major cortical neuron type, exogenous NA elicits only modest variations of membrane potential, but triggers intense cAMP-protein kinase A (PKA) signals through activation of β1 adrenoceptors [START_REF] Mccormick | Actions of norepinephrine in the cerebral cortex and thalamus: implications for function of the central noradrenergic system[END_REF][START_REF] Castro | Type 4 phosphodiesterase plays different integrating roles in different cellular domains in pyramidal cortical neurons[END_REF][START_REF] Nomura | Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex[END_REF]). Likewise, imaging of genetically-encoded optical sensors for cAMP-PKA activity provides a sensitive readout of NA effects and allows detecting endogenous NA transmission events [START_REF] Nomura | Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex[END_REF][START_REF] Ma | A Highly Sensitive A-Kinase Activity Reporter for Imaging Neuromodulatory Events in Awake Mice[END_REF][START_REF] Oe | Distinct temporal integration of noradrenaline signaling by astrocytic second messengers during vigilance[END_REF].

The use of channelrhodopsin2 (ChR2) photostimulation to trigger neurotransmitter release [START_REF] Nagel | Channelrhodopsin-2, a directly light-gated cation-selective membrane channel[END_REF], Zhang et al. 2007) is compatible with 2-photon imaging of NA effects, due to the virtual absence of ChR2 activation upon 2-photon laser scanning [START_REF] Rickgauer | Two-photon excitation of channelrhodopsin-2 at saturation[END_REF]Tank 2009, Schoenenberger et al. 2011). We thus combined 2-photon imaging of pyramidal cells expressing fluorescent PKA activity sensors with 1-photon stimulation of ChR2-expressing LC fibers to characterize catecholaminergic transmission dynamics in the neocortex. We found that burst photostimulation of LC fibers resulted in transient and repeatable activation of PKA in pyramidal cells, and used selective antagonists to characterize catecholamine receptors and reuptake transporters involved in this response, in brain slice and in vivo.

RESULTS

Expression of channelrhodopsin in LC fibers and of AKAR3EV in cortical neurons

In order to stimulate specifically LC fibers, we selectively expressed a fusion protein consisting of ChR2 and YFP in LC neurons using conditional viral transduction in DBH-Cre mice (see Methods and Nomura et al. 2014). Eight weeks after virus injection, immunohistochemistry revealed that ChR2-YFP was selectively expressed in a large proportion of DBH-positive neurons in LCs of both hemispheres (Fig. 1A), but not in other catecholaminergic nuclei (not shown, but see [START_REF] Nomura | Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex[END_REF]. We found that 87.0 ± 2.7 % of DBH-positive neurons in the LC expressed GFP (n=214, N=3 mice), consistent with earlier observations [START_REF] Nomura | Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex[END_REF]. Conversely, only 4.9 ± 0.9 % of GFP-positive cells were DBH-negative (n=197, N=3).

ChR2-YFP was efficiently targeted to the plasma membrane of TH-positive somata (Fig. 1A).

In the parietal cortex, ChR2-YFP-positive LC fibers coursed throughout all layers (Fig. 1B), consistent with their wide distribution in the cortical mantle [START_REF] Nomura | Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex[END_REF]. These results

show that the present protocol enables selective and efficient expression of ChR2-YFP in the soma and axonal processes of LC neurons.

To monitor cortical responses to photostimulation of ChR2-expressing LC fibers, we also injected recombinant viruses encoding PKA activity sensors in the parietal cortex after LC transduction (see Methods). For imaging in cortical slices, we used a Sindbis virus to express the AKAR3EV sensor [START_REF] Komatsu | Development of an optimized backbone of FRET biosensors for kinases and GTPases[END_REF]. AKAR3EV is based on FRET between two GFP mutants, whose brightness facilitates the imaging in diffractive slices from myelinated adult tissue. Ten hours after virus injection, AKAR3EV fluorescence was observed throughout cortical layers II to VI, with AKAR3EV-expressing neurons observed as far as 300 µm away from the injection site (Fig. 1C). For imaging cortical neurons in vivo, we used an AAV to express the single-chromophore, GFP-based GAkdYmut sensor [START_REF] Bonnot | Single-fluorophore biosensors based on conformation-sensitive GFP variants[END_REF], because its large dynamic range enables the detection of small changes in PKA activity and singlechromophore sensors allow two-photon imaging without the depth-dependent re-absorption problem of FRET sensors. Five weeks after AAV injection, GAkdYmut was widely expressed in layer V pyramidal neurons typically exhibiting a prominent apical dendrite (Fig. 1D), allowing 2-photon imaging of GAkdYmut-positive neurons in the depth of the cortex in vivo (Fig. 1E). Co-injection of recombinant viruses encoding the red fluorophores mCherry or TurboRFP (see Methods, Fig. 1C and Fig. 1D-E), allowed visualizing viral transduction areas in cortical slices or in vivo, respectively, with minimal excitation of ChR2. These results show that the present approaches result in efficient cortical expression of both ChR2 in LC fibers and PKA sensors in neurons.

Photostimulation of LC fibers elicits PKA activation in pyramidal neurons

The Sindbis virus preferentially transduces neocortical pyramidal neurons and allows overnight expression of biosensors without conspicuous alteration of neuronal physiology [START_REF] Lendvai | Experience-dependent plasticity of dendritic spines in the developing rat barrel cortex in vivo[END_REF], Gervasi et al. 2007[START_REF] Drobac | Calcium imaging in single neurons from brain slices using bioluminescent reporters[END_REF][START_REF] Hu | VIP, CRF, and PACAP act at distinct receptors to elicit different cAMP/PKA dynamics in the neocortex[END_REF]. We imaged PKA dynamics in AKAR3EV-expressing layers II/III and V pyramidal cells from cortical slices, which both respond to exogenous catecholamines [START_REF] Nomura | Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex[END_REF], and focused primarily on layer V pyramidal neurons. In a series of pilot experiments, photostimulation patterns were tested for their ability to evoke PKA signals, based on the in vivo burst firing of LC neurons that discharge action potentials at a maximal frequency of 10 Hz (Aston-Jones andBloom 1981a, 1981b), which is compatible with ChR2 kinetic properties [START_REF] Mattis | Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins[END_REF]. We thus applied trains of light pulses of variable length, at a maximal frequency of 10 Hz, for different overall durations. Most protocols elicited transient PKA activation (see examples in Fig. S1). However, photostimulation consisting of 5 ms light pulses applied at 10 Hz during 5 s yielded robust and reproducible responses (Fig. 2A), and was used in all subsequent experiments in brain slices.

Unless otherwise stated, responses were measured at the soma of pyramidal neurons. At the end of photostimulation series, saturation of the AKAR3EV sensor was obtained by bath application of a maximally effective concentration of the adenylate cyclase activator forskolin (FSK, 12.5 μM; [START_REF] Gervasi | Dynamics of protein kinase A signaling at the membrane, in the cytosol, and in the nucleus of neurons in mouse brain slices[END_REF]) (Fig. 2A). Photostimulation induced clearly detectable peaks of PKA activity in 93 ± 7 % of individual layer II/III (n=34 out of 37, N=4 slices from 4 mice), and in 86 ± 14 % of individual layer V (n=18 out of 21, N=3 slices from 3 mice) pyramidal neurons.

Responses were transient and were consistently observed at each stimulus upon repetitive stimulation (interval between photostimulation trains, ~10 min, see examples in Fig. 2A and Fig S2). Despite individual variability (Fig. S3), mean responses of layer V pyramidal cells to five successive photostimulus trains exhibited roughly stable amplitudes that ranged between 86 ± 14 % and 100 ± 10 % of the first response (n=18 cells, N=3 slices from 3 mice; Fig. 2A, Fig. S3 and Table 1). Essentially similar results were obtained in the dendrites of these neurons (Fig. S4), although larger mean amplitudes were observed in dendrites than in somata (Fig. S4 and Table 1). The increase of the FRET ratio elicited by the first photostimulation in the soma of these neurons was 7.4 ± 1.2 %, which represents roughly one third of their response to FSK (25.7 ± 2.0 % increase of FRET ratio, Table 1). For comparison, PKA responses of cortical pyramidal cells to bath application of a saturating dose of NA reach 60 % of the response to FSK [START_REF] Nomura | Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex[END_REF], corresponding to only twice the response amplitude measured here upon LC fiber photostimulation. This suggests that a significant proportion of the catecholamine receptors expressed in pyramidal cells are activated upon burst photostimulation of LC fibers. Responses of layer V pyramidal cells peaked within 1.5 min after photostimulation and decayed with time constants ranging from 140 ± 15 s to 178 ± 18 s (Table 1). Response kinetics did not significantly differ between the five photostimulation trains (Table 1).

Essentially similar results were obtained in layer II/III pyramidal neurons, except that response amplitude gradually declined upon repetitive stimulation (Fig. S2 and Table 1). We thus focused on somata of layer V pyramidal neurons in subsequent brain slice experiments. These results suggest that selective photostimulation of cortical LC fibers triggers the release of endogenous catecholamines, which robustly activate cAMP/PKA signaling in pyramidal neurons, thereby allowing the pharmacological characterization of their responses.

PKA dynamics evoked by LC fibers photostimulation are mediated by NA receptors

LC fibers can release both NA and DA in the forebrain [START_REF] Devoto | Evidence for co-release of noradrenaline and dopamine from noradrenergic neurons in the cerebral cortex[END_REF], 2005, 2008, 2015[START_REF] Kempadoo | Dopamine release from the locus coeruleus to the dorsal hippocampus promotes spatial learning and memory[END_REF], and both NA and DA receptors are widely expressed in the neocortex, beyond the restricted distribution of dopaminergic fibers [START_REF] Ariano | Dopamine receptor distribution in the rat CNS: elucidation using antipeptide antisera directed against D1A and D3 subtypes[END_REF][START_REF] Khan | Differential regional and cellular distribution of dopamine D2-like receptors: an immunocytochemical study of subtype-specific antibodies in rat and human brain[END_REF][START_REF] Luedtke | Immunoblot and immunohistochemical comparison of murine monoclonal antibodies specific for the rat D1a and D1b dopamine receptor subtypes[END_REF][START_REF] Rivera | Cellular localization and distribution of dopamine D(4) receptors in the rat cerebral cortex and their relationship with the cortical dopaminergic and noradrenergic nerve terminal networks[END_REF][START_REF] Oda | Dopamine D5 receptor immunoreactivity is differentially distributed in GABAergic interneurons and pyramidal cells in the rat medial prefrontal cortex[END_REF][START_REF] Nomura | Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex[END_REF]). In the parietal cortex, DA fibers are scarce and restricted to layer VI, but Gs-coupled D1-5 and Gicoupled D2-like DA receptors are nonetheless functionally expressed and coupled to cAMP/PKA signaling in layers II-V pyramidal cells [START_REF] Nomura | Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex[END_REF]. We thus characterized receptors involved in light-induced responses using specific antagonists of Gsand Gi-coupled NA and DA receptors. Following two photostimulation trials in control condition to assess the stability of the PKA response, the third photostimulation was performed in the presence of antagonist pre-applied for at least 10 min. Response amplitudes thereafter are expressed as percentage of the first response amplitude. Perfusion of the specific β1-adrenergic receptor antagonist CGP20712 (100 nM) almost completely prevented PKA activation (Fig. 2B, Table 2). The amplitude of the residual response was 12 ± 3 % of the first response (N=4, n=18). Conversely, application of the D1/D5 receptor antagonist SCH23390 (1 µM) did not significantly modify the response (117 ± 10 %, N=3, n=25; Table 2). These results indicate that light-induced PKA activation in pyramidal cells was essentially mediated by β1-adrenergic receptors, without detectable contribution of the D1/D5 receptors functionally expressed in these cells [START_REF] Nomura | Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex[END_REF]. We next tested whether responses to photostimulation also involve Gi-coupled receptors by applying the α2 adrenoceptor antagonist yohimbine (1 µM) and the D2-like dopaminergic receptor antagonist haloperidol (10 µM). Application of yohimbine resulted in an increase of response amplitude (143 ± 15 %, N=3, n=21; Fig. 2B, Table 2), whereas haloperidol did not significantly alter the response (87 ± 10 %, N=3, n=21; Table 2). Hence, PKA activation upon photostimulation of LC fibers is mediated by Gs-coupled β1 adrenoceptors and is dampened by co-activation of Gi-coupled α2 adrenoceptors. This suggests that NA released by LC fibers activates both receptor types in pyramidal cells, as also observed upon application of exogenous NA [START_REF] Nomura | Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex[END_REF]. Conversely, these results rule out the involvement of DA receptors in PKA responses of pyramidal cells to burst photostimulation of LC fibers.

Cortical NA transmission is critically controlled by catecholamine re-uptake

The clearance of released catecholamines involves their recapture into catecholaminergic axons or neighboring cells by various transporters showing low selectivity for NA vs. DA [START_REF] Grundemann | Molecular identification of the corticosterone-sensitive extraneuronal catecholamine transporter[END_REF][START_REF] Torres | Plasma membrane monoamine transporters: structure, regulation and function[END_REF]. We tested the role of the NA transporter (NET), DA transporter (DAT) and extraneuronal transporters of the organic cation transporter (OCT) family in light-induced PKA responses. We found that application of the specific NET inhibitor reboxetine (100 nM) largely increased the response to photostimulation, whose amplitude was more than twice that of the response to the first photostimulation (251 ± 23 %, N=5, n=27; Fig. 2C). This was accompanied by an increase of the response time-to-peak, without significant modification of decay kinetics (Table 2). In contrast, neither the specific DAT inhibitor GBR12783 (100 nM) nor the OCT inhibitor corticosterone (100 µM, [START_REF] Grundemann | Molecular identification of the corticosterone-sensitive extraneuronal catecholamine transporter[END_REF], significantly modified the amplitude of the PKA response (GBR12783: 86 ± 8 %, N=3, n=20; corticosterone: 90 ± 8 %, N=3, n=24; Table 2). These results indicate that the effects of NA released upon burst photostimulation of LC fibers are strongly reduced by reuptake through NET transporters. Conversely, DAT and OCT were not involved in shaping cortical catecholaminergic transmission from LC fibers.

Cortical NA transmission in vivo

In order to validate, in the intact neocortex, the main results obtained in brain slice, we used in vivo photostimulation of LC fibers and imaging of pyramidal neurons expressing the GAkdYmut PKA sensor and TurboRFP (see Methods and Fig. 1). We monitored GAkdYmut fluorescence intensity in dendrites and somata during time windows corresponding to three consecutive bursts of photostimulation (interval between bursts, 5 min, Fig. 3A). The photostimulation pattern used in slices (5 ms pulse at 10 Hz for 5 s) failed to evoke consistent responses in vivo (Fig. S5), presumably because side illumination through the cranial window (see Methods) and tissue light scattering properties resulted in suboptimal ChR2 activation in depth of the cortex. We thus increased photostimulation (5 ms at 20 Hz for 10 s) and observed clear responses, which consisted in transient GAkdYmut fluorescence increases in dendrites (see example in Fig. 3A). Mean responses of dendritic ROIs reached 5.7 ± 0.7 % of baseline GAkdYmut fluorescence (n=960 ROIs, 12 stimulation trials performed in N=6 mice). No change in dendritic TurboRFP fluorescence was observed in the same experiments (Fig. S6).

In order to establish a criterion to select regions of interest (ROIs) for further quantitative analyses of responses, fluorescence changes of each dendritic ROI were submitted to a statistical test to determine their individual responsiveness to photostimulation (see Methods).

Only ROIs whose post-stimulus fluorescence significantly differed from baseline were considered as responsive. Among the 90 dendritic ROIs shown in Fig. 3A, we found that 60, 67 and 58 ROIs significantly responded to the 1 st , 2 nd and 3 rd successive stimulation trials, respectively, with 42.2 % of these 90 ROIs responding to all three photostimulation trials (Fig. S7). Responses of individual dendritic ROIs showed ROI-to-ROI and stimulus-to-stimulus variability (Fig. S7), but averaged responses of pooled dendritic ROIs had stable amplitude upon repetitive photostimulation (see example in Fig. 3A). Although responses to photostimulation were also detectable in somatic ROIs (Fig. 3A), they were much less frequent compared to dendrites. Indeed, using the same statistical criterion as above, we found that 10, 7 and 8 out of 35 somatic ROIs significantly responded to the 1st, 2nd and 3rd successive stimulation trials, respectively, with only 5.7 % of these 35 somatic ROIs responding to all three photostimulation trials (Fig. S8). Furthermore, somatic fluorescence changes were of much smaller amplitude than dendritic ones (p<0.001, Mann whitney test, Fig. S8), consistent with the differential responsiveness of these subcellular domains to β-adrenoceptor-induced cAMP/PKA signaling (Castro et al. 2011). When considering all responsive dendritic ROIs over the three trials shown in Fig. 3A and Fig. S7, fluorescence increases were larger than 2 % of baseline in 99.5 % of these ROIs (n=59+67+58= 184 out of 185 traces, see above).

Conversely, among fluorescence responses higher than 2 % (225 traces), 81.8 % of traces passed our statistical test. Thus, in the rest of this in vivo study, we only considered dendritic fluorescence changes larger than 2 % of baseline for analyses of light-induced responses.

Among ROIs imaged in control conditions (n=960 ROIs, 12 stimulation trials performed in N=6 mice), those showing fluorescence changes > 2 % (76 ± 3 % of ROIs) were selected to calculate mean response kinetics, yielding onset (τ = 14.7 ± 4.5 s) and decay (τ = 154.1 ± 31.0 s) time constants. We then imaged GAkdYmut fluorescence in dendrites to characterize the pharmacological properties of responses to effective photostimulation (5 ms pulse, 20 Hz, 10 s) of LC fibers in vivo. In each experiment, photostimulation was performed first in control condition, then 15 min after i.p. injection of saline, and finally after i.p. injection of drug.

Response amplitudes remained stable after saline injection (103 ± 4 % of control, n=520 ROIs, N=6), but markedly changed after drug administration (Fig. 3B-C). Indeed, the response was virtually abolished 15 min after injection of the β-adrenoceptor antagonist propranolol (4 mg/kg, -11 ± 5 % of control, n=330 ROIs, N=3) and only partially recovered 30 min after propranolol injection (24 ± 5 % of control, n=330 ROIs, N=3, Fig. 3B). Conversely, response amplitude was largely enhanced 15 min after injection of reboxetine (10 mg/kg, 182 ± 14 % of control, n=190, N=3), reaching a maximum 20 min after injection (250 ± 20 % of control, n=125, N=2) before declining 30 min after reboxetine treatment (214 ± 13 % of control, n=190, N=3, Fig. 3C). These results confirm, in the cortex in vivo, that burst photostimulation of LC fibers triggers the release of NA, which activates PKA signaling in pyramidal neurons via βadrenoceptors, and whose effects are limited by reuptake through NET transporters.

DISCUSSION (1474 words)

We combined imaging of PKA sensors expressed in pyramidal neurons with photostimulation of ChR2-expressing LC fibers in the neocortex, in acute slices and in vivo. We found that burst photostimulation of LC fibers elicited fluorescence changes of the sensors, indicative of PKA activation in somata and dendrites of individual pyramidal cells. These responses were transient and their amplitude was stable upon repetitive stimulation. The use of selective antagonists showed that responses were mediated by 1 adrenoceptors and dampened by co-activation of 2 adrenoceptors, but did not involve DA receptors, showing that NA was the major catecholamine released from LC fibers. Responses to photostimulation were largely increased upon NET inhibition, indicating that the clearance of NA released from LC fibers is primarily achieved by reuptake through this transporter. Our results demonstrate that the present optogenetic tools provide sensitive and reliable means to analyze quantitatively the dynamics of NA transmission in the brain at cellular and subcellular levels.

Burst photostimulation of LC fibers triggers PKA activation

Biosensors of the AKAR series, which include AKAR3EV and GAkdYmut, are based on conformational changes upon phosphorylation of a PKA substrate domain and selectively report PKA activity in cellulo [START_REF] Zhang | Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering[END_REF]and 2005[START_REF] Allen | Subcellular dynamics of protein kinase A activity visualized by FRETbased reporters[END_REF], Gervasi et al. 2007[START_REF] Hu | VIP, CRF, and PACAP act at distinct receptors to elicit different cAMP/PKA dynamics in the neocortex[END_REF][START_REF] Bonnot | Single-fluorophore biosensors based on conformation-sensitive GFP variants[END_REF]. Our results thus indicate that burst photostimulation of LC fibers evoked robust cAMP/PKA signaling transients, clearly detectable in real time at the level of single pyramidal neurons and their dendrites, in slices and in vivo. Light-induced responses in slices reached one-third the amplitude of responses to FSK, indicating that NA released by LC fibers is able to activate a significant proportion of somatodendritic 1 adrenoceptors present at the membrane of pyramidal neurons, despite reuptake by NET. For comparison, the maximal effect of exogenous NA is about two-third of the FSK effect on cortical pyramidal cells [START_REF] Nomura | Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex[END_REF], suggesting that NA released upon burst photostimulation in slices reached the membrane of these neurons at a mean concentration close to its EC50 (25 nM, [START_REF] Nomura | Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex[END_REF]. Smaller light-induced responses were observed in vivo, reaching only ~8 % of the FSK effect previously measured in slice (74 % increase of GAkdYmut fluorescence, [START_REF] Bonnot | Single-fluorophore biosensors based on conformation-sensitive GFP variants[END_REF], consistent with less efficient photostimulation of LC fibers in vivo. Indeed, in these latter conditions (see Methods), illumination angle, light reflection/refraction at cranial glass window interfaces, as well as light scattering and absorption through blood-perfused tissue, likely resulted in low illumination intensity of LC fibers in the imaging field in layer II/III. Dendritic responses were larger than somatic responses both in slices and in vivo. The differential responsiveness of these cellular domains to β-adrenoceptor-induced cAMP/PKA signaling in cortical pyramidal cells is documented, and attributed to the effect of phosphodiesterase type 4 that prevents diffusion of cAMP from the submembrane space to the cytosol, thereby favoring domains with a high surface to volume ratio, such as dendrites [START_REF] Castro | Type 4 phosphodiesterase plays different integrating roles in different cellular domains in pyramidal cortical neurons[END_REF]. Responses were time-locked on the photostimulus, and imaging at high temporal resolution in vivo (6 Hz) revealed rapid onset (τ = 15 s) and slower decay (τ = 154 s) kinetics, in good correspondence with our results in brain slices. Prior observations indicate that the time-course of light-induced responses of the biosensors followed that of the PKA signal in pyramidal cells: (i) responses of AKAR probes to cAMP photo-uncaging peak within 30 s [START_REF] Zhang | Insulin disrupts beta-adrenergic signalling to protein kinase A in adipocytes[END_REF], Gervasi et al. 2007), (ii) responses of AKAR probes and of cAMP biosensors exhibit similar onset kinetics upon activation of Gs-coupled receptors [START_REF] Castro | Type 4 phosphodiesterase plays different integrating roles in different cellular domains in pyramidal cortical neurons[END_REF]), (iii) relaxation kinetics of AKAR probes (i.e. their dephosphorylation by cellular phosphatases) are faster than the decay of the present responses to photostimulation [START_REF] Allen | Subcellular dynamics of protein kinase A activity visualized by FRETbased reporters[END_REF][START_REF] Dunn | Imaging of cAMP levels and protein kinase A activity reveals that retinal waves drive oscillations in second-messenger cascades[END_REF], Gervasi et al. 2007).

Hence, our results indicate that burst activation of LC fibers for a few seconds rapidly triggers robust, minutes-lasting cAMP/PKA signals in cortical pyramidal cells.

PKA dynamics evoked by burst photostimulation rely on NA but not DA transmission

The activation of PKA in pyramidal cells was essentially mediated by β1 adrenoceptors, consistent with NA release from LC fibers and with the effect of exogenous NA [START_REF] Nomura | Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex[END_REF]. The α2 antagonist yohimbine increased the amplitude of light-induced responses by 140%, an effect larger than that observed on responses to exogenous NA (117%, [START_REF] Nomura | Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex[END_REF]. This suggests that recruitment of α2 adrenoceptors dampens the response both by inhibiting adenylate cyclase in pyramidal cells and by acting at LC fibers to decrease NA release, as classically described [START_REF] Starke | Presynaptic autoreceptors in the third decade: focus on alpha2-adrenoceptors[END_REF]. The NET inhibitor reboxetine dramatically enhanced the amplitude of light-induced responses, but did not change their decay kinetics.

This indicates that NA reuptake by NET limits the number of receptors activated upon NA release by minimizing the spatial extent of NA diffusion, but is not involved in limiting the duration of NA transmission events. We exclude a role of DAT and OCT in shaping lightinduced responses, but cannot rule out a contribution of the corticosterone-insensitive uptake2 transporter PMAT [START_REF] Engel | Identification and characterization of a novel monoamine transporter in the human brain[END_REF][START_REF] Engel | Interaction of organic cations with a newly identified plasma membrane monoamine transporter[END_REF] in NA clearance, since the fluorescence of its antagonist decynium-22 [START_REF] Inyushin | Membrane potential and pH-dependent accumulation of decynium-22 (1,1'-diethyl-2,2'-cyanine iodide) fluorescence through OCT transporters in astrocytes[END_REF]) precluded its use in our imaging study. [START_REF] Devoto | Evidence for co-release of noradrenaline and dopamine from noradrenergic neurons in the cerebral cortex[END_REF][START_REF] Devoto | Stimulation of the locus coeruleus elicits noradrenaline and dopamine release in the medial prefrontal and parietal cortex[END_REF][START_REF] Devoto | 6-Hydroxydopamine lesion in the ventral tegmental area fails to reduce extracellular dopamine in the cerebral cortex[END_REF][START_REF] Devoto | Selective inhibition of dopamine-betahydroxylase enhances dopamine release from noradrenergic terminals in the medial prefrontal cortex[END_REF] have shown that electrical stimulation of the LC in vivo results in cortical release of both NA and its biosynthetic precursor DA. However, although both D1/D5 and D2-like receptors are functionally coupled to adenylate cyclase in pyramidal cells [START_REF] Nomura | Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex[END_REF], we failed to detect their involvement in PKA responses to photostimulation in cortical slices. This discrepancy presumably relates to the low potency of DA (notably a ~20 fold higher EC50 than NA) at stimulating PKA in cortical pyramidal cells [START_REF] Nomura | Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex[END_REF] may favor DA release and thus lead to the recruitment of DA receptors coupled to adenylate cyclase. Indeed, in these latter conditions, DA may accumulate in vesicles pending depletion of vesicular DBH that is co-released with NA [START_REF] Weinshilboum | Proportional release of norepinephrine and dopamine-β-hydroxylase from sympathetic nerves[END_REF], or may be released by reverse transport from the axoplasm upon inversion of ion gradients [START_REF] Levi | Carrier-mediated release of neurotransmitters[END_REF]Raiteri 1993, Sitte and[START_REF] Sitte | The reverse operation of Na(+)/Cl(-)-coupled neurotransmitter transporters--why amphetamines take two to tango[END_REF]. It is noteworthy that our in vivo data, i.e. the complete block of light-induced responses by the β-adrenergic antagonist propranolol, do not rule out DA receptor activation, since propranolol also prevents D1/D5 receptor-mediated effects of exogenous DA on pyramidal cells [START_REF] Nomura | Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex[END_REF]). Hence, further work is required to assess and characterize NA and DA co-transmission from cortical LC fibers.

Phasic noradrenergic transmission and burst firing of LC neurons

The firing of LC neurons consists either of single spikes emitted at low frequency (tonic mode), or of bursts of action potentials (phasic mode) that involve synchronization of the population of LC neurons (Aston-Jones andBloom 1981a, 1981b). The burst firing of LC neurons is associated with a peak of NA release and has important behavioral correlates (Florin-Lechner 1996, Aston-Jones and Cohen 2005). Our results obtained upon bulk photostimulation indicate that a single episode of synchronized burst firing in LC neurons has rapid and long lasting effects on the cortical network, and that phasic NA transmission operates with higher intensity and temporal precision than generally thought of a neuromodulatory system relying on volume transmission. Light induced responses exhibited cell-to-cell and dendrite-to-dendrite variability, as observed upon exogenous NA application [START_REF] Nomura | Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex[END_REF], presumably reflecting variations in distance to NA release sites, involvement of α2 adrenoceptors, and basal level of cAMP/PKA activity. Nonetheless, population responses were robust and reproducible.

Gs-coupled receptors enhance neuronal excitability by decreasing PKA-sensitive membrane potassium currents with onset kinetics similar to those of AKAR probes responses (Gervasi et al. 2007[START_REF] Hu | VIP, CRF, and PACAP act at distinct receptors to elicit different cAMP/PKA dynamics in the neocortex[END_REF]). Based on these latter studies, the present light-induced PKA responses (~30 % of FSK effect) would translate into a ~50 % reduction of PKA-sensitive potassium currents in pyramidal neurons. Hence, a single episode of synchronized burst firing in LC neurons likely induces a rapid and substantial increase of the excitability of pyramidal neuron populations with potentially important acute and long-term effects on the function of the cortical network.

Imaging the cellular dynamics of catecholaminergic transmission

The detectability of endogenous catecholaminergic transmission in vivo has been recently demonstrated using fluorescent cAMP or PKA activity sensors expressed in neurons or glia [START_REF] Goto | Circuit-dependent striatal PKA and ERK signaling underlies rapid behavioral shift in mating reaction of male mice[END_REF][START_REF] Ma | A Highly Sensitive A-Kinase Activity Reporter for Imaging Neuromodulatory Events in Awake Mice[END_REF][START_REF] Oe | Distinct temporal integration of noradrenaline signaling by astrocytic second messengers during vigilance[END_REF]). Here we show that 2-photon imaging of FRET or single-wavelength PKA sensors allows the quantitative analysis of key properties (intensity, kinetics, receptors and reuptake transporters) of NA transmission events in cortical neurons and their dendrites, in brain slice and in vivo. Our results confirm that any optical sensor of cAMP/PKA signaling is suitable for the imaging of endogenous NA transmission, pending sufficient dynamic range. In this regard, the bright AKAR3EV FRET sensor proved useful for the imaging in diffractive adult slice, despite smaller dynamic range than GAkdYmut (ΔR/R0: ~25% vs. ΔF/F0: 74 %, respectively, in response to FSK in similar experimental conditions, see Table 1 and [START_REF] Bonnot | Single-fluorophore biosensors based on conformation-sensitive GFP variants[END_REF]. Conversely, GAkdYmut enabled fast acquisition (375 x 375 µm frame at 6.2 Hz) to analyze PKA response kinetics with high resolution in vivo. This single-wavelength green sensor is well-adapted to 2-photon imaging, as fluorescence can be collected in its entire emission spectrum for increased sensitivity, and is easily compatible with other sensors for multicolor imaging, as exemplified here by the dual monitoring of GAkdYmut and TurboRFP in vivo. It is thus likely that GAkdYmut can be combined with singlewavelength cAMP or Ca 2+ red sensors with large dynamic range (e.g. [START_REF] Dana | Sensitive red protein calcium indicators for imaging neural activity[END_REF][START_REF] Ohta | Red fluorescent cAMP indicator with increased affinity and expanded dynamic range[END_REF][START_REF] Oe | Distinct temporal integration of noradrenaline signaling by astrocytic second messengers during vigilance[END_REF] for future examination of endogenous catecholaminergic transmission dynamics and their impact on network electrical activity. 2: Pharmacological properties of responses to photostimulation of LC fibers in cortical slices. Photostimulation consisted of 5 ms light pulses at 10 Hz for 5 s, and PKA activity was measured in somata of layer V pyramidal cells from 3 to 5 independent brain slices. * and † correspond to values significantly different from the values obtained for the first and second stimulation, respectively.

.

MATERIALS AND METHODS

Animals

Experiments were in accordance with the European Communities Council Directive 86/609/062, approved by the Charles Darwin ethics committee (project 01907.1) and the OIST Institutional Animal Care and Use Committee, and performed in accredited facilities.

Transgenic DBH-Cre mice were a gift from Bruno Giros [MMRRC line: Tg(Dbhcre)KH212Gsat/Mmucd, stock number 032081-UCD [START_REF] Gong | Targeting Cre recombinase to specific neuron populations with bacterial artificial chromosome constructs[END_REF]]. Genotyping of DBH-Cre mice was done by PCR with primers: forward AATGGCAGAGTGGGGTTGGG, reverse CGGCAAACGGACAGAAGCATT (225 bp). Animals were maintained in a 12 h light-12 h dark cycle, in stable conditions of temperature (22°C), with food and water available ad libitum.

Data were collected from 29 mice aged 5-7-weeks at the time of first viral injection, including 9 females and 13 males for brain slice experiments, and 7 females for in vivo experiments.

Recombinant viruses

The adeno-associated virus (AAV) driving Cre-dependent expression of a fusion protein containing channelrhodopsin 2 (ChR2) and the yellow fluorophore YFP (AAV2/1-EF1a-DIO-hChR2(H134R)-EYFP-WPRE-HGHpA) was produced from Addgene plasmid #20298 (a gift from Karl Deisseroth) at vector core facilities of Nantes University (France, titer: 3x10 11 gc/ml) and Pennsylvania University (USA, titer: 8x10 12 gc/ml). The Sindbis virus encoding the AKAR3EV PKA activity sensor [START_REF] Komatsu | Development of an optimized backbone of FRET biosensors for kinases and GTPases[END_REF]) was produced as described [START_REF] Hepp | Phosphodiesterase type 2 and the homeostasis of cyclic GMP in living thalamic neurons[END_REF], SIN-AKAR3EV titer: 1x10 9 ip/ml) and mixed with a Sindbis virus encoding the red fluorophore mCherry (SIN-mCherry, gift from Katia Boutourlinski, UMR8246 Paris) at a ratio 40:1. The AAV encoding the GAkdYmut PKA activity sensor [START_REF] Bonnot | Single-fluorophore biosensors based on conformation-sensitive GFP variants[END_REF]) was custom made and produced by the vector core facility of Pennsylvania University (AAV2/1-hSyn-GAkdYmut-hGH, titer: 4x10 14 gc/ml), and mixed with AAV2/1-hSyn-TurboRFP-WPRE encoding the red fluorophore TurboRFP (titer: 4x10 13 gc/ml, same supplier) at a ratio 1:1.

Expression of ChR2 and AKAR3EV for optogenetic experiments in cortical slices

Site-and cell-type-specific expression of ChR2 in NA neurons was achieved by viral transfer into the LC of DBH-Cre mice, exactly as described [START_REF] Nomura | Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex[END_REF]. Mice (1-month-old)

were anesthetized with isoflurane and the AAV-ChR2-YFP suspension (titer: 3x10 11 gc/ml) injected bilaterally at coordinates from bregma: anteroposterior (AP) -5.45 mm, mediolateral (ML) ± 1 mm, dorsoventral (DV) -3.65 mm, through an internal canula at a rate of 0.1 μl/min for 10 min (1 µl/site). For postoperative care, ropivacaine (10 mg/ml) was added dropwise on the skin suture, and mice received buprenorphine (0.1 mg/kg). Eight to ten weeks after AAV-ChR2-YFP injection, mice were anesthetized with 2 % isoflurane and the SIN-AKAR3EV/SIN-mCherry viral suspension mix was injected bilaterally in the primary sensory area of the parietal cortex at coordinates from bregma: AP -1.5 mm, ML ± 2 mm, DV -0.4-0.5 mm, through a glass pipette at a rate of 0.1 µl/min (2 µl/site). Mice received postoperative care as described above.

Ten to twelve hours after Sindbis virus injection, mice were deeply anesthetized with 10 mg/kg ketamine and 0.1 mg/kg xylazine before transcardiac perfusion with 20 ml ice cold high- NaHCO3, 20 D-glucose, 5 Na pyruvate (mM), supplemented with 2 mM kynurenic acid and 50 nM minocycline hydrochloride, and bubbled with 5% CO2/95% O2. Slices were then allowed to recover at room temperature for 1 hour. Slices were transferred into a recording chamber perfused with ACSF (2 ml/min, 30 °C, pH 7.4) bubbled with 95% O2 and 5% CO2. Slices were inspected for mCherry expression using wide-field fluorescence to localize viral transduction area with minimal excitation of ChR2.

Expression of ChR2 and GAkdYmut for optogenetic experiments in vivo

ChR2 was expressed in NA neurons essentially as described above. The AAV-ChR2-YFP suspension (titer: 8x10 12 gc/ml) was injected bilaterally into the LC of 5 to 7-week-old DBH-Cre mice at a rate of 10 nl/min (350 nl/site). Three weeks after AAV-ChR2-YFP injection, anesthesia was induced with a mixture of medetomidine (0.3 mg/kg), midazolam (4 mg/kg) and butorphanol (5 mg/kg), and a 5 mm craniotomy was performed under 2% isoflurane anesthesia.

Then, 70-140 nl of the AAV-GAkdYmut/AAV-TurboRFP suspension mix was injected in layer V of the parietal cortex at coordinates from bregma: AP -1.5 mm, ML 1.7 mm, DV -0.6-0.7 mm, at a rate of 10 nl/min. A chronic cranial window (5 mm glass coverslip) was next mounted as described [START_REF] Roome | Chronic cranial window with access port for repeated cellular manipulations, drug application, and electrophysiology[END_REF]. At the end of the surgery, mice received atipamezole (0.3 mg/kg) for recovery from anesthesia, and postoperative care as described above. Five to eight weeks after the last AAV injection, mice were head-fixed for imaging experiments performed under anesthesia with 1% isoflurane.

Optical stimulation and imaging in cortical slices

Two-photon images were obtained with a custom built two-photon laser scanning microscope described earlier [START_REF] Castro | Type 4 phosphodiesterase plays different integrating roles in different cellular domains in pyramidal cortical neurons[END_REF][START_REF] Bonnot | Single-fluorophore biosensors based on conformation-sensitive GFP variants[END_REF], and using a 60X (0.9 NA, Olympus)

water-immersion objective. Sampling rate was at 1.4 frame / s. For imaging of AKAR3EV, Instruments). Sampling rate was 30.9 frame/s.

ChR2 photostimulation was done with a collimated blue LED (450-495 nm, BDX, X-Cite XLED1, Lumen Dynamics) illuminating the area of the chronic cranial window with an angle of 30° from the glass surface. The power at the window glass surface was 0.35 mW/mm².

Photomultipliers were manually shut off during photostimulation.

Movies (512 x 512 pixel per frame, corresponding to a field of view of 375 µm x 375 µm) were analyzed with custom made MATLAB code and ImageJ. The 30.9 Hz imaging data was temporally binned to 6.2 Hz. ROIs were selected from the GAkdYmut channel and relative fluorescence change ∆𝐹 𝐹 (𝑡) was calculated as: )] + 𝐵

Histology

The expression of ChR2-YFP and dopamine-β-hydroxylase was examined on fixed brain slices using a polyclonal rabbit anti DBH (1/1000, AB1538, Merck-Millipore) and a polyclonal chicken anti-GFP antibody (1/2000, GFP-1020, Aves Labs), as described [START_REF] Nomura | Noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex[END_REF].

Secondary antibodies were Alexa Fluor 488 goat anti chicken (1/1000, Life Technologies A11039) and goat anti Rabbit-Alexa 555 (1/1000, Life Technologies A-21428). Slices were mounted in fluoromount-G (Clinisciences). Images were acquired using a confocal laser scanning microscope (SP5, Leica) or using wide-field epifluorescence (DMR microscope, Leica) and processed with ImageJ. Following in vivo imaging experiments, brains were fixed, sliced in 60 µM sections, and images were acquired using a wide-field fluorescence microscope (Nikon Eclipse, Nikon).

Drugs

Drugs were purchased from Tocris or Sigma-Aldrich. Drugs were bath applied on brain slices:

CGP20712 hydrochloride (100 nM), SCH23390 hydrochloride (1µM); Yohimbine (1 µM,)

Haloperidol (10 µM), Forskolin (12.5 µM), Reboxetine mysylate (100 nM), GBR12783 dihydrochloride (100 nM), Corticosterone (100 µM). For in vivo pharmacology, saline, Propranolol (4 mg/kg), Reboxetine mesylate (10 mg/ kg) were injected intraperitoneally.

Statistical analyses

In this report, N represents the number of brain slices or mice tested while n represents the number of cells or ROIs on which measurements were performed. Data are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed with MATLAB.

Unless otherwise stated, one-way ANOVA followed by Tukey's test was used to determine statistical significance, except for analyses of single ROI responsiveness in vivo.

  , which suggests in turn two hypotheses regarding DA transmission from cortical LC fibers. (i) DA transmission may occur through DA receptors preferentially coupled to phospholipase C, as documented for D1, D2 and D5 receptors (reviewed by Beaulieu and Gainetdinov 2011), thereby escaping our present detection using PKA biosensors. (ii)Alternatively, burst photostimulation in cortical slices may elicit only minimal DA release, whereas intense and prolonged activation of LC fibers in vivo (as performed byDevoto et al.)

FIGURE LEGENDS Figure 1 .

 LEGENDS1 FIGURE LEGENDS

Figure 2 .

 2 Figure 2. Imaging NA transmission triggered by photostimulation of LC fibers in cortical slices Two-photon imaging of layer V pyramidal neurons expressing the AKAR3EV sensor. (A) Upper: The greyscale image shows the F535 intensity and pseudocolor images show variations of the F535/F480 ratio value (coded by pixel hue) and F535 intensity (coded by pixel intensity), in pyramidal cells during the course of the experiment. Middle: variations of the F535/F480 emission ratio measured at the soma of these individual pyramidal neurons (n=7, grey traces, mean trace in red), in response to burst photostimulation (arrowheads) and to bath application of forskolin (FSK, 12.5 µM). Arrows indicate time points corresponding to pseudocolor images in upper panel. Lower: Results obtained in n=18 layer V pyramidal cells from N=3 slices, from one mouse, each. Differences are not significant. (B) Effects of β1 (CGP20712, 100 nM) and 2 (yohimbine, 1 µM) adrenoceptor antagonists, and of the NA transporter inhibitor reboxetine, (100 nM) on responses to photostimulation. Left: responses of individual pyramidal neurons (grey traces, mean trace in red) in the presence or absence of drug. Right, from top to bottom: results obtained in N=4 and n=18, N=3 and n=25, N=5 and n=27. * significantly different from the response to the first photostimulation.

Figure 3 .

 3 Figure 3. Imaging cortical NA transmission in vivo upon LC fibers photostimulation Two-photon imaging of cortical neurons expressing the GAkdYmut sensor. (A) Upper: The greyscale images (275 x 275 µm) show the 2-photon imaging plane (168 µm below dura in layer 2/3) with yellow circles delineating ROIs around GAkdYmut-positive dendrites and somata. Middle: Raster plots show GAkdYmut fluorescence intensity (calibration bar: ΔF/F) of these ROIs before and after each of three consecutive bursts of photostimulation (darker

}

  based on fluorescence resonance energy transfer (FRET) between a blue donor (ECFP) and a yellow acceptor (YPet), 2-photon excitation of ECFP was performed at 850 nm with a power of 30 mW. ECFP and YPet fluorescence signals (peaks 480 and 535 nm, respectively) were filtered (Chroma Technology: E700 SP, Semrock: FF01-479/40, FF01-542/50), separated with a dichroic beamsplitter (FF506-Di02-25x36 Semrock) and simultaneously detected in two channels (H9305 photomultipliers, Hamamatsu). Images were acquired every minute prior to ChR2 photostimulation, and then every 30 s following photostimulation.ChR2 photostimulation consisted in 470 nm light pulses delivered by a LED (LEDD1B, Thorlabs) through a 400 µm diameter fiber optic cable (ThorLabs) controlled with micromanipulators (ROE-200, Sutter Instrument). The light power at the tip of the optical fiber was 10 mW. The fiber tip was placed just above the slice surface and positioned 400 µm away from the center of the imaging field with an angle of 30° from the slice surface. Light pulses and a shutter (DSS25 and VDM1000, UNIBLITZ) protecting photomultipliers from light interference during photostimulation were controlled by custom software written in LabVIEW (National Instruments).The 512x512-pixel images were exported using MATLAB (The Mathworks), and analyzed with custom macros derived from ImageJ (U.S. National Institute of Health). Fluorescence intensity of regions of interest (ROIs) was calculated for each time point from average intensity z-projection of 3 frames by averaging pixel intensity. Fluorescence changes were measured as the ratio R=F535/F480. Pseudocolor hue saturation value (HSV) encoding of fluorescence intensity and ratio was performed using MATLAB custom procedures written by Hirokazu Tanimoto (Yokohama City University). Decay time-constants were obtained using the exp-XOffset fit function of IgorPro6 (WaveMetrics) using the equation:𝑦 0 + 𝐴𝑒𝑥𝑝 { -(𝑥-𝑥 0 )Optical stimulation and imaging in the cortex in vivoA combined wide-field/two-photon microscope (MOM, Sutter Instruments) with a femtosecond-pulsed Ti:sapphire laser (Vision II, Coherent) was used. To increase the point spread function of excitation the back aperture of the 25X water immersion objective (Olympus) was underfilled (spatial resolution 1 µm x 1 µm x 4 µm). The collar of the objective was adjusted to correct for the window glass thickness (170 µm). Simultaneous excitation of GAkdYmut (GFP-based single fluorophore sensor,[START_REF] Bonnot | Single-fluorophore biosensors based on conformation-sensitive GFP variants[END_REF] and TurboRFP was performed at 950 nm with a typical power of 5-11 mW. Fluorescence was detected in two channels by GaAsP photomultipliers (Hamamatsu) in spectral windows 490-550 nm (GAkdYmut) and 600-700 nm (TurboRFP), separated by a 560 nm dichroic mirror (all Semrock). The microscope was controlled by a commercial software (MScan, Sutter

,

  where 𝐹 𝑏𝑎𝑠𝑒𝑙𝑖𝑛𝑒 is the average fluorescence intensity during a 30 s interval ending 5 s before photostimulation. Onset and decay time-constants were obtained by fitting responses with the function:

  photostimulation). Response amplitude measured over the interval [20.0;52.4 s] (n=201 data points) was then compared to baseline signal (301 data points) using a Kolmogorov-Smirnov test. Throughout this manuscript, a p-value below 0.05 was considered statistically significant.

Table 1 : Properties of PKA responses obtained in pyramidal neurons from cortical slices upon five consecutive photostimulation episodes.

 1 Each photostimulation episode consisted of 5 ms light pulses at 10 Hz for 5 s. AKAR3EV imaging: R=F535/F480. Time to peak refers to the time between the beginning of the photostimulation and the maximum of the response. The value has been calculated for each response and the mean value has been replaced by a 30s long time interval to take in account the acquisition rate (see Methods). The decay time  was calculated by fitting the decay of the response with a single exponential. Time to peak and decay were calculated for responses in somata. Experiments were performed on 4 and 3 independent brain slices for layers II/III and V, respectively. *and † indicate values significantly different from those obtained for the first and second stimulation, respectively. NM: not measured.

	Photo-stimulation	1		2		3		4		5		Forskolin
	Cortical layer	II/III	V	II/III	V	II/III	V	II/III	V	II/III	V	II/III	V
	Amplitude in somata (R/R0 %)	7.3 ± 0.5 (n=34)	7.4 ± 1.2 (n=18)	6.0* ± 0.4 (n=34)	6.1 ± 0.9 (n=18)	4.9* † ± 0.3 (n=34)	5.6 ± 0.8 (n=18)	4.6* † ± 0.4 (n=34)	5.7 ± 0.9 (n=18)	4.5* † ± 0.3 (n=34)	6.4 ± 1.0 (n=18)	20.5 ± 0.9 (n=34)	25.7 ± 2.0 (n=18)
	Normalized
	amplitude in somata (%	100 ± 7.3 (n=34)	100 ± 9.8 (n=18)	77.6* ± 5.6 (n=34)	91.4 ± 11.5 (n=18)	58.36* ± 5.6 (n=34)	99.4 ± 19.9 (n=18)	59.7* ± 5.0 (n=34)	85.8 ± 13.6 (n=18)	53.1* ± 4.7 (n=34)	99.6 ± 12.9 (n=34)	NM	NM
	first stim)
	Amplitude in dendrites (R/R0 %)	NM	10,2 ± 1,8 (n=31)	NM	8,5 ± 1,5 (n=31)	NM	8,1 ± 1,5 (n=31)	NM	8,2 ± 1,5 (n=31)	NM	7,9 ± 1,4 (n=31)	NM	28 ± 5 (n=31)
	Time to peak range (s)	[60-90] (n=24)	[60-90] (n=17)	[60-90] (n=24)	[60-90] (n=17)	[60-90] (n=24)	[60-90] (n=17)	[60-90] (n=24)	[60-90] (n=16)	[60-90] (n=24)	[60-90] (n=17)	NM	NM
	Decay ( in s)	221.7 ± 17.1 (n=20)	178.4 ± 17.7 (n=12)	220.8 ± 26.4 (n=20)	147.4 ± 10.6 (n=12)	223.6 ± 27.4 (n=19)	172.2 ± 27.8 (n=12)	232.9 ± 24.4 (n=20)	159.7 ± 15.6 (n=12)	212.6 ± 28.3 (n=20)	140.5 ± 15.2 (n=12)	NM	NM

Table

This work was supported by grants from Ecole des Neurosciences de Paris ("Network for Viral Transfer"), Fondation pour la Recherche sur le Cerveau / Rotary Club de France, Agence Nationale de la Recherche ("IHU Institut de Neurosciences Translationelles de Paris", ANR-10-IAIHU-06), and the OIST Graduate University. Shinobu Nomura was recipient of a Nakajima Foundation fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Normalized amplitude

Photostimulus number

Supplementary Figure 2 1.25 Supplementary Figure 6


Marcus Metri Corrêa

Marília Costa Cavalcanti

Costa Dário

Fernando Primo

Rolim Cartaxo

Jean Neto

Rômulo Martins

Cezar Simões

Antonio Menezes
email: rmenezes@ufpe.br

Dantas Celso

Isaque Antonino
email: antonio.antonino@ufpe.br

Souza De

Lívia Regina Mendes

Santos Dos

Medeiros

Marcus Metri Corrêa
email: marcus.metri@gmail.com

; Marília

Costa Cavalcan

; Dário

Cos Primo

Fernando Cartaxo
email: fernandocartaxo@yahoo.com.br

Rolim Neto

Jean Manuel Martins
email: jean.martins@univ-grenoble-alpes.fr

Rômulo Simões

Cezar Menez

Antonio Celso

Dantas Antonin

Souza Mendes

; Lívia

Regina Dos

Santos Medeiros

Wastewater reuse in irrigation: short-term effect on soil carbon and nitrogen stocks in Brazilian semi-arid region

Keywords: carbon and nitrogen sequestration, greenhouse effects, land use change, soils

établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Reúso de água residual na irrigação: efeito de curto-prazo sobre os estoques de carbono e nitrogênio do solo no semiárido brasileiro RESUMO O principal processo natural que se contrapõe ao Efeito Estufa é chamado de sequestro de carbono, um fenômeno que essencialmente busca estabelecer um equilíbrio dinâmico entre a emissão dos gases do efeito estufa (GEE) a partir da imobilização de seus elementos, especialmente do C e N. O presente trabalho teve como objetivo principal avaliar os estoques de C e N em solo da região semiárida do Brasil submetido a irrigação com água residual tratada de esgoto doméstico. Para isso, o estoque de carbono e nitrogênio do solo soram comparados para quatro diferentes usos: caatinga nativa aberta (ONC); irrigação com água residual tratada (TSEI); irrigação com água superficial (SWI); e agricultura de sequeiro (TRA). A hipótese levantada na pesquisa foi de que o reuso de efluentes tratados na agricultura, além de ser uma alternativa que pode mitigar a problemática de escassez hídrica da região semiárida, também é capaz de influenciar a capacidade de estocar C e N no solo devido a sua carga orgânica. Os resultados indicaram que entre os diferentes usos de solos avaliados, os estoques de C e N no solo foi maior na ONC diminuindo com o uso do solo para a agricultura. O maior acúmulo de C e N na caatinga deve-se a presença de serrapilheira, não sendo detectado, no presente estudo, influência da irrigação com efluente de esgoto tratado. Conclui-se que o aporte de resíduos de vegetação na superfície do solo vem a ser o fator de maior contribuição para a estocagem de carbono e nitrogênio nos solos estudados.

INTRODUCTION

Although climate variation is part of the evolution of the planet, human activities significantly potentiate greenhouse gas (GHG) emissions and hence global warming [START_REF] Zhou | Investigating interior driving factors and cross-industrial linkages of carbon emission efficiency in China's construction industry: Based on Super-SBM DEA and GVAR model[END_REF]. Among the factors that contribute most to CO2 emissions are the combustion of fossil fuels (IPCC, 2013) and the conversion of native vegetation areas to areas for human use [START_REF] Kopittke | Global changes in soil stocks of carbon, nitrogen, phosphorus, and sulphur as influenced by long-term agricultural production[END_REF]. The current challenge is to seek to restore GHG concentrations in the atmosphere, and researchers around the world are turning their attention to two main questions: how to reduce GHG emissions and how to immobilize them.

In recent decades, terrestrial ecosystems have been considered as important as oceans in absorbing and storing C and N from the atmosphere, thereby minimizing the problem of the greenhouse effect. The largest C compartment of terrestrial ecosystems is the soil, where it is estimated that up to 30 cm deep it is about twice the amount of C present in the atmosphere as CO2 and up to four times that present in plant biomass [START_REF] Powlson | Soil management in relation to sustainable agriculture and ecosystem services[END_REF]. Therefore, it is clear that any increase or decrease in soil carbon stock has a significant impact on the atmospheric concentration of CO2 affecting the global climate [START_REF] Gao | Effect of different tillage systems on aggregate structure and inner distribution of organic carbon[END_REF]. Land use changes can also alter the stock of nitrogen (N) in the soil that is closely linked to the Soil Organic Carbon (SOC) stock [START_REF] Heyn | Soil organic C and N stocks in the first rotation of poplar plantations in Germany[END_REF].

Conversely, good management practices can increase soil organic matter content and contribute positively to soil C and N stocks [START_REF] Wells | Differences in soil organic carbon and soil erosion for native pasture and minimum till agricultural management systems[END_REF], in addition to reducing CO2 concentration in the atmosphere and potentially mitigating global climate change [START_REF] Gao | Effect of different tillage systems on aggregate structure and inner distribution of organic carbon[END_REF]. In Brazil, 77% of CO2 emissions are due to land-use change and the areas most affected are the Cerrado and Amazon biomes [START_REF] Lapola | Pervasive transition of the Brazilian landuse system[END_REF]. There is some research on the influence of land use changes on soil C and N stocks in the semi-arid region of Brazil [START_REF] Sacramento | Soil carbon and nitrogen stocks in traditional agricultural and agroforestry systems in the semiarid region of Brazil[END_REF][START_REF] Santana | Carbon and nitrogen stocks of soils under different land uses in Pernambuco state, Brazil[END_REF][START_REF] Santos | Land use changes the soil carbon stocks, microbial biomass and fatty acid methyl ester (FAME) in Brazilian semiarid área[END_REF], but there are information gaps that have yet to be filled, particular if the reuse of treated effluents in agriculture is Rev. Ambient. Água vol. 16 n. 1, e2623 -Taubaté 2021 considered as a sustainable management practice in agriculture. The National Semi-Arid Institute (INSA) has been recommending irrigation of forage palm (Opuntia stricta) with wastewater to provide food security in the livestock activity in the region [START_REF] Araújo | Palma forrageira: plantio e manejo[END_REF]. The reuse of domestic effluents as an alternative source of water in agriculture has been recommended in many studies as a means of increasing water availability in arid and semi-arid regions and addressing water scarcity [START_REF] Hamilton | Waste-water irrigation: the state of play[END_REF]Feinerman and Tsur, 2014;[START_REF] Bedbabis | Long-term effects of irrigation with treated municipal wastewater on soil. yield and olive oil quality[END_REF][START_REF] Martinez | Domestic wastewaters reuse reclaimed by an improved horizontal subsurface-flow constructed wetland: A case study in the southeast of Spain[END_REF] and to lessen the use of mineral fertilizers due to the presence of organic material [START_REF] Marinho | Application of reclaimed wastewater in the irrigation of rosebushes[END_REF].

Therefore, the use of effluents in agriculture can be not only a solution for water, but also an important ally for the mitigation of CO2 concentration in the atmosphere due to the organic material load present. With proper management, some of the carbon can be retained in the soil profile or used for biomass formation, which characterizes the mechanism of carbon sequestration from the atmosphere to the Earth's crust. However, it is known that in order to develop more systematically sustainable management practices and proposals, it is essential to have information on the impacts (positive or negative) on soils and their stocks of C, N and other nutrients, caused by the incorporation of new resources and technologies in anthropogenic activities, such as the treatment and reuse of domestic effluent in agriculture.

Given this scenario, this research is based on the assumption that the adoption of new techniques in agricultural systems, such as the agricultural reuse of wastewater, can influence the capacity of soils to accumulate C, N and other nutrients. The objective of this work was to assess the stocks of C and N in a Planosol soil, located in the municipality of Santana do Seridó, state of Rio Grande do Norte, semi-arid region of Brazil, under four different uses: Open Native Caatinga (ONC); Treated Sewage Effluent Irrigation (TSEI); Surface Water Irrigation (SWI); and Traditional Rainfed Agriculture (TRA).

MATERIALS AND METHODS

Field Site

The research was conducted in the municipality of Santana do Seridó, located in the southcentral zone of the state of Rio Grande do Norte (6°45'58'' S; 36°44'0'' W). The municipality covers approximately 188.4 km² and has a population of 2,680 habitants in 2019 (Figure 1). It is characterized by the Caatinga biome, with a semi-arid climate, classified as Bsh according to Köppen and Geiger, with prolonged droughts, low rainfall, annual average of 559 mm, and low hydric potential.

Land Use

In the experimental area, the soil was classified as Planosol, morphologically described and classified according to WRB (FAO, 2014), and four different land use types were identified: 1) Open Native Caatinga vegetation -ONC (6°45'56.2" S; 36°43'49.9" W), identified according to the criteria established by the Brazilian Vegetation Technician's Guide (IBGE, 2012); 2) Treated-Sewage Effluent Irrigation -TSEI (6°45'56.9" S; 36°43'55.7" W), agriculture with forage palm (Opuntia stricta) irrigated with treated domestic sewage effluent for animal feed; 3) Surface Water Irrigation -SWI (6°46'01.4" S; 36°44'00.7" W), agriculture with conventional sprinkler irrigation of tomatoes (Solanum lycopersicum L.), irrigated with surface water; and 4) Traditional Rainfed Agriculture -TRA (6°45'56.1" S; 36°43'54.7" W), traditional rainfed agriculture, subsistence crops, mainly maize (Zea mays L.) and beans (Phaseolus vulgaris L.), planted during the rainy season with minimum inputs, as shown in Figure 2.

A survey conducted at the site revealed that soil management in the study areas had been in place for at least four years prior to the date of collection of samples.

Sampling and estimation of soil carbon, nitrogen, calcium, magnesium, potassium and sodium contents

A total of 32 soil samples were collected for studies on the four land uses, resulting in eight samples per zone. All soil samples collected were packaged in plastic bags, properly labeled Rev. Ambient. Água vol. 16 n. 1, e2623 -Taubaté 2021 and sent to the laboratory for processing and analysis. At each site, six soil samples with volumetric rings (core method) were taken to determine soil bulk density, following the methodology of [START_REF] Blake | Bulk Density[END_REF].

At the laboratory, the soil samples were air-dried and sieved in 2 mm mesh size. An aliquot was kept for soil texture characterization, following the methodology presented by [START_REF] Embrapa | Centro Nacional de Pesquisa de Solos[END_REF], and another aliquot was grinded in a ball mill until it passed through a 100 mesh (0.150 mm). For the quantification of Total Organic Carbon (TOC) levels in the soil, the wet oxidation methodology of [START_REF] Yeomans | A rapid and precise method for routine determination of organic carbon in soil[END_REF] was used. For the Total Nitrogen (TN) concentration of the soil, the Kjeldahl [START_REF] Embrapa | Centro Nacional de Pesquisa de Solos[END_REF] method was used. Stocks were calculated on the basis of C and N concentrations and soil volume and density [START_REF] Veldkamp | Organic Carbon Turnover in Three Tropical Soils under Pasture after Deforestation[END_REF]) Equation 1:

Stock = (T)(Ds)(e)(1-Rv) 10 (1)
Where Stock represents the mass of C or N per unit area equivalent layer (Mg ha -1 ); T is the total C or N content in the sampled layer (g kg -1 ); Ds is the bulk density (kg dm -3 ); e = the thickness of the considered layer (cm) and Rv is the fraction of rock fragments (> 2mm).

The contents of Na and K were extracted with a solution of Mehlich-1 and determined by flame spectrophotometry [START_REF] Embrapa | Centro Nacional de Pesquisa de Solos[END_REF] and for Ca and Mg, the sample extracts obtained with 1 mol L -1 KCl were subjected to atomic absorption [START_REF] Embrapa | Centro Nacional de Pesquisa de Solos[END_REF]. Phosphorus was determined by colorimetry by reading the intensity of the color of the phosphomolybdic complex produced by the reduction of molybdate with ascorbic acid. The pH of the samples of soil was determined by direct electrochemical measurement of the effective concentration of H + ions in the soil solution using a combined electrode immersed in a soil/water suspension in a ratio of 1:2.5. To calculate the Sodium Adsorption Ratio (SAR), the concentrations of Na, Ca and Mg determined were used and the following equation was applied Equation 2:

𝑆𝐴𝑅 = 𝑁𝑎 √ 𝐶𝑎+𝑀𝑔 2 (2)
Where all concentrations were expressed as mmol Kg -1 .

Statistical analysis

The results of the TOC and TN contents and stock variables for all soils were subjected to an analysis of variance (ANOVA) followed by a test of comparison of means (Tukey, 5%). The statistical program used for the analyzes was Assistant 7.7 pt.

RESULTS AND DISCUSSION

The particle-size distribution of the soil samples collected under the different land uses did not show any significant difference, indicating that they were of the same soil class (Table 1), i.e., sand, thus verifying the predominance of a sand fraction. Sandy soils have a porosity that ensures an important infiltration rate. In addition, soil texture influences its chemical characteristics, such as lower organic matter content and decomposition rate, and low nutrient storage capacity. [START_REF] Sugihara | Effects of land management CO2 flux and soil C stock in two Tanzanian croplands with contrasting soil texture[END_REF] assessed carbon dynamics in two tropical growing areas in Tanzania with soils of contrasting textures and found that clay soils stored much more carbon than sandy soils. The authors attributed this to the possibility of carbon loss through leaching and macrofauna activity, but this is due to the fact that clays are capable of forming clay-organic complexes, which are less likely to decompose, providing greater physical protection for carbon. Bulk density is considered an attribute that can vary with different management practices. However, as with grain size and despite different land uses, no significant differences were identified between bulk density data for the studied areas, including the open native Caatinga area -ONC. Mean values obtained from four replicates for Particle-Size Distribution (granulometry) and three replicates for Bulk Density ± one standard deviation.

Source: Author.

Table 2 shows the total organic carbon (TOC) and total nitrogen (TN) contents for the different land uses. In the 0-10 cm layer, the native Caatinga vegetation (ONC) had a higher TOC content than the other areas used for agriculture, without significant differences for TN. Similar results were found in the Planosols of the municipality of Jataúba-PE, where concentrations were 8.7 and 8.9 g kg -1 C in conserved areas at depths of 0-14 and 0-17 cm, while in the degraded environment they were 5.6 and 3.6 g kg -1 C at depths of 0-9 and 0-20 cm, respectively [START_REF] Galindo | Relações solo-vegetação em áreas sob processo de desertificação no município de Jataúba-PE[END_REF]. The presence of higher C concentrations in open native Caatinga vegetation compared to agriculture areas, confirms the overall behavior that the replacement of forested areas by agriculture areas tends to decrease soil C and N concentrations [START_REF] Wei | Global pattern of soil carbon losses due to the conversion of forests to agricultural land[END_REF][START_REF] Kunlanit | Land-Use Changes Influencing C Sequestration and Quality in Topsoil and Subsoil[END_REF]. Non-significant differences in N content were also observed by [START_REF] Santana | Carbon and nitrogen stocks of soils under different land uses in Pernambuco state, Brazil[END_REF] who attributed this behavior to higher losses of C than N when land use changes occur. The results obtained here corroborate those of [START_REF] Santana | Carbon and nitrogen stocks of soils under different land uses in Pernambuco state, Brazil[END_REF], which resulted in a lower C/N ratio of agricultural areas compared to native open Caatinga.

In the Caatinga, TOC levels decrease considerably with increasing depth. This behavior is typical of the situation under native vegetation, because the high input of plant residues on the soil surface favours slow and progressive decomposition, which guarantees the constant incorporation of organic matter into the soil. However, the opposite situation was verified for TN levels, which is probably due to the type of plant residues incorporated (Table 2). As it is a Caatinga-type vegetation, the input of organic waste is poor in N, since, in the semiarid conditions, low soil moisture leads to lower microbial activity near the soil surface, which corroborates the considerations of López- [START_REF] López-Merino | Glomalin accumulated in seagrass sediments reveals past alterations in soil quality due to land-use change[END_REF] and [START_REF] Merino | Soil organic matter and phosphorus dynamics after low intensity prescribed burning in forests and shrubland[END_REF]. Similarly, the higher C content in the topsoil may be related to the virtual absence of microbial activity, with the incorporation of the most recalcitrant C compounds through physical and chemical degradation of the deposited plant residues [START_REF] Franzluebbers | Determination of microbial biomass and nitrogen mineralization following rewetting of dried soils[END_REF] Mean values obtained from four replicates ± one standard deviation. Means followed by the same letter do not differ statistically from each other for the same soil depth.

Source: Author.

Although high, the soil TOC content in Caatinga may correspond to OM that is not incorporated in the mineral fractions of the soil, thus becoming constantly dependent on the input of residues for its maintenance. During the drought period there would be indeed an increase in the content of soil elements, while during the rainy season, with the activity of the soil microbiota, there would be decomposition of these residues and mineralization of C. In this context, low levels of TOC and TN would be justifiable in the agricultural soils studied (Table 2), since in these cultivated areas, especially palm groves, there is little or no replacement of crop residues.

In relation to the additional contributions of organic compounds, caused by the application of irrigation with treated effluents, contrary to what was expected, the total carbon concentrations in the TSE-irrigated soil were significantly lower than those in other areas studied at a depth of 0-10 cm, although this tends to be in balance with the areas of increasing depth (Table 2). A similar result was described by [START_REF] Leuther | Structure and hydraulic properties in soils under long-term irrigation with treated wastewater[END_REF] in a study conducted in Israel under similar conditions. They found total C and N concentrations in samples from a soil without irrigation to be significantly higher than in a soil irrigated with treated effluent at a depth of 0-20cm, with carbon values of 1.74 and 0.83 g 100 g -1 and nitrogen values of 0.17 and 0.10 g 100 g -1 , respectively. These authors attributed these results to the contribution of organic residue from trees present in the non-irrigated area, without identifying any influence of irrigation water quality on soil carbon (C) and nitrogen (N) concentrations.

In the present research, the low quantities of irrigation water applied, due to the fact that the forage palm is a xerophilous plant, combined with the implementation period (4 years) of the reuse project, which can be considered low to promote changes in the organic fraction of the soil, may justify the insignificance of the effect of TSE on soil carbon (C) and nitrogen (N) concentrations. [START_REF] Haynes | Labile organic matter fractions as central components of the quality of agricultural soils: an overview[END_REF] suggests that short-to mediumterm crop management is not conducive to significant changes in TOC content. Therefore, in the experimental conditions of the present work, it can be deduced that the contents found would be more reacted to the organomineral complexes of the clay fraction, without irrigation or soil cover interfering with the C and N contents. The results found for the presence of C in soils reinforce what [START_REF] Oliveira | Características físicas e carbono orgânico de solos sob diferentes tipos de uso da terra[END_REF] verified in their research, showing that the soil surface layers are more sensitive to carbon variations due to the action of microorganisms linked to soil organic matter. [START_REF] Kopittke | Global changes in soil stocks of carbon, nitrogen, phosphorus, and sulphur as influenced by long-term agricultural production[END_REF] argue that C content tends to decrease in agricultural areas compared to native vegetation, since the conversion of native vegetation to crops reduces the input of plant debris and increases soil temperature, accelerating the decomposition of soil organic matter by microorganisms. [START_REF] Machado | Carbono do solo e a mitigação da mudança climática global[END_REF] points out that surface C levels tend to decrease, but show little change at greater depths.

The highest C/N ratio was observed in Caatinga soils, certainly due to the higher input of plant residues into the soil, while the lowest C/N ratio was found in the TSE irrigated soil (Table 2). The lower the C/N ratio, the greater the decomposition process of organic material. An increase in enzymatic activity has been reported in soils irrigated with treated wastewater [START_REF] Adrover | Chemical properties and biological activity in soils of Mallorca following twenty years of treated wastewater irrigation[END_REF], suggesting that irrigation with wastewater can stimulate the activity of microorganisms in the biochemical cycle of elements such as C, N and P, thus accelerating the mineralization of soil organic matter. This could explain the fact that, in this study, the area irrigated with TSE had the lowest values of C/N (more available nitrogen) compared to the other experimental sites. However, in the TSE irrigation site, lower organic matter contents can be found than in the surface water irrigation site. As mentioned above, it is likely that the lower C/N ratio favoured rapid mineralization, leading to a decrease in the organic matter content of the soil.

Table 2, which TOC and TN stocks in the soils of the different sites studied, indicates that TOC stock in Caatinga exceeds those of the other sites. It was noted that the average TOC and TN stocks for the surface layer of soils are much lower than those verified by [START_REF] Rossato | Caracterização molecular de populações de palmeiras do gênero Butia do Rio Grande do Sul através de marcadores ISSR[END_REF]. They found 86 Mg ha -1 and 54 Mg ha -1 of TOC stocks, respectively, in an area of native vegetation and in a corn culture area, sown manually without the use of agricultural input. These authors found also N stocks of 9 Mg ha -1 and 6 Mg ha -1 , respectively, in the same sites. A comparative analysis of the carbon stocks of different Brazilian biomes was carried out by [START_REF] Fidalgo | Estoque de carbono nos solos do Brasil[END_REF], verifying that the average TOC accumulation for the soils of the Caatinga Biome is 23.7 Mg ha -1 . [START_REF] Tiessen | Organic matter stability and nutrient availability under temperate and tropical conditions[END_REF] estimated the carbon stock for the soils of this region at 20 Mg ha -1 for the 0-20 cm deep layer, and [START_REF] Fraga | Declines of organic nutrient pools in tropical semi-arid soils under subsistence farming[END_REF] found, in a hyperxerophilic Caatinga, values of 17.9 Mg ha -1 and 28.6 Mg ha -1 for the 0-7.5 cm and 7.5-15 cm layers, respectively. These values corroborate the data found in this study for the Caatinga area.

For the other zones studied, the changes resulting from the anthropization process are certainly at the origin of the variation of the soil carbon stock compared to the local native vegetation (Caatinga), with a decrease varying between 44 and 62%. The effect of different cropping systems is highlighted in the study by [START_REF] Giongo | Carbono no sistema solo-planta no Semiárido brasileiro[END_REF], which assessed the carbon stock in preserved Caatinga, altered Caatinga, pastures with Buffel grass and irrigated cropping in a Yellow Argisol, obtaining decreasing values for the TOC stock in the respective order cited, with reductions in the range of 23 to 56%. [START_REF] Giongo | Carbono no sistema solo-planta no Semiárido brasileiro[END_REF] explained these reductions by the assumption that the high temperatures and intensity of sunshine, characteristic of the semiarid climate, associated with high water availability (by irrigation), increase the entropy of the system, resulting in a reduction of the soil carbon stock. In other words, because the rate of mineralization is highly dependent on the soil's water potential, the constant moisture level associated with high temperatures promotes rapid mineralization of organic material added to the soil, a fact also stated by [START_REF] Artiola | Longterm influence of liquid sewage sludge on the organic carbon and nitrogen content of a furrow-irrigated desert soil[END_REF]. Thus, effluent irrigation has the potential to modify the recycling processes of C and N by increasing soil moisture to levels that stimulate the O.M. decomposition activity of soil microorganisms. These claims may explain the lower values of content and stocks of these nutrients in irrigated areas, especially those irrigated with TSE. It can also be deduced that the deforestation of the native vegetation and of Rev. Ambient. Água vol. 16 n. 1, e2623 -Taubaté 2021 soil in rotation for cultivation in the areas studied has led to alteration of the soil structure and intense loss of C from soil to the atmosphere, considerably reducing the capacity of retention of this element in the soil. Coupled with this, it is likely that little or no input of plant residues in cropping situations will not allow for nutrient recovery and accumulation in the soils studied here.

In Table 3, different from what was observed in the levels of C and N, it can be seen that the soil irrigated with TSE had higher levels of Na, reaching, at a depth of 10-20 cm, an average value about 10 times higher than that observed in the Caatinga soil, also differing from the treatment with surface water irrigation. Similar results were observed by [START_REF] Barreto | Changes in chemical attributes of a fluvent cultivated with castor bean and irrigated with wastewater[END_REF] in their study on wastewater irrigation, which increased Na + concentration over the whole profile of soil. Indeed, unlike other cations, Na + has a low affinity for the soil exchange complex found mainly in the soil solution thus facilitating its leachability. Consequently, the Na + added from wastewater moves easily, justifying its higher concentrations with increasing depth [START_REF] Tarchouna | Effects of long-term irrigation with treated wastewater. Part 1: Evolution of soil physico-chemical properties[END_REF]. [START_REF] Barreto | Changes in chemical attributes of a fluvent cultivated with castor bean and irrigated with wastewater[END_REF] reinforce the above by stating that the rate of water application, the sodium content of the effluent, the absorption capacity of the crop and the soil texture can have a direct influence on the accumulation of this cation along the soil profile.

The high concentration of salts in soil, which may be due to the natural characteristics of the environment itself or to human activities, is capable of affecting the physical and chemical properties of the soil, making it difficult for plants to absorb water. It is noteworthy that high salt concentration in irrigated areas is common, since soluble salts present in irrigation water are added to the soil solution. This is not otherwise the case in the situations studied here, where irrigation water comes from treated sewage and also from a local surface reservoir potentially rich in salts due to the edaphoclimatic characteristics of the semi-arid region.

Salinization probably has the greatest negative impact on irrigated soils in arid and semiarid regions [START_REF] Diaw | Assessing of the Suitability for Irrigation Water and Their Repercussions on Land Degradation Process in Delta and Lower Senegal River Valley[END_REF], and this risk can be assessed by the soil Sodium Adsorption Ratio (SAR). For the soils in question, analyses indicate a SAR of irrigated soil higher with TSE than with the other waters (Table 3). [START_REF] Medeiros | Utilização de água residuária de origem doméstica na agricultura: Estudo das alterações químicas do solo[END_REF] also found in their studies that a soil that was irrigated had an increased SAR, both when using clean water or wastewater. A similar result was observed by [START_REF] Leuther | Structure and hydraulic properties in soils under long-term irrigation with treated wastewater[END_REF], who reported that soil sodicity was the most affected factor when comparing non irrigated and irrigated areas with freshwater and treated effluent, with the highest SAR value for plots irrigated with TSE. Although sodicity increased in the soil irrigated with TSE, soils from all treatments can be classified as not affected by salt, with a SAR <6 [START_REF] Rengasamy | Soil processes affecting crop production in salt-affected soils[END_REF]. This is due to the fact that the generally very poor sandy clay soils, as in this study, tend to be non-saline and will not have structural problems related to SAR due to their natural high permeability, which facilitates salt leaching during the rainy season. Clay soils, on the other hand, tend to accumulate salts and are rich in sodium, with structural problems that can occur if SAR increases.

For P, K, Ca and Mg analyses of the soils, in general, the highest results were found in the area irrigated with surface water, followed by the area irrigated with TSE. The excess of soluble salts in soil solution resulting from the accumulation of Na + , Ca 2+ , Mg 2+ and K + in the irrigated soil horizons can be due to a combination of several factors, the main ones being soil management (mineral fertilization) and the presence of these elements in wastewater effluents or irrigation water [START_REF] Qadir | Crop and irrigation management strategies for saline-sodic soils and waters aime at environmentally sustainable agriculture[END_REF][START_REF] Ribeiro | Origem e Classificação dos Solos Afetados por Sais[END_REF]. The average soil phosphorus content differs between treatments, particularly in the area irrigated with water, which presented the highest P content. [START_REF] Kouraa | Reuse of urban wastewater by combined stabilization pond system in Benslimane (Morocco)[END_REF] evaluated the irrigation of potatoes and lettuce with raw sewage, treated wastewater and drinking water, noting that in one cropping year there was no change in the phosphorus content of the cultivated soils. The authors also state that for changes in soil chemical characteristics to take place, several years of irrigation are necessary, since the dynamics of this element are very slow. On the other hand, [START_REF] References | Some environmental problems associated with the use of treated wastewater for irrigation in Jordan[END_REF] contradicted the study mentioned above, because in only five months of growing eggplant irrigated with a treated effluent containing 28 mg L -1 of PO4 -, they found a significant increase in soil phosphorus contents. This variation in P content between studies reinforces what has been said about the influences, not only of the quality of water used for irrigation, but also of irrigation management, vegetation requirements, and the soil and climate characteristics of the region. With regard to the pH of soils, the values found are close to neutral in all treatments. These results corroborate those obtained by [START_REF] Barreto | Changes in chemical attributes of a fluvent cultivated with castor bean and irrigated with wastewater[END_REF], who did not observe any change in the pH of soils irrigated with treated effluents.

Rev. Ambient. Água vol. 16 n. 1, e2623 -Taubaté 2021

CONCLUSIONS

-Land use had a significant influence on soil carbon and no significant influence on total nitrogen, with native vegetation in Caatinga presenting the highest TOC content compared to the three other areas used for agriculture.

-TOC contents tended to decrease with depth, and the mean TOC and TN stocks for the 0-20 cm layer were 28.43 Mg ha -1 and 5.62 Mg ha -1 for the open Caatinga and 13.61 Mg ha -1 and 4.50 Mg ha -1 for the TSE irrigation area, respectively.

-The highest C/N ratio was found in Caatinga soils, certainly due to the higher natural input of plant residues in the soil, while the lowest C/N ratio was observed in the soil irrigated with TSE suggesting that irrigation with wastewater can stimulate the activity of soil microorganisms in the biochemical cycle of elements such as C, N and P, thus accelerating the mineralization of soil organic matter.

-The changes in the physicochemical properties of soils due to medium-term irrigation with TSE were mainly observed for significant changes in SAR, reaching, at a depth of 10-20 cm, an average value about 10 times greater than that observed in the Caatinga soil, also differing from the treatment with surface water irrigation.

-The four years of irrigation with TSE in the soil proved to be insufficient to significantly alter the soil TOC and TN stocks in the soil, and under the experimental conditions observed, the treated wastewater reuse technology behaved more like a source of GHGs than a sink.

Finally, it's necessary that long-term research be carried out with the objective of verifying if the reuse of domestic sewage in agriculture (in isolated or action) can combat global warming and provide water security needed in semiarid regions.

Figure 1 .

 1 Figure 1. Study area and its location in the state of Rio Grande do Norte, Brazil.

Figure 2 .

 2 Figure 2. Land uses studied. A) Open Native Caatinga (ONC); B) Treated Sewage Effluent Irrigation (TSEI); C) Surface Water Irrigation (SWI) and D) Traditional Rainfed Agriculture (TRA).

  Rev. Ambient. Água vol. 16 n. 1, e2623 -Taubaté 2021

Table 1 .

 1 Sand, silt and clay contents and density values at two depths in the four studied sites.

	Land Use/Depth	Particle Size Distribution (%) Sand Silt Clay	Bulk Density (kg m -3 )
		TSEI
	0-10 cm	84.06 ±1.14 9.82 ±0.77 6.12 ±0.83	1.59 ±0.03
	10-20 cm	82.85 ±1.75 9.60 ±1.63 7.56 ±1.00	1.69 ±0.04
		SWI
	0-10 cm	86.18 ±0.83 7.69 ±1.06 6.13 ±0.45	1.63 ±0.04
	10-20 cm	84.85 ±1.37 8.10 ±1.11 7.05 ±2.39	1.67 ±0.05
		TRA
	0-10 cm	83.52 ±2.63 10.52 ±1.55 5.96 ±1.18	1.55 ±0.04
	10-20 cm	80.17 ±1.85 11.60 ±1.50 8.24 ±1.42	1.62 ±0.05
		ONC
	0-10 cm	81.83 ±0.44 12.47 ±0.99 5.70 ±0.61	1.63 ±0.06
	10-20 cm	81.05 ±0.74 13.63 ±1.22 5.32 ±0.80	1.65 ±0.03

Table 2 .

 2 Soil C and N contents, Ratio C/N, O.M. content, Soil TOC and TN stocks at two depths in the four studied sites.

	Land Use	TOC (g.kg -1 )	TN (g.kg -1 )	C:N (g.kg -1 )	O.M. (Organic Matter) (g.kg -1 )	TOC Stock (Mg.ha -1 )	TN Stock (Mg.ha -1 )
				0-10 cm
	TSEI	3.71 ±1.11 b	1.08 ±0.26 a	3.52 ±0.83 a	6.38 ±1.90	7.87 ±2.34 b	2.29 ±0.55 a
	SWI	5.38 ±0.69 ab	1.00 ±0.21 a	5.52 ±0.80 a	9.25 ±1.20	11.73 ±1.51 ab	2.18 ±0.46 a
	TRA	5.45 ±1.37 ab	0.85 ±0.11 a	6.55 ±1.91 a	9.37 ±2.35	11.28 ±2.83 b	1.76 ±0.23 a
	ONC	9.70 ±3.07 a	0.98 ±0.63 a	13.28 ±7.95 a	16.69 ±5.29	21.15 ±6.70 a	2.14 ±1.38 a
				10-20 cm
	TSEI	2.54 ±0.90 a	0.98 ±0.22 a	2.60 ±0.76 a	4.36 ±1.54	5.74 ±4.06 a	2.21 ±0.98 a
	SWI	3.67 ±0.25 a	1.03 ±0.23 a	3.76 ±0.84 a	6.30 ±0.42	8.18 ±1.09 a	2.30 ±1.02 a
	TRA	4.41 ±0.64 a	1.05 ±0.18 a	4.29 ±0.77 a	7.58 ±1.10	9.57 ±2.78 a	2.28 ±0.78 a
	ONC	3.31 ±1.05 a	1.58 ±0.65 a	2.59 ±1.36 a	5.70 ±1.81	7.28 ±4.63 a	3.48 ±2.86 a

Table 3 .

 3 Chemical characterization of soil at two soil depths under different uses in Santana do Seridó / RN.Mean values obtained from four replicates ± one standard deviation. Means followed by the same letter do not differ statistically from each other for the same soil depth.

				Elements
	Land Use/Depths	P (mg kg -1 )	K (mg kg -1 )	Na (mg kg -1 )	Ca (mg kg -1 )	Mg (mg kg -1 )	pH	SAR
				0-10 cm
	TSEI	18.06 ±9.55 b 75.00 ±20.27 c 141.00 ±47.88 a 276.00 ±64.30 b 114.00 ±20.98 b 7.2 ±0.65 a 2.6 ±0.94 a
	SWI	34.74 ±13.13 a 160.50 ±51.68 a 57.00 ±33.80 b 524.00 ±53.73 a 158.00 ±9.10 a 7.3 ±0.34 a 0.8 ±0.48 b
	TRA	9.68 ±7.46 c	112.00 ±7.35 b	20.00 ±4.56 c	410.00 ±88.59 a 101.00 ±13.88 b 7.0 ±0.08 a 0.3 ±0.09 b
	ONC	9.57±2.98 c	109.50 ±9.10 b	24.00 ±9.38 c	508.0 ±144.53 a 92.00 ±21.07 b 6.3 ±0.07 b 0.4 ±0.10 b
				10-20 cm
	TSEI	13.07 ±7.69 b	80.5 ±24.95 c	215.00 ±93.65 a 274.00 ±41.59 b 119.00 ±29.35 b 7.9 ±0.24 a 3.8 ±1.37 a
	SWI	28.79 ±21.96 a 140.00 ±46.45 a 82.00 ±63.27 b 500.00 ±42.65 a 154.00 ±42.31 a 7.9 ±0.25 a 1.2±1.00 b
	TRA	5.08±3.39 c	121.00 ±20.07 b 23.00 ±5.39 c	379.00 ±60.63 b 113.00 ±22.92 b 7.2 ±0.29 b 0.4 ±0.10 b
	ONC	4.07 ±1.95 c	113.00 ±9.43 b	22.00 ±0.87 c	357.0 ±103.93 b 94.00 ±24.80 c 6.6 ±0.33 c 0.4 ±0.05 b

Source:

Author.

Palavras-chave: efeito estufa, sequestro de carbono e nitrogênio, solos, variação do uso da terra.

Rev. Ambient. Água vol. 16 n. 1, e2623 -Taubaté 2021

ACKNOWLEDGMENTS

The authors of this article would like to thank the CNPq, CAPES and FACEPE for the scholarships awarded to students and researchers as well as for the financial support provided by the following research projects/programs grants: Universal MCTI/CNPq Nº 14/2014, MCTI/CNPq Nº 19/2017 Nexus, MCTI/CNPq/CAPES/FAPS Nº 16/2014, FACEPE 04/2017, National Observatory of Water and Carbon Dynamics in the Caatinga Biome -NOWCDCB, FACEPE (APQ-0498-3.07/17), CNPq (465764/2014-2), CAPES (88887.136369/2017-00); and CAPES PrInt (88881.318207/2019-01; 88887.371204/2019-00). The authors also thank the Instituto Nacional do Semi Árido (INSA) for providing the structures and materials for the research activities of this study. IGE is part of the Labex OSUG@2020 (ANR10 LABX56).