Astrocytes mediate cerebral blood flow and neuronal response to cocaine in prefrontal cortex

Cocaine affects both cerebral blood vessels and neuronal activity in brain. Cocaine can also disrupt astrocytes, which are involved in neurovascular coupling process that modulates cerebral hemodynamics in response to neuronal activity. However, separating neuronal and astrocytic effects from cocaine’s direct vasoactive effects is challenging, partially due to limitations of neuroimaging techniques to differentiate vascular from neuronal and glial effects at high temporal and spatial resolutions. Here, we used a newly-developed multi-channel fluorescence and optical coherence Doppler microscope (fl-ODM) that allows for simultaneous measurements of neuronal and astrocytic activities alongside their vascular interactions in vivo to address this challenge. Using green and red genetically-encoded Ca2+ indicators differentially expressed in astrocytes and neurons, fl-ODM enabled concomitant imaging of large-scale astrocytic and neuronal Ca2+ fluorescence and 3D cerebral blood flow velocity (CBFv) in vascular networks in the mouse cortex. We assessed cocaine’s effects in the prefrontal cortex (PFC) and found that the CBFv changes triggered by cocaine were temporally correlated with astrocytic Ca2 + A activity. Chemogenetic inhibition of astrocytes during the baseline state resulted in blood vessel dilation and CBFv increases but did not affect neuronal activity, suggesting modulation of spontaneous blood vessel’s vascular tone by astrocytes. Chemogenetic inhibition of astrocytes during cocaine challenge prevented its vasoconstricting effects alongside the CBFv decreases but also attenuated the neuronal Ca2+ N increases triggered by cocaine. These results document a role of astrocytes both in regulating vascular tone of blood flow at baseline and for mediating the vasoconstricting responses to cocaine as well as its neuronal activation in the PFC. Strategies to inhibit astrocytic activity could offer promise for ameliorating vascular and neuronal toxicity from cocaine misuse.


Introduction
Cocaine directly affects both cerebral vessels and neuronal activity in the brain. Glia, in particular, astrocytes, are involved in neurovascular coupling (NVC) -a process that modulates cerebral hemodynamics in response to changes in neuronal activity, which is disrupted by cocaine 1 . Astrocytes interact with neurons and the surrounding blood vessels through their endfeet and processes that ensheath blood vessels and synapses 2 . Astrocyte processes terminate the action of glutamate released by neurons via the glutamate-glutamine cycle 3 and mediate CBF by modulating NVC 3 . Clinical studies have reported neuronal, astrocytic and vascular pathological alterations in the brain of cocaine users, including neuronal loss, reduction of glial brillary acidic protein (GFAP)-immunopositive astrocytes and reactive and degenerative changes of the cerebral micro vasculature 4 . Astrocytes have also been shown to restore synaptic glutamate homeostasis in the NAcore after repeated cocaine exposure in rodents, and their manipulation can attenuate relapse after cocaine withdrawal 5 indicative of cocaine's affects in neuro-glio-vascular (NGV) processes.
Cocaine's effects on cerebral blood vessels versus those in neurons and astrocytes are confounded by their interactions and change with chronicity of cocaine exposures. Thus, distinguishing these effects ideally requires simultaneous multi-parameter measurements performed longitudinally in vivo. Moreover, studying the role of astrocytes in brain function is di cult because their removal causes neuronal death 3 .
Thus, much of what we know about astrocyte function has resulted from studies of isolated mammalian astrocytes in vitro, which cannot inform on how they interact with neurons and the surrounding vessels. Additionally astrocytes are hard to study with electrophysiological tools due to their slow changes in membrane potentials 3 . However, since astrocyte activation involves intracellular Ca 2+ increases, the detection of astrocytic Ca 2+ (Ca 2 + A ) signaling can be used to monitor their activity. Electrophysiological and Ca 2+ imaging studies using rodent brain slices have led to new insights into astrocyte-neuron interactions and to astrocytes' role in the activity of neuronal networks 6-8 . Also, with advances in in vivo imaging techniques such as two-photon microscopy (TPM), astrocyte activity and its correlation with cerebral blood ow are now monitored in living brains 9 . However, studies on Ca 2 + A signaling have been mostly conducted at the single-astrocyte level within a small eld of view (FOV) accessible such as by TPM rather than over larger astrocyte populations, from which synchronized Ca 2 + A signaling arises 10,11 .
Indeed, the behavior of synchronized Ca 2 + A signaling from large-scale astrocyte populations (e.g., astrocyte ensembles) is not well understood and neither is their functional role in NGV interactions.
Genetically encoded Ca 2+ uorescence indicators (GECIs) such as green GCaMP6f enabled us to image cortical neuronal or astrocytic Ca 2+ uorescence signals in separate groups of animals 12,13 . Recently, jRGECO1a, a sensitive red GECI was used to image neuronal activity [14][15][16] . Therefore, if combined with a green GECI (e.g., GCaMP6f), both indicators could be used to simultaneously image neuronal and astrocytic activities in the same animal allowing for studying their interactions. To achieve this, we developed a spectrally resolved uorescence imaging system capable of distinguishing two-color uorescence emissions.
Advances in optical coherence tomography (OCT) for 3D vascular imaging have led to OCT angiography (OCTA or OCA) to visualize the vasculature [17][18][19][20] and optical coherence Doppler tomography (ODT) 20,21 for quantitative CBF velocity (CBFv) imaging. We reported simultaneous imaging with ultrahigh-resolution OCA (µOCA) and ODT (µODT) based on phase-intensity-multiplexing to concomitantly obtain 3D microangiography and quantitative CBFv measures at capillary resolution 21,22 . 3D µODT measures intrinsic Doppler effect of moving red blood cells to image CBFv circumventing the need for a contrast agent. µODT allows 3D imaging of CBFv in arteries, veins, and capillaries 23 with high sensitivity (< 20µm/s) and a large eld of view (e.g., 3x2.4x1.4mm 3 ). Such an ultrahigh-resolution CBFv imaging technique provides a powerful tool to study the role of astrocytes in NGV interactions and to investigate cocaine's effect.
Here, we applied a novel multi-channel uorescence and µOCA/µODT microscope ( -ODM), which enabled us to concomitantly image large-scale astrocytic and neuronal Ca 2+ uorescence and 3D CBFv in vascular networks of the mouse cortex. We used -ODM to acquire genetically encoded Ca 2+ uorescence images of cellular activities in neurons (Ca 2 + N with jRGECO1a 14 ) and in astrocytes (Ca 2 + A with GCaMP6f 12 ) alongside the CBFv effects in response to cocaine in the prefrontal cortex (PFC) of GFAP-cre mice 12 in vivo. We hypothesized that cocaine enhances Ca 2 + A accumulation 24 , increasing the vulnerability of the brain to ischemia and thus jeopardizing neuronal activities in the PFC. To assess the role of astrocytes in modulating NGV interactions in responses to cocaine, we used chemogenetics (Designer Receptors Exclusively Activated by Designer Drugs or DREADDs) to inhibit Ca 2 + A accumulation (e.g., GFAP-DREADDs(Gi)), which we hypothesized would reduce cocaine-induced neuronal Ca 2 + N activity and ameliorate CBF decrease in PFC.
To de ne the mechanisms by which astrocytic and neuronal activities are involved in NGV interactions in vivo, we used viral injection to express genetically encoded Ca 2+ indicators in mice in a cell-speci c manner. Figure 1b illustrates our approach to express astrocytic Ca 2+ A and neuronal Ca 2+ N in the PFC in vivo. The use of GFAP-cre mouse with delivery of two mixed viral vectors including 50% AAV5.CAG.Flex.GCaMP6f.WPRE.SV40 (#100835, Add-gene) and 50% AAV1.Syn.NES-jRGECO1a.WPRE.SV40 (100854, Add-gene) allowed us to express GCaMP6f for Ca 2+ A uorescence and jRGECO1a for Ca 2+ N uorescence in the cortex. Prior to imaging, a cranial window was implanted above the PFC as illustrated in Fig. 1c (see Method Section for details). Figures 1d1-d3 show simultaneous in vivo images of Ca 2+ A (d1) and Ca 2+ N (d2) uorescence from PFC of a GFAP-cre mouse at ~4wks after viral injection of synapsin jRGECO1a for neurons and cre-GCaMP6f for astrocytes. Ex vivo double staining with GFAP antibody to label astrocytes and NeuN antibody to label neurons con rmed astrocyte-(d1') and neuron-(d2') speci c Ca 2+ expressions. Astrocytes also modulate neuro-vascular coupling (NVC) through their process endfeet forming close interactions with neurons and microvessels 3 . Our ex vivo images (Figs. 1e-g) show that astrocytes (labeled by GFAP, green) ensheathe neurons (labeled by NeuN, red) and microvessels (red dashed lines -GFAP of astrocyte endfeet) in the mouse cortex (e, g). followed by a downshoot. Similarly, cocaine increased Ca 2+ A to a maximum of 3.87%±0.23% (m=5) at t p−A =10.6±0.4min, peaking slightly behind the t p−N of Ca 2+ N (p=0.008, m=5). However, Ca 2+ A did not return to baseline until t r−A =50.6±5.6min (m=5), indicating a longer-lasting effect of cocaine on astrocytes than on neurons (p=0.01, m=5). Meanwhile, cocaine decreased CBFv to -22.8%±5.1% (m=5) below baseline at t p−V =16.6±2.2min followed by a gradual recovery by t r−V =58.6±5.8min, also indicative of a long-lasting CBFv decrease presumably due to vasoconstriction.

Cocaine increased neuronal Ca
In addition, -ODM allowed us to concomitantly measure cocaine-induced transient CBFv changes in arteries, veins and capillaries. Figures 2d-e show time-lapse µODT images and the ratio changes over the baseline of a smaller volume (2×0.3×1.2mm 3 , marked by a dished box in Fig. 2c') in the mouse PFC acquired to quantify dynamic ow changes (2min/volume) from baseline (t<-4min) to t>50min after cocaine (1mg/kg, i.v., t=0min). The 3D µODT image in Supplemental Fig.S1 shows that the selected ROIs included pial ows in layer 1 and deep ows in layers 4-5. Under iso urane anesthesia, all three vessel compartments (f) showed ow decreases after cocaine injection, among which arteriolar (AF) and venular (VF) ows dropped −19.8%±6.3% (p<0.001, m=4; t=8-36min) and −29.3%±4.5% (p<0.001, m=4; t=6-36min), respectively, followed by a gradual recovery to their baselines at 49.5±4.6min and 45.5±3.2min, respectively. Although capillary ows (CF) showed an overall decrease to -13.5%±3.0% that peaked at 20±2.9min, individual ow changes varied, with increases over 7% in some capillaries and decreases over −35% in others. The heterogeneity in the capillary responses highlights the importance of measuring ow in multiple capillaries instead of isolated vessels when studying NGV interactions and its responses to cocaine.

Astrocytic Ca 2+
A , but not neuronal Ca 2+ N , correlated with cocaine-induced CBFv decreases Figure 2 illustrates the increases in Ca 2+ N and Ca 2+ A and the decreases in CBFv triggered by cocaine in the mouse PFC. Unlike the short-lasting dynamic change in Ca 2+ N uorescence (e.g., <30min), cocaine induced long-lasting changes in Ca 2+ A and CBFv (e.g., 50-58min). To analyze the temporal relationship among these cellular and vascular changes, we studied 6 additional mice to examine the effects of acute cocaine (1mg/kg, i.v.) on neuronal Ca 2+ N, astrocytic Ca 2+ A and microvascular CBFv dynamic responses in the PFC. Figure 3 summarizes the cocaine-induced mean changes in Ca 2+ N , Ca 2+ A and CBFv within different vascular compartments (e.g., arteries, veins and capillaries) acquired from seven animals. The mean time-course changes in Fig. 3a indicate that cocaine induced a ΔCa 2+ N increase of 2.46±0.88% at t p−N =8.2±2.1min followed by recovery to baseline at t r−N =28.8±3.5min. Similarly, ΔCa 2+ A increased to 2.97±0.43% at t p−A =12.1±2.2min, but the effect was long-lasting and did not return to baseline until t r−A =59.5±8.0min. In parallel cocaine decreased mean vascular ΔCBFv to -25.1±4.9% at t p−V =21.0±2.9min, which slowly recovered to baseline at t r−V =64.0±7.5min, with a similar duration to that of ΔCa 2+ A . Statistical comparisons of the response duration to cocaine between ΔCa 2+ N , ΔCa 2+ A and ΔCBFv are summarized in Fig. 3b, which indicates that the response duration of Ca 2+ A and CBFv to cocaine was signi cantly longer (~2 folds) than that of Ca 2+ N (P*=0,005 and P*=0.002, respectively, n=7). No signi cant difference was found between t r−A and t r−V (P=0.62, n=7). The temporal correlations between cocaine-induced ΔCa 2+ A (t), ΔCa 2+ N (t) and ΔCBFv(t), were computed and the data derived from all 7 mice is illustrated in Supplemental Fig.S2. The results in Fig. 3c show a strong correlation between

Activation of GFAP-DREADDs(Gi) inhibited astrocytic Ca 2+ A and induced vasodilation and increased CBFv during baseline
DREADDS is a chemogenic approach that enables subtype selective activation (Gq) or silencing (Gi) of cellular signaling (e.g., astrocytes) via clozapine activation 25 . A cocktail of two viruses consisting of 0.4µl AAV5.CAG.Flex.GCaMP6f.WPRE.SV40 and 0.4µl AAV5.GFAP.hM4D(Gi)-mCherry was injected into the PFC of GFAP-cre mice. Figure 4 shows in vivo results using Gi-coupled DREADDS(hM4Di) expression in astrocytes (referred as GFAP-DREADDS(Gi)) into the mouse PFC. After 4-6wks from injection, time-lapse images of Ca 2+ A uorescence and cerebrovascular networks in the PFC were continuously acquired before and after clozapine injection (0.8mg/kg, i.p.) at t=0min for over 40min. Clozapine instead of clozapine-N-oxide (CNO) was used to activate DREADDS(Gi) because a recent study showed that clozapine (the CNO metabolite) rather than CNO itself stimulates the DREADDS receptor 25 . Figure 4b is a representative ratio ΔCa 2+ A image of a mouse PFC post clozapine (t=25min) over its baseline (t=-5min), showing Ca 2+ A decreases within the blue region of brain tissue along with vasodilation (red tracks in vascular trees, Fig. 4a). Details of vascular responses to clozapine activation of DREADDS(Gi) can be visualized in Supplemental movie VS1 and ratio image shown in Supplemental Fig.S3. Figure 4d summarizes the mean changes in vascular diameters and CBFv as a function of time post clozapine administration across animals (m=5 ROI/parameter/animal, n=5 mice). DREADD(Gi) activation resulted in vasodilation, with an increase of Δφ=9.17%±0.96% in mean vessel diameters (black trace) from baseline (t=-5min, Δφ=0.22%±0.35%) and an increase in ΔCBFv of 10.51%±2.95% at 25min post clozapine compared to baseline (t=-5min, ΔCBFv=0.21%±1.18%). The time course of ΔCa 2+ A in response to astrocyte inhibition is shown in Fig. 4e, revealing a signi cant decrease after 5min following clozapine injection (p<0.05); at t=25min post clozapine, ΔCa 2+ A decreased −3.0%±1.0% over its baseline (t=-5min, ΔCa 2+ A =0.02%±0.16%). These results provide evidence that astrocytic signaling modulates vascular tone at baseline, thus adjusting CBFv within the brain.

GFAP-DREADD (Gi) inhibition of astrocytic Ca 2+
A activation attenuated the decreases in CBFv and the neuronal activation triggered by acute cocaine To examine whether inhibition of astrocytic signaling (Ca 2+ A ) could block the CBFv decrease due to cocaine's vasoconstricting effects, we imaged the vascular responses to cocaine in the PFC without and with GFAP-DREADDS(Gi) activation by clozapine. Animal preparations were as described in the experiment shown in Fig. 4 but extended to assess the effects of acute cocaine. For this experiment, two sets of imaging sessions were conducted with at least 2hr separation between two sequential cocaine injections: images were acquired from 10min prior (baseline) to 60min after the rst cocaine injection (1mg/kg, i.v.); this was followed by a>40min no intervention period after which a clozapine injection (0.1mg/kg, 0.16ml) was given and 30min later the second in vivo imaging session was initiated and included a 10min baseline prior to and 60min following a second cocaine injection (1mg/kg, iv).

Astrocytic Ca 2+
A uorescence and CBFv images were continuously recorded prior to and following each cocaine injection (n=3). In an additional group of animals (n=4) we assessed whether inhibition of astrocytes in uenced the neuronal response to cocaine by imaging neuronal Ca 2+ N and astrocytic Ca 2 uorescence and CBFv changes in response to cocaine without and with GFAP-DREADD(Gi) activation by clozapine.

Figure 5(a-b) show the temporal responses of mean astrocytic Ca 2+
A to cocaine before and after DREADD(Gi) activation, in which the insets illustrate the corresponding representative ratio images of

While GFAP-DREADD(Gi) targets astrocytes and inhibits cocaine-induced Ca 2+
A increase (Fig. 5a), it was unclear whether it could indirectly modify neuronal responses to cocaine. Figure 5(g-h) shows the temporal responses of mean neuronal Ca 2+ N to cocaine before and after DREADD(Gi) activation to inhibit Ca 2+ A . Unlike the long-lasting response of astrocytes to cocaine (>60min shown in Fig. 5a) the neuronal Ca 2+ N increase returned to baseline at 30min after cocaine as illustrated in Fig. 5g. The full-width-halfmaximum duration of cocaine-induced Ca 2+ N increase was reduced from τ g =11.02±1.85min to τ h =2.24±0.62min after DREADD(Gi) activation. The ΔCa 2+ N rate decreased from 1.11%±0.29%/min to 0.11%±0.1%/min after GFAP-DREADD(Gi) activation (Fig. 5i), thus indicating a signi cant reduction of cocaine-induced neuronal activation (p*=0.02, n=4). Taking together with the effect of DREADDS(Gi)'s blockade on cocaine-induced CBFv decreases (Fig. 5f), this result indicates that cocaine's effects on CBFv and neuronal activity (Ca 2+ N ) were modulated by Ca 2+ A signaling. Thus, inhibition of astrocytic activity might help alleviate cocaine associated PFC dysfunction resulting from improper tissue perfusion and the decrease in neuronal reactivity might help reduce compulsive drug taking,

Discussion
Neuroimaging has advanced our understanding of the brain but there is need for tools with cellular and capillary resolutions capable of distinguishing signaling from distinct cell types alongside the dynamics of the vascular responses in order to investigate the roles of astrocytes and neurons on cocaine's effects in the neuro-glio-vascular (NGV) circuit. Here we integrate uorescence imaging and ultrahigh-resolution ODT to form multi-channel uorescence and optical coherence Doppler microscopy ( -ODM), and apply it to study cocaine effects on the NGV circuit within mouse PFC. We explored how acute cocaine affects astrocytic Ca 2+ A and neuronal Ca 2+ N activities and CBFv, and tested the hypothesis that cocaine induces Ca 2+ A accumulation resulting in vasoconstriction and CBFv decreases that disrupt neurovascular coupling. We then investigated the role of Ca 2+ A in cocaine elicited vasoconstriction, CBFv decrease and Ca 2+ N change, and tested the hypothesis that reducing Ca 2+ A via DREADDS(Gi) could relieve acute cocaine induced vasoconstriction, CBFv decreases and Ca 2+ N increases.

1) Cocaine-induced Ca 2+ A accumulation resulted in vasoconstriction and CBF decreases that disrupted neurovascular coupling
Astrocytic Ca 2+ A signaling cascades are involved in the communication between neurons and astrocytes, and astrocyte-to-astrocyte communicate via Ca 2+ waves that propagate signaling over a large range 3 .

Ca 2+
A may also regulate CBF independently of synaptic activity 26 and activation of Ca 2+ A may release glutamate to regulate synaptic homeostasis 5 . These ndings highlight the role of astrocytes in modulating neuronal function and hemodynamics.
Prior studies from our group and others have shown that astrocytes contribute to vasodilation during neurovascular coupling and to vasoconstriction that subsequently restores vascular tone 12,27 . However, studies on the contribution of astrocytes to cocaine's effects on the brain have mostly focused on its synaptic and circuitry regulation associated with its rewarding and addictive effects 28 . Only few studies have investigated the effects of acute cocaine on astrocyte activity, including an in vitro study that used brain slices from the nucleus accumbens incubated with cocaine that reported increases in Ca 2+ transients in astrocytes 29 . To our knowledge, there is no in vivo study that has simultaneously imaged Ca 2+ N and Ca 2+ A uorescence alongside CBFv changes in response to cocaine, thus the hybrid -ODM imaging platform reported here uniquely enables us to characterize how astrocyte and neuronal networks in the PFC interact and mediate the associated local neurovascular responses to cocaine.
Cocaine directly affects both cerebral blood vessels and neuronal activity in the brain [30][31][32][33] . Glia, in particular astrocytes, are involved in neurovascular coupling, which modulates hemodynamics in response to changes in neuronal activity 34 . Neurovascular coupling can be disrupted by use of addictive drugs such as cocaine and by disease processes such as Alzheimer's disease and other dementias. The ability to distinguish neuronal from vascular effects remains a challenge, partially due to technical limitations of neuroimaging techniques to differentiate vascular from neuronal and glial effects at high spatiotemporal resolutions. Here, we applied -ODM to study cocaine's effects on the neurovascular network and on neuronal and astrocytic activities in the PFC. Speci cally, we simultaneously imaged activations of Ca 2+ N , Ca 2+ A uorescence and local CBFv changes elicited by an acute cocaine challenge (1mg/kg, i.v.). Findings revealed a temporal association between cocaine-induced CBFv decreases (due to vasoconstriction) and the long-lasting astrocytic activation. Analysis of temporal correlations showed that cocaine-induced Ca 2+ A increases had strong negative correlations with the CBFv decreases. These ndings are in agreement with our recent results obtained in separate groups of animals 35 . In that study we used single virus containing GCaMP6f (not jRGECO1a) delivered into the somatosensory cortex and reported that cocaine-induced neuronal Ca 2+ N increases recovered by 30min followed by a downshoot similar to our observations in this study but in the PFC (Fig. 3a above). In our previous study we also showed that cocaine-induced Ca 2+ N changes were inversely correlated with temporal changes in tissue oxygenation 35 . The duration of neuronal activation in response to cocaine observed here and in our prior study is consistent with the pharmacokinetics of intravenous cocaine in the brain 36-38 and to the duration of striatal dopamine increases 39 . The inverse association between Ca 2+ N and tissue oxygenation indicates that cocaine's effects on neuronal activation and deactivation underly the changes in tissue oxygenation and might also underlie the reductions in brain glucose metabolism reported during cocaine withdrawal 40,41 . Although we had observed that the lasting increases in Ca 2+ A induced by cocaine were associated with vasoconstriction as assessed via quanti cation of vessel diameter changes 35 , the measurements of Ca 2+ A and Ca 2+ N changes were done in separate groups of mice, whereas in the current study the -ODM enabled us to assess the dynamic Ca 2+ A and Ca 2+ N responses simultaneously in the same animals. This was crucial for assessing the role of astrocytes in mediating the neuronal responses to cocaine alongside vascular function. It also allowed us to control for group variability. For example, in our prior study using separate groups of mice, the amplitude of cocaineinduced Ca 2+ A was 3 fold lower than that of the Ca 2+ N response 35 , whereas in the current study that measured them in the same animals there were no differences between the peak values of Ca 2+ A and Ca 2+ N (i.e., 2.97±0.42% vs 2.46±0.42%, p=0.82). This discrepancy likely re ects differences between the groups (i.e., WT vs GFAP-cre mice) in our prior study. Nevertheless, this study extended our previous ndings and showed for the rst time that inhibition of Ca 2+ A also attenuated cocaine-induced Ca 2+ N increases.

2) Reducing Ca 2+ A via DREADDS(Gi) relieved cocaine-induced vasoconstriction, CBFv decreases and Ca 2+ A increases
At baseline the inhibition of astrocytic activation decreased Ca 2+ A and resulted in vasodilation and CBFv increases but did not affect neurons in PFC. This suggests that at baseline astrocytes, but not neurons, mediate vascular tone, which is consistent with previous reports with two-photon microscopy (Takano et al, 2006). During a cocaine challenge, the inhibition of astrocytic activation prevented the CBFv decreases triggered by cocaine-induced vasoconstriction. Different from baseline, inhibition of astrocyte activation also blunted neuronal activation by cocaine. Together, these ndings indicate the involvement of astrocytes in mediating cocaine's effects on vasoconstriction and in modulating the neuronal responses to cocaine. Though the mechanisms by which astrocytes affect neuronal responses to cocaine are unclear it is possible that it could involve astrocytes' role in terminating the action of glutamate released by neurons via the glutamate-glutamine cycle 3 and in restoring synaptic glutamate homeostasis after cocaine exposure 5,24 . Moreover, manipulating astrocyte function attenuate relapse after cocaine withdrawal 5,42,43 .
Human studies have reported neuronal, astrocytic and vascular pathology in the brain of individuals with cocaine use disorder (as well as other drugs of abuse) that encompassed neuronal loss, reduction of glial brillary acidic protein (GFAP)-immunopositive astrocytes and reactive and degenerative changes of cerebral microvessels 4 . These observations imply that drugs including cocaine initiate a cascade of interacting toxic processes in the NGV circuit that are likely to contribute to the cognitive and behavioral changes observed in drug users.Intracellular Ca 2+ increases are associated with cell death 3 ; thus the cocaine-induced cellular Ca 2+ increases that we observed are clinically relevant, particularly since they occur in parallel with CBF decreases and hypoxia 40 . The use of a new -ODM enabled us to separate cocaine's effects on astrocytes, neurons and vascular networks to underpin their contributions to PFC dysfunction induced by cocaine. We focused on the PFC since clinical studies provide ample evidence of PFC dysfunction in drug users 1,31 that is implicated on the loss of control over drug taking 44 . Relevant to our ndings is a recent report that mediation of Ca 2+ A signaling ameliorated neuronal death and reduced behavioral de cits after ischemic stroke 45 . We had also reported that nifedipine (Ca 2+ antagonist and vasodilator 46 ) prevented cocaine-induced CBF decreases and neuronal Ca 2+ increases in PFC and reduced cocaine intake. In that study we interpreted nifedipine's actions to indicate neuronal effects, our current ndings suggest that blockade of L-type Ca 2+ channels in astrocytes are likely to be involved 47,48,49,50 . Our current ndings provide new insights into cocaine's effects on the NGV interactions that may provide new targets for development of novel addiction treatments.
A limitation for our study was that experiments were conducted in anesthetized mice using iso urane to avoid artifacts from animal motion during imaging. Iso urane-induced vasodilation might have facilitated the detection of cocaine-induced vasoconstriction, especially in capillaries and its anesthetic effects might have attenuated the sensitivity of neurons 52 and perhaps also of astrocytes to cocaine.
Another limitation was that in order to compare cocaine's effects with and without blockade of astrocytic activation by DREADDs(Gi), each animal was administered cocaine twice, which might have resulted in tolerance. In our study we gave the second cocaine dose 2hrs after the rst one, for we had previously shown that the hemodynamic responses to cocaine in PFC between two cocaine doses separated by a 2hr interval did not differ from one another 51 . Nonetheless, we cannot completely rule out the potential effects of tolerance to the second cocaine dose. Another limitation was the viral delivery protocol, which only allowed delivery of two types of viruses into the PFC to minimize risk of animal losses from viral injections and to ensure su cient amount of viruses (e.g., 0.4ul per virus) to be expressed in each cell type (e.g., neuron, astrocytes) for uorescence detection. The complexity of the studies also restricted the sample sizes of animals investigated, which precluded us to compare male and female responses to cocaine. Finally, the mechanisms by which astrocytes mediate vasoconstriction or neuronal responses to cocaine are unclear.
In conclusion, we observed that at baseline astrocytes mediated vascular tone but did not change neuronal activity whereas during a cocaine challenge they not only prevented the CBFv decreases but also attenuated cocaine-induced Ca 2+ N increases in PFC, Our ndings provide further evidence for the role of astrocytes in modulating NGV interactions in responses to cocaine both via direct effects in cerebral blood vessels and indirectly via their modulation of neuronal reactivity. Strategies to inhibit astrocytic activity could be promising in addressing vascular and neuronal toxicity from cocaine misuse. In addition, our ndings demonstrate the capabilities of -ODM for distinguishing activities of different cells (e.g., neuron, astrocytes) and its compatibility with other imaging tools for simultaneous monitoring of hemodynamics (or other processes).

Methods
Animals: All experiments were carried out according to National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee of Stony Brook University. GFAP-cre mice, obtained from Jackson Laboratory and maintained as a heterozygous line, were used for experiments when they reached the age between postnatal 60-70 days (P60-P70). All the information regarding the generation and genotyping of this line is available at https://www.jax.org/strain/024098. The mCherry (50479-AAV5, Addgene) was injected into the same PFC region. During viral injection, mice were anesthetized with inhalation of 2% iso urane mixed with pure oxygen and their heads mounted on a stereotaxic frame while we monitored their physiology. After completion of the procedure, the mice were monitored daily for a few days to ensure that they fully recovered from the surgery.
Cranial window implantation: A region of interest on the mouse PFC (A /P: +2.5; M/L: 0.5; D/V: -0.5 mm) was selected, where the cortical bone was rst thinned using a dental drill and then carefully removed, leaving the dura intact. The explored brain region was treated with dexamethasone sodium phosphate (50989-437-12, VEDCO) and then immediately covered by a 3.5×4.5mm 2 coverslip and sealed with biocompatible glue. Dental cement was spread around the edges of the coverslip to further secure its attachment with the skull for repeated imaging.
In vivo time-lapse -ODM imaging: Mice were anesthetized using inhalational iso urane (1.5%~2.5%) and the head mounted onto a stereotaxic frame. A custom -µODM developed in our lab was used to simultaneously image the CBFv networks and Ca 2+ N and Ca 2+ A uorescence in a spectral-multiplex, timesharing mode in the PFC (Fig. 1). For astrocytic GCaMP6f-Ca 2+ A and neuronal jRGECO1a-Ca 2+ N uorescence imaging, light beams from 10ms-duration pulsed narrow-band blue LED at 488nm and yellowish-green LED at 560nm of a light engine (Aura III, Lumencor) were combined in a light guide to illuminate a modi ed uorescence microscope (FN1 Nikon) for excitation. By spectral multiplexing, the epi uorescence cube C2 selectively allowed green Ca 2+ A (500-540nm) and red Ca 2+ N (574-670nm) emission from the mouse cortex (3⋅4mm 2 ) to be acquired by a sCMOS camera (Zyla 5.5, Andor) in a timesharing mode synchronized with the excitation pulses at up to 80fps. The recorded Ca 2+ A and Ca 2+ N activity was quanti ed as the relative uorescence change (∆F/F). For vascular imaging, a full-size 3D µODT image at 1.3um of mouse CBFv networks (2.4⋅2⋅1.2mm 3 ) was acquired in ∼15min, and time-lapse µODT images over a smaller volume (e.g., 2⋅0.3⋅1.2mm 3 ) were acquired per 45s or less for tracking ow dynamic changes (∆CBFv) along with Ca 2+ A and Ca 2+ N activations. Similarly, the ow network change was quanti ed as the ratio image (∆CBFv/CBFv). Immunohistochemistry: After in vivo imaging studies, the mouse was perfused transcardially with 0.1M PBS, followed by xation with 4% paraformaldehyde in 0.1M PBS. The frozen brain was sliced to 40~50µm in thickness. For GCaMP6f signal enhancement to astrocytes, the antibody [chicken anti-GFP (1:200) antibody] to green uorescence was used as the primary antibody followed by an Alexa Fluor 488 anti-chicken for GFP (1:200) conjugated secondary antibody. To identify GFAP-DREADDs(Gi) expression into astrocytes, antibody was used to enhance green uorescence emission in astrocytes, but no immunostaining was used for GFAP-DREADDS(Gi). The jRGECO1a expression to neurons was imaged with a confocal uorescence microscope (A1, Zeiss) without immunostaining for uorescence enhancement.
Statistics: All data are presented as mean±s.e.m. Data were analyzed by one-way or two-way mixed model analysis of variance (ANOVAs) and the Holm-Sidak method was used for post-hoc analysis.   Comparisons of cocaine's effects on neuronal Ca 2+ N , astrocytic Ca 2+ A uorescence and vascular CBFv in the PFC (n=7 mice). a) Mean ΔCa 2+ A (green), ΔCa 2+ N (red) and vascular ΔCBFv (black) responses to cocaine (1mg/kg, i.v.). b) Comparisons of return time to baseline between ΔCa 2+ A , ΔCa 2+ N and ΔCBFv, showing that the Ca 2+ A and CBFv responses to cocaine lasted signi cantly longer than that of Ca 2+ N , while there was no difference between ΔCa 2+ A and ΔCBFv (p=0.62, n=7). c) Temporal correlations of Ca 2+ A and Ca 2+ N transient changes vs CBFv changes in response to cocaine. Figure 4 a-c) Ratio images of cerebrovessels and of Ca 2+ A and Ca 2+ N uorescence at t=25min after GFAP-DREADDS(Gi) activation via clozapine vs their baselines at t=-5min, indicating vasodilation (red edges in a) and Ca 2+ A uorescence decrease (blue area in b) but no effects on Ca 2+ N (in c) in PFC; d-f) Mean time courses of Df increase or vasodilation and DCBFv increase in d) and Ca 2+ A decrease in e) but no Ca 2+ N change in f) after DREADDS(Gi) activation (n=5 mice). g) Correlation analyses of ΔCa 2+ A (t) and ΔCa 2+ N (t) vs. ΔΦ(t); h) A comparison of correlation coe cients of ΔCa 2+ A (t) vs ΔΦ(t) (green bar) and ΔCa 2+ N (t) vs ΔΦ(t) (red bar), showing a signi cant difference (*p=0.01).

Figure 5
Inhibiting Ca 2+ A with GFAP-DREADD (Gi) blocked cocaine-induced vasoconstriction and CBFv reduction and blunted the neuronal activation in response to cocaine in PFC. a-b) Cocaine-induced mean astrocytic

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