Ventral tegmental area astrocytes orchestrate avoidance and approach behavior

The ventral tegmental area (VTA) is a heterogeneous midbrain structure, containing neurons and astrocytes, that coordinates behaviors by integrating activity from numerous afferents. Within neuron-astrocyte networks, astrocytes control signals from distinct afferents in a circuit-specific manner, but whether this capacity scales up to drive motivated behavior has been undetermined. Using genetic and optical dissection strategies we report that VTA astrocytes tune glutamatergic signaling selectively on local inhibitory neurons to drive a functional circuit for learned avoidance. In this circuit, astrocytes facilitate excitation of VTA GABA neurons to increase inhibition of dopamine neurons, eliciting real-time and learned avoidance behavior that is sufficient to impede expression of preference for reward. Loss of one glutamate transporter (GLT-1) from VTA astrocytes selectively blocks these avoidance behaviors and spares preference for reward. Thus, VTA astrocytes selectively regulate excitation of local GABA neurons to drive a distinct avoidance circuit that opposes approach behavior.

T he ability to coordinate approach and avoidance actions in different environments can determine individual survival.Avoidance learning is an adaptive mechanism, but can become maladaptive and lead to the development and persistence of many specific anxiety disorders including post-traumatic stress and social anxiety disorders 1 .Dopamine signaling from the ventral tegmental area (VTA) mediates approach and avoidance learning 2 .However, the VTA is a heterogeneous structure containing astrocytes and neurons that receive mixed and distributed signals from various independent afferents conveying information for cues that drive both approach and avoidance 3,4 .For example, both aversive stimuli and reward expectation increase VTA GABA neuron firing and can inhibit dopamine neurons, leading to avoidance behaviors [5][6][7][8][9] .A mechanism capable of separating different signals underlying reward and aversion within the VTA is, therefore, necessary to appropriately coordinate approach and avoidance, respectively.By selectively affecting the activity of different neurons, VTA astrocytes may be positioned to coordinate avoidance and approach behaviors.
In the present study, we examined the role of astrocytes on VTA circuit activity and behavior.We found that VTA astrocytes (1) selectively facilitate glutamatergic excitation of local GABA neurons through a mechanism that requires astrocytic GLT-1; (2) generate a subsequent GABA neuron-dependent inhibition of dopamine neurons; and (3) consequently drive learned avoidance that is sufficient to block conditioned place preference for cocaine.Conditional mutants lacking GLT-1 in VTA astrocytes fail to express the tested avoidance behaviors, but maintain preference for reward.These experiments establish that VTA astrocytes selectively leverage glutamatergic excitation of local GABA neurons to dynamically define a privileged neuronastrocyte circuit underlying behaviors associated with aversion, but not reward.

Results
Astrocytes modulate excitation of VTA GABA neurons.To investigate the mechanism by which astrocytes regulate the circuitry of the VTA we obtained ex vivo recordings of VTA astrocytes (Fig. 1a).We first determined that neuronal afferent b Recording from a VTA astrocyte showing the current generated by electrical stimulation (black; 50 Hz, 20 pulses), the current remaining after blocking action potentials with 250 µM lidocaine (red), and after washing out lidocaine (green).c Summary of the currents before (black) and after lidocaine application (red; paired t-test t (5) = 2.679, P = 0.04, n = 6 cells, 4 mice).d Current generated in a VTA astrocyte by electrical stimulation of afferents before (black) and after adding 15 µm TFB-TBOA (red).e Summary of the currents generated by electrical stimulation in VTA astrocytes before (black) and after (red) addition of TFB-TBOA (paired t-test t (4) = 2.159, P = 0.04, n = 5 cells, 5 mice).*: P < 0.05 stimulation elicits action potential-dependent currents in VTA astrocytes (Fig. 1, and Supplementary Fig. 1).Electrical stimulation of the VTA during whole-cell voltage-clamp recordings of astrocytes resulted in a lidocaine-sensitive inward current (Fig. 1b, c).A significant portion of the inward current (35 ± 11%) was sensitive to the glutamate transporter blocker, TFB-TBOA (15 µM), indicating that, similar to other reports 14 , neuronal afferent input activates glutamate receptors and glutamate transporter activity on VTA astrocytes (Fig. 1d, e, and Supplementary Fig. 1).
We investigated whether local GABA neurons are subject to modulation by VTA astrocytes.We found that during voltageclamp recordings of VTA GABA neurons (Supplementary Fig. 4a-e), electrically-stimulated excitatory postsynaptic currents (EPSCs) increased in peak amplitude when the electrical stimulation was paired with ChR2 photoactivation on VTA astrocytes (GLT-1 f/f , Elec = 22.0 ± 3.7 pA, Elec + ChR2 = 32.4 ± 6.6 pA; paired t-test t (5) = 3.147, P = 0.025, n = 6 cells, 3 mice) with no effect on inhibitory postsynaptic currents (IPSCs) (GLT-1 f/f , Elec = 44.8± 18.1 pA, Elec + ChR2 = 40.4± 22.1 pA; paired t-test t (4) = 0.8756, P = 0.43, n = 5 cells, 3 mice; data not shown for GLT-1 f/f mice).When the same cells were recorded in cellattached mode, electrical stimulation of all afferents in the brain slice (no blockers were present) elicited a net pause in firing.When the electrical stimulation was paired with photoactivation of astrocytes, the pause duration decreased, which is consistent with an increase in EPSC amplitude observed in voltage-clamp mode (Fig. 2e, f).Astrocyte photoactivation had no effect on the intrinsic properties of neurons (Supplementary Fig. 5).
GABA neurons mediate increased dopamine neuron inhibition.Given the surprising result of a glutamate transporter mediating the increase of an outward current in dopamine neurons, we next determined whether the outward current was indeed an IPSC mediated by an inhibitory input.We tested the sensitivity of the outward current to the GABA A receptor antagonist, bicuculline (30 µM).Both the electrically-induced IPSC, and the peak amplitude increase elicited by pairing electrical stimulation with photoactivation of astrocytes, were abolished by addition of bicuculline, indicating that all outward currents were mediated by GABA A receptor activation (paired t-test t (4) = 3.682, P = 0.0015, n = 5 cells, 3 mice; Fig. 4a-c).However, only the increase in IPSC amplitude, not the electrically-elicited outward current per se, was sensitive to glutamate receptor antagonism (10 NBQX + 100 AP5, paired t-test t (7) = 3.101, P = 0.0173, n = 8 cells, 4 mice; Fig. 4d, e).Since (1) ChR2 activation increased the EPSC amplitude on VTA GABA neurons, and (2) the increased IPSC on dopamine neurons was dependent on glutamate receptor activation, dopamine neurons may be inhibited via a circuit mechanism involving depolarization of GABA neurons.

VTA astrocytes drive real-time avoidance mediated by GABA.
Previous studies demonstrate that direct optogenetic depolarization of VTA GABA neurons inhibits the activity of neighboring dopamine neurons and elicits avoidance behavior 6,7 .Our recordings demonstrate that VTA astrocytes can increase the response of GABA neurons to active excitatory afferent input, leading to increased inhibition of dopamine neurons.We, therefore, aimed to determine whether ChR2 activation of astrocytes would directly drive avoidance behavior.Extending our same optogenetic strategy to behavioral assays, we found that photoactivation of VTA astrocytes induced real-time avoidance in control mice in a real-time place preference (RTPP) assay (GLT-1 +/+ , paired t-test t (17) = 11.56,P = 0.0001, n = 18 mice; Fig. 6b, e; Supplementary Fig. 10, and Supplementary Movie 1).The effect of astrocyte activation was reversible since mice could be manipulated to avoid either side of the chamber by changing which side was paired with laser stimulation (Fig. 6b, c).The avoidance behavior was also dependent on astrocytic GLT-1 since conditional knockout restricted to VTA astrocytes (GLT-1 cKO VTA Astrocyte , gfaABC1D::ChR2 VTA ) eliminated the real-time avoidance elicited by photoactivating VTA astrocytes (paired t-test t (10) = 0.1399, P = 0.98, n = 11 mice; Fig. 6d, e).
We then confirmed that GABA neuron depolarization is necessary for eliciting real-time place avoidance by optogenetically hyperpolarizing VTA GABA neurons concurrently with ChR2 activation of VTA astrocytes.Similar to the increase in IPSC amplitude in dopamine neurons, real-time avoidance elicited by photoactivation of VTA astrocytes from VGAT Cre :: NpHR VTA mice was blocked by concurrently hyperpolarizing local GABA neurons (paired t-test t (6) = 3.642, eNpHR + ChR2; P = 0.01, n = 7 mice; Fig. 6g, h, and Supplementary Fig. 9).Therefore, astrocyte activation reliably induced real-time avoidance behavior through astrocytic GLT-1-dependent and GABA neuron depolarization-dependent mechanisms that are consistent with the circuit pathway established in our ex vivo experiments.
VTA astrocytic GLT-1 is necessary for open field avoidance.To determine the impact of VTA astrocytic GLT-1 function on endogenous behavior we tested whether selective deletion of GLT-1 in VTA astrocytes is sufficient to skew an animal's natural behavior in an open field test as an ethological assay that indexes approach-avoidance conflict.Mice with GLT-1 cKO restricted to VTA astrocytes (GLT-1 cKO VTA Astrocyte ) displayed a decreased tendency to remain in the peripheral zone near the walls of the chamber, and increased tendency to explore the center of the open field, relative to littermate controls (unpaired t-test t (16) = 2.449, P = 0.026, n = 9 mice; Fig. 8b, c).Disrupted approachavoidance behavior in GLT-1 cKO VTA Astrocyte mice indicates that VTA astrocytes are an integral component of a circuit within the VTA capable of coordinating approach and avoidance behaviors.

Discussion
Our experiments identify, through a series of genetic and optogenetic strategies in slice and in vivo, a specialized VTA microcircuit that is regulated by astrocytes to drive avoidance behavior.We observed that local astrocytes within the VTA circuit modulate glutamate availability via GLT-1 to dynamically regulate active GABAergic inhibition of dopamine neurons (Fig. 9).As a result, VTA astrocytes induced robust, time-locked behavioral avoidance de novo.Rapid manipulation of astrocyte glutamate transport in the VTA served as a powerful stimulus that, when paired with cocaine, was sufficient to impede behavioral expression of cocaine CPP.Thus, despite cocaine's many systemic effects, local astrocyte manipulation within the VTA was sufficient to override expression of preference for a highly salient cocaine-associated cue.These experiments suggest that midbrain a c d  astrocytes have the capacity to drive behavior, and not merely modulate the gain or polarity of an ongoing behavior.They underscore a computational role for astrocytes in the VTA network, and provide the first evidence that VTA astrocytes can selectively and reliably determine complex motivated behavior.The complexity of afferents arriving at the VTA points to its importance as a nexus where signals related to sensory experience, valence, and behavioral state intersect with associative processes to cue motivated behavior.For example, separate projections from the bed nucleus of the stria terminalis (BNST) to the VTA mediate both aversive and rewarding signals that promote and attenuate anxiety phenotypes, respectively 42 .But, the BNST provides equivalent projections to both GABA and dopamine neurons within the VTA 43 .Afferents from the pedunculopontine nucleus to both VTA GABA and dopamine neurons mediate approach behaviors like conditioned place preference for reward 44,45 .The VTA is therefore capable of parsing avoidance signals from different afferents to drive avoidance behavior and appropriately coordinate approach and avoidance.But a distinct circuit for avoidance capable of sufficiently opposing approach had not been described.
Inhibition exerts a powerful regulatory influence on dopamine neurons 46 , and prior studies establish that direct depolarization of VTA GABA neurons elicits real-time avoidance but fails to disrupt conditioned responses to reward-predictive cues 6,7 .Our experiments reliably reproduce the real-time avoidance results solely through optogenetic activation of VTA astrocytes.Furthermore, they illustrate that a single astrocytic mechanism can facilitate dopamine neuron inhibition, induces both active and learned avoidance, and interferes with expression of cocaine CPP.All rely on GLT-1 expression at VTA astrocytes; when VTA astrocytes lack functional GLT-1, cued conditioning fails to produce learned avoidance, and also fails to block expression of cocaine CPP (Fig. 7g, h).The selective influence of astrocyte mechanisms on avoidance but not approach in the dopaminergic VTA circuit reinforces the view that the microcircuits mediating avoidance and approach are functionally separate within the VTA, but converge at the level of inputs to dopamine neurons 9 .
Our results demonstrate that VTA astrocytes can exert opposing effects on GABA and dopamine neurons through serial excitation of GABA neurons and subsequent inhibition of dopamine neurons (Fig. 9).The unexpected lack of direct astrocyte-mediated glutamatergic effects at dopamine neurons concurrent with those observed at GABA neurons highlight the separate circuits for avoidance and approach, and may reflect a partitioning mechanism akin to the basal ganglia wherein distinct subpopulations of astrocytes are functionally linked to either D1-type or D2-type receptor-expressing populations of spiny projection neurons 17 (but see also ref. 47 ).One possibility is that astrocytic glutamate transport is differentially recruited by GABA and dopamine neurons in the VTA.Astrocyte populations that express distinct complements of transporters and receptors have been reported in the hippocampus 16,48 , and may similarly tile the VTA to partition neuron types and their synapses.Additionally, GLT-1 is expressed in both astrocytes and neurons 49 .Conditional genetic deletion of GLT-1 from neurons blunts the effects of amphetamine, and, in direct contrast to the VTA astrocyte GLT-1 knockout (Fig. 8), has no effect on open field behavior 50 .This indicates that, at least for our open field test results, any potential reduction in neuronal GLT-1 expression due to low-level leak expression of gfaABC1D-driven Cre in neurons would likely not affect our results.Characterizing the molecular diversity, morphology and distribution of astrocytes within the VTA will be required to fully mine the dimensions of their computational capacity 51 .
Astrocytes discriminate the activity of discrete synapses originating from different afferents 14,15,20 .Recent work points to the unique molecular and physiological profiles of ventral midbrain astrocytes relative to their telencephalic counterparts 51 , highlighting the need for approaches tailored to suit the functional dynamics of the system under study 52 .With this in mind, we implemented an astrocyte manipulation protocol aligned to the temporal dynamics of VTA dopamine neurons (where bursting occurs on the order of hundreds of milliseconds) and associated behaviors.Expression of ChR2 specifically in VTA astrocytes exploits the tight association between astrocyte cation permeability and GLT-1 function [25][26][27][28][29][30]35,37 . By enaging chiefly ion gradients, optical stimulation recruits native astrocyte machinery in electrochemically realistic processes.This approach succeeds in expressing astrocyte-specific, temporally controlled dynamics that make few assumptions about the relatively unknown cellular processes of VTA astrocytes 34 .Finally, the use of visible light wavelengths minimizes reactive gliosis, which often confounds modulatory dynamics with long-lasting inflammatory processes 53 .It is notable that in vivo astrocyte manipulation produced real-time aversion that was readily reversed and spatially manipulated (Fig. 6b, c), signifying that ChR2 activation of astrocytes reflects dynamic modulation rather than long-lasting reactive processes.Optogenetic manipulation of astrocytes also affects only those afferents that are active during ChR2 photoactivation (Fig. 2, 3, and Supplementary Fig. 8) 14 .Therefore, during our conditioned place paradigms, only those neuronal inputs that are active during the paired conditioning session will be facilitated during laser activation.
As a core symptom of anxiety disorders, maladaptive avoidance is considered to be an underlying mechanism maintaining anxiety 1 .Avoidance is a decision to refrain from potential negative outcomes, which could come at the expense of potential rewards.It is in this context that our finding is most relevant; VTA astrocyte GLT-1 can be manipulated to change inhibitory dynamics on dopamine neurons that interfere with the expression of conditioned place preference for reward.It is significant that the conditional loss of GLT-1 eradicates dynamical control of inhibition in the VTA and concurrently reduces avoidance behavior, but preserves approach to reward.Thus, separable pathways within the VTA drive behavioral responses to reward and aversion 9 , and may be subject to distinct pathophysiologies that culminate in maladaptive learning processes 1 .VTA astrocytes may, therefore, reflect a specific target for therapeutics, which have the added advantage of producing effects limited to activated synapses.
Our results establish a previously unknown, intra-VTA neuron-astrocyte circuit mechanism specific to avoidance behavior through which astrocytes exert fast, dynamic control of dopamine neuron firing by modulating afferent glutamatergic drive on GABA neurons (glutamatergic afferents → astrocyte GLT-1 → GABA neuron EPSP → dopamine neuron IPSP → avoidance; Fig. 9).We conclude that VTA astrocytes orchestrate temporal control of dopamine neuron firing, rendering them a decisive element in the circuit computation of avoidance, and promising targets for new therapeutic strategies to intervene in pathological disorders of behavior.

Methods
Animals.Male and female C57BL/6J mice, Vgat-ires-Cre mice (016962 Stock) and GLT-1 f/f (GLT-1 flox/flox ) 39 mice were used.GLT-1 f/f mice (Slc1A2tm1.1Pros;MGI: 5752263) were obtained from the founder colony at Boston Children's Hospital.All procedures were approved by the University of Texas at San Antonio Institutional Animal care and Use Committees in accordance with the National Institutes of Health guidelines.
Creation of AAV-TAS-gfaABC1D-ChR2-EYFP.With the creation of an abbreviated GFAP promoter that maintained astrocyte specificity 38 , expression is amplified by using a transcriptional amplification strategy (TAS).This includes cloning a mini-CMV promoter driving expression of Gal4 binding domain fused with the p65 transcriptional activation domain on the opposite strand, and 5 concatenated Gal4 binding sites between the two promoters, mini-CMV and gfaABC1D 54 .Using PCR, the TAS-gfaABC1D enhancer and promoter was amplified and isolated with AscI/SalI sites on the 5' and 3' ends, respectively.This fragment was subcloned into a AAV-CaMKII-ChR2-EYFP by digesting the plasmid with MluI/SalI to remove the CaMKII promoter and have compatible cohesive ends with the new promoter.The AAV vector was packaged and pseudo-typed with AAV5 at the UNC vector core.
All mice were implanted with custom-made fiber optic cannulas 100 µm above the injection site.Custom-made cannulas were constructed using 1.25 mm diameter ferrule, 200 µm fiber optic cable with NA 0.39 and a two-part epoxy.The fiber optic cannulas were secured to the skull of each mouse using miniature screws and dental cement.Mice were singly housed and allowed to recover for 12 days before behavioral experiments were performed.A total of 3 mice were excluded from analysis for having virus and/or fiber optics outside of the VTA.
Immunohistochemistry. Once electrophysiology recordings were completed, the slices were incubated in 4% paraformaldehyde (PFA) for 30 min and moved to phosphate buffered solution (PBS) with .02%sodium azide for long-term storage at 4 °C.
Mice used for behavioral experiments were anesthetized and intracardially perfused with ice-cold PBS, followed by 4% PFA.The brains were removed and placed in 4% PFA overnight at 4 °C.The brains were next rinsed with PBS and 100 µM coronal slices containing VTA were collected.The slices were placed in PBS with .02%sodium azide for long-term storage at 4 °C.
Flow cytometry.The VTA was carefully sectioned from GLT-1 f/f and cKO mice in 1X Hanks's Balanced Salt Solution (HBSS).The tissue was minced and incubated in pronase and repeatedly triturated every 20 min for 1 h.DNASE (100 units/ml, Worthington) was added to the HBSS containing pronase solution to prevent cell aggregation.After 1 h the cell suspension was diluted with 1× HBSS and centrifuged at 500×g for 5 min.The pellet was resuspended in HBSS and filtered through a 100 µm filter.The cells were counted, and the filtrate was centrifuged and resuspended in Phosphate Buffer Saline (PBS).
The cells were resuspended in 4% paraformaldehyde (PFA; Sigma) for 15 min and subsequently washed with 100 mM glycine (Sigma) in PBS to remove any traces of PFA.After centrifugation, the cells were resuspended in 5% donkey serum in 0.3% Triton-X for 30 min to block and permeabilize the cell membrane.The cells were then incubated with primary antibodies: mouse anti-GFAP (1:1000, G3893), rabbit anti EEAT-2 (1:1000, AB41621) followed by secondary antibodies: donkey anti mouse Alexa 488 (1:1000, A21202), donkey anti-rabbit Alexa 555 (1:1000, A31572) for 20 and 15 min, respectively.Cells were analyzed in a BD LSR II flow equipped with three excitation lasers (488, 555, and 633 nm).Briefly, events were selected based on their scatter properties indicative of cells, then selected for single cells, and selected based on fluorescence intensities.All gates were determined based on unstained, single-color and fluorescent minus 1 GLT-1 f/f of mouse brain cells.Gating analysis was done using FlowJo software (Treestar).
Open field test.Mice were placed in a custom-made plastic rectangular box (50 × 30 cm) and the box was divided into a center and outer zone with the outer zone being 7 cm from the wall.Mice were allowed to freely explore for 30 min.Anymaze (Stoelting) video tracking system was used to track the distance each mouse traveled and how much time they spent in each zone for all behavioral experiments.
Real-time place preference (RTPP).Mice were placed in a custom-made plastic rectangular box (50 cm × 30 cm) with no distinguishing cues.One side of the chamber was randomly designated to be the side where the lasers would be activated.Individual mice were initially placed in the chamber and allowed to explore before beginning the experiment.During the 30-minute session the laser would be activated (1 Hz: 0.5 s pulse duration, light intensity between 15 and 20 mW) each time the mouse entered the laser-paired side of the chamber, and would be turned off when the mouse crossed back into the non-stimulation side of the chamber.The difference score was calculated by taking the time the mouse spent in the Laser side minus time in the No Laser side.
Mice expressing ChR2 or eYFP in astrocytes were connected to a 473 nm laser using a 1 × 1 rotary joint.For mice expressing ChR2 in astrocytes and NpHR3.0 in GABA cells, we used a 2 × 1 rotary joint connected to 405 nm and 593 nm laser.This allowed us to activate either ChR2 or NpHR3.0 individually or together.The same set up was used for all RTPP experiments.
Laser conditioned place avoidance (Laser CPA).Laser CPA experiments were performed over a four-day period using a two-chamber conditioning apparatus (Med Associates) with each chamber consisting of a black chamber with a vertical metal bar floor, and a white chamber with a mesh floor.One mouse with a strong initial preference for either chamber (defined as a mouse spending more than 75% of the time in either chamber) was discarded from analysis.On day 1 (pre-test), mice were placed in one of the two sides and allowed to freely explore the entire apparatus for 30 min.During days 2 and 3 (conditioning) mice were confined to one of the two chambers (counterbalanced across all mice) and received optical stimulation (0.5 sec pulse, 18 mW) once a minute for 30 min, or no stimulation.During the no stimulation session mice were connected the same as during the laser session, but the laser was off.A minimum of 4 h after the first training session mice were placed into the other side of the chamber and the treatment reversed.Day 4 (post-test), mice were allowed to freely roam the apparatus for 30 min.The difference score was calculated by taking the time each mouse spent on the laser-paired side in the post-test minus the time spent in the pre-test.

Cocaine conditioned place preference (cocaine CPP). A modified version of
Laser CPA and the same chamber were used to perform cocaine CPP experiments.The pre-test and the post-test were performed identically, but the conditioning sessions were changed.During days 2 and 3 of conditioning, mice were confined to one of the two chambers (counterbalanced across all mice) and received an i.p. cocaine (15 mg/kg) injection paired with optical stimulation (0.5 sec pulse, 18 mW) once a minute for 30 min, or i.p. saline injection with no laser stimulation.During both conditioning sessions mice were tethered to the patch cable.The difference score was calculated by taking the time each mouse spent on the cocaine/laserpaired side in the post-test minus the time spent in the pre-test.
Brain slice preparation and electrophysiology.After completion of behavioral experiments, adult 10 to 14 week old mice were deeply anesthetized with isoflurane followed by rapid decapitation.The brain was quickly removed and chilled in icecold cutting solution containing (in mM): 110 cholineCl, 2.5 KCl, 1.25 NaH 2 PO 4 , 7 MgCl 2 , 0.5 CaCl 2 , 10 dextrose, 25 NaHCO 3 , and oxygenated with 95% O2 -5% CO 2 .250 µm horizontal brain slices containing VTA were incubated for 30 min at 35 °C in a chamber filled with artificial cerebrospinal fluid (aCSF) and stored at room temperature for the remainder of the day.Slices were transferred to a recording chamber where they continuously received aCSF (33-35 °C) at a rate of 2 ml per minute by a gravity-feed system.aCSF was composed of (in mM): 126 NaCl, 2.5 KCl, 1.25 NaH 2 PO 4 , 2 MgCl 2 , 2 CaCl 2 , 10 dextrose, 25 NaHCO 3 , was equilibrated using 95% O 2 -5%CO 2 and had a pH of 7.2.
Midbrain neurons and astrocytes were visualized using gradient contrast illumination through a ×40 water-immersion lens attached to an Olympus BX51 upright microscope.The VTA was defined as neurons medial to the medial terminal nucleus of the accessory optic tract.Neurons were considered dopaminergic based on the following electrophysiological criteria: have a hyperpolarization-activated inward current, slow spontaneous firing frequency ≤5 Hz and an action potential half-width ≥1.5 ms.Neurons were considered GABAergic based on the following electrophysiological criteria: continuous fast firing without spike frequency accommodation upon depolarizing current injection, fast spontaneous firing frequency >10 Hz and an action potential halfwidth ≤1.0 ms.Dopamine (TH-positive) and GABA (GABA-positive) neurons filled with biocytin were further identified immunocytochemically (Supplementary Fig. 4).Astrocytes expressed GFP or eYFP and were visualized with a ×40 or ×60 water-immersion objective on a fluorescent microscope capable of detecting GFP/eYFP.
Postsynaptic currents (PSC) from excitatory and inhibitory afferents were evoked with no blockers present by placing a 250 µm bipolar stimulating electrode within 500 µm of the neuron being recorded and applying a single pulse (0.05 ms).PSCs were isolated by voltage clamping the cells at −55 mV for EPSCs and at + 10 mV for IPSCs.The effect astrocytes had on synaptic activity was tested by photoactivating ChR2 on astrocytes for 500 ms (constant stimulation) and evoking PSCs 400 ms into the optical stimulation period.ChR2 on astrocytes was stimulated using a 473 or 405 nm laser (10-20 mW).Afferent-mediated currents on astrocytes were obtained by applying 50 Hz electrical stimulation (20 pulses, 0.05 ms pulse width).Whole-cell recordings were made using a Multiclamp 700B amplifier (Molecular Devices).Signals were digitized at 50 kHz and saved to a hard drive for analysis using Axograph X (Axograph).
Statistics.A student's two-tailed t-test was used when comparing between two groups.A One-way ANOVA with Tukey's post-hoc test was used for comparisons across multiple groups.A P-value <0.05 was used as our significance level for all tests.Power analysis showed greater than 80% power in all analyses.
Reporting summary.Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.

Fig. 1
Fig.1Neuronal afferent activity changes glutamate transport in VTA astrocytes.a Schematic showing the microcircuitry of the VTA and recordings performed.b Recording from a VTA astrocyte showing the current generated by electrical stimulation (black; 50 Hz, 20 pulses), the current remaining after blocking action potentials with 250 µM lidocaine (red), and after washing out lidocaine (green).c Summary of the currents before (black) and after lidocaine application (red; paired t-test t (5) = 2.679, P = 0.04, n = 6 cells, 4 mice).d Current generated in a VTA astrocyte by electrical stimulation of afferents before (black) and after adding 15 µm TFB-TBOA (red).e Summary of the currents generated by electrical stimulation in VTA astrocytes before (black) and after (red) addition of TFB-TBOA (paired t-test t (4) = 2.159, P = 0.04, n = 5 cells, 5 mice).*: P < 0.05

Fig. 8 Fig. 9 A
Fig. 8 GLT-1 removal from VTA astrocytes disrupts endogenous avoidance behavior.a Schematic depicting experimental design.b Track plots from a GLT-1 f/f mouse with only a fluorescent reporter (eYFP) on astrocytes displaying normal thigmotaxis in an open field (left), and a GLT-1 cKO VTA Astrocyte (GLT-1 f/f , gfaABC1D::Cre VTA ) mouse with increased tendency to explore the center of the open field (right).c, d GLT-1 cKO VTA Astrocyte mice spent more time exploring the center of the open field without affecting locomotor activity (d; unpaired t-test t (16) = 1.981,P = 0.065, n = 9 mice).**p < 0.01; Error bars indicate ± SEM