Understanding the function of glutamate transporters has broad implications for explaining how neurons integrate information and relay it through complex neuronal circuits. Most of what is currently known about glutamate transporters, specifically their ability to maintain glutamate homeostasis and limit glutamate diffusion away from the synaptic cleft, is based on studies of glial glutamate transporters. By contrast, little is known about the functional implications of neuronal glutamate transporters. The neuronal glutamate transporter EAAC1 is widely expressed throughout the brain, particularly in the striatum, the primary input nucleus of the basal ganglia, a region implicated with movement execution and reward. Here, we show that EAAC1 limits synaptic excitation onto a population of striatal medium spiny neurons identified for their expression of D1 dopamine receptors (D1-MSNs). In these cells, EAAC1 also contributes to strengthen lateral inhibition from other D1-MSNs. Together, these effects contribute to reduce the gain of the input-output relationship and increase the offset at increasing levels of synaptic inhibition in D1-MSNs. By reducing the sensitivity and dynamic range of action potential firing in D1-MSNs, EAAC1 limits the propensity of mice to rapidly switch between behaviors associated with different reward probabilities. Together, these findings shed light on some important molecular and cellular mechanisms implicated with behavior flexibility in mice.

Glutamate transporters preserve the spatial specificity of synaptic transmission by limiting glutamate diffusion away from the synaptic cleft, and prevent excitotoxicity by keeping the extracellular concentration of glutamate at low nanomolar levels. Glutamate transporters are abundantly expressed in astrocytes, and previous estimates have been obtained about their surface expression in astrocytes of the rat hippocampus and cerebellum. Analogous estimates for the mouse hippocampus are currently not available. In this work, we derive the surface density of astrocytic glutamate transporters in mice of different ages via quantitative dot blot. We find that the surface density of glial glutamate transporters is similar in 7-8 week old mice and rats. In mice, the levels of glutamate transporters increase until about 6 months of age and then begin to decline slowly. Our data, obtained from a combination of experimental and modeling approaches, point to the existence of stark differences in the density of expression of glutamate transporters across different sub-cellular compartments, indicating that the extent to which astrocytes limit extrasynaptic glutamate diffusion depends not only on their level of synaptic coverage, but also on the identity of the astrocyte compartment in contact with the synapse. Together, these findings provide information on how heterogeneity in the spatial distribution of glutamate transporters in the plasma membrane of hippocampal astrocytes my alter glutamate receptor activation out of the synaptic cleft.

Glutamate transporters preserve the spatial specificity of synaptic transmission by limiting gluta-mate diffusion away from the synaptic cleft, and prevent excitotoxicity by keeping the extracellular concentration of glutamate at low nanomolar levels. Glutamate transporters are abundantly expressed in astrocytes. Previous estimates in the rat hippocampus suggest that the surface density of glutamate transporters in astrocytic membranes is ~ 10, 800 μm−2. Here, we estimate their surface density in astrocytic membranes of the mouse hippocampus, at different ages. By using realistic 3D Monte Carlo reaction-diffusion models, we show that varying the local glutamate transporter expression in astrocytes can alter profoundly the activation of extrasynaptic AMPA and NMDA receptors. Our findings show that the average density of astrocyte membranes and their surface density of glutamate transporters is higher in mice compared to rats, and increases with mouse age. There are stark differences in the density of expression of these molecules in different sub-cellular compartments, indicating that the extent to which astrocytes limit extrasynaptic glutamate diffusion depends not only on the level of astrocytic coverage, but also on the identity of the astrocyte compartment in contact with the synapse. Together, these findings provide information on the spatial distribution of glutamate transporters in the mouse hippocampus, which can be used in mathematical models of the spatiotemporal profile of extracellular glutamate after synaptic release.The astrocyte membrane density in the hippocampal neuropil is higher than in rats.The surface density of glutamate transporters in mouse astrocytes is higher than in rats and varies widely across different sub-cellular compartments.The identify of the astrocyte compartment in contact with a synapse, not only the extent of astrocytic coverage, is a main determinant of glutamate spillover and extrasynaptic receptor activation.

18 Understanding the function of glutamate transporters has broad implications for explaining how 19 neurons integrate information and relay it through complex neuronal circuits. Most of what is currently 20 known about glutamate transporters, specifically their ability to maintain glutamate homeostasis and 21 limit glutamate diffusion away from the synaptic cleft, is based on studies of glial glutamate 22 transporters. By contrast, little is known about the functional implications of neuronal glutamate 23 transporters. The neuronal glutamate transporter EAAC1 is widely expressed throughout the brain, 24 particularly in the striatum, the primary input nucleus of the basal ganglia, a region implicated with 25 movement execution and reward. Here, we show that EAAC1 limits synaptic excitation onto a 26 population of striatal medium spiny neurons identified for their expression of D1 dopamine receptors 27 (D1-MSNs). In these cells, EAAC1 also contributes to strengthen lateral inhibition from other D1-MSNs. 28 Together, these effects contribute to reduce the gain of the input-output relationship and increase the 29 offset at increasing levels of synaptic inhibition in D1-MSNs. By reducing the sensitivity and dynamic 30 range of action potential firing in D1-MSNs, EAAC1 limits the propensity of mice to rapidly switch 31 between behaviors associated with different reward probabilities. Together, these findings shed light on 32 some important molecular and cellular mechanisms implicated with behavior flexibility in mice. 33 Introduction 34 The neuronal glutamate transporter EAAC1, encoded by the Slc1a1 gene, is distributed broadly 35 throughout the brain (Rothstein et al. 1994; Shashidharan et al. 1997), with plasma membrane surface 36 density values thought to be significantly lower than those of astrocytic glutamate transporters 37 (Holmseth et al. 2012). Glutamate binding to glutamate transporters activates currents that can be 38 easily recorded in heterologous expression systems and astrocytes, where the density of expression of 39 these molecules is high (Wadiche, Amara, and Kavanaugh 1995). In most neurons, recording glutamate 40 transporter mediated currents continues to be elusive (Holmseth et al. 2012). This finding sparked some 41 doubts on the functional relevance of neuronal glutamate transporters like EAAC1 in the brain 42 (Holmseth et al. 2012). Despite this concern, multiple lines of evidence point to the fact that EAAC1 is an 43 important player in the regulation of synaptic function and behavior. For example, in the hippocampus, 44 EAAC1 limits NMDA receptor activation and increases GABA release (Diamond 2001; Scimemi, Tian, and 45 Diamond 2009; Mathews and Diamond 2003). In the dorsolateral striatum (DLS), EAAC1 limits activation 46 of group I metabotropic glutamate receptors (mGluRI) and is associated with increased execution of 47 stereotyped motor behaviors (Bellini et al. 2018). While most of these works relied on the use of 48 Slc1a1 mice (Peghini, Janzen, and Stoffel 1997), other studies that used overexpression models of 49 EAAC1 showed behavioral abnormalities, including increased anxiety-like behaviors (Delgado-Acevedo 50 et al. 2019; Escobar et al. 2021) and reduced responses to amphetamine-induced hyperlocomotion (Zike 51 et al. 2017). The emerging picture is that maintaining optimal levels of EAAC1 expression is key for 52 proper execution of a wide range of complex motor behaviors and habitual actions, many of which are 53 critically dependent on the activity of the DLS (Gremel and Costa 2013; Yin, Knowlton, and Balleine 54 2006, 2004). 55 What continues to puzzle the field is how we can reconcile the obvious behavioral abnormalities of 56 Slc1a1 mice with the apparent inability to record EAAC1-mediated currents in neurons. A possible way 57 out of this conundrum comes from considerations of the biophysical properties and sub-cellular 58 distribution of this neuronal transporter. First, EAAC1 has a very low single channel conductance (∼0.3 59 fS) (Grewer et al. 2000) and is not evenly distributed along the plasma membrane (Cheng et al. 2002; 60 Conti et al. 1998; Rothstein et al. 1994; He et al. 2000). Second, the fact that currents generated at a 61 distance from the soma are dramatically reduced by electrotonic filtering and attenuation could make 62 them particularly challenging to identify using somatic patch-clamp recordings (Tonnesen et al. 2014; 63 Svoboda, Tank, and Denk 1996; Rall 1959). Third, tentative estimates of the average surface density of 64 expression EAAC1 obtained from immunoblot experiments do not provide information on the local 65 density of expression of EAAC1 in dendritic spines and axonal boutons, where EAAC1 is thought to be 66 confined. Fourth, the amplitude of local EAAC1-mediated currents does not provide a direct readout of 67 the number of receptors that EAAC1 might protect from glutamate spillover (Scimemi, Tian, and 68 Diamond 2009). Together, these findings suggest that the inability to record EAAC1-mediated currents 69 from the soma should not be interpreted as evidence of a lack of any functional role for glutamate 70 uptake via EAAC1. 71 Interestingly, qualitative pre-, and post-embedding ultrastructural works show that EAAC1 has a 72 distinctive punctate peri-synaptic and post-synaptic expression in glutamatergic neurons, attributed to 73 the presence of a specific domain in the C-terminal region that controls the targeting of EAAC1 to 74 dendritic spines and shafts (Cheng et al. 2002; He et al. 2000). EAAC1 is also expressed in a population of 75 GABAergic neurons identified by their expression of glutamic acid decarboxylase, the biosynthetic 76 enzyme for GABA (Conti et al. 1998). In these neurons, EAAC1 shows a clustered expression in a subset 77 of axonal presynaptic terminals (He et al. 2000; Rothstein et al. 1994), although these findings have 78 been brought into question (Holmseth et al. 2012). In this case, the inconsistent results may be due to 79 the limited sensitivity and specificity of antibodies directed against EAAC1 used for immunolabeling 80 studies, which is a historically challenging, if not insurmountable issue. Other labeling strategies based 81 on the use of pharmacological tools have been hampered by the fact that there continues to be no drug 82 that targets EAAC1 without also affecting other glial glutamate transporters (Shimamoto et al. 1998; 83 Tsukada et al. 2005). For these reasons, functional studies combined with genetic approaches currently 84 represent an ideal tool to fill current gaps of knowledge on the functional roles of EAAC1 on synaptic 85 communication in different brain areas. 86 Multiple lines of evidence suggest the existence of potential association between loss of function of 87 EAAC1, altered synaptic activity in the striatum, and increased compulsive behaviors. First, the striatum 88 is one of the brain regions with the most abundant expression of EAAC1. Second, hyperactivity of striatal 89 circuits is associated with the emergence of compulsive behaviors (Gasso et al. 2015; Gilbert et al. 2008; 90 Menzies, Williams, et al. 2008; Abramowitz, Taylor, and McKay 2009; Carmin et al. 2002). Third, several 91 genome-wide studies identified loss-of-function variants and single nucleotide polymorphisms of 92 SLC1A1, the homolog gene encoding EAAC1 in humans diagnosed with OCD, autism spectrum disorder 93 (ASD) and attention deficit/hyperactivity disorder (ADHD) (Veenstra-VanderWeele et al. 2012; 94 Wendland et al. 2009; Porton et al. 2013; Stewart et al. 2007; Stewart, Mayerfeld, et al. 2013; Stewart, 95 Yu, et al. 2013; Gadow et al. 2010; Brune et al. 2008). 96 One of the hypotheses that has been brought forward to explain how EAAC1 may limit striatal 97 hyperactivity is that impairing glutamate uptake via EAAC1 might lead to an increased ambient 98 glutamate concentration in the striatum, which in turn would trigger hyperactivity (Porton et al. 2013). 99 This interpretation is difficult to reconcile with evidence that EAAC1 consitutes only ∼5% of all glutamate 100 transporters (Holmseth et al. 2012). Even if all EAAC1 molecules lost their ability to bind and transport 101 glutamate, under steady-state conditions, the remaining ∼95% of glutamate transporters in glial cells 102 would be able to keep the extracellular glutamate concentration at low nanomolar levels (Herman and 103 Jahr 2007; Chiu and Jahr 2017). Accordingly, experimental data show that loss of EAAC1 does not change 104 the extracellular glutamate concentration (Rothstein et al. 1996), a result supported also by our own 105 previous works in the striatum (Bellini et al. 2018) and hippocampus (Scimemi, Tian, and Diamond 106 2009). An alternative hypothesis comes from computational models of neural dynamics in cortico107 striatal-thalamic networks, which suggest that loss of EAAC1 might contribute to striatal hyperactivity by 108 changing the relative strength of synaptic excitation and inhibition (E/I) locally, in some or all medium 109 spiny neurons (MSNs), the main long-range projection neurons in the striatum (Rǎdulescu et al. 2017). 110 According to this model, local changes in E/I of striatal MSNs can alter the firing rates of neurons not 111 only in the striatum but also in larger neural networks that include the cortex and thalamus, bringing 112 them in a regime of hyperactivity (Rǎdulescu et al. 2017). Although these theoretical inferences provide 113 potential explanations of how local changes in E/I may lead to changes in the activity of more complex 114 neural networks and behaviors, an experimental investigation of how EAAC1 alters E/I in different 115 populations of striatal MSNs has not been previously performed. 116 Here, we show that EAAC1 limits synaptic excitation and strengthens synaptic inhibition in the DLS 117 (i.e., reduces E/I), by modulating the strength of excitatory synaptic transmission onto D1 dopamine 118 recepto