Excitatory amino acid transporters (EAATs) are abundantly expressed by astrocytes, rapidly remove glutamate from the extracellular environment, and restrict the temporal and spatial extent of glutamate signaling. Studies probing EAAT function suggest that their capacity to remove glutamate is large and does not saturate, even with substantial glutamate challenges. In contrast, we report that neuronal activity rapidly and reversibly modulates EAAT-dependent glutamate transport. To date, no physiological manipulation has shown changes in functional glutamate uptake in a nonpathological state. Using iGluSnFr-based glutamate imaging and electrophysiology in the adult mouse cortex, we show that glutamate uptake is slowed up to threefold following bursts of neuronal activity. The slowing of glutamate uptake depends on the frequency and duration of presynaptic neuronal activity but is independent of the amount of glutamate released. The modulation of glutamate uptake is brief, returning to normal within 50 ms after stimulation ceases. Interestingly, the slowing of glutamate uptake is specific to activated synapses, even within the domain of an individual astrocyte. Activity-induced slowing of glutamate uptake, and the increased persistence of glutamate in the extracellular space, is reflected by increased decay times of neuronal NR2A-mediated NMDA currents. These results show that astrocytic clearance of extracellular glutamate is slowed in a temporally and spatially specific manner following bursts of neuronal activity Ն30 Hz and that these changes affect the neuronal response to released glutamate. This suggests a previously unreported form of neuron-astrocyte interaction.

Developing cortical GABAergic interneurons rely on genetic programs, neuronal activity, and environmental cues to construct inhibitory circuits during early postnatal development. Disruption of these events can cause long-term changes in cortical inhibition and may be involved in neurological disorders associated with inhibitory circuit dysfunction. We hypothesized that tonic glutamate signaling in the neonatal cortex contributes to, and is necessary for, the maturation of cortical interneurons. To test this hypothesis, we used mice of both sexes to quantify extracellular glutamate concentrations in the cortex during development, measure ambient glutamate-mediated activation of developing cortical interneurons, and manipulate tonic glutamate signaling using subtype-specific NMDA receptor antagonists in vitro and in vivo. We report that ambient glutamate levels are high (Ϸ100 nM) in the neonatal cortex and decrease (to Ϸ50 nM) during the first weeks of life, coincident with increases in astrocytic glutamate uptake. Consistent with elevated ambient glutamate, putative parvalbumin-positive interneurons in the cortex (identified using G42:GAD1-eGFP reporter mice) exhibit a transient, tonic NMDA current at the end of the first postnatal week. GluN2C/GluN2D-containing NMDA receptors mediate the majority of this current and contribute to the resting membrane potential and intrinsic properties of developing putative parvalbumin interneurons. Pharmacological blockade of GluN2C/GluN2D-containing NMDA receptors in vivo during the period of tonic interneuron activation, but not later, leads to lasting decreases in interneuron morphological complexity and causes deficits in cortical inhibition later in life. These results demonstrate that dynamic ambient glutamate signaling contributes to cortical interneuron maturation via tonic activation of GluN2C/ GluN2D-containing NMDA receptors. Inhibitory GABAergic interneurons make up 20% of cortical neurons and are critical to controlling cortical network activity. Dysfunction of cortical inhibition is associated with multiple neurological disorders, including epilepsy. Establishing inhibitory cortical networks requires in utero proliferation, differentiation, and migration of immature GABAergic interneurons, and subsequent postnatal morphological maturation and circuit integration. Here, we demonstrate that ambient glutamate provides tonic activation of immature, putative parvalbumin-positive GABAergic interneurons in the neonatal cortex via high-affinity NMDA receptors. When this activation is blocked, GABAergic interneuron maturation is disrupted, and cortical networks exhibit lasting abnormal hyperexcitability. We conclude that temporally precise activation of developing cortical interneurons by ambient glutamate is critically important for establishing normal cortical inhibition.

Glutamate uptake by astrocytes controls the time course of glutamate in the extracellular space and affects neurotransmission, synaptogenesis, and circuit development. Astrocytic glutamate uptake has been shown to undergo post-natal maturation in the hippocampus, but has been largely unexplored in other brain regions. Notably, glutamate uptake has never been examined in the developing neocortex. In these studies, we investigated the development of astrocytic glutamate transport, intrinsic membrane properties, and control of neuronal NMDA receptor activation in the developing neocortex. Using astrocytic and neuronal electrophysiology, immunofluorescence, and Western blot analysis we show that: 1) glutamate uptake in the neonatal neocortex is slow relative to neonatal hippocampus; 2) astrocytes in the neonatal neocortex undergo a significant maturation of intrinsic membrane properties; 3) slow glutamate uptake is accompanied by lower expression of both GLT-1 and GLAST; 4) glutamate uptake is less dependent on GLT-1 in neonatal neocortex than in neonatal hippocampus, and 5) the slow glutamate uptake we report in the neonatal neocortex corresponds to minimal astrocytic control of neuronal NMDA receptor activation. Taken together, our results clearly show fundamental differences between astrocytic maturation in the developing neocortex and hippocampus, and corresponding changes in how astrocytes control glutamate signaling.

Appetite is driven by nutritional state, environmental cues, mood, and reward pathways. Environmental cues strongly influence feeding behavior, as they can dramatically induce or diminish the drive to consume food despite homeostatic state. Here, we have uncovered an excitatory neuronal population in the basal forebrain that is activated by food-odor related stimuli, and potently drives hypophagia. Notably, we found that the basal forebrain directly integrates environmental sensory cues to govern feeding behavior, and that basal forebrain signaling, mediated through projections to the lateral hypothalamus, promotes selective avoidance of food and food-related stimuli. Together, these findings reveal a novel role for the excitatory basal forebrain in regulating appetite suppression through food avoidance mechanisms, highlighting a key function for this structure as a potent integrator of sensory information towards governing consummatory behaviors.

The role of transcription factors during astrocyte development and their subsequent effects on neuronal development has been well studied. Less is known about astrocytes contributions towards circuits and behavior in the adult brain. Astrocytes play important roles in synaptic development and modulation, however their contributions towards neuronal sensory function and maintenance of neuronal circuit architecture remain unclear. Here, we show that loss of the transcription factor Sox9 results in both anatomical and functional changes in adult mouse olfactory bulb (OB) astrocytes, affecting sensory processing. Indeed, astrocyte-specific deletion of Sox9 in the OB results in decreased odor detection thresholds and discrimination and it is associated with aberrant neuronal sensory response maps. At functional level, loss of astrocytic Sox9 impairs the electrophysiological properties of mitral and tufted neurons. RNA-sequencing analysis reveals widespread changes in the gene expression profiles of OB astrocytes. In particular, we observe reduced GLT-1 expression and consequential alterations in glutamate transport. Our findings reveal that astrocytes are required for physiological sensory processing and we identify astrocytic Sox9 as an essential transcriptional regulator of mature astrocyte function in the mouse OB.