High-affinity uptake of glutamate from the synaptic cleft plays a crucial role in regulating neuronal activity in physiological and pathological conditions. We have used affinity-purified specific polyclonal antibodies raised against a synthetic peptide corresponding to the C-terminal region of rabbit and rat EAAC1, a glutamate (Glu) transporter believed to be exclusively neuronal, to investigate its cellular and subcellular localization and whether it is expressed exclusively in glutamatergic cells of infragranular layers, as suggested by previous studies. Light microscopic studies revealed that EAAC1 immunoreactivity (ir) is localized to neurons and punctate elements in the neuropil. EAAC1-positive neurons were more numerous in layers II-III and V-VI, i.e. throughout all projection layers. Most EAAC1-positive neurons were pyramidal, although nonpyramidal cells were also observed. Some EAAC1-positive non-pyramidal neurons stained positively with an antiserum to GAD, thus demonstrating that EAAC1 is not confined to glutamatergic neurons. Non-neuronal EAAC1-positive cells were also observed in the white matter, and some of them stained positively with an antiserum to GFAP. Ultrastructural studies showed that EAAC1-ir was in neuronal cell bodies, dendrites and dendritic spines, but not in axon terminals, i.e. exclusively postsynaptic. Analysis of the type of axon terminals synapsing on EAAC1-ir profiles showed that 97% of them formed asymmetric contacts, thus indicating that EAAC1 is located at the very sites of excitatory amino acid release. Unexpectedly, EAAC1-ir was also found in a few astrocytic processes located in both the gray and the white matter. The localization of EAAC1 may explain the pathological symptoms that follow EAAC knockout (seizures and mild toxicity), as seizures could be due to the loss of EAAC1-mediated fine regulation of neuronal excitability at axodendritic and axospinous synapses, whereas the mild toxicity may be related to the functional inactivation of astrocytic EAAC1.

Migraine is a common disabling brain disorder. A subtype of migraine with aura (familial hemiplegic migraine type 2: FHM2) is caused by loss‐of‐function mutations in α₂ Na⁺,K⁺ATPase (α₂NKA), an isoform almost exclusively expressed in astrocytes in adult brain. Cortical spreading depression (CSD), the phenomenon that underlies migraine aura and activates migraine headache mechanisms, is facilitated in heterozygous FHM2‐knockin mice with reduced expression of α₂NKA. The mechanisms underlying an increased susceptibility to CSD in FHM2 are unknown. Here, we show reduced rates of glutamate and K⁺ clearance by cortical astrocytes during neuronal activity and reduced density of GLT‐1a glutamate transporters in cortical perisynaptic astrocytic processes in heterozygous FHM2‐knockin mice, demonstrating key physiological roles of α₂NKA and supporting tight coupling with GLT‐1a. Using ceftriaxone treatment of FHM2 mutants and partial inhibition of glutamate transporters in wild‐type mice, we obtain evidence that defective glutamate clearance can account for most of the facilitation of CSD initiation in FHM2‐knockin mice, pointing to excessive glutamatergic transmission as a key mechanism underlying the vulnerability to CSD ignition in migraine. Synopsis FHM2 is a rare monogenic form of migraine with aura caused by loss‐of‐function mutations in the astrocytic α2 Na,K ATPase. Investigating the mechanisms underlying the facilitation of cortical spreading depression (CSD) in a genetic mouse model of FHM2 uncovers insights into migraine pathophysiology. The rates of clearance of glutamate and K⁺ released during cortical activity are both reduced in heterozygous FHM2‐knockin mice with 50% reduced expression of the α2 Na,K ATPase (NKA). In FHM2‐knockin mice, the membrane density of the glutamate transporter GLT‐1 is about 50% reduced in astrocytic processes surrounding cortical excitatory synapses, but is unaltered in axon terminals. The relative impairment of glutamate clearance in FHM2‐knockin mice is activity dependent. Most of the facilitation of CSD initiation and a large fraction of the facilitation of CSD propagation in FHM2‐knockin mice are due to the defective glutamate clearance by cortical astrocytes.

The signaling diversity of GABAergic interneurons to post-synaptic neurons is crucial to generate the functional heterogeneity that characterizes brain circuits. Whether this diversity applies to other brain cells, such as the glial cells astrocytes, remains unexplored. Using optogenetics and two-photon functional imaging in the adult mouse neocortex, we here reveal that parvalbumin- and somatostatin-expressing interneurons, two key interneuron classes in the brain, differentially signal to astrocytes inducing weak and robust GABA_B receptor-mediated Ca²⁺ elevations, respectively. Furthermore, the astrocyte response depresses upon parvalbumin interneuron repetitive stimulations and potentiates upon somatostatin interneuron repetitive stimulations, revealing a distinguished astrocyte plasticity. Remarkably, the potentiated response crucially depends on the neuropeptide somatostatin, released by somatostatin interneurons, which activates somatostatin receptors at astrocytic processes. Our study unveils, in the living brain, a hitherto unidentified signaling specificity between interneuron subtypes and astrocytes opening a new perspective into the role of astrocytes as non-neuronal components of inhibitory circuits.