The Effect of GLT-1 Upregulation on Extracellular Glutamate Dynamics.

Pharmacological upregulation of glutamate transporter-1 (GLT-1), commonly achieved using the beta-lactam antibiotic ceftriaxone, represents a promising therapeutic strategy to accelerate glutamate uptake and prevent excitotoxic damage in neurological conditions. While excitotoxicity is indeed implicated in numerous brain diseases, it is typically restricted to select vulnerable brain regions, particularly in early disease stages. In healthy brain tissue, the speed of glutamate uptake is not constant and rather varies in both an activity- and region-dependent manner. Despite the widespread use of ceftriaxone in disease models, very little is known about how such treatments impact functional measures of glutamate uptake in healthy tissue, and whether GLT-1 upregulation can mask the naturally occurring activity-dependent and regional heterogeneities in uptake. Here, we used two different compounds, ceftriaxone and LDN/OSU-0212320 (LDN), to upregulate GLT-1 in healthy wild-type mice. We then used real-time imaging of the glutamate biosensor iGluSnFR to investigate functional consequences of GLT-1 upregulation on activity- and regional-dependent variations in glutamate uptake capacity. We found that while both ceftriaxone and LDN increased GLT-1 expression in multiple brain regions, they did not prevent activity-dependent slowing of glutamate clearance nor did they speed basal clearance rates, even in areas characterized by slow uptake (e.g., striatum). Unexpectedly, ceftriaxone but not LDN decreased glutamate release in the cortex, suggesting that ceftriaxone may alter release properties independent of its effects on GLT-1 expression. In sum, our data demonstrate the complexities of glutamate uptake by showing that GLT-1 expression does not necessarily translate to accelerated uptake. Furthermore, these data suggest that the mechanisms underlying activity- and regional-dependent differences in glutamate dynamics are independent of GLT-1 expression levels.

Hippocampal Synaptic Dysfunction in a Mouse Model of Huntington Disease Is Not Alleviated by Ceftriaxone Treatment.

Glutamate transporters, particularly glutamate transporter 1 (GLT-1), help to prevent the adverse effects associated with glutamate toxicity by rapidly clearing glutamate from the extracellular space. Since GLT-1 expression and/or function are reduced in many neurodegenerative diseases, upregulation of GLT-1 is a favorable approach to treat the symptoms of these diseases. Ceftriaxone, a ß-lactam antibiotic reported to increase GLT-1 expression, can exert neuroprotective effects in a variety of neurodegenerative diseases; however, many of these diseases do not exhibit uniform brain pathology. In contrast, as a drug that readily crosses the blood-brain barrier, ceftriaxone administration is likely to increase GLT-1 levels globally throughout the neuroaxis. In Huntington disease (HD), low GLT-1 expression is observed in the striatum in postmortem tissue and animal models. While ceftriaxone was reported to increase striatal GLT-1 and ameliorate the motor symptoms in a mouse model of HD, the extrastriatal effects of ceftriaxone in HD are unknown. Using electrophysiology and high-speed imaging of the glutamate biosensor iGluSnFR, we quantified real-time glutamate dynamics and synaptic plasticity in the hippocampus of the Q175FDN mouse model of HD, following intraperitoneal injections of either saline or ceftriaxone. We observed an activity-dependent increase in extracellular glutamate accumulation within the HD hippocampus, which was not the result of reduced GLT-1 expression. Surprisingly, ceftriaxone had little effect on glutamate clearance rates and negatively impacted synaptic plasticity. These data provide evidence for glutamate dysregulation in the HD hippocampus but also caution the use of ceftriaxone as a treatment for HD.

Region- and Activity-Dependent Regulation of Extracellular Glutamate.

Transporter-mediated glutamate uptake plays an essential role in shaping synaptic neurotransmission. The rapid removal of synaptically released glutamate ensures the high temporal dynamics characteristic of fast excitatory chemical neurotransmission and prevents the overexcitation of extrasynaptic NMDA receptors that have been implicated in synaptic plasticity impairments and cell death. Despite clear regional differences in plasticity and excitotoxic thresholds, few studies have compared extracellular glutamate dynamics across different brain regions and in response to a range of neural activity including plasticity-inducing stimuli. Here, we used the rapid extracellular fluorescent glutamate sensor iGluSnFR (intensity-based glutamate-sensing fluorescent reporter) and high-speed imaging (205 frames per second) to quantify relative differences in glutamate clearance rates over a wide range of presynaptic activity in situ in the hippocampus, cortex, and striatum of male C57/BL6NCrl mice. We found that the hippocampus was significantly more efficient than the cortex and striatum at clearing synaptically released glutamate and that this efficiency could be attributed, at least in part, to faster glutamate diffusion away from the release site. In addition, we found that pharmacological inhibition of GLT-1, the brain's most abundant glutamate transporter, slowed clearance rates to only a fraction (∼20-25%) of the effect induced by nonselective transporter blockade, regardless of the brain region and the duration of presynaptic activity. In all, our data reveal clear regional differences in glutamate dynamics after neural activity and suggest that non-GLT-1 transporters can make a large contribution to the rate of glutamate clearance in the hippocampus, cortex, and striatum.SIGNIFICANCE STATEMENT Glutamate is the brain's most abundant neurotransmitter, and although essential for rapid cell-cell communication, too much glutamate can negatively impact cellular health. Extracellular glutamate levels are tightly regulated by membrane-bound transporters that rapidly remove the glutamate that is released during neural activity, thereby shaping both the spatial and temporal dynamics of excitatory neurotransmission. Using high-speed imaging of an optical sensor of extracellular glutamate, we show that glutamate dynamics vary widely from one brain region to the next and are highly dependent on the duration of synaptic activity. Our data demonstrate the heterogeneous nature of glutamate regulation in the brain and suggest that such regional differences can dramatically affect both the localization and duration of postsynaptic receptor activation during synaptic neurotransmission.