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Astrocytes extend serotonin-mediated effects on cortical inhibition

Date

2020-08-11

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Thesis

Degree Level

Doctoral

Abstract

Astrocytes, while long thought of as passive players in cell signaling, are now known to be playing crucial roles in coordinating and modulating activity of cortical networks. A single astrocyte has the capacity to contact over 100,000 synapses, release gliotransmitters that potently affect neuron and interneuron activity, change network excitability and synaptic transmission via alterations in potassium (K+) handling, and possess receptors for neuromodulators; such as serotonin (5HT). 5HT has established effects on cortical interneurons and thus affects inhibition. However, a number of issues exist that would prevent the efficient and rapid action of 5HT directly on interneurons. First, reuptake transporters would decrease the concentration of the neuromodulator in the extracellular space. Secondly, degradation by enzymes would have a similar effect of the 5HT concentration. Thirdly, diffusion is in and of itself a slow process that would limit the rapid response needed and observed in 5HT actions. Finally, astrocytes create a physical barrier that prevents the overflow of transmitters in the synapses and thus limits the diffusion capability of 5HT. Therefore, we hypothesize that astrocytes are ideally positioned and equipped to be extending the 5HT-mediated effects on cortical inhibitory networks. Several techniques were performed with acutely isolated mouse brain slices to test our hypothesis. Extracellular field recordings in somatosensory cortex were employed using a paired-pulse stimulation paradigm which allowed for selective examination of whole network inhibitory responses. We also used ion-selective microelectrodes to study the contribution of K+ homeostasis to the 5HT-mediated cortical response. Finally, we used whole-cell patch clamp technique of both neurons (to directly measure inhibitory currents) and astrocytes (to selectively dialyze the syncytium using calcium chelation) to provide further evidence for an astrocytic role in 5HT-mediated inhibitory responses. Paired-pulse field recordings indicated that 5HT was decreasing evoked inhibition. This was achieved by decreasing the first EPSP (P1) amplitude and increasing the second EPSP (P2) amplitude. To assess the contribution of astrocytic gliotransmitters ATP and adenosine, we used the antagonists Ab129 and SCH 58261 to inhibit the P2Y1 and A2A receptors respectively. These receptors have established roles in modulating cortical interneurons and were thus of interest to this study. We also employed the GAPDH antagonist, which selectively disrupts astrocyte metabolism (glycolysis preferred in astrocytes; oxidative phosphorylation preferred in neurons) to assess the role of astrocytes in the 5HT-mediated decrease in evoked inhibition. All antagonists displayed robust blockade of the 5HT-mediated response on cortical inhibition. Interestingly, A2A antagonism was the only one to block the 5HT effects on both P1 and P2. Ion-selective recordings implicated both A2A receptors and potassium inward rectifiers (Kir) in the 5HT-mediated response on cortical networks. A ten-pulse stimulation produced an evoked K+ response. The decay/recovery tau of this evoked response was measured and found to be decreased in the presence of 5HT. Purinergic antagonists and disruption of potassium handling both affected this response. We also looked at field recordings produced by the ten-pulse stimulation and found that A2A receptors and Kir blockade inhibit the 5HT-mediated decrease in cortical adaptation and fEPSP amplitude. Finally, whole cell patch clamp of neurons and astrocytes provides strong verification for previous findings. Normally, 5HT increased frequency and amplitude of spontaneous inhibitory post-synaptic currents (sIPSCs) and decreased amplitude of evoked inhibitory post-synaptic currents (eIPSCs) in neurons. It was found that A2A blockade completely inhibited the 5HT effects on sIPSCs, but not eIPSCs. Kir inhibition, however, antagonized the 5HT-mediated decrease in eIPSC amplitude, but did not affect the 5HT effects on sIPSCs. Whole-cell patch clamp of astrocytes dialyzed with a calcium chelator, BAPTA, indicate the 5HT-mediated effect on evoked inhibition (in extracellular field recording model) relies on astrocytic calcium. Taken together, the evidence suggests that astrocytes participate in 5HT-mediated effects on cortical inhibition. Not only are purinergic signaling and potassium homeostasis - cornerstones of astrocyte function - implicated in the 5HT response, but direct calcium chelation of astrocytes inhibited the 5HT effect on evoked inhibition. These studies highlight the role of astrocytes in propagating 5HT neuromodulatory effects rapidly in the somatosensory cortex to aid in filtering and shaping sensory input.

Description

Keywords

astrocytes, serotonin

Citation

Degree

Doctor of Philosophy (Ph.D.)

Department

Pharmacology

Program

Pharmacology

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DOI

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