Astrocyte HIF-2α supports learning in a passive avoidance paradigm under hypoxic stress

Abstract

Background:

The brain is extensively vascularized, uses ~20% of the body’s oxygen, and is highly sensitive to changes in oxygen. While synaptic plasticity and memory are impaired in healthy individuals by exposure to mild hypoxia, aged individuals appear to be even more sensitive. Aging is associated with progressive failure in pulmonary and cardiovascular systems, exposing the aged to both chronic and superimposed acute hypoxia. The HIF proteins, the “master regulators” of the cellular response to hypoxia, are robustly expressed in neurons and astrocytes. Astrocytes support neurons and synaptic plasticity via complex metabolic and trophic mechanisms. The activity of HIF proteins in the brain is diminished with aging, and the increased exposure to chronic and acute hypoxia with aging combined with diminished HIF activity may impair synaptic plasticity.

Purpose:

Herein, we test the hypothesis that astrocyte HIF supports synaptic plasticity and learning upon hypoxia.

Materials and Methods:

An Astrocyte-specific HIF loss-of-function model was employed, where knock-out of HIF-1α or HIF-2α in GFAP expressing cells was accomplished by cre-mediated recombination. Animals were tested for behavioral (open field and rotarod), learning (passive avoidance paradigm), and electrophysiological (long term potentiation) responses to mild hypoxic challenge.

Results:

In an astrocyte-specific HIF loss-of-function model followed by mild hypoxia, we identified that the depletion of HIF-2α resulted in an impaired passive avoidance learning performance. This was accompanied by an attenuated response to induction in long-term potentiation (LTP), suggesting that the hippocampal circuitry was perturbed upon hypoxic exposure following HIF-2α loss in astrocytes, and not due to hippocampal cell death. We investigated HIF-regulated trophic and metabolic target genes and found that they were not regulated by HIF-2α, suggesting that these specific targets may not be involved in mediating the phenotypes observed.

Conclusion:

Together, these results point to a role for HIF-2α in the astrocyte’s regulatory role in synaptic plasticity and learning under hypoxia and suggest that even mild, acute hypoxic challenges can impair cognitive performance in the aged population who harbor impaired HIF function.

Keywords::

• astrocyte
• hypoxia
• HIF
• learning
• LTP
• tripartite synapse
• memory
• glia
• cognitive function

Acknowledgments

The authors thank Ying Wang, PhD, for help in the characterization of the physiology and recombination of the animal models described herein, thank Ted Abel, PhD, at the University of Pennsylvania, Smilow Center for Translational Research (Philadelphia, PA, USA) for consultation on the passive avoidance paradigm and for helpful conversations on the other behavioral and LTP tests, and specially thank Laurie Levine at the Division for Laboratory Animal Resources at SBU for help in animal maintenance, breeding, and caretaking. The authors would like to thank Lucia Roa-Peña, MD, and Yan Ji, BS, MBA, from the Histopathology Core at the Department of Pathology, Stony Brook University, for help in processing tissue and optimizing the cleaved caspase-3 antibody and immunohistochemistry conditions. The authors would like to thank the laboratory of Dr. Holly Colognato for the development of protocols to establish mixed glial cultures used for generation of primary cortical astrocytes. This research was sponsored by the Department of Anesthesiology, Stony Brook University, and the Research Foundation of the State University of New York at Stony Brook, Stony Brook, NY, USA.

Author contributions

TFF, CVL, and EC designed the research strategy. EC, KC, JS, JP, and MC performed gross physiological assessments, behavioral tests (open field, rotarod, and passive avoidance), and characterized recombination in the animal models described herein. EC and JS performed the electrophysiological measurements of LTP. All data were analyzed by TFF and CVL who discussed the interpretation with all investigators. CVL in cooperation with KM, DMM, AD, and BC cultured astrocyte primary cell cultures for the analysis of HIF expression from the animal models described. CVL and AC cultured and analyzed astrocyte primary cell cultures for HIF target gene expression. CVL and MK optimized astrocyte in vitro cultures and transfection protocols. CVL and RL tested cell death via cleaved caspase-3 staining. CVL managed mouse colonies. TFF, CVL, and MK wrote, edited, and constructed the manuscript with input and discussion from all other authors. All authors contributed to data analysis, drafting and revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.

Disclosure

CVL was partly supported by a postdoctoral fellowship awarded by the National Science Foundation’s workforce development program, the Alliance for Graduate Education in the Professoriate-Transformation grant (HRD-1311318), through the Stony Brook University Center for Inclusive Education, Stony Brook, NY, USA. The authors report no other conflicts of interest in this work.