Assessing neuronal structure and function in a mouse model of therapeutic cooling

Johns Hopkins University, Baltimore MD

Investigators

Abstract

In recent years, mild therapeutic cooling, to about 32°C for 8-48 hours, has been shown to attenuate hypoxic brain injury that occurs following cardiac arrest or ischemic stroke. In addition, induced cardiac arrest necessitated by aortic arch surgery is now often accompanied by strong therapeutic cooling, to about 16°C for~60 min. Despite a wealth of preclinical and clinical data on the neuroprotective benefits of therapeutic brain cooling, little is known about how this process affects the structure and function of neurons and hence the extent and quality of recovery. Here we seek to answer the question: What are the transient and persistent changes in neuronal structure and function that accompany mouse models of therapeutic cooling? We find that intraperitoneal AMP injection coupled with an ambient temperature of 15°C or 30°C results in reduced heart rate and a stable core temperature of ~16°C or 32°C, respectively, in adult mice. These mild or strong torpid states were maintained for 1 or 8 hours and were rapidly reversed upon rewarming. In Thy1-M mice, which express EGFP sparsely in layer 5 pyramidal neurons, we have implanted cranial windows overlying the somatosensory cortex for time-lapse in vivo imaging to measure spine density and dynamics before, during and after induced hypothermia. Our pilot data with both mild and strong cooling show a selective loss of long thin spines that lasts for 1-2 days. In addition, we observe damage to apical dendrites with blebbing and distal retraction, which resolved within 7 days. Here, we propose to complete and extend these studies as a first step towards determining whether mild or strong therapeutic cooling alters neocortical function. Aim 1. What changes in the fine structure of neocortical neurons are evoked by mild or strong therapeutic cooling and do these changes persist? We shall use two-photon microscopy together with EGFP-expressing transgenic mice to produce time-lapse images of layer 5 pyramidal neurons, somatostatin interneurons and parvalbumin interneurons. This technique allows us to measure the dynamic aspects of neuronal structure that are inaccessible to fixed tissue studies such as spine turnover and dendritic retraction, blebbing and regrowth. Aim 2. Does mild or strong therapeutic cooling produce lasting changes in the electrophysiological function of identified neocortical neurons? We shall prepare ex vivo slices of the neocortex 1 and 7 days after mild or strong cooling to perform whole-cell recordings from identified layer 5 pyramidal cells as well as somatostatin and parvalbumin interneurons to assess basal firing rate, intrinsic excitability and the kinetic properties of miniature and evoked IPSCs and EPSCs. Together, these measurements will allow us to detect cooling-evoked transient or persistent changes to neocortical circuits.