Assessment of Brain Functional Activity Using a Miniaturized Head-Mounted Scanning Photoacoustic Imaging System in Awake and Freely Moving Rats

Understanding the relationship between brain function and natural behavior remains a significant challenge in neuroscience because there are very few convincing imaging/recording tools available for the evaluation of awake and freely moving animals. Here, we employed a miniaturized head-mounted scanning photoacoustic imaging (hmPAI) system to image real-time cortical dynamics. A compact photoacoustic (PA) probe based on four in-house optical fiber pads and a single custom-made 48-MHz focused ultrasound transducer was designed to enable focused dark-field PA imaging, and miniature linear motors were included to enable two-dimensional (2D) scanning. The total dimensions and weight of the proposed hmPAI system are only approximately 50 × 64 × 48 mm and 58.7 g (excluding cables). Our ex vivo phantom experimental tests revealed that a spatial resolution of approximately 0.225 mm could be achieved at a depth of 9 mm. Our in vivo results further revealed that the diameters of cortical vessels draining into the superior sagittal sinus (SSS) could be clearly imaged and continuously observed in both anesthetized rats and awake, freely moving rats. Statistical analysis showed that the full width at half maximum (FWHM) of the PA A-line signals (relative to the blood vessel diameter) was significantly increased in the selected SSS-drained cortical vessels of awake rats (0.58 ± 0.17 mm) compared with those of anesthetized rats (0.31 ± 0.09 mm) (p < 0.01, paired t-test). In addition, the number of pixels in PA B-scan images (relative to the cerebral blood volume (CBV)) was also significantly increased in the selected SSS-drained blood vessels of awake rats (107.66 ± 23.02 pixels) compared with those of anesthetized rats (81.99 ± 21.52 pixels) (p < 0.01, paired t-test). This outcome may result from a more active brain in awake rats than in anesthetized rats, which caused cerebral blood vessels to transport more blood to meet the increased nutrient demand of the tissue, resulting in an obvious increase in blood vessel volume. This hmPAI system was further validated for utility in the brains of awake and freely moving rats, showing that their natural behavior was unimpaired during vascular imaging, thereby providing novel opportunities for studies of behavior, cognition, and preclinical models of brain diseases.

Tempol Reverses the Negative Effects of Morphine on Arterial Blood-Gas Chemistry and Tissue Oxygen Saturation in Freely-Moving Rats

We have reported that pretreatment with the clinically approved superoxide dismutase mimetic, Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), blunts the cardiorespiratory depressant responses elicited by a subsequent injection of fentanyl, in halothane-anesthetized rats. The objective of the present study was to determine whether Tempol is able to reverse the effects of morphine on arterial blood-gas (ABG) chemistry in freely-moving Sprague Dawley rats. The intravenous injection of morphine (10 mg/kg) elicited substantial decreases in pH, pO2 and sO2 that were accompanied by substantial increases in pCO2 and Alveolar-arterial gradient, which results in diminished gas-exchange within the lungs. Intravenous injection of a 60 mg/kg dose of Tempol 15 min after the injection of morphine caused minor improvements in pO2 and pCO2 but not in other ABG parameters. In contrast, the 100 mg/kg dose of Tempol caused an immediate and sustained reversal of the negative effects of morphine on arterial blood pH, pCO2, pO2, sO2 and Alveolar-arterial gradient. In other rats, we used pulse oximetry to determine that the 100 mg/kg dose of Tempol, but not the 60 mg/kg dose elicited a rapid and sustained reversal of the negative effects of morphine (10 mg/kg, IV) on tissue O2 saturation (SpO2). The injection of morphine caused a relatively minor fall in mean arterial blood pressure that was somewhat exacerbated by Tempol. These findings demonstrate that Tempol can reverse the negative effects of morphine on ABG chemistry in freely-moving rats paving the way of structure-activity and mechanisms of action studies with the host of Tempol analogues that are commercially available.

In Vivo Multi-Day Calcium Imaging of CA1 Hippocampus in Freely Moving Rats Reveals a High Preponderance of Place Cells and No Detectable Photobleaching

Calcium imaging using GCaMP calcium indicators and miniature microscopes has been used to image cellular populations during long timescales and in different task phases, as well as to determine neuronal circuit topology and organization. Because the hippocampus (HPC) is essential for many tasks of memory, spatial navigation, and learning, calcium imaging of large populations of HPC neurons can provide new insight on cell changes and organization over time during these tasks. To our knowledge, all reported HPC in vivo calcium imaging experiments have been done in mouse. However, rats have many behavioral and physiological experimental advantages over mice, and, due to their larger size, rats are able to support larger implants, thereby enabling the recording of a greater number of cells. In this paper, we present the first in vivo calcium imaging from CA1 hippocampus in freely moving rats. Using GCaMP7c and the UCLA Miniscope, we demonstrate that hundreds of cells (mean 240+-90 cells per session, maximum 428 cells) can reliably be visualized and held across weeks, and that calcium events in these cells are correlated with periods of movement. We additionally show proof of method by showing that an extremely high percent of place cells (82.3%+-8.1%, far surpassing the percent seen during mouse calcium imaging) can be recorded on a navigational task, and that these place cells enable accurately decoding of animal position. Finally, we show that calcium imaging is rats is not prone to photobleaching during hour-long recordings and that cells can be reliably recorded for an hour or more per session. A detailed protocol for this technique, including notes on the numerous parameter changes needed to use Ca2+ in rats, is included in the Materials and Methods section, and implications of these advancements are discussed.