David M. Rector

Spatio-temporal mapping of rat whisker barrels with fast scattered light signals

Optical techniques offer a number of potential advantages for imaging dynamic spatio-temporal patterns of activity in neural tissue. The methods provide the wide field of view required to image population activation across networks, while allowing resolution of the detailed structure of individual cells. Optical probes can provide high temporal resolution without penetrating the tissue surface. However, functional optical imaging has been constrained by the small size of the signals and the sluggish nature of the metabolic and hemodynamic responses that are the basis of most existing methods. Here, we employ both high-speed CCD cameras and high-sensitivity photodiodes to optimize resolution in both space and time, together with dark-field illumination in the near-infrared, to record fast intrinsic scattering signals from rat somatosensory cortex in vivo. Optical responses tracked the physiological activation of cortical columns elicited by single whisker twitches. High-speed imaging produced maps that were initially restricted in space to individual barrels, and then spread over time. Photodiode recordings disclosed 400-600 Hz oscillatory responses, tightly correlated in frequency and phase to those seen in simultaneous electrical recordings. Imaging based on fast intrinsic light scattering signals eventually could provide high resolution dynamic movies of neural networks in action.

Scattered-Light Imaging in Vivo Tracks Fast and Slow Processes of Neurophysiological Activation

We imaged fast optical changes associated with evoked neural activation in the dorsal brainstem of anesthetized rats, using a novel imaging device. The imager consisted of a gradient-index (GRIN) lens, a microscope objective, and a miniature charged-coupled device (CCD) video camera. We placed the probe in contact with tissue above cardiorespiratory areas of the nucleus of the solitary tract and illuminated the tissue with 780-nm light through flexible fibers around the probe perimeter. The focus depth was adjusted by moving the camera and microscope objective relative to the fixed GRIN lens. Back-scattered light images were relayed through the GRIN lens to the CCD camera. Video frames were digitized at 100 frames per second, along with tracheal pressure, arterial blood pressure, and electrocardiogram signals recorded at 1 kHz per channel. A macroelectrode placed under the GRIN lens recorded field potentials from the imaged area. Aortic, vagal, and superior laryngeal nerves were dissected free of surrounding tissue within the neck. Separate shocks to each dissected nerve elicited evoked electrical responses and caused localized optical activity patterns. The optical response was modeled by four distinct temporal components corresponding to putative physical mechanisms underlying scattered light changes. Region-of-interest analysis revealed image areas which were dominated by one or more of the different time-course components, some of which were also optimally recorded at different tissue depths. Two slow optical components appear to correspond to hemodynamic responses to metabolic demand associated with activation. Two fast optical components paralleled electrical evoked responses.

Optical lever recording of displacements from activated lobster nerve bundles and Nitella internodes

An optical lever was designed for studying physical displacements associated with electrophysiological activation of lobster nerve bundles. Stimulation current pulses generated a compound action potential volley, and upward physical displacements of <1 nm were recorded. The swelling displacement propagated in the same direction as the action potential volley, occurred simultaneously with the action potentials, and required 10 ms to relax after the electrical potential was restored. For comparison with previous reports, we also recorded the displacement of Nitella internodes associated with electrical stimulation. We found that a rapid swelling displacement (approximately 10 nm) was followed by a larger, slow-shrinking displacement (approximately 100 nm).

Hippocampal activity during transient respiratory events in the freely behaving cat.

We measured dorsal hippocampal activity accompanying sighs and apnea using reflectance imaging and electrophysiologic measures in freely behaving cats. Reflected 660-nm light from a 1-mm2 area of CA1 was captured during sighs and apnea at 25 Hz through a coherent image conduit coupled to a charge coupled device camera. Sighs and apnea frequently coincided with state transitions. Thus, state transitions without apnea or sighs were separately assessed to control for state-related activity changes. All dorsal hippocampal sites showed discrete regions of activation and inactivation during transient respiratory events. Imaged hippocampal activity increased 1-3 s before the enhanced inspiratory effort associated with sighs, and before resumption of breathing after apnea. State transitions lacking sighs and apnea did not elicit analogous optical activity patterns. The suprasylvian cortex, a control for site, showed no significant overall reflectance changes during phasic respiratory events, and no discrete regions of activation or inactivation. Spectral estimates of hippocampal electroencephalographic activity from 0-12 Hz showed significantly increased power at 3-4 Hz rhythmical slow activity before sighs and apnea, and increased 5-6 Hz rhythmical slow activity power during apnea, before resumption of breathing. Imaged activity and broadband hippocampal electroencephalogram power decreased during sighs. We propose that increased hippocampal activity before sigh onset and apnea termination indicates a role for the hippocampus in initiating inspiratory effort during transient respiratory events.

Imaging of hippocampal neural activity in freely behaving animals.

We examined intrinsic activity of neurons in the cat dorsal hippocampus following stimulation of the posterior medial hypothalamus and following a 2.5 mg/kg intravenous administration of cocaine. Posterior hypothalamic stimulation elicited banding of optical activity in the dorsal hippocampus, suggestive of spatial organization of neural activity underlying synchronous rhythmical slow wave activity. Cocaine administration resulted in a major decline in reflected light at 700 nm, possibly representing diminished oxygenation to the underlying tissue. The activity was examined by a new technique which uses principles of reflectance at specific bandwidths of light during cellular activity, and allows acquisition of images in the freely behaving animal. The imaging device consists of a 1.6 mm diameter coherent optic fiber bundle of 25 microns fibers attached to a 128 x 128 pixel charge-coupled device (CCD) array which can be cooled to reduce thermal noise. The target is illuminated by a light emitting diode array producing light at a number of operator-selectable narrow bandwidths which permits assessment of physiological characteristics of neural tissue. The CCD output is digitized, stored by computer, and the resulting images are averaged to obtain a greater signal-to-noise ratio. The procedure shows considerable promise for providing on-line, real-time indications of activity in localized brain regions.

A focusing image probe for assessing neural activity in vivo

We describe a compact, focusing image probe to record rapid optical changes from neural tissue. A gradient index (GRIN) lens served as a relay lens from tissue to a microscope objective which projected an image onto a CCD camera. The microscope objective and camera assembly was adjusted independently from the GRIN lens, allowing focus changes without disturbing the probe/tissue interface; firm contact minimized movement and specular reflectance. Fiber optics around the probe perimeter provided diffuse illumination from a 780 nm laser, or 660 and 560 nm light emitting diodes. To characterize depth-of-field, we imaged a black suture through increasing tissue thicknesses. Light modulation by the suture remained detectable down to 900 microm using 780 nm illumination. We acquired images from cardiorespiratory areas of the rat dorsal medulla, at different depths and illumination wavelengths. Images illuminated at 560 nm were dominated by vasculature flow patterns, while 660 nm illumination revealed different spatial patterns which preceded vascular flow by 40 ms and may represent cardiac-related neural activity. Using 780 nm light, image sequences triggered by the cardiac R-wave showed vascular perfusion changes with delayed and broader responses at deeper levels. Electrical stimulation within the vagal bundle caused fast optical changes which track the electrical response, with a different spatial distribution from hemodynamic signals.

A miniature CCD video camera for high-sensitivity light measurements in freely behaving animals

We developed a miniaturized, high-sensitivity camera that can be placed in areas of difficult access in freely behaving animals for neural tissue imaging. The device consists of a charged coupled device (CCD) chip, a coherent image conduit and miniature light emitting diodes (LEDs). An amplifier circuit is constructed on the camera chip and nine wires are attached for external connections. Placement of LEDs around the image conduit perimeter provides dark-field illumination, which increases detection of cellular-related light scattering changes and doubles the depth-of-view over conventional reflectance imaging procedures. The device has been successfully used to record from several deep brain structures, including the ventral medullary surface of sleeping and waking cats. The procedure allows assessment of light scattering changes that result from neural activity or detection of vital dyes to metabolic or voltage-induced activation.

Imaging the dorsal hippocampus" light reflectance relationships to electroencephalographic patterns during sleep

We assessed the correspondence of 660 nm light reflectance changes from the dorsal hippocampus with slow wave electroencephalographic (EEG) activity during quiet sleep (QS) and rapid eye movement (REM) sleep in four cats. An optic probe, attached to a charge-coupled-device (CCD) video camera, was placed on the dorsal hippocampal surface to collect reflectance images simultaneously with EEG, which was measured by macroelectrodes placed around the probe circumference. Spectral estimates of EEG and light reflectance amplitude indicated that reflectance changes occurred in a similar frequency range as EEG changes. Dividing the image into 10 subregions revealed that reflectance changes at the rhythmical slow wave activity band (RSA, 4-6 Hz) persisted in localized regions during QS and REM sleep, but regional changes showed considerable wave-by-wave independence between areas and from slow wave electrical activity. Peak frequencies for reflectance changes corresponded to fast RSA frequencies observed in the EEG. Optical changes most likely derive from fast-acting physical phenomena, rather than from alterations in blood perfusion, and provide increased spatial resolution over that offered by electrical measurements.

Physiological and ventral medullary surface activity during hypovolemia.

The objective was to determine ventral medullary surface responses to blood loss sufficient to induce shock. We examined changes in scattered light from rostral and intermediate areas of the ventral medullary surface in four intact, drug-free cats during acute hypovolemia. Scattered light images, collected during 660 and 560 nm illumination to measure cellular activity and hemodynamic aspects, respectively, were digitized at 50 frames/s during baseline, and during withdrawal of 20-30% blood volume. Hypovolemia elicited a profound hypotension and eventual bradycardia. In all cats, a modest increase in ventral medullary surface reflectance (activity decline) accompanied initial blood loss; as hypovolemia continued, and blood pressure declined, reflectance switched to a decline (activity increase), with the lowest reflectance occurring at maximal blood loss. Hypovolemia elicited multiple transient physiologic behaviors, including tachycardia, tachypnea, intermittent isolated and sustained bursts of enhanced inspiratory efforts, and extensor activation of the somatic musculature. The phasic physiological behaviors during hypovolemia were accompanied by partial recovery of medullary surface reflectance and blood pressure towards baseline values; however, reflectance continued to decrease as blood pressure progressively fell after these recovery efforts. Patterns of reflectance were not uniform over areas examined; isolated regions of enhanced or diminished reflectance appeared upon the overall images. Optical signals indicating hemodynamic changes followed the neural activity patterns, but not precisely. Regions within the ventral surface are responsive to hypovolemia, and to transient behaviors associated with momentary restoration of blood pressure; these ventral surface areas may assume essential roles in the systemic response to hypovolemic-induced shock.

Relationships between Hippocampal Activity and Breathing Patterns

Single cell discharge, EEG activity, and optical changes accompanying alterations in breathing patterns, as well as the knowledge that respiratory musculature is heavily involved in movement and other behavioral acts, implicate hippocampal regions in some aspects of breathing control. The control is unlikely to reside in oscillatory breathing movements, because such patterns emerge in preparations retaining only the medulla (and perhaps only the spinal cord). However, momentary changes in breathing patterns induced by affect, startle, whole-body movement changes, or compensatory ventilatory changes mediated by rostral brain regions likely depend on hippocampal action in aspects of control. Hippocampal activity was enhanced prior to sighs, and this enhancement was accompanied by increased slow theta activity. Theta frequency increased during apnea, prior to return of breathing. Consideration of hippocampal contributions to breathing control should be viewed in the context that significant interactions exist between blood pressure changes and ventilation, and that modest breathing challenges, such as exposure to hypercapnia or to increased resistive loads, bring into action a vast array of brain regions involving nearly every level of the neuraxis.