Gina R. Poe

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.

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.

Concurrent reflectance imaging and microdialysis in the freely behaving cat

We present a method to perform simultaneous microdialysis with light reflectance imaging of neural activity in a discrete brain region of the freely behaving animal. We applied this method to the dorsal hippocampus of freely behaving cats to (1) measure extracellular glutamate and reflectance variations across a sleep-waking cycle, (2) assess spatially coherent neural activity changes accompanying local perfusion of cocaine and (3) measure local changes in cell volume induced by infusion of hyper- and hypo-osmotic solutions. Higher extracellular glutamate concentrations corresponded to higher imaged neural activity. Sequential images showed that cocaine perfusion elicited a propagating reflectance change as cocaine reached the tissue. Microperfusion of hypo-osmotic solution ( - 100 mOsm), which increases cell volume, decreased reflectance. Microperfusion of hyperosmotic sucrose solutions, which reduce cell volume, increased reflectance in a dose-dependent manner. The data indicate that reflectance imaging can measure changes in cell volume, and could, thus, measure neural activity through activity/cell volume corollaries. Combining microdialysis and optical imaging enables investigation of the neurochemical bases of spontaneous neural activity patterns within discrete brain nuclei.

Activity changes of the cat paraventricular hypothalamus during phasic respiratory events

We monitored the spatiotemporal organization of cellular activity in the medial paraventricular hypothalamus during spontaneously-occurring periods of increased inspiratory effort followed by prolonged respiratory pauses (sigh/apnea) in the freely-behaving cat. Paraventricular hypothalamic activity was assayed by video images of light captured with a stereotaxically-placed fibre optic probe. Respiratory activity was measured through electromyographic wire electrodes placed in the diaphragm. Sigh/apnea events appeared in all behavioural states, and especially during quiet sleep. Overall paraventricular hypothalamic activity declined transiently, with the onset of decline coinciding with the beginning of the sigh inspiratory effort, reached a nadir at apnea onset 4.4+0.5 s from the beginning of the sigh, increased during the course of the apnea, and subsequently rebounded above baseline to peak at 10.9+2.5 s after sigh onset. Scattered, small areas of the imaged region were activated or depressed independently of the overall image values. The data suggest that paraventricular hypothalamic activity changes dynamically during phasic respiratory events, and may contribute to the progression of the sigh/apnea. We speculate that the medial paraventricular hypothalamus influences breathing patterns through projections to parabrachial respiratory phase-shift regions, and that longer-latency influences may also be exerted indirectly through blood pressure effects from paraventricular hypothalamic projections to medullary cardiovascular nuclei. Additionally, the paraventricular hypothalamus may convey respiratory influences from other rostral structures, such as the hippocampus.

Sleep Is for Forgetting

It is possible that one of the essential functions of sleep is to take out the garbage, as it were, erasing and “forgetting” information built up throughout the day that would clutter the synaptic network that defines us. It may also be that this cleanup function of sleep is a general principle of neuroscience, applicable to every creature with a nervous system.

Imaging of hippocampal and neocortical neural activity following intravenous cocaine administration in freely behaving cats

We examined spatial-temporal patterns of neural activity, as inferred from 700 nm light reflectance, from the dorsal hippocampus and surrounding neocortex in seven freely behaving cats following 1.5, 2.5, 3.5 and 5.0 mg/kg intravenous cocaine administration. Images were acquired using a new technique which gathered reflected light from cortical and subcortical structures. Cardiac and respiratory patterning, collected simultaneously with optical images, revealed increased rates and diminished variation after intravenous cocaine administration. Cocaine increased reflectance correlates of hippocampal neural activity in a dose-dependent fashion over a 120 min period, with a lengthening time-to-peak effect (22-76 min). The largest dose resulted in an initial decrease, followed by the greatest enhancement in neuronal activity. Correlates of neural activation in the neocortex displayed an inverse dose-response curve to that found in the hippocampus; the time-to-peak effect was shorter (6-43 min) and the maximal change was reduced. Regional patches and bands of activation occurred during the period of the cocaine response, and were more pronounced in the hippocampus than the neocortex. Procaine, administered in a similar dose, slightly increased neural activity for 10 min in both the hippocampus and neocortex, and elicited a small increase in respiration. Cocaine induces a pronounced enhancement of neural activation in the neocortex and dorsal hippocampus; the time course of activation in the hippocampus parallels an increased respiratory pattern and outlasts the neocortical response. We speculate that hippocampal activation may be related to the profound respiratory acceleration found in response to cocaine.

Fiber optic imaging of subcortical neural tissue in freely behaving animals.

Changes in voltage potentials have been used as representative measures of activity from single neurons, as well as from large populations of neurons. However, electrophysiological techniques are limited in the number of individual cells that can be recorded simultaneously. Correlates of neural activity are also measured in two or three dimensional space with procedures such as Positron Emission Tomography or Magnetic Resonance Imaging. However, the spatial resolution of these procedures is insufficient to observe single neurons or small groups of cells, and the temporal resolution is inadequate to examine many functional aspects.

Rostral brain regions contributing to respiratory control

Publisher Summary Respiratory muscles are used for a variety of behaviors in addition to the primary function of air exchange. These behaviors require input from a number of brain structures onto motorneuron pools for muscles mediating respiration, and include descending influences from limbic, periaqueductal grey, and cortical regions. Respiratory output neurons are also influenced by blood pressure variations; cardiovascular activity is, in turn, mediated by descending limbic projections, as well as by local activity on the ventral medullary surface. The rostral ventral medullary surface shows a marked regional activation to blood pressure lowering, and a profound decline in activity with blood pressure elevation over widespread areas, suggesting local influences on enhanced breathing during depressor challenges and suppressed breathing during pressor stimuli. The influence of limbic projections on breathing patterns may be especially pronounced during transient events, such as startle or affective activities. Dorsal hippocampal regions show pronounced regional activation changes during momentary respiratory events, such as sighs and apnea, with onset of activity preceding resumption of breathing.

State-dependent cellular activity patterns of the cat paraventricular hypothalamus measured by reflectance imaging

Activity within the cat paraventricular hypothalamus (PVH) during sleep and waking states was measured by quantifying intrinsic tissue reflectivity. A fiber optic probe consisting of a 1.0 mm coherent image conduit, surrounded by plastic fibers which conducted 660 nm source light, was attached to a charge-coupled device camera, and positioned over the PVH in five cats. Electrodes for assessing state variables, including electroencephalographic activity, eye movement, and somatic muscle tone were also placed. After surgical recovery, reflected light intensity was measured continuously at 2.5 Hz during spontaneously varying sleep/waking states. Sequential state transitions from active waking to quiet waking, quiet sleep and active sleep were accompanied by progressively increased levels of PVH activity. Overall activity was highest during active sleep, and decreased markedly upon awakening. Moment-to-moment activity oscillated in the 0-0.1 Hz range, especially during active sleep and active waking; this oscillation diminished during quiet sleep. Distinct sub-regions of enhanced or diminished activity emerged within the imaged area in a state-dependent manner. We conclude that PVH activity changes with behavioral state in a regionally specific manner, and that overall activity increases during quiet sleep, and is even more enhanced in active sleep. PVH activation could be expected to stimulate pituitary release of adrenocorticotropic hormone (ACTH) and affect input to autonomic regulatory sites. Since ACTH and corticotropin releasing factor elicit arousal, and since the PVH projects to other brain areas which modulate state, we speculate that the PVH plays a role in shaping characteristics of sleep/waking states.