Mechanisms underlying state dependent surface-evoked response patterns.

Cortical evoked response potentials (ERPs) display a rich set of waveforms that are both context and state dependent. However, the mechanisms that underlie state dependent ERP patterns are unclear. Determining those mechanisms through analysis of single trial ERP waveform signatures may provide insight into the regulation of cortical column state and the roles that sleep plays in cortical function. We implanted rats with electroencephalogram (EEG) and electromyogram (EMG) electrodes to record ERPs and to assess sleep/wake states continuously during 1-2 s random auditory clicks. Individual cortical auditory ERPs were sorted into one of eight behavioral states, and fell into three categories based on amplitude and latency characteristics. ERPs within waking and rapid eye movement (REM) sleep were predominantly low amplitude and short latency. Approximately 50% of ERPs during light quiet sleep (quiet sleep 1 and quiet sleep 2) exhibited low amplitude, short latency responses, and the remaining ERPs had high amplitude, long latency responses. This distribution was characteristic of EEG fluctuations during low frequency delta waves. Significantly more individual ERPs showed very low amplitudes during deep quiet sleep (quiet sleep 3 and quiet sleep 4), resulting in a lower average ERP. These results support the hypothesis that evoked response amplitudes and waveform patterns follow specific EEG patterns. Since evoked response characteristics distribute differently across states, they could aid our understanding of sleep mechanisms through state-related and local neural signaling.

Tumor necrosis factor alpha: activity dependent expression and promotion of cortical column sleep in rats.

Cortical surface evoked potentials (SEPs) are larger during sleep and characterize a sleep-like state in cortical columns. Since tumor necrosis factor alpha (TNF) may be involved in sleep regulation and is produced as a consequence of waking activity, we tested the hypothesis that direct application of TNF to the cortex will induce a sleep-like state within cortical columns and enhance SEP amplitudes. We found that microinjection of TNF onto the surface of the rat somatosensory cortex enhanced whisker stimulation-induced SEP amplitude relative to a control heat-inactivated TNF microinjection. We also determined if whisker stimulation enhanced endogenous TNF expression. TNF immunoreactivity (IR) was visualized after 2 h of deflection of a single whisker on each side. The number of TNF-IR cells increased in layers II-IV of the activated somatosensory barrel column. In two separate studies, unilateral deflection of multiple whiskers for 2 h increased the number of TNF-IR cells in layers II-V in columns that also exhibited enhanced cellular ongogene (Fos-IR). TNF-IR also colocalized with NeuN-IR suggesting that TNF expression was in neurons. Collectively these data are consistent with the hypotheses that TNF is produced in response to neural activity and in turn enhances the probability of a local sleep-like state as determined by increases in SEP amplitudes.

Optically teasing apart neural swelling and depolarization.

We measured birefringence, 90 degree scattered light, and voltage sensitive dye changes from lobster walking leg nerves. Systematic application of key chemical agents revealed separate cellular mechanisms underlying fast optical signals. Each agent exhibited mixed effects, some having a greater effect on cellular swelling and refractive index, and some altering membrane potential. Birefringence changes were tightly correlated with voltage sensitive dye signals and were perturbed by those agents that altered membrane potential. Signals from light scattered at 90 degrees corroborated the hypothesis that large angle scattering signals arise from changes in the interstitial spaces and were perturbed by those agents that altered cellular swelling and refractive index. We conclude that multiple cellular mechanisms can be exploited to measure rapid optical signals. Since birefringence produces much larger changes than scattering, the use of polarized light might lead to improvements in imaging neural activity with high temporal resolution, especially since birefringence changes corresponded closely to membrane potential.

Cerebellar fastigial nuclei activity during blood pressure challenges.

The cerebellar fastigial nuclei (FN) assist in regulating compensatory responses to large blood pressure changes and show structural injury and functional impairment to cardiovascular challenges in syndromes with sleep-disordered breathing. The patterned time course of FN responses to elevation or lowering of blood pressure and location of responsive regions within the nuclei are unclear. We evaluated FN neural activity in six anesthetized rats using optical imaging procedures during elevation and lowering of arterial pressure by phenylephrine and nitroprusside, respectively. Hypertension diminished optical correlates of FN neural activity, while measures of activity increased to hypotension, with peak neural responses occurring 5-10 s later than peak blood pressure changes. Blood pressure responses were followed by heart rate changes, and peak respiratory rates developed even later, in close temporal proximity to FN activity patterns. Although overall topographical response trends were similar, regional patterns of altered neural activity appeared to both hypertension and hypotension. The extent of neural change was greater during recovery from hypertension than for hypotension at high-dose levels. Blood pressure levels saturated with increasing phenylephrine doses, while FN activity continued to decline. No saturation appeared in heart or respiratory rate trends. The findings suggest that the FN compensate for large blood pressure changes by sympathoexcitatory and inhibitory processes, which accompany late-developing somatic or respiratory adjustments.

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.

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.

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.

Ventral medullary neuronal responses to peripheral chemoreceptor stimulation.

Recent findings suggest that carotid chemoreceptor input into the ventral medullary surface intermediate area during hypoxia is inhibitory (Gozal et al., (1994) Neurosci. Lett. 178, 73-76. However, systemic hypoxia is a complex stimulus, and effects of carotid chemoreceptor stimulation per se on intermediate ventral medullary surface neuronal activity are difficult to isolate. Therefore, we studied neural activation of the intermediate ventral medullary surface during peripheral chemoreceptor stimulation by intravenous sodium cyanide using optical procedures in seven pentobarbital-anesthetized cats. Control recordings were also acquired in the suprasylvian cortex of three cats. Images of reflected 660 nm light were collected at l/s with a charge-coupled device camera, triggered by the cardiac R wave, after 0.0, 0.5, 2, 5, 10, 20 and 40 micrograms/kg i.v. sodium cyanide administration before and following carotid sinus denervation. Sodium cyanide doses > 5 micrograms/kg significantly increased ventilation, an effect which was eliminated following carotid sinus denervation. A pronounced, dose-dependent activity decrease within the intermediate ventral medullary surface occurred within seconds of sodium cyanide administration, with subsequent return to baseline. Carotid sinus denervation eliminated rapid-onset neural responses to all sodium cyanide doses. However, at the 40 micrograms/kg dose, a smaller, slower onset (25 s), activity decrease occurred both pre- and postdenervation. In the neocortex, the sodium cyanide-induced fast responses were absent. Intravenous cyanide, acting via a carotid sinus nerve pathway, results in a dose-dependent decrease in neural activity within the intermediate ventral medullary surface of cats. High-dose sodium cyanide also appears to decrease intermediate ventral medullary surface neural activity directly.

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.

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.

Optical imaging of the ventral medullary surface of cats: hypoxia-induced differences in neural activation.

Large-array optical recording procedures provide the potential to examine simultaneous activity of large numbers of neurons. We applied this technique to examine regional neuronal activation on the ventral medullary surface (VMS) of cats during hypoxic challenges. VMS was exposed through a ventral surgical approach in eight adult cats under pentobarbital sodium anesthesia. Arterial pressure, end-tidal CO2, costal diaphragmatic electromyograms, and electrocardiograms were continuously monitored. A coherent image conduit with 12-microns-fiber resolution was attached to a charge-coupled device camera and positioned over the VMS. Reflected 700-nm light was digitized continuously at 2- to 3-s intervals during baseline period, hypoxic (6, 9, and 12% O2 in N2) exposure, and recovery. Forty images within each epoch were averaged and subtracted from baseline. Regional differences within the image were determined by analysis of variance procedures (alpha = 0.05). In caudal VMS, hypoxic challenges with 12% O2 consistently induced a regional diminution in reflected light (increased neural activity) that was rapid in onset and persisted for approximately 20 min after termination of exposure, well beyond the duration of discernible ventilatory alterations. In contrast, the same challenge resulted in decreased neural activity of similar duration in rostral VMS areas. Challenges with lower inspired concentrations of O2 reversed the pattern of diminished neural activity in rostral regions and led to a dose-dependent increase in neural activity, a dependency also observed in caudal VMS. We conclude that caudal VMS neurons demonstrate a unidirectional dose-dependent response pattern to hypoxic stimuli, whereas rostral VMS regions exhibit a bidirectional response to increasing hypoxic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

State influences on ventral medullary surface and physiological responses to sodium cyanide challenges.

Intravenous sodium cyanide (NaCN) administration lowers ventral medullary surface (VMS) activity in anesthetized cats. Sleep states modify spontaneous and blood pressure-evoked VMS activity and may alter VMS responses to chemoreceptor input. We studied VMS activation during peripheral chemoreceptor stimulation by intravenous NaCN using optical procedures in six cats instrumented for recording sleep physiology during sham saline and control site trials. Images of scattered 660-nm light were collected at 50 frames/s with an optical device after 80-100 microg total bolus intravenous NaCN delivery during waking and sleep states. Cyanide elicited an initial ventilatory decline, followed by large inspiratory efforts and an increase in respiratory rate, except in rapid eye movement sleep, in which an initial breathing increase occurred. NaCN evoked a pronounced decrease in VMS activity in all states; control sites and sham injections showed little effect. The activity decline was faster in rapid eye movement sleep, and the activity nadir occurred later in waking. Sleep states alter the time course but not the extent of decline in VMS activity.

Dew-point hygrometry system for measurement of evaporative water loss in infants.

Evaporation of water from the skin is an important mechanism in thermal homeostasis. Resistance hygrometry, in which the water vapor pressure gradient above the skin surface is calculated, has been the measurement method of choice in the majority of pediatric investigations. However, resistance hygrometry is influenced by changes in ambient conditions such as relative humidity, surface temperature, and convection currents. We have developed a ventilated capsule method that minimized these potential sources of measurement error and that allowed second-by-second, long-term, continuous measurements of evaporative water loss in sleeping infants. Air with a controlled reference humidity (dew-point temperature = 0 degree C) is delivered to a small, lightweight skin capsule and mixed with the vapor on the surface of the skin. The dew point of the resulting mixture is measured by using a chilled mirror dew-point hygrometer. The system indicates leaks, is mobile, and is accurate within 2%, as determined by gravimetric calibration. Examples from a recording of a 13-wk-old full-term infant obtained by using the system give evaporative water loss rates of approximately 0.02 mgH2O.cm-2.min-1 for normothermic baseline conditions and values up to 0.4 mgH2O.cm-2. min-1 when the subject was being warmed. The system is effective for clinical investigations that require dynamic measurements of water loss.

Low-cost acquisition of video images simultaneously with 240 electrophysiological signals.

We developed a low-cost system for simultaneous collection and storage of physiological and video signals. The system samples and multiplexes up to 240 low-bandwidth analog channels with a camera video signal, and outputs a standard composite video signal containing analog and video data. The combined signals can be stored on video tape or can be digitized by an inexpensive framegrabber. The circuitry separates horizontal synchronizing pulses from a camera output; the pulses increment a counter that sequentially selects each electrophysiological channel on a sample-and-hold multiplexer. The intensity of each horizontal scan line from the multiplexer output represents the amplitude of one sample of each physiological channel. This signal is then multiplexed with the video signal, such that a portion of each video horizontal line represents the physiological data. The combined output is stored together, providing a means for synchronizing the two signals during analysis. The design allows easy coordination of electrophysiological events with video images from a standard video camera, avoiding the necessity for separate analog to digital circuitry for physiological and video signal storage on computer media, as well as the need for complex synchronization of the data from different media.

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.

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.

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.

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.

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.

Afferent contributions to intermediate area of the cat ventral medullary surface during mild hypoxia.

The intermediate area of the cat ventral medullary surface activates to mild hypoxia. Carotid body and vagal afferent contributions to this response were examined by recording activity levels, measured as changes in scattered 660 nm light, from the medullary surface in 7 anesthetized, spontaneously breathing cats following 12% O2 in N2 ventilatory challenge. A miniaturized video camera collected images synchronous with the peak of cardiac R wave at 1/s, from a 3.2 mm diameter area, before, and following bilateral carotid sinus denervation (CSD) and vagotomy. In intact animals, hypoxia increased activity; however, greater increases in activity levels followed CSD, while vagotomy elicited a marked reduction of the response. Thus, carotid body afferents exert inhibitory or disfacilitatory influences on intermediate area neurons, while the vagus appears to play an excitatory role.