Hippocampal activity during transient respiratory events in the freely behaving cat

~ Pergamon 0306-4522(95)00525-0

Neuroscience Vol. 72, No. 1, pp. 3948, 1996 Elsevier Science Ltd

Copyright © 1996 IBRO Printed in Great Britain. All rights reserved

0306-4522/96 $15.00 + 0.00

H I P P O C A M P A L ACTIVITY D U R I N G T R A N S I E N T R E S P I R A T O R Y EVENTS IN THE F R E E L Y B E H A V I N G CAT

G. R. POE,* M. P. K R I S T E N S E N , * D. M. R E C T O R * and R. M. HARPERt :~

*Interdepartmental Program in Neuroscience and i'Department of Neurobiology, University of California Los Angeles School of Medicine, Los Angeles,

CA 90095-1763, U.S.A.

Abstract--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-mm 2 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.

Key words: sleep state, rhythmical slow activity, respiration, imaging, reflectance.

Transient respiratory events, such as apnea and sighs, are normal constituents of breathing in intact animals, even in the absence of blood gas alterations. 3'26 Al though rhythmic breathing can be maintained with only an intact brainstem, 18 transient respiratory events often depend on participation of rostral brain mechanisms. Selected affective processes, such as those produced by noxious or novel stimuli, are mediated through limbic structures, and can elicit apnea or apneusis. 4 Affective contributions to respiratory musculature are demonstrated in patients with pyramidal tract lesions who cannot breathe upon command, but exhibit spontaneous sighs and apnea, and can move the diaphragm and other respiratory musculature with affective excitation, such as laughter. 24

A variety of evidence supports a role for the hippocampus, a limbic structure mediating selected affective behaviors,19 in aspects of respiratory control. A small proport ion of human hippocampal neurons shows discharge modulat ion with the respiratory

:~To whom correspondence should be addressed. Abbreviations: CCD, charge coupled device; dEMGRMs,

running root means square of diaphragmatic EMG; ECG, electrocardiogram; EEG, electroencephalogram; EMG, electromyogram; REM, rapid eye movement; RSA, rhythmical slow activity.

cycle. 14 In a variety of species, electrical stimulation of particular limbic structures, including the dorsal hippocampus, elicits marked changes in respiration, including apnea, untimely inspiration, and respiratory rate alterations. 2'12'33 Phasic respiratory changes occur spontaneously in the intact animal, primarily at transitions between waking and sleep, or during active waking behaviors, such as orienting and periods of rapid eye movement (REM) sleep. 23

This study examined whether hippocampal acti- vat ion coincided with spontaneously occurring phasic respiratory events during sleep and waking states as well as state transitions. We assessed dorsal hippocampal activity associated with sighs and apnea in the freely behaving cat using optical and electrophysiological measures.

Reflectance imaging procedures provide a two- dimensional map of neural activity in intact preparations over extended time periods. 15'27 Measures of reflected light can detect changes in neural activity, since neural activation modifies tissue light scattering properties through physical mechanisms, such as increased blood, gliai and/or neuronal vo lume) °'13'21"36 Optical and structural changes in neural tissue occur on the time order of simultaneously recorded membrane potential changes, evoked potentials, and action potentials. 10.21.36 Although chemical and electrical stimulation produces reflectance image changes in

39

40 G. R. Poe et al.

intact preparat ions, 3~ the relationship between individual action potentials and imaged changes has yet to be tested. Current technology allows spatially coherent measurements of imaged activity change at 25 Hz from a 1 mm 2 area of tissue. 32

We used reflectance imaging to assess regional h ippocampal activation during sighs and apnea, while an at tached electrode provided measures of h ippocampal electrical activity. Recordings from overlying neocortical areas and from the hippocampus during periods lacking phasic respiratory

events served as controls.

EXPERIMENTAL PROCEDURES

Surgical procedures

Seven adult cats, 2.1-3.3 kg (acquired from the Division of Laboratory Animal Medicine, UCLA), were anesthetized with sodium pentobarbital for chronic placement of electrodes and the optical recording device. Stainless steel wires were placed into the lateral costal diaphragm to monitor electromyographic (EMG) and electrocardiographic (ECG) activity. To assess behavioral state, wire electrodes were also placed into the nuchal musculature to measure neck muscle tonus, and screw electrodes were placed over the bony orbit to record eye movements, as well as in the wall of the medial frontal sinus to record neocortical electroencephalographic

(EEG) activity. A screw in the frontal cranium served as ground reference.

To assess hippocampal and suprasylvian cortical activity, an optical recording device, comprised of a coherent image conduit coupled to a charge coupled device (CCD) camera (VLSI Vision, Edinburgh, U.K.) with 16 × 20#m pixel size, 3°'3t was placed over the CAI field of the dorsal hippocampus in four cats, and over the suprasylvian cortex in three cats. The coherent image conduit consisted of approximately 10,000 parallel 6 or 12/~m diameter fibers encased in glass cladding for a total conduit diameter of 1.6mm. Flexible optic fibers, attached to a light emitting diode for tissue illumination at 660 _ 10 nm, surrounded the coherent image conduit. Four macrowire electrodes were interspersed among the flexible fibers and extended 0.4 mm beyond the end of the probe to assess hippocampal EEG activity.

A column of neocortical tissue above the hippocampus was removed by aspiration, and the hippocampal surface or overlying suprasylvian cortex was visually identified before positioning the optic probe (stereotactic coordinates: 5 A 2.5; L 6.0; H 9.0, hippocampus, and A 2.5; L 6.0; H 14.0, neocortex). The optic device was secured to the cranium with miniature stainless steel screws and dental acrylic. Post mortem histological examination verified probe placements.

Recording procedures

One week after surgical recovery, cats were placed in a 60 cm x 60 cm x 60 cm chamber, to which they had been previously adapted, and leads were attached from a commutator to the animal. The leads and commutator allowed the

inter-peak-interval

j . . . . . . . . . . . . . . . . . . . : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ap a n th l sigh amplitude

--sto{ peak N I I I I I I

Pea~ ill

A L

Late Peak/ j Apnea Peak

t Early / L/A

"~' peak activity amplitudes

I .

/ Nadir

Fig. 1. Image and respiratory parameters. Schematic diagram of a respiratory trace (dEMG~s) , shows a sigh followed by an apnea and the accompanying optical activity changes ( - A R % ) . Parameters used to assess the timing latencies and extent of activation of the optical recording include latencies to peak activity from the onset of sighs or apnea, and the amplitudes of activity peaks and nadirs, measured from

initial baseline levels.

Hippocampal activity during respiratory events 41

cat free movement within the recording chamber. Illumina- tion levels were set such that reflected light intensity produced two-thirds the maximal video signal amplitude. Images of reflected light were amplified, digitized with 8-bit resolution at video rates of 25 or 30 Hz, and stored on digital media and videotape with the electrophysiological recordings. We optimized the dynamic range of the video signal by adjusting the video signal black level to half the

amplitude of the dimmest plxel value. Gain was set so that the brightest pixel was approximately 200 in a range of 0-255. Calibrations determined that the optimization pro- cedure increased the CCD dynamic range by a factor of 6.6, allowing assessment of a 0.06% change in absolute tissue reflectance from a single pixel.

Physiological signals were routed to a Grass 78 polygraph and a signal processor, where they were amplified and

Hippocampus

Neocortex

Time (s) CAI Regions

m I m m Sigh

Apne~

Fig. 2. Imaged activity changes over time. Individual examples of averaged activity changes ( - A R %) in the dorsal hippocampus (top) and neocortex (middle) during a sigh/apnea event, and from the CA 1 regions of four cats during sigh/apnea. Red arrows indicate sigh onset. Traces represent overall averaged activity changes over time, whereas each 120-by-80 pixel image is composed of frames averaged over 0.08 s (20 frames), subtracted from an initial baseline averaged image, and color-coded for significant changes in activity. Yellow-to-white areas indicate regions of increased activity, with white representing a 1% increase from baseline. Blue-to-black regions indicate a similarly scaled decrease in activity, whereas green pixels represent no significant activity change (ct = 0.05). Images (lower panel) depict the difference between CA1 areas activated, for each cat, during peak activation of the sigh portion of a sigh/apnea event versus

approximately 3 s after onset of the subsequent apnea. Scale bar = 200 #m.

42 G . R . Poe et al.

band-pass filtered in accordance with Nyquist frequency requirements. The raw diaphragmatic E M G signal was processed with a running root means square calculation (dEMGRM s, time c o n s t a n t = 0.3 s). Electrophysiological data were simultaneously written on paper, stored on videotape, and digitized on to a computer disk (hippocampal and cortical EEG, 250 Hz). Recordings spanned at least 2 cycles of active waking, quiet sleep and REM sleep, which were later scored from electrophysiological records according to s tandard state classification criteria for cats. 37

Respiratory event criteria

Inspiratory peaks were identified with a max imum amplitude detection algorithm, and amplitude values were con- verted to z-scores by dividing the absolute amplitude of the peak by the mean peak amplitude of the surrounding 20 breaths. Sighs were defined as : -scored inspiratory amplitudes exceeding the mean peak inspiratory amplitude by 2 times the S.D. Inter-breath intervals were z-scored by dividing each inter-peak interval by the mean inter-peak interval of the 20-breath epoch. Apnea were identified when a z-scored inter-breath interval exceeded the mean by 2.5 x S.D. The onset and cessation of inspiratory efforts were identified by a threshold detection algorithm set to 1 S.D. above baseline dEMGRM s signal levels. Apnea lengths were measured between inspiratory offset and subsequent onset (Fig. 1).

State classification of respiratory events

Sighs and state-matched control periods lacking phasic respiratory events typically included brief (1 s) arousals in quiet sleep (nine of 12 events) or transitions between waking and quiet sleep. Isolated apnea did not take place during state transitions, but arose during either REM sleep (five of nine events) or active waking (four events). The majority of sigh/apnea events took place during transition from waking to quiet sleep (four of 15 events), or during brief arousals in transition from stage 1 to stage 2 quiet sleep (eight of 15 events). Sigh/apnea also occurred during active waking, transition from quiet sleep to waking and transition from REM sleep to waking.

Image signal processing

Overall activity changes. Video frames were analysed during an interval spanning from 12 s before, to 28 s after the onset of a sigh or apnea, and from state-matched control periods. State-matched control periods were aligned by the onset of state transitions matching that of sighs. With reflectance imaging, areas with enhanced or suppressed activity appear as regions which are darker or lighter, respectively. 3° Activity changes during phasic respiratory events and control periods were calculated by subtracting the overall reflectance value of each frame from the overall reflectance value of the first frame in the 40-s period ( - A R ) . Reflectance values were normalized across trials and recordings by dividing the activity change value of each frame by the average reflectance value of the first frame, thereby correcting for differences in initial reflectance levels. Moment - to -moment normalized activity changes were averaged across a min imum of nine trials within a classification (e.g. sighs, sigh/apnea, apnea, or state-matched controls).

Temporal relations between imaged activity and respiratory events. We measured the amplitude of optical signal peaks and their timing in relation to phasic respiratory parameters (Fig. 1) to determine whether neural activity changes preceded or followed respiratory events, the persistence of hippocampal activation relative to the length of the respiratory events, and the differences between the extent of activation associated with sighs, apnea and sigh/apnea.

We performed a repeated A N O V A to evaluate respiratory parameter relationships in relation to the timing or amplitude of hippocampal activity peaks, and multiple

regression analyses to assess the interdependence of respiratory and hippocampal activity parameters. A sign test was used to assess timing relationships between the onset of activity changes and respiratory events.

Images of activity change. A N O V A (c~ = 0.05) were em- ployed to evaluate regional differences within the imaged field across time. Sequential frames from the 40-s span of respiratory or control events were partitioned into succes- sive 0.8-s blocks (20 or 24 frames/block from data collected at 25 and 30 Hz, respectively). Images within each block were averaged, pixel-by-pixel, and subtracted from the first block average in the sequence. Subtracted images were pseudocolored to show activity changes where significant.

Hippocampal electroencephalogram signal analysis

Hippocampal EEG activity during the respiratory event was divided into sequential epochs of 3 s each, demeaned and cosine tapered, and subjected to fast Fourier trans- forms. Power in 32 frequency bins from 0 to 12.5 Hz was plotted over time. Power estimates were grouped and averaged by respiratory event type (e.g. apnea) within each animal, and normalized and averaged across animals. Samples from state-matched control periods were analyzed in the same fashion as those which included respiratory events. A N O V A procedures and post hoc tests with repeated measures were applied to assess changes in hippocampal EEG across sigh and/or apnea periods. Slow

o.30~ ~[]][K ~<. o.oo~i~~Ill~B ~

< -o.12-t ! ~

.. 1 0 s

Fig. 3. Mean overall imaged activity changes. Average hippocampal neural activity changes ( - -AR%) , thickness indicates mean _+ S.E.M. before and after onset of all respiratory events from all animals: sighs, sighs followed by apnea, and isolated apnea. The solid vertical line through the sigh and sigh/apnea traces indicates inspiratory onset during the sigh, and the dashed line through sigh/apnea and apnea indicates apnea onset. Respiratory traces below the averages depict single, representative events. The control trace represents average activity during state transitions, such as occurred during sighs, but without the respiratory event. The dotted vertical line through the control trace

indicates onset of the state change.


(3 ~, Hz) and fast (5-6 Hz) rhythmical slow activity (RSA) was identified according to conventions established by others. 7

ation of apnea (Fig. 6) showed distinct spectral power and frequency changes that were not related solely to state transition influences.

RESULTS

Reflectance changes with respiratory events

Sighs, and the resumption of breathing after apnea, were preceded (1-3 s) by significantly increased optical measures of dorsal hippocampal activity (Figs 2, 3). Comparable activity changes did not appear in the neocortex (Fig. 2) or in state-matched control periods lacking sighs and apnea (Fig. 3). Regions activated prior to sighs differed from regions activated prior to apnea (Fig. 2). The unique timing and amplitude characteristics of activity changes associated with sigh/apnea events showed that sigh/apnea were not simply additive combinations of isolated sigh and apnea events (Fig. 5). Finally, hippocampal EEG measures of activity prior to sighs and the termin-

Images of regional act&ation

Images collected from the dorsal hippocampus during a representative sigh/apnea event are shown in the top panel of Fig. 2. The neocortical field (middle panel) displayed uniform activity levels across the image that were not temporally linked with sighs and/or apnea. Within the image field, specific hippocampal regions exhibited increased activity prior to a sigh, and these regions differed from areas showing increased activity during apnea prior to resumption of breathing. An example of such regional differences during a sigh/apnea event for each hippocampal placement is shown in Fig. 2 (lowest panel).

Average traces of hippocampal activity changes associated with control state transitions, isolated

Respiratory Cycle Cardiac Cycle

500 ms 100 ms

Hippocampus

~, , i i I . ~ l l~ l t l l l J l m . t ~ . . . . . . I[ "q HI W' ',Nt I

Neocortex

-0.12 Fig. 4. Summary respiratory, cardiac and reflectance traces. Summary traces (mean __+ S.E.M., n = 50) of control respiratory and cardiac periods and associated reflectance changes ( -AR %) in the hippocampus and neocortex. Average reflectance traces were triggered by onset of normal inspirations or by the cardiac

R-wave.

44 G.R. Poe et al.

sighs, isolated apnea, and sigh/apnea conditions are shown in Fig. 3. Isolated sighs were consistently preceded 1.5 s (_+ 3, p < 0.01) by transiently increased activity, followed by an activity decline during the sigh, and a return to baseline after several seconds.

The resumption of breathing after isolated apnea (mean apnea duration, 3 s) was preceded 1.2 s ( _ 0.4, p < 0.05) by increased activity. Hippocampal activity peaked 1.5 s ( _ 0.7) after resumption of breathing at twice the activation amplitude achieved during isolated sighs, then gradually declined to baseline over the subsequent 11 s. This effect was enhanced during REM sleep, accounting for much of the variability at the peak.

Sighs followed by apnea combined components of both sigh and apnea responses. A rise in hippocampal activity consistently preceded sigh onset (p < 0.01). Activity began to increase 3.2 s ( _ 0.4) prior to sigh onset, and began to decline during the sigh, before apnea onset (p < 0.01). During apnea, a second burst of hippocampal activation began 2.2 s (___ 0.3) before breathing resumed (p <0.01), and peaked 9.5s ( + 1.0) after initiation of apnea. Hippocampal activity returned to baseline levels approximately 10 s after termination of phasic respiratory events.

The averaged activity of sighs and state-matched control periods was obtained during a combination of state transitions, and showed no overall shift in activity levels. However, during sigh/apnea, when the majority of transitions were from waking to quiet sleep, and stage 1 to 2 quiet sleep (sigh/apnea trace, Fig. 3), post-transition activity levels remained elev- ated above pre-sigh values, as previously reported. 27

Reflectance changes with heartbeats and normal breathing

Alterations in reflectance during periods of normal breathing and cardiac cycles were smaller (by a factor of 10) than changes associated with sighs and apnea (Fig. 4), and the few significantly affected pixels (~'°~o of total) exhibited a diffuse distribution.

Comparisons of peak activity and timing

With the exception of the first imaged activation peak, time from the onset of the respiratory event to the peak or nadir hippocampal response was significantly longer during sigh/apnea than sighs or apnea alone (Fig. 5). In addition, peak activity amplitude values were significantly higher during sigh/apnea than sighs alone, although intervening nadir values were similar. Peak activity amplitudes during isolated apnea were comparable to those associated with the apnea of sigh/apnea events.

Sigh amplitude and apnea duration did not correlate with peak hippocampal activity changes (p = n.s.). However, more extensive diaphragmatic movements during sighs correlated with shorter latencies to the nadir of hippocampal activity (r = - 0 . 6 7 , p <0.05); and longer pauses in diaphragmatic activity were correlated with longer

delays to peak hippocampal activity after apnea onset (r = 0.96, p < 0.05, isolated apnea; r = 0.50, p = 0.09 sigh/apnea).

Hippocampal electroencephalogram frequency profiles

Hippocampal EEG characteristics during sighs, apnea and sigh/apnea are summarized in Fig. 6. All changes described below were significant (p < 0.05), unless otherwise noted.

Sighs. Power declined in all but slow (3-4 Hz) and fast (5-6 Hz) RSA frequencies before isolated sighs, then declined across all frequencies during the sigh, and increased at slow and fast RSA frequencies upon resumption of normal breathing. State-matched control periods (not shown) displayed decreased power at all frequencies during transient arousals, with no selective sparing or alteration in RSA frequency power.

Apnea. Since isolated apnea occurred only in REM sleep and active waking, spectral power was concen- trated in a narrow, mid-RSA band (4.5-5.25 Hz).

Latencies to Peak td ,

0.45-

0.30-

~" 0.15-

~ 0.00-

-0.15-

-0.30-

Peak Activity Amplitudes

E N

/ Sigh/Apnea

L

m Apnea

A ?--q SEM Sigh

Fig. 5. Summary of overall imaged activity parameters. Latencies to peak activity and peak activity amplitudes of sighs, sigh/apnea, and apnea. Mean values are indicated by the filled and patterned bars, while solid white boxes indicate the standard error of the mean (S.E.M.). The abscissae indicate data at the early peak after initiation of the sigh (E), nadir (N) and second peak (L) of the biphasic response associated with sighs and sigh/apnea events. The peak latency and amplitude from initiation of the apnea (A) were measured for both sigh/apnea and isolated apnea. Asterisks indicate significant differences (*p < 0.05;

**p < 0.01).

Hippocampal activity during respiratory events

Sigh/Apnea Apnea Sigh 45

; e .

" " o u J

-~ i k / I f

/ \ / \ J %

Freq. (Hz) Fig. 6. Hippocampal changes. Hippocampal electroencephalographic spectral activity from 0 to 12 Hz during isolated sighs, isolated apnea and sigh/apnea and corresponding enlargements of the RSA frequency range (3~5 Hz). Normalized spectral power densities are indicated in pseudocolored, linear scale (white = maximum). Time is indicated on the abscissa and frequency on the ordinate. Data were averaged across all animals and all events within each classification. Onset of each respiratory event is indicated

by the letters "a" (apnea) and "s" (sighs). Scale bar = I epoch (3 s).

This band broadened to include slow RSA (3-4.5 Hz) before apnea onset. At apnea onset, the dominant RSA frequency shifted upwards by 1 Hz. Power at other frequencies did not change (p = n.s.) over the course of the apnea.

Sigh/apnea. Power increased at slow RSA frequencies before the sigh and before the apnea. As with isolated apnea, dominant RSA frequency increased 1 Hz during apnea and the resumption of breathing. Power at frequencies 0-3 Hz and 6-12 Hz did not significantly change (p = n.s.) across sigh/apnea.

State effects on hippocampal electroencephalogram. Behavioral state significantly affected slow electrical activity (p < 0.01) during all conditions. Before apnea onset (isolated apnea or sigh/apnea), slow RSA power was substantially diminished during REM sleep, compared to active waking and quiet sleep, but before apnea termination, fast RSA power was es- pecially increased during REM sleep, compared to other states. However, multivariate analyses revealed respiratory event related activity changes which could not be explained by state effects.

D I S C U S S I O N

Changes in hippocampal activation patterns ac- companied transient respiratory events. Increased activity preceded sighs and resumption of breathing during apnea. Areas of the image that activated prior to sighs differed consistently from the areas primarily activated during apnea, before apnea termination. The differences in timing and amplitude of peak activity changes between events suggest that sigh/apnea activation patterns are not simply the result of combining activity profiles of isolated sighs and apnea. Hippocampal EEG analysis revealed shifts in 3-6 Hz RSA power accompanying respiratory event-related optical activity changes. Slow RSA (3-4 Hz) power increased relative to other frequencies prior to sighs, whereas peak RSA power moved to higher frequencies during apnea. State transitions without transient respiratory events did not elicit the reproducible pattern, amplitude and time course of hippocampal activity changes accompanying sighs and apnea.

46 G.R. Poe et al.

Anatomic evidence o f limbic involvement in respiratory events

Functional evidence exists for vagal afferent activity reaching the hippocampus, 29 and components of the hippocampal apnea and sigh responses may be associated with such sensory input. Several anatomical pathways could also mediate forebrain influences on transient respiratory events. The dorsal hippocampus projects to the septal nuclei and hypothalamus through the fornix, and hypothalamic neurons project to the midbrain tegmentum, a pre-motor structure involved with somatic motor and visceral reactions.l The paraventricular nucleus of the hypothalamus, which receives input from the CA1 via the subiculum and septal nuclei, 35 projects directly to midbrain and brainstem regions which modulate diaphragmatic activity 2~,34 and inspiratory/expiratory phase switching, 6'H essential to phasic respiratory events. A functional role for the paraventricular nucleus of the hypothalamus in phasic respiratory events is supported by separate studies which demonstrated transient overall activity changes in medial portions of the paraventricular region preceding the termination of apnea during sigh/apnea events, z° Spontaneous sighs and apnea during sleep are not executed through pyramidal pathways in humans, 24 and may be mediated through these limbic projections.

Ruit and Neafsey 33 found that, within hippocampal/entorhinal areas, stimulation of the CAl region most reliably produced respiratory responses with low (10/xA) stimulation currents in the anesthetized rat. Latencies to respiratory change ranged from 1-10 s from stimulus onset, and respiratory patterns returned to baseline levels before offset of the 60 s stimulus train. Their findings are comparable to the 1-3 s latencies we observed between onset of spontaneous hippocampal activation and sigh onset or apnea termination, and to the continued spontaneous hippocampal activation which extended l0 s beyond resumption of regular breathing patterns.

\ Concomitant optical and hippocampal electroencephalogram changes during sighs and apnea

Both optical and RSA changes preceded the increased respiratory efforts of sighs and termination of apnea. Slow RSA frequency power increased prior to sighs, as specific regions of the hippocampus were activated, whereas during apnea, the dominant RSA frequency shifted upwards 1 Hz, and different CA1 regions were activated. The finding of regional activity changes in parallel with frequency shifts is not unprecedented. Bragin et al. s and Bullock et al. 9 demonstrated sharp demarcations between hippocampal areas producing theta and gamma frequency activity in the awake, freely moving rat, suggesting a functional anatomical distinction between fields involved with various activity frequencies.

Slow and fast RSA amplitude and frequency

modulations are associated with different types of behaviors and appear to be produced by different neurotransmitter systems. 3s The findings of increased slow RSA amplitude preceding sighs, and increased fast RSA amplitude before apnea termination suggest separate neural mechanisms underlying the altered diaphragmatic activity associated with these respiratory events. In addition, differences in optical activity found in distinct CA1 regions during sighs and apnea imply separate anatomical substrates. Finally, our results show that slow and fast RSA, as well as subregional hippocampal activity, may be differentially modulated by state, since slow RSA changes prior to apnea were attenuated during REM sleep as compared with waking, whereas fast RSA and optical changes during apnea were enhanced.

Technical considerations: sources o f reflectance change

Cellular swelling. A primary source of reflectance changes observed during neural activation is the alteration in cell volume accompanying voltage changes in neurons and glia. Studies demonstrating increased neural volume with stimulation of neural activity date from the late nineteenth century. 22 Tis- sue light scattering properties are altered by cell swelling through a decrease in the refractive index of the membrane, l° MacVicar and Hochman 21 showed increased light transmission in the hippocampal slice upon electrical stimulation, which could be blocked by agents inhibiting cell swelling. Cell volume changes induced by hypo-osmotic and hyper-osmotic solution perfusion also alter 660nm light reflectance. 2s Thus, changes in cell volume, coincident with neural activity, can be measured through reflectance alterations.

In tracranial pressure, blood gases and blood volume. Small reflectance alterations in the hippocampus and neocortex with the cardiac and respiratory cycle may result from pressure artifacts. Intracranial pressure varies with thoracic movements. 16 However, despite extensive thoracic pressure changes occurring with the regular, deep inspiratory efforts of quiet sleep, intracranial pressure changes during this period ex- erted minimal effects on hippocampal signals, and cannot account for the finding that hippocampal reflectance declines precede, by several seconds, the increased inspiratory efforts of sighs and cessation of apnea. In addition, reflectance changes continued during extended apnea (a period of minimal thoracic movement), persisted several seconds after breathing resumed, and were more extensive during termination of apnea than preceding sighs, although sighs would create larger thoracic pressure changes than would resumption of normal respiratory patterns. Finally, the absence of significant correlations between sigh amplitude and peak reflectance changes suggest that thoracic pressure does not directly relate to reflectance.

The absence of respiratory event-related activity changes in the neocortex indicates that mechanisms


other than alterations in blood 0 2 and C O 2 levels account for the reflectance changes found in this study. Hippocampal activity began to fall within 1 s of sigh onset, too short a period for changes in blood perfusion effects. Apnea durat ion did not correlate with peak activation amplitude, although longer apnea should correspond to higher tissue CO2 saturation.

Blood perfusion is altered by neural activity and may contribute to the reflectance changes observed with phasic respiratory events. However, reflectance is altered by neural activity in the absence of vascular perfusion changes, such as during stimulation in the blood-free slice preparation 21 and during stimulation in intact preparations when blood perfusion alterations are pharmacologically blocked. 17 Reflectance is also altered at rates sufficiently rapid to follow action potentials. 10,36 Collectively, the evidence suggests that reflectance changes primarily result f rom alterations in cell volume coincident with neural activation.

CONCLUSIONS

Overall and regional hippocampal activity in-

creased before spontaneous sighs, and was ac- companied by increased slow RSA power. Different regions increased activity, and RSA frequency be- came faster during apnea, before the resumption of breathing. These findings suggest a hippocampal influence on transient respiratory events across states. The extent of activation events was much larger, and of different spatial distribution, than activation associated with normal breaths. The neocortex did not show changes analogous to respiratory-related acti- vat ion in the dorsal hippocampus, and comparable changes did not occur in state-matched control periods. The specificity of areas initiating activity changes with sighs and apnea suggests that distinct CA1 regions are differentially involved in phasic respiratory events.

Acknowledgements--This research was supported by HL-22418. G.P. is supported by a Howard Hughes Medi- cal Institute Predoctoral Fellowship. D.R. is supported by predoctoral fellowship NIDR DE 07212. We would like to thank Paula Moore and Douglas Nitz for their assistance.

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(Accepted 20 October 1995)

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Hippocampal activity during transient respiratory events in the freely behaving cat