Basal forebrain acetylcholine release during REM sleep is significantly greater than during waking

Cholinergic neurons of the basal forebrain supply the neocortex with ACh and play a major role in regulating behavioral arousal and cortical electroencephalographic activation. Cortical ACh release is greatest during waking and rapid eye movement (REM) sleep and reduced during non-REM (NREM) sleep. Loss of basal forebrain cholinergic neurons contributes to sleep disruption and to the cognitive deficits of many neurological disorders. ACh release within the basal forebrain previously has not been quantified during sleep. This study used in vivo microdialysis to test the hypothesis that basal forebrain ACh release varies as a function of sleep and waking. Cats were trained to sleep in a head-stable position, and dialysis samples were collected during polygraphically defined states of waking, NREM sleep, and REM sleep. Results from 22 experiments in four animals demonstrated that means +/- SE ACh release (pmol/10 min) was greatest during REM sleep (0.77 +/- 0.07), intermediate during waking (0.58 +/- 0.03), and lowest during NREM sleep (0.34 +/- 0.01). The finding that, during REM sleep, basal forebrain ACh release is significantly elevated over waking levels suggests a differential role for basal forebrain ACh during REM sleep and waking.     

Dialysis delivery of an adenosine A2A agonist into the pontine reticular formation of C57BL/6J mouse increases pontine acetylcholine release and sleep

In vivo microdialysis in C57BL/6J (B6) mouse was used to test the hypothesis that activating adenosine A(2A) receptors in the pontine reticular formation (PRF) increases acetylcholine (ACh) release and rapid eye movement (REM) sleep. Eight concentrations of the adenosine A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS 21680; CGS) were delivered to the PRF and ACh in the PRF was quantified. ACh release was significantly increased by dialysis with 3 mum CGS and significantly decreased by dialysis with 10 and 100 microm CGS. Co-administration of the adenosine A(2A) receptor antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; 30 nM) blocked the CGS-induced increase in ACh release. In a second series of experiments, CGS (3 microm) was delivered by dialysis to the PRF for 2 h while recording sleep and wakefulness. CGS significantly decreased time in wakefulness (-51% in h 1; -54% in h 2), increased time in non-rapid eye movement (NREM) sleep (90% in h 1; 151% in h 2), and increased both time in REM sleep (331% in h 2) and the number of REM sleep episodes (488% in h 2). The enhancement of REM sleep is consistent with the interpretation that adenosine A(2A) receptors in the PRF of the B6 mouse contribute to REM sleep regulation, in part, by increasing ACh release in the PRF. A(2A) receptor activation may promote NREM sleep via GABAergic inhibition of arousal promoting neurons in the PRF.     

Formation and Dynamics of Waves in a Cortical Model of Cholinergic Modulation

Acetylcholine (ACh) is a regulator of neural excitability and one of the neurochemical substrates of sleep. Amongst the cellular effects induced by cholinergic modulation are a reduction in spike-frequency adaptation (SFA) and a shift in the phase response curve (PRC). We demonstrate in a biophysical model how changes in neural excitability and network structure interact to create three distinct functional regimes: localized asynchronous, traveling asynchronous, and traveling synchronous. Our results qualitatively match those observed experimentally. Cortical activity during slow wave sleep (SWS) differs from that during REM sleep or waking states. During SWS there are traveling patterns of activity in the cortex; in other states stationary patterns occur. Our model is a network composed of Hodgkin-Huxley type neurons with a M-current regulated by ACh. Regulation of ACh level can account for dynamical changes between functional regimes. Reduction of the magnitude of this current recreates the reduction in SFA the shift from a type 2 to a type 1 PRC observed in the presence of ACh. When SFA is minimal (in waking or REM sleep state, high ACh) patterns of activity are localized and easily pinned by network inhomogeneities. When SFA is present (decreasing ACh), traveling waves of activity naturally arise. A further decrease in ACh leads to a high degree of synchrony within traveling waves. We also show that the level of ACh determines how sensitive network activity is to synaptic heterogeneity. These regimes may have a profound functional significance as stationary patterns may play a role in the proper encoding of external input as memory and traveling waves could lead to synaptic regularization, giving unique insights into the role and significance of ACh in determining patterns of cortical activity and functional differences arising from the patterns.     

Effects of a New Thyrotropin Releasing Hormone Analogue, YM-14673, on the In Vivo Release of Acetylcholine as Measured by Intracerebral Dialysis in Rats

The effects of a new thyrotropin releasing hormone (TRH) analogue, YM-14673 (N alpha-[[(S)-4-oxo-2-azetidinyl]carbonyl]-L-histidyl-L-prolinamide dihydrate), on the release of acetylcholine (ACh) in free-moving rats were examined in vivo by intracerebral dialysis. In the frontal cortex, YM-14673 (0.1-0.3 mg/kg) caused a significant dose-dependent increase in the extracellular levels of ACh, suggesting that YM-14673 stimulated the ACh release. These actions of YM-14673 were about 50 times more potent than those of TRH. On the other hand, extracellular levels of ACh in caudate nucleus were not changed following injection of YM-14673 even at 3 mg/kg. TRH and methamphetamine also increased the release of ACh in frontal cortex. Haloperidol prevented the increase in the methamphetamine-induced release of ACh, whereas the increased release of ACh produced by YM-14673 was partially antagonized by haloperidol. These results suggest that the dopaminergic system affects the facilitatory effects on the ACh release in the frontal cortex and that the stimulatory effect of YM-14673 on the frontal cholinergic neurons is partially mediated by dopaminergic neurons.     

In Vivo Production and Release of Acetylcholine from Primary Fibroblasts Genetically Modified to Express Choline Acetyltransferase

Abstract: Primary rat fibroblasts genetically modified to express Drosophila choline acetyltransferase (dChAT) synthesize and release acetylcholine (ACh) in vitro. The ACh produced from the transduced fibroblasts was found to be enhanced by increasing amounts of choline chloride in the culture media. These dChAT-expressing cells were then implanted into the intact hippocampus of adult rats and in vivo microdialysis was performed 7–10 days after grafting to assess the ability of the cells to produce ACh and respond to exogenous choline in vivo. Samples collected from anesthetized rats revealed fourfold higher levels of ACh around dChAT grafts than from either non-grafted or control-grafted hippocampi. Localized choline infusion (200 μ) through the dialysis probes was found to induce a selective twofold increase in ACh release only from the dChAT-expressing fibroblasts. These results indicate not only that dChAT-expressing fibroblasts continue to synthesize and secrete ACh for at least 10 days after intracerebral grafting, but that the levels of ACh can be manipulated in vivo. The ability to regulate products within genetically modified cells in vivo may provide a powerful avenue for exploring the role of discrete substances within the CNS.     

Biogenic amines in the regulation of wakefulness and sleep

Neurophysiological, neurochemical and neuropharmacological evidence indicates that cerebral monoamines are important regulators of wakefulness and sleep besides cerebral amino acid-ergic and peptidergic systems. The cerebral monoamines noradrenaline, dopamine and acetylcholine are positively involved in electroencephalographic aspects of waking and paradoxical or REM sleep. A high level of noradrenergic transmission facilitates waking, and a lower, moderate level facilitates REM sleep. Serotonin is involved in the regulation of synthesis, storage and release of sleep inducing factors, and in the gating mechanisms of REM sleep. Histamine neurons play a role in the regulation of vigilance during waking state. These neurotransmitter systems are important targets for drug actions.     

Arginine-vasopressin fragment 4-9 stimulates the acetylcholine release in hippocampus of freely-moving rats.

We examined the effects of arginine-vasopressin (AVP) C-terminal fragment 4-9, which facilitates learning and memory, on the extracellular acetylcholine (ACh) release in hippocampus of freely-moving rats using the microdialysis technique. Following administration of AVP4-9, p-Glu-Asn-Cys[Cys]-Pro-Arg-Gly-NH2, through the dialysis probe into the hippocampus, ACh levels in dialysates from the hippocampus increased markedly in dose and time dependent manner at 2-2.5 and 2.5-3 hr. AVP1-9, the parent peptide, has a similar enhancing effect on ACh release as AVP4-9. Stimulated ACh release by AVP4-9 was significantly inhibited by V1-selective receptor antagonist ([1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-(O-methyl)-tyrosine]AVP), but not by V2-selective antagonist ([1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-D-Ile, 4-Ile]AVP). From these observations, it is demonstrated that AVP4-9 stimulates the ACh release in rat hippocampus via mediating V1-like vasopressin receptors.     

Rapid changes in glutamate levels in the posterior hypothalamus across sleep-wake states in freely behaving rats

The histamine-containing posterior hypothalamic region (PH-TMN) plays a key role in sleep-wake regulation. We investigated rapid changes in glutamate release in the PH-TMN across the sleep-wake cycle with a glutamate biosensor that allows the measurement of glutamate levels at 1- to 4-s resolution. In the PH-TMN, glutamate levels increased in active waking (AW) and rapid eye movement (REM) sleep compared with quiet waking and nonrapid eye movement (NREM) sleep. There was a rapid (0.6 +/- 1.8 s) and progressive increase in glutamate levels at REM sleep onset. A reduction in glutamate levels consistently preceded the offset of REM sleep by 8 +/- 3 s. Short-duration sleep deprivation resulted in a progressive increase in glutamate levels in the PH-TMN, perifornical-lateral hypothalamus (PF-LH), and cortex. We found that in the PF-LH, glutamate levels took a longer time to return to basal values compared with the time it took for glutamate levels to increase to peak values during AW onset. This is in contrast to other regions we studied in which the return to baseline values after AW was quicker than their rise with waking onset. In summary, we demonstrated an increase in glutamate levels in the PH-TMN with REM/AW onset and a drop in glutamate levels before the offset of REM. High temporal resolution measurement of glutamate levels reveals dynamic changes in release linked to the initiation and termination of REM sleep.     

Cataplexy-active neurons in the hypothalamus: implications for the role of histamine in sleep and waking behavior

Noradrenergic, serotonergic, and histaminergic neurons are continuously active during waking, reduce discharge during NREM sleep, and cease discharge during REM sleep. Cataplexy, a symptom associated with narcolepsy, is a waking state in which muscle tone is lost, as it is in REM sleep, while environmental awareness continues, as in alert waking. In prior work, we reported that, during cataplexy, noradrenergic neurons cease discharge, and serotonergic neurons greatly reduce activity. We now report that, in contrast to these other monoaminergic "REM-off" cell groups, histamine neurons are active in cataplexy at a level similar to or greater than that in quiet waking. We hypothesize that the activity of histamine cells is linked to the maintenance of waking, in contrast to activity in noradrenergic and serotonergic neurons, which is more tightly coupled to the maintenance of muscle tone in waking and its loss in REM sleep and cataplexy.     

Effects of exposure to low level radiofrequency fields on acetylcholine release in hippocampus of freely moving rats

Some central cholinergic effects have been reported in animals after acute exposure to radiofrequency electromagnetic field at low intensity. We studied acetylcholine (ACh) release in the brain of freely moving rats exposed for 1 h during the day to a 2.45 GHz continuous wave radiofrequency field (RF) (2 or 4 mW/cm(2)) or exposed for 1 or 14 h during the night to a 800 MHz field modulated at 32 Hz (AM 200 mW/cm(2)). Measurements were performed by microdialysis using a membrane implanted through the upper CA1 region of the hippocampus. After irradiation with the 2.45 GHz RF, rats exposed at 2 mW/cm(2) did not show a significant modification of Ach release, whereas those exposed at 4 mW/cm(2) showed a significant 40% decrease in mean ACh release from hippocampus. This decrease was maximal at 5 h post exposure. Exposure to the 800 MHz RF for 1 h did not cause any significant effect, but exposure for 14 hrs induced a significant 43% decrease in ACh release during the period 11 p.m.-4 a.m. compared to control rats. In the control group we observed an increase of ACh release at the beginning of the night, which was linked to the waking period of rats. This normal increase was disturbed in rats exposed overnight to the 800 MHz RF. This work indicates that neurochemical modification of the hippocampal cholinergic system can be observed during and after an exposure to low intensity RF.     

Pregnenolone sulfate and aging of cognitive functions: behavioral, neurochemical, and morphological investigations

Neurosteroids are a subclass of steroids that can be synthesized in the central nervous system independently of peripheral sources. Several neurosteroids influence cognitive functions. Indeed, in senescent animals we have previously demonstrated a significant correlation between the cerebral concentration of pregnenolone sulfate (PREG-S) and cognitive performance. Indeed, rats with memory impairments exhibited low PREG-S concentrations compared to animals with correct memory performance. Furthermore, these memory deficits can be reversed by intracerebral infusions of PREG-S. Neurotransmitter systems modulated by this neurosteroid were unknown until our recent report of an enhancement of acetylcholine (ACh) release in basolateral amygdala, cortex, and hippocampus induced by central administrations of PREG-S. Central ACh neurotransmission is involved in the regulation of memory processes and is affected in normal aging and in human neurodegenerative pathologies like Alzheimer's disease. ACh neurotransmission is also involved in the modulation of sleep-wakefulness cycle and relationships between paradoxical sleep and memory are well documented in the literature. PREG-S infused at the level of ACh cell bodies induces a dramatic increase of paradoxical sleep in young animals. Cognitive dysfunctions, particularly those observed in Alzheimer's disease, have also been related to alterations of cerebral plasticity. Among these mechanisms, neurogenesis has been recently studied. Preliminary data suggest that PREG-S central infusions dramatically increase neurogenesis. Taken together these data suggest that PREG-S can influence cognitive processes, particularly in senescent subjects, through a modulation of ACh neurotransmission associated with paradoxical sleep modifications; furthermore our recent data suggest a role for neurosteroids in the modulation of hippocampal neurogenesis.     

Opposite variations of extracellular concentrations of 5-hydroxyindoleacetic acid (5-HIAA) measured by voltammetry of axonal terminals and cell bodies of the dorsal raphe nucleus through the sleep-wake cycle

Differential pulse voltammetry was used for 5-hydroxyindoleacetic acid (5-HIAA) detection in the rat caudate (n. Cd) and Raphe Dorsalis (n. RD) nuclei, in chronic experimental conditions. In the anterior and ventral part of n. RD, large increases in the extracellular concentrations of 5-HIAA were reported during slow wave sleep (SWS) and paradoxical sleep (PS) whereas a decrease occurred during waking. These variations could reflect the dendritic release of serotonin. In n. Cd, opposite variations of the extracellular concentrations of 5-HIAA were observed i.e. increase during waking state and decrease during SWS and PS.     

Radio frequency electromagnetic field exposure in humans: Estimation of SAR distribution in the brain,effects on sleep and heart rate

In two previous studies we demonstrated that radiofrequency electromagnetic fields (RF EMF) similar to those emitted by digital radiotelephone handsets affect brain physiology of healthy young subjects exposed to RF EMF (900 MHz; spatial peak specific absorption rate [SAR] 1 W/kg) either during sleep or during the waking period preceding sleep. In the first experiment, subjects were exposed intermittently during an 8 h nighttime sleep episode and in the second experiment, unilaterally for 30 min prior to a 3 h daytime sleep episode. Here we report an extended analysis of the two studies as well as the detailed dosimetry of the brain areas, including the assessment of the exposure variability and uncertainties. The latter enabled a more in depth analysis and discussion of the findings. Compared to the control condition with sham exposure, spectral power of the non-rapid eye movement sleep electroencephalogram (EEG) was initially increased in the 9-14 Hz range in both experiments. No topographical differences with respect to the effect of RF EMF exposure were observed in the two experiments. Even unilateral exposure during waking induced a similar effect in both hemispheres. Exposure during sleep reduced waking after sleep onset and affected heart rate variability. Exposure prior to sleep reduced heart rate during waking and stage 1 sleep. The lack of asymmetries in the effects on sleep EEG, independent of bi- or unilateral exposure of the cortex, may indicate involvement of subcortical bilateral projections to the cortex in the generation of brain function changes, especially since the exposure of the thalamus was similar in both experiments (approx. 0.1 W/kg).     

Nitric oxide in B6 mouse and nitric oxide-sensitive soluble guanylate cyclase in cat modulate acetylcholine release in pontine reticular formation.

ACh regulates arousal, and the present study was designed to provide insight into the neurochemical mechanisms modulating ACh release in the pontine reticular formation. Nitric oxide (NO)-releasing beads microinjected into the pontine reticular formation of C57BL/6J (B6) mice significantly (P < 0.0001) increased ACh release. Microdialysis delivery of the NO donor N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)-ethanamine (NOC-12) to the mouse pontine reticular formation also caused a concentration-dependent increase in ACh release (P < 0.001). These are the first neurochemical data showing that ACh release in the pontine reticular formation of the B6 mouse is modulated by NO. The signal transduction cascade through which NO modulates ACh release in the pontine reticular formation has not previously been characterized. Therefore, an additional series of studies quantified the effects of a soluble guanylate cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), on ACh release in the cat medial pontine reticular formation. During naturally occurring states of sleep and wakefulness, but not anesthesia, ODQ caused a significant (P < 0.001) decrease in ACh release. These results show for the first time that NO modulates ACh in the medial pontine reticular formation of the cat via an NO-sensitive sGC signal transduction cascade. Isoflurane and halothane anesthesia have been shown to decrease ACh release in the medial pontine reticular formation. The finding that ODQ did not alter ACh release during isoflurane or halothane anesthesia demonstrates that these anesthetics disrupt the NO-sensitive sGC-cGMP pathway. Considered together, results from the mouse and cat indicate that NO modulates ACh release in arousal-promoting regions of the pontine reticular formation via an NO-sensitive sGC-cGMP pathway.     

Determination of Acetylcholine Release in the Striatum of Anesthetized Rats Using In Vivo Microdialysis and a Radioimmunoassay

A vertical-type in vivo microdialysis probe and a sensitive, specific radioimmunoassay (RIA) were used to study the mechanism of acetylcholine (ACh) release in the striatum of anesthetized rats. Without the use of physostigmine, a cholinesterase inhibitor, our RIA could still detect the amount of ACh present in the perfusate (5.6 +/- 0.6 fmol/min, n = 16). Tetrodotoxin (1 microM) produced a significant decrease in the amount of ACh collected in the perfusate, suggesting that basal ACh determined under the present experimental conditions was related to cholinergic neural activity. Atropine (0.1-1 microM) applied topically via the dialysis probe did not affect the amount of ACh recovered in the perfusate in the absence of physostigmine. Addition of physostigmine (10 microM) to the perfusion fluid produced about a 100-fold increase in the amount of ACh collected. In the presence of physostigmine, topical administration of atropine and pirenzepine (0.01-1 microM) through a dialysis probe produced a further three- to fourfold increase in ACh output, whereas a slight increase was produced by AF-DX 116 at the highest concentration (1 microM). These results indicate that presynaptic modulation of ACh release in the striatum does not occur under basal conditions, and that presynaptic M1 muscarinic receptors are involved in the modulation of ACh release when the ACh concentration is raised under certain conditions.     

Endogenous Dopamine Facilitates Striatalln Vivo Acetylcholine Release by Acting on D1 Receptors Localized in the Striatum

Intrastriatal application of the D1 antagonist SCH 23390 by two procedures, reverse dialysis (20 microM) and local injection (0.45 nmol per striatum), elicited a reduction in acetylcholine (ACh) release superimposable on that induced by systemic administration. The novel selective D1 antagonist SCH 39166 produced a similar decreasing effect on striatal ACh release on local injection (0.45 nmol per striatum). On the other hand, local application of SCH 23390 into the frontal cortices (0.45 nmol per side) failed to alter striatal ACh overflow, indicating that the drug does not diffuse out of its injection site to any significant extent. The dopamine release inducer d-amphetamine (2 mg/kg s.c.) and the dopamine uptake inhibitor cocaine raised ACh release like the D1 agonists. These effects were completely blocked by 10 microM SCH 23390 applied by reverse dialysis. The results suggest that D1 receptors regulating ACh release are located in the striatum.     

Sleep initiation and initial sleep intensity: interactions of homeostatic and circadian mechanisms

Sleep initiation and sleep intensity in humans show a dissimilar time course. The propensity of sleep initiation (PSI), as measured by the multiple sleep latency test, remains at a relatively constant level throughout the habitual period of waking or exhibits a midafternoon peak. When waking is extended into the sleep period, PSI rises rapidly within a few hours. In contrast, sleep intensity, as measured by electroencephalographic slow-wave activity during naps, shows a gradual increase during the period of habitual waking. In the two-process model of sleep regulation, it corresponds to the rising limb of the homeostatic Process S. We propose that PSI is determined by the difference between Process S and the threshold H defining sleep onset, which is modulated by the circadian process C. In contrast to a previous version of the model, the parameters of H (amplitude, phase, skewness) differ from those of threshold L, which defines sleep termination. The present model is able to simulate the time course of PSI under baseline conditions as well as following recovery sleep after extended sleep deprivation. The simulations suggest that during the regular period of waking, a circadian process counteracts the increasing sleep propensity induced by a homeostatic process. Data obtained in the rat indicate that during the circadian period of predominant waking, a circadian process prevents a major intrusion of sleep.     

Acetylcholine release detected by trans-striatal dialysis in freely moving rats correlates with spontaneous motor activity

In vivo acetylcholine (ACh) release from the anterior, middle, or posterior striatum and the level of spontaneous motor activity were determined simultaneously using trans-striatal dialysis in freely moving rats. Spontaneous ACh release from the middle striatum was higher than from either the anterior or posterior striatum and correlated with the level of spontaneous motor activity. These findings indicate that drug-induced striatal ACh release could be modified by changes of the level of motor activity in freely moving rats.     

Melanin concentrating hormone induces hippocampal acetylcholine release via the medial septum in rats

Among various actions of melanin concentrating hormone (MCH), its memory function has been focused in animal studies. Although MCH neurons project to various areas in the brain, one main target site of MCH is hippocampal formation for memory consolidation. Recent immunohistochemical study shows that MCH neurons directly project to the hippocampal formation and may indirectly affect the hippocampus through the medial septum nucleus (MS). It has been reported that sleep is necessary for memory and that hippocampal acetylcholine (ACh) release is indispensable for memory consolidation. However, there is no report how MCH actually influences the hippocampal ACh effluxes in accordance with the sleep–wake cycle changes. Thus, we investigated the modulatory function of intracerebroventricular (icv) injection of MCH on the sleep–wake cycle and ACh release using microdialysis techniques. Icv injection of MCH significantly increased the rapid eye movement (REM) and non-REM episode time and the hippocampal, not cortical, ACh effluxes. There was a significant correlation between REM episode time and hippocampal ACh effluxes, but not between REM episode time and cortical ACh effluxes. Microinjection of MCH into the MS increased the hippocampal ACh effluxes with no influence on the REM episode time. It appears that the effect sites of icv MCH for prolongation of REM episode time may be other neuronal areas than the cholinergic neurons in the MS. We conclude that MCH actually increases the hippocampal ACh release at least in part through the MS in rats.     

State-Dependent Changes in Auditory Sensory Gating in Different Cortical Areas in Rats

Sensory gating is a process in which the brain’s response to a repetitive stimulus is attenuated; it is thought to contribute to information processing by enabling organisms to filter extraneous sensory inputs from the environment. To date, sensory gating has typically been used to determine whether brain function is impaired, such as in individuals with schizophrenia or addiction. In healthy subjects, sensory gating is sensitive to a subject’s behavioral state, such as acute stress and attention. The cortical response to sensory stimulation significantly decreases during sleep; however, information processing continues throughout sleep, and an auditory evoked potential (AEP) can be elicited by sound. It is not known whether sensory gating changes during sleep. Sleep is a non-uniform process in the whole brain with regional differences in neural activities. Thus, another question arises concerning whether sensory gating changes are uniform in different brain areas from waking to sleep. To address these questions, we used the sound stimuli of a Conditioning-testing paradigm to examine sensory gating during waking, rapid eye movement (REM) sleep and Non-REM (NREM) sleep in different cortical areas in rats. We demonstrated the following: 1. Auditory sensory gating was affected by vigilant states in the frontal and parietal areas but not in the occipital areas. 2. Auditory sensory gating decreased in NREM sleep but not REM sleep from waking in the frontal and parietal areas. 3. The decreased sensory gating in the frontal and parietal areas during NREM sleep was the result of a significant increase in the test sound amplitude.