Bradycardia In Rabbits Research Articles

Mast Cell Degranulation and Adenosine Release:Acupoint Specificity for Effect of Electroacupuncture on Pituitrin-Induced Acute Heart Bradycardia in Rabbits.

Acupuncture is a medical modality based on the theory of traditional Chinese medicine, and its effect is relatively dependent on acupoint specificity. However, there is little knowledge on acupoint specificity versus acupuncture outcomes because of the deficiency of rigorous investigation on this topic, which has impeded the growing legitimacy of acupuncture in the mainstream of medicine as an evidence-based therapy. Therefore, it is of utmost importance to clarify this critical issue. The present study aims to verify the phenomenon of acupoint specificity in acupuncture-induced cardiovascular regulation and explore the biological mechanism by measuring mast cells' degranulation and adenosine release. This study was conducted to explore the specificity of acupoints in an acute bradycardia rabbit model. After electroacupuncture (EA) stimulation at PC6, PC control (con) 1, PC con 2, LU7, LI11, and nonacupoint, only the PC6 group showed a significant improvement in relative heart rate as compared to that of the model group. There was no significant difference between the relative heart rate of other EA groups and that of the model group. Historical results also showed that the ratio of degranulated mast cells in PC6 was significantly higher than other acupoints and control points. From the results of high-performance liquid chromatography (HPLC), a transient elevation of adenosine concentration during EA was only observed on acupoints and control points (P < 0.05) along the pericardium meridian. The EA-induced adjustment on acute bradycardia exhibits a relative specificity of acupoints, which may be related to mast cell degranulation and adenosine release in local acupoint areas. Increased degranulation of mast cells and augmentation of adenosine release during EA may be the mechanisms for PC6 having significantly better acupuncture effects than other acupoints and nonacupoints.

Mast cell activation in the acupoint is important for the electroacupuncture effect against pituitrin-induced bradycardia in rabbits

This research was conducted to verify the structural and functional characteristics of mast cells in the electroacupuncture (EA) effects on bradycardia. First, we examined the mast cell density at PC 6, adjacent acupoint LU 7, and a non-acupoint. We tested the effects of EA at PC 6 on heart rate (HR) and blood pressure (BP) in rabbits with pituitrin-induced bradycardia. We also injected sodium cromolyn (Cro), a mast cell membrane stabilizer, at PC 6 30 min before EA to investigate if it affected the EA effects. The results showed that in both PC 6 and LU 7, the mast cell densities were higher than in the non-acupoint (P < 0.05). EA could induce mast cell degranulation at PC 6, which could be suppressed by sodium cromolyn (P < 0.05). EA improved HR, though the change was relatively small in the initial stage with a significant change at 35 min after modelling (P < 0.05). BP significantly improved at 10 min after the onset of pituitrin-induced bradycardia (P < 0.05). The EA effects on both HR and BP were suppressed by sodium cromolyn (P < 0.05). Therefore, we concluded that mast cells in the acupoint are important for the EA effects against pituitrin-induced bradycardia in rabbits.

Effect of infrared laser on pituitrin-induced bradycardia in rabbits

To observe the effect of infrared laser stimulation on Neiguan (PC 6) for bradycardia of rabbits induced by injection of pituitrin. 10.6 microm CO(2) infrared laser, 650 nm semiconductor red laser and the compound laser of their aggregation were sent out by a compound laser sanatory instrument to stimulate Neiguan (PC 6) respectively for the purpose of analyzing the differences of the effects exerted by infrared laser and red laser as well as the changes of the effects after their aggregation. After 30 min and 40 min of the injection of pituitrin, the heart rate showed no significant difference between 10.6 microm CO(2) infrared laser group and the normal control group (P>0.05). However, the heart rate showed significant difference between the 650 nm semiconductor red laser group and the normal control group (P<0.05). After 30 min of the injection of pituitrin, the heart rate of the compound laser group were significantly different to that of the untreated group (P<0.05), and it was not significantly different to that of the normal control group (P>0.05). However, the heart rate of non-acupoint stimulated by the compound laser group showed no significant difference to that of the untreated group (P>0.05), and it was significantly different to that of the normal control group (P<0.05). Stimulation on Neiguan with 10.6 microm CO(2) infrared laser can evidently improve the bradycardia induced by injection of pituitrin, while the 650 nm semiconductor red laser has no such function, but they have coordinating effect. The thermal effect produced by CO(2) infrared laser stimulating acupoint is probably beneficial for semiconductor laser penetrating skin as well as the biochemical reactions it carries. The improvement of heart rate produced by the laser stimulation on Neiguan (PC 6) has relative specificity.

Effect of acute inhibition of nitric oxide synthesis byl‐NAME on cardiovascular responses following peripheral autonomic blockade in rabbits

The pressor and chronotropic responses to acute inhibition of nitric oxide synthase enzyme by N(G)-nitro-L-arginine methyl ester (L-NAME) were studied in anaesthetized rabbits with intact autonomic nervous system (ANS) activity. Also, they were investigated when administration of L-NAME was preceded by peripheral autonomic blockade. Autonomic blockade had different forms: ganglionic (hexamethonium-induced), post-ganglionic beta-adrenergic blockade (propranolol induced), parasympathetic blockade (atropine induced), and complete autonomic blockade by coadministration of hexamethonium and atropine simultaneously. L-NAME injected intravenously (10 mg/kg) in animals with intact and blocked autonomic activity induced a pressor response. This pressor response was accompanied by bradycardia in rabbits with either intact autonomic activity or hexamethonium-induced ganglionic blockade. L-NAME exerted no effect on heart rate in animals with beta-adrenergic blockade or parasympathetic blockade. In rabbits with complete autonomic blockade, L-NAME evoked tachycardia. These experiments indicate that L-NAME-induced hypertension is not relying only on ANS. Also, L-NAME-induced tachycardia in rabbits treated with atropine plus hexamethonium suggests other humoral mechanisms that may be involved in the L-NAME induced chronotropic response.

Opposite to α 2-adrenergic agonists, an imidazoline I 1 selective compound does not influence reflex bradycardia in rabbits

This work aimed to study the respective effects of central α 2-adrenergic receptors (α 2-AR S) and I 1 imidazoline receptors (I 1Rs) in the facilitatory effects of imidazoline-like drugs on the reflex bradycardia (RB). Experiments were performed in anaesthetized rabbits. The reflex bradycardic response was induced by phenylephrine injected i.v. LNP 509, rilmenidine and dexmedetomidine were administered intracisternally (i.c.). LNP509 (1 mg/kg, i.c.), a ligand highly selective for I 1Rs, induced hypotension (54 ± 3 vs. 93 ± 2 mm Hg) and bradycardia (260 ± 13 vs. 322 ± 13 beats/min) ( p < 0.05, n = 5) but did not affect RB. Rilmenidine (1 μg/kg, i.c.), a hybrid ligand which binds to both I 1 and α 2-AR S, also decreased arterial pressure (61 ± 2 vs. 101 ± 2 mm Hg) and heart rate (260 ± 4 vs. 308 ± 8) ( p < 0.01, n = 5); it potentiated the RB (maximum R–R interval: 284 ± 17 vs. 196 ± 6 ms) ( p < 0.05, n = 5). Dexmedetomidine (1 μg/kg, i.c.), a ligand selective for α 2-ARs, reduced blood pressure (53 ± 3 vs. 104 ± 2 mm Hg) and heart rate (246 ± 4 vs. 312 ± 8 beats/min) ( p < 0.05, n = 5) and potentiated the RB (maximum R–R interval: 518 ± 38 vs. 194 ± 4 ms) ( p < 0.05, n = 5). The potentiation of RB was much greater than that observed with rilmenidine and was significantly prevented by L-NNA injected centrally. This study shows that: (i) an exclusive action on I 1Rs which decreases arterial pressure, does not potentiate the RB ii) activation of α 2-ARs potentiates the RB (iii) the R–R prolongation caused by α 2-ARs stimulation is prevented by central NOS inhibition.

Potassium Channel Subunit Remodeling in Rabbits Exposed to Long-Term Bradycardia or Tachycardia

Sustained heart rate abnormalities produce electrical remodeling and susceptibility to arrhythmia. Uncontrolled tachycardia produces heart failure and ventricular tachyarrhythmia susceptibility, whereas bradycardia promotes spontaneous torsade de pointes (TdP). This study compared arrhythmic phenotypes and molecular electrophysiological remodeling produced by tachycardia versus bradycardia in rabbits. We evaluated mRNA and protein expression of subunits underlying rapid (IKr) and slow (IKs) delayed-rectifier and transient-outward K+ currents in ventricular tissues from sinus rhythm control rabbits and rabbits with AV block submitted to 3-week ventricular pacing either at 60 to 90 bpm (bradypaced) or at 350 to 370 bpm (tachypaced). QT intervals at matched ventricular pacing rates were longer in bradypaced than tachypaced rabbits (eg, by approximately 50% at 60 bpm; P<0.01). KvLQT1 and minK mRNA and protein levels were downregulated in both bradypaced and tachypaced rabbits, whereas ERG was significantly downregulated in bradypaced rabbits only. Kv4.3 and Kv1.4 were downregulated by tachypacing only. Patch-clamp experiments showed that IKs was reduced in both but IKr was decreased in bradypaced rabbits only. Continuous monitoring revealed spontaneous TdP in 75% of bradypaced but only isolated ventricular ectopy in tachypaced rabbits. Administration of dofetilide (0.02 mg/kg) to mimic IKr downregulation produced ultimately lethal TdP in all tachypaced rabbits. Sustained tachycardia and bradycardia downregulate IKs subunits, but bradycardia also suppresses ERG/IKr, causing prominent repolarization delays and spontaneous TdP. Susceptibility of tachycardia/heart failure rabbits to malignant tachyarrhythmias is induced by exposure to IKr blockers. These results point to a crucial role for delayed-rectifier subunit remodeling in TdP susceptibility associated with rate-related cardiac remodeling.

Role of the medial prefrontal cortex in the development of conditioned bradycardia in rabbits with lesions of the cerebellar vermis.

The effects on the expression of conditioned bradycardia of pairing an early (fourth postnatal day) cerebellar vermal lesion with a lesion of the medial prefrontal cortex (mPFC) were studied in adult New Zealand rabbits. In the conditioning procedure, an auditory stimulus (5 s, 1000 Hz) served as a conditioning stimulus (CS) and a train of electrical impulses applied to the ear (500 ms, 100 Hz, 1.5 mA) was used as the unconditioned stimulus (US). Heart rate (HR) responses exhibited by rabbits with the early double lesion (PFCBs) during orientation (CS-alone) and conditioning (CS-US paired) were analyzed and compared with those shown by unoperated controls as well as by a group of animals in which a cerebellar lesion alone had been performed on the fourth postnatal day (CBs). In all the experimental groups vermal lesions were localized in the cortex of lobules V-VII and the underlying white matter. As for mPFC ablation, the lesioned area involved the agranular precentral region (Brodmann's area 8), the anterior cingulate cortex (Brodmann's area 24) and the prelimbic area (Brodmann's area 32). All the experimental animals had a normal baseline HR as well as a marked orientation response, both comparable with those exhibited by controls. In contrast, while CB rabbits showed an increase in the amplitude of the conditioned bradycardic response when compared with controls, the HR conditioned response of PFCB animals was comparable to that exhibited by controls. These results suggest that, since the double lesion produces a conditioned bradycardia similar to that of the controls, the increase in the amplitude of this response observed after early cerebellar removal may depend on the mPFC which, in the absence of specific cerebellar circuits, is unable to produce a properly calibrated HR conditioned response.

Characterization of Single-Unit Activity in the Mediodorsal Nucleus of the Thalamus during Expression of Differential Heart Rate Conditioning in the Rabbit

Much recent evidence has shown that the thalamic–prefrontal axis is involved in Pavlovian conditioning in rabbits. However, while single cell activity in the prefrontal cortex has been previously studied during classical conditioning in rabbits, that of its thalamic projection nucleus, the mediodorsal (MD) nucleus, has not. Consequently, in the present research we recorded neuronal activity from individual cells in MD during expression of conditioned bradycardia in rabbits that received differential Pavlovian conditioning in which tones served as conditioned stimuli and periorbital shock served as unconditioned stimuli. The pattern of firing in MD was similar to that evoked in the medial prefrontal cortex (mPFC). Of 84 cells sampled, approximately 35% showed CS-evoked activity. Ninety percent of these cells showed increases in activity, while the remainder were biphasic, showing an initial increase followed by a decrease. Also, like the mPFC, some cells showed initial increases, which declined during CS presentation, while others showed gradual increases which reached their maximum at CS offset. Also some cells were responsive to the CS+and others to the CS−. Thus, MD cells, like mPFC cells, are somewhat heterogenous with regard to responding to conditional stimuli, although, unlike the mPFC, no strictly inhibitory cells were found.

Subicular lesions disrupt but do not abolish classically conditioned bradycardia in rabbits.

Subicular lesions disrupt but do not abolish classically conditioned bradycardia in rabbits.

Subicular lesions disrupt but do not abolish classically conditioned bradycardia in rabbits.

Rabbits and rats received horseradish peroxidase injections in the medial prefrontal cortex, and retrograde labeling was examined in the hippocampus (HC) and subicular complex (SC). Labeled cells were observed in HC and SC in the rat, but only in the SC of the rabbit. In a second experiment, separate groups of rabbits with sham, SC, or cortical control lesions were subjected to differential classical heart rate conditioning, in which 4-s, 75-db tones served as conditioned stimuli and a 3-mA paraorbital shock was the unconditioned stimulus. Although conditioned bradycardia was obtained in animals with SC lesions, it was slower to develop and was much shorter in duration than in the cortical and sham control groups. In the animals with SC lesions, the bradycardiac response was quickly replaced with tachycardia, suggesting a sympathetic bias in these animals.

On the generality of conditioned bradycardia in rabbits: Assessment of CS and US modality

To determine whether the magnitude of heart rate (HR) slowing induced by classical conditioning contingencies is comparable under a broad range of stimulus conditions, experiments were conducted in which rabbits were exposed to tones, increases in illumination, or vibratory stimuli as conditioned stimuli (CSs) and in which paraorbital electric shocks, corneal airpuffs, or intraoral pulses of water served as unconditioned stimuli (USs). The results indicated that conditioned bradycardia was elicited by all three CSs. Moreover, when a corneal airpuff served as the US, small but reliable CS-evoked HR decelerations also occurred. Finally, CS-evoked HR slowing also occurred in response to a tone CS employed in an appetitive task, in which water was the US. These findings suggest that HR slowing is a general phenomenon that occurs when rabbits are exposed to signals that systematically predict aversive or appetitive consequences according to a Pavlovian conditioning paradigm.

Ibotenic acid lesions in the amygdaloid central nucleus but not in the lateral subthalamic area prevent the acquisition of differential Pavlovian conditioning of bradycardia in rabbits

The present study examined the effect of ibotenic acid lesions in the amygdaloid central nucleus (ACe) or in the lateral zona incerta of the subthalamus (LZI) on the acquisition of differential Pavlovian conditioning of bradycardia in rabbits. Previous work has shown that bilateral electrolytic lesions in either ACe or LZI abolished the retention of conditioned heart rate (HR) responses. In order to determine whether these findings were due to destruction of cells intrinsic to ACe or LZI, ibotenic acid lesions were placed bilaterally in either structure or in control sites. Following recovery, animals were subjected to differential Pavlovian conditioning in which one tone (CS+) was paired with periorbital shock and a second tone (CS−) was presented alone. It was found that destruction of cell bodies in ACe, but not LZI, prevented the acquisition of the differential bradycardiac conditioned response. In addition, ACe lesions did not interfere with baseline HR, the HR orienting response, the HR unconditioned response to shock, or the concomitantly conditioned corneoretinal potential. The results of this study suggest that destruction of cells intrinsic to ACe selectively prevents the acquisition of differentially conditioned HR, and perhaps other conditioned responses related to conditioned arousal, but does not affect unlearned HR responses or specific somatomotor conditioned responses.

Single-unit activity in the dorsomedial prefrontal cortex during the expression of discriminative bradycardia in rabbits

Much recent evidence suggests that the medial prefrontal cortex (PFCm) participates in the development and/or expression of learned, primary bradycardiac adjustments in rabbits. Accordingly, the present experiments were undertaken to determine whether single-unit activity in the precentral agranular and anterior cingulate regions of the PFCm is systematically affected by differential aversive Pavlovian conditioning and, if so, whether such activity is correlated with concomitant heart rate (HR) variables. In these studies, extracellular recordings were made during a training procedure that involved two distinctive, tone conditioned stimuli (CSs); one of these (CS+) was often paired with eye-shock (unconditioned stimulus, US), whereas the other (CS-) was never so paired. Of the 100 spontaneously-active cells whose activity was so evaluated, 74 were found to be tone-responsive and could be classified as belonging to one of 5 subpopulations on the basis of their specific patterns of evoked changes in discharge; additional data suggested that these cells were also generally responsive to the eye-shock US. Regarding the effects of training, the vast majority of cells in these subpopulations showed greater evoked activity changes on CS+, as opposed to CS-, trials, irrespective of the sign (i.e. increase and/or decrease) of their responses. This finding was not confounded by differences in baseline activity preceding CS+ vs CS- trials, which was generally characterized as being both irregular and unrelated to concomitant HR variables; nor could it be attributed to unconditioned responses biases. Thus, the discriminative activity patterns of these cells appeared to reflect the differential Pavlovian contingencies in effect. Moreover, approximately half of these cells exhibited tone-evoked activity changes that were reliably correlated with concomitant HR changes on a trial-by-trial basis. Consequently, the present data indicate that training-induced changes in the CS-evoked activity of PFCm cells are significantly related to aversively conditioned bradycardia in rabbits.

Studies on the mechanism of the cardiovascular effect of intraventricular 5-hydroxytryptamine in rabbits

An attempt was made to investigate the effect of intracerebroventricular 5-hydroxytryptamine (5-HT) on the cardiovascular system in urethane-anesthetized rabbits and to elucidate the mechanism of its action. 5-HT given into a lateral ventricle caused clearly a dose-dependent decrease in both arterial blood pressure and in heart rate. The bradycardia and hypotension induced by 5-HT were significantly attenuated by the prior injection of ketanserin, cyproheptadine or clonidine. Pretreatment of atropine with bilateral vagotomy did not affect both bradycardia and hypotension. Propranolol weakened markedly the bradycardia of 5-HT but did not influence the depressor response of 5-HT. These experimental results suggest that intraventricular 5-HT cause the hypotension and bradycardia in rabbits through the stimulation of serotonergic receptors in brain, which is seemed to be associated to inhibition of sympathetic tone.

Sinoaortic denervation does not prevent differential Pavlovian conditioning of bradycardia in rabbits

Recent findings suggest that descending projections from the amygdaloid central nucleus of the solitary tract (NTS) may modulate the baroreceptor reflex and thereby facilitate the expression of the bradycardiac conditioned response (CR) in rabbits 18,21. The purpose of the present study was to examine the role of the afferent limb of the baroreceptor reflex in differential Pavlovian conditioning of bradycardia in rabbits. Animals received either aortic denervation, sinoaortic denervation or sham denervation. After recovery from surgery, animals received one differential Pavlovian conditioning session per day over the nex 6 days. Sinoaortic denervation abolished the baroreceptor reflex as assessed by intravenous injections of phenylephrine. In addition, sinoaortic denervation increased baseline heart rate (HR), altered the topography of the HR unconditioned response, but did not abolish the HR orienting response or prevent the acquisition of bradycardiac CRs. The findings of the present study suggest that afferent barosensory input is not necessary for the expression of the HR CR in rabbits. However, descending ACE projections may stil play a role in the HC CR by directly affecting NTS neurons.

The role of amygdaloid central nucleus in the retention of differential pavlovian conditioning of bradycardia in rabbits

The present study examined the role of the amygdaloid central nucleus (ACE) in the retention of differential pavlovian conditioning of bradycardia in rabbits. Electrodes were implanted bilaterally in ACE or in control sites just dorsal and rostral to ACE. Following recovery, animals were subjected to differential pavlovian conditioning in which one tone (CS+) was paired with periorbital shock and a second tone (CS-) was presented alone. Subsequent electrolytic lesions abolished the heart rate (HR) conditioned response (CR), yet had no effect on HR orienting response, unconditioned response, or baseline. In a follow-up experiment, corneoretinal potential (CRP) and HR were recorded. Bilateral ACE lesions profoundly attenuated or abolished the HR CR without abolishing CRP CRs. The major finding of this study is that bilateral lesions of ACE selectively attenuate the HR CR while not necessarily abolishing other CRs.

The role of the medial geniculate region in differential Pavlovian conditioning of bradycardia in rabbits

The present study examined the role of the medial geniculate region (MGN) in differential Pavlovian conditioning of bradycardia and corneo-retinal potential (CRP) to acoustic stimuli in rabbits. Injections of horseradish peroxidase into the amygdala central nucleus, an area that mediates the bradycardia-conditioned reponse (CR), produced cell body and fiber labeling at the ventral and medial borders of the MGN. Then, bilateral electrolytic lesions were made at the medial border of the MGN or in control sites dorsal and/or rostral to the MGN. Ten days after surgery, lesioned and unoperated control animals were subjected to 7 days of differential Pavlovian conditioning. In the control lesion and unoperated groups, the CS+ consistently elicited larger bradycardia responses than the CS−. However, animals with bilateral lesions in the medial MGN did not demonstrate differential bradycardia CRs. Bradycardia response magnitude in MGN lesion animals was not affected. Evidence of CRP differential conditioning was present in each group. The present findings suggest that a region just medial to the MGN is involved in bradycardia differential conditioning in rabbits. The fact that bradycardia responses were still present after medial MGN lesions suggests that other auditory regions may also be involved in the mediation of the bradycardia CR.

Lateral subthalamic area as mediator of classically conditioned bradycardia in rabbits.

This study was conducted to examine whether a central efferent pathway passing through the lateral zona incerta (LZI) of the subthalamus selectively mediates the bradycardia conditioned response (CR) in rabbits. Electrodes were implanted bilaterally in LZI or in control sites just dorsal or ventral to LZI. Two days following surgery, animals were subjected to Pavlovian conditioning or to pseudoconditioning. Subsequent bilateral electrolytic lesions did not influence the heart rate (HR) orienting response, unconditioned response, baseline, or lack of response to pseudoconditioning. Bilateral LZI lesions alone abolished the HR CR. In a follow-up experiment, the corneoretinal potential (CRP) CR and HR were recorded. Bilateral LZI lesions following conditioning to a criterion of 65% CRP CRs abolished the HR CR without affecting CRP CRs. The present findings indicate that LZI is part of an efferent pathway that selectively mediates the HR CR in rabbits.

Lateral subthalamic area as mediator of classically conditioned bradycardia in rabbits.

This study was conducted to examine whether a central efferent pathway passing through the lateral zona incerta (LZI) of the subthalamus selectively mediates the bradycardia conditioned response (CR) in rabbits. Electrodes were implanted bilaterally in LZI or in control sites just dorsal or ventral to LZI. Two days following surgery, animals were subjected to Pavlovian conditioning or to pseudoconditioning. Subsequent bilateral electrolytic lesions did not influence the heart rate (HR) orienting response, unconditioned response, baseline, or lack of response to pseudoconditioning. Bilateral LZI lesions alone abolished the HR CR. In a follow-up experiment, the corneoretinal potential (CRP) CR and HR were recorded. Bilateral LZI lesions following conditioning to a criterion of 65% CRP CRs abolished the HR CR without affecting CRP CRs. The present findings indicate that LZI is part of an efferent pathway that selectively mediates the HR CR in rabbits.

Electrical stimulation of insular cortex elicits cardiac inhibition but insular lesions do not abolish conditioned bradycardia in rabbits

Conscious rabbits received electrical stimulation of insular and more posterior perirhinal cortex through chronically implanted electrodes. Active sites for cardiovascular responses were found in both anterior and posterior insular cortex as well as more posterior perirhinal regions. Although differential response topographies occurred related to anterior versus posterior insular cortex, all heart rate responses consisted of bradycardia. Pharmacological manipulations revealed that this bradycardia was due to a combination of vagal and sympatho-inhibitory mechanisms. Some posterior sites yielded pressor responses, and bradycardia which was sensitive to phentolamine, suggesting that the bradycardia in these instances was due to activation of the baroreceptor reflex. All other blood pressure changes were depressor responses. In a second experiment two different groups of rabbits with lesions of either anterior or posterior agranular insular cortex were compared with a third group of animals with sham lesions in a differential Pavlovian conditioning experiment. No lesion completely abolished the classically conditioned bradycardia associated with tone/shock contingencies. However, anterior insular lesions attenuated the magnitude of the conditioned bradycardia compared to the posterior and sham lesions. Control experiments suggested that this attenuation was due to the lesion's effects on the conditioned stimulus/unconditioned stimulus association and not to its effects on unconditioned responding to the conditioned stimulus or unconditioned stimulus alone.