Anodal Block Research Articles

Nociceptive C fibre input to the primary somatosensory cortex (SI). A field potential study in the rat

In the present study, noxious thermal stimulation of the skin with short pulses of CO 2-laser radiation was used to identify a cutaneous nociceptive C fibre input to SI and investigate the organization of this input in halothane-nitrous oxide anaesthetized rats. Noxious CO 2-laser stimulation of the glabrous skin of the hindpaw consistently evoked late surface positive field potentials in SI (average onset latency 226 ms, peak latency 296 ms). It was demonstrated that these late potentials were evoked by an input from nociceptive C fibres, using a combination of latency measurements, anodal block of A fibre conduction and graded intensities of stimulation. Compared to the tactile evoked potentials in SI, the nociceptive C fibre evoked potentials were more widespread and exhibited a crude somatotopical organization. Intracortically, both tactile and nociceptive C fibre evoked potentials reversed polarity and exhibited a peak negativity in laminae III–IV. The nociceptive C fibre evoked potentials exhibited an additional peak negativity in laminae V–VI. The latter potential had a different time course as compared to the nociceptive C fibre potential evoked in laminae III–IV. In conclusion, an input from cutaneous nociceptive C fibres to SI was demonstrated for the first time in animal experiments. The input to SI from tactile receptors and cutaneous C nociceptors were differently organized.

Selective contribution of two types of carotid sinus baroreceptors to the control of blood pressure.

This study was performed to determine if selective elimination of afferent input from two different types of previously described baroreceptors altered the ability of the dog to regulate blood pressure (BP), examining specifically if there was differential loss of baroreceptor control of tonic levels of baseline pressure versus dynamic changes in pressure. In the first series of experiments in this study, anodal block of the carotid sinus nerve was used to selectively block afferent input in a sequence from large-diameter A-fiber carotid baroreceptors (mostly type I) to smaller A-fiber and nonmyelinated C-fiber baroreceptors (mostly type II). In the second series of experiments, anesthetic block of the carotid sinus nerve with bupivacaine was used to selectively eliminate afferent input in reverse order from anodal block, first blocking input from baroreceptors with small afferent fibers and then additionally eliminating input from the larger-diameter A-fiber baroreceptors. The effects of selective elimination of each baroreceptor type were determined by monitoring baseline BP during constant carotid sinus pressure (CSP) perfusion of a vascularly isolated carotid sinus (tonic control) and obtaining baroreflex sensitivity (slope) during ramp pressure stimulations of the carotid sinus (dynamic control) under various blocking conditions. Low levels of anodal block significantly attenuated baroreflex sensitivity (-0.84 +/- 0.11 versus -0.63 +/- 0.10 mm Hg BP/mm Hg CSP) at levels of block that had no effect on tonic baseline BP (158.41 +/- 9.5 versus 160.7 +/- 9.5 mm Hg BP). In contrast, low levels of bupivacaine block produced significant increases in tonic BP (158.8 +/- 6.4 versus 169.0 +/- 6.5 mm Hg BP), whereas there was no effect on dynamic baroreflex sensitivity (-0.85 +/- 0.08 versus -0.73 +/- 0.08 mm Hg BP/mm Hg CSP). Thus, blocking large A-fiber baroreceptors resulted in significant decreases in baroreflex sensitivity without changes in baseline levels of BP, indicating primarily an attenuation in dynamic baroreflex regulation. Blocking of smaller A-fiber and unmyelinated C-fiber baroreceptors resulted in smaller decreases in baroreflex sensitivity and significant elevations in baseline BP, indicating a loss of tonic control of pressure. These results suggest that the two types of baroreceptors contribute differently to the regulation of blood pressure.

ANODAL BLOCK V ANODAL STIMULATION

Anodal block and stimulation are poorly documented electrophysiologic phenomenon. Median and superficial radial nerves are examined in a prospective study to explore the significance of anodal block in routine nerve conduction studies. In addition, the anode's ability to stimulate the peripheral nervous system is evaluated. A monopolar stimulation technique is employed to achieve pure anode-generated responses. Additionally, a similar monopolar cathode stimulation technique is utilized and found to be equivalent to the traditional bipolar cathode stimulation. Based on the findings in this investigation, anodal block does not appear to occur during routine nerve conduction studies; however, transposition of the anode and cathode is clinically significant because the increased distance between the cathode and recording electrode results in predictably prolonged latencies. With higher levels of stimulus intensity, sensory, motor and F wave responses are generated by anodal stimulation in all cases. The actual mechanism of anodal stimulation remains uncertain and requires further study. Predicated on the results of this investigation, it appears that anodal block is an unlikely occurrence during routine electrodiagnostic medicine evaluations.

The reflex bradycardia evoked by brain ischemia and its relation to aortic A- and C-fiber baroreceptors

In urethane-anesthetized rabbits with the right aortic nerve (RAN) sectioned, we examined the reflex heart rate (HR) responses during brain ischemia for approximately 30 s, by applying the anodal block to the unsectioned left aortic nerve (LAN) and, subsequently, by denervating the LAN. The maximum decreases in HR occurred at around 30 s after the onset of brain ischemia. The anodal block used in this study selectively inhibited the aortic A-fiber conduction but did not inhibit the volley of aortic C-fibers. The maximum HR fall responses to brain ischemia with and without the anodal block were 143 ± 7 and 183 ± 7 beats/min, respectively. When the maximum value of HR fall during brain ischemia in the absence of aortic nerve signals was subtracted from these two values, the reflex HR fall responses to brain ischemia with aortic C and A baroreceptor activation were 98 ± 7 and 43 ± 8 beats/min, respectively. In another series of experiments used for the same techniques, the reflex fall in HR seen during brain ischemia with the anodal block was totally abolished by vagotomy. The results indicate that the ability of aortic C baroreceptors becomes more prominent on the magnitude of brain ischemia-induced reflex bradycardia as compared to that of aortic A baroreceptors.

Contractions of frog tonus fibers and their modification by length changes

Isometric and isotonic contractions of the tonus fibers of the frog were recorded using anodal block of the nerve fibers of the twitch fibers. Repetitive stimulation produced a contraction with a very slow rising phase because the individual responses were very weak. The first two or three stimuli usually did not give a visible response at all. However, if the twitch fibers were also stimulated, the responses of the tonus fibers were many times stronger and faster, but only under isotonic conditions. This indicates that the large increase in the responses of the tonus fibers was produced by the passive shortening caused by the contraction of the twitch fibers. A strong and fast response of the tonus fibers was also obtained if during stimulation of the tonus fibers the muscle was made to shorten by diminishing the load. It is suggested that the enormous effect of shortening is due to the regenerating action of shortening previously demonstrated.

Group I afferent fibers: effects on cardiorespiratory system

In anesthetized cats, we examined cardiorespiratory activity during excitation of large afferent fibers from muscle proprioceptors. We found that selective stimulation of group I fibers with electric impulses at 200-300 Hz induces an increase in pulmonary ventilation from control value (mean +/- SE) of 486 +/- 8 to a maximum of 544 +/- 8 ml/min and an increase in mean systemic arterial pressure from control value of 151 +/- 2 to a maximum of 160 +/- 2 mmHg. Neither of these increases was produced by the same stimulation when applied during anodal block of volleys of group I fibers. Hyperpnea could be obtained independently from changes in cardiovascular activity, and the pressor response could be obtained during artificial ventilation at constant tidal volume after curarization. Consequently, it appears that respiratory and cardiovascular responses to stimulation of group I fibers can be independent of each other.

Climbing fibres projecting to cat cerebellar anterior lobe activated by cutaneous A and C fibres.

1. Climbing fibre responses evoked on stimulation of the ipsilateral superficial radial nerve were examined in the forelimb area of the C3 zone in the barbiturate-anaesthetized cat. Climbing fibre responses were recorded in sixty-five Purkinje cells and as field potentials from the surface of the cerebellum. 2. In addition to the previously described A beta-fibre-evoked climbing fibre response, late climbing fibre responses were consistently evoked in all Purkinje cells studied when C fibres were stimulated. The latencies of the A beta- and C-fibre-evoked climbing fibre responses were 11-20 ms and 110-220 ms, respectively. In most experiments climbing fibre responses with an intermediate latency (20-30 ms) were evoked. It was demonstrated that this response depended on A delta fibres. 3. The long-latency climbing fibre response generated by electrical stimulation at C-fibre strength was evoked also during selective anodal block of conduction in A fibres (Brown & Hamman, 1972). Hence, impulses in C fibres were sufficient for generation of climbing fibre responses. 4. The distribution within the forelimb area of the C3 zone of the A beta- and C-fibre-evoked climbing fibre field potential was similar. No climbing fibre response was evoked in this area of the C3 zone by stimulation of A and C fibres in the contralateral superficial radial nerve or in the plantar nerves of the hind limbs. 5. It can be concluded that climbing fibres projecting to the forelimb area of the C3 zone in the cerebellum receive a somatotopically organized input from both A beta and C fibres.

Stimulation of cat cutaneous nociceptive C fibres causing tonic and synchronous activity in climbing fibres.

1. The input from cutaneous nociceptors to climbing fibres projecting to the forelimb area of the C3 zone in the cerebellar anterior lobe was examined in barbiturate-anaesthetized cats. Climbing fibre responses were simultaneously recorded in single Purkinje cells and as field potentials from the cerebellar surface close to these cells. 2. The cutaneous receptive field of the climbing fibres studied were located on the ipsilateral forelimb. All climbing fibres were activated by both non-noxious tactile stimulation and noxious pinch of the skin. The location of the receptive field and the distribution of sensitivity in the receptive field appeared to be identical for noxious and tactile stimuli. 3. A phasic response in the climbing fibres was evoked by either short- or long-lasting non-noxious pressure applied to their cutaneous receptive field. By contrast, all climbing fibres studied were strongly and tonically activated (up to 4-11 Hz for the duration of the stimulation) by sustained noxious pinch in the most sensitive area of their receptive fields. 4. Experiments with anodal block of impulse conduction in myelinated fibres indicated that a major input to climbing fibres during sustained noxious pinch originates from nociceptive C fibres. 5. Sustained noxious pinch of the skin evoked large field potentials on the cerebellar surface. These field potentials were evoked simultaneously with climbing fibre responses in single Purkinje cells and were due to synchronous activation of many climbing fibres. These field potentials and discharges in single climbing fibres were elicited from the same area of the skin suggesting that many of the synchronously discharging climbing fibres have the same receptive field on the skin.

Field potentials evoked in rat primary somatosensory cortex (SI) by impulses in cutaneous Aβ- and C-fibres

A projection of cutaneous C-afferent fibres to the contralateral primary somatosensory cortex (SI) was examined in the halothaneanaesthetized rat. Field potentials evoked by electrical stimulation of the right sural nerve were recorded at the surface of and intracortically within the left SI cortex. A late surface positive potential (latency 110–190 ms, mean 136 ms) was evoked by sural stimulation at a strength that activated A- and C-fibres. A selective anodal block of impulse conduction in A-fibres proximal to the stimulating electrodes showed that impulses in C-fibres generate the late potential also in the absence of a preceding A-fibre input. Stimulation of the sural nerve at two sites caused a shift in latency of the late potential corresponding to a conduction velocity of the primary afferent fibres of less than 1.0 m/s. The distribution of the C-fibre-evoked potential in SI was similar to that of the Aβ-fibre evoked ‘primary’ potential suggesting that the investigated projection of cutaneous C-fibres to SI has a somatotopic organization.

Evidence implicating descending fibers in self-stimulation of the medial forebrain bundle

The role of ascending and descending fibers in self-stimulation of the lateral hypothalamus and ventral tegmental area in the rat was assessed by noting whether anodal hyperpolarization of one of these sites could reduce the rewarding effect of stimulating the other site. Strength-duration curves were obtained by psychophysical means, with one of the depth electrodes serving as the cathode and the other as the anode. It was anticipated that at long pulse durations, conduction in some of the fibers stimulated at the cathode would be blocked at the anode. At shorter durations, the anodal hyperpolarization should have dissipated before the arrival of the action potentials triggered by the cathode. Thus, the predicted effect of the block was to bend the strength-duration curves obtained with two depth electrodes upward at long pulse durations, provided that the anode lay between the cathode and the efferent stages of the pathway responsible for the rewarding effect. To control for possible differences in the density of the reward substrate in the lateral hypothalamic and ventral tegmental areas, the strength-duration curves obtained with a given cathode and a depth anode were compared to curves obtained with the same cathode but with an anode consisting of a set of skull screws. It was expected that the concentrated current entering from the depth anode would much more effectively block conduction in the medial forebrain bundle than the diffuse current entering from the large, distant skull screws. The predicted change in the shape of the strength-duration curves was observed only when the ventral tegmental electrode served as the anode and the lateral hypothalamic electrode as the cathode. This is consistent with the notion that in at least some of the neurons responsible for the rewarding effect, action potentials elicited by the lateral hypothalamic electrode had to pass through the ventral tegmental area in order to reach the efferent stages of the reward pathway. In the simplest anatomical arrangement consonant with this view, the somata of these cells lie in the forebrain and give rise to descending axons. As a test of the hypothesis that anodal block was responsible for changing the shape of the strength-duration curve obtained with the ventral tegmental anode, a psychophysical version of the collision test was used to determine whether the tips of the lateral hypothalamic and ventral tegmental electrodes were indeed linked by a common set of reward-related fibers.(ABSTRACT TRUNCATED AT 400 WORDS)

Attenuation of the negative chronotropic effect of myelinated fibers by nonmyelinated fibers in the vagus nerve of the rabbit.

In the adult rabbit, both myelinated and nonmyelinated efferent fibers travel from the cervical vagus nerve to the sinoauricular node. The myelinated fibers convey the negative chronotropic effect. The influence of the nonmyelinated fibers was studied. Stimulation (4/sec) of the myelinated fibers, while the compound action potential was monitored continuously, caused a slowing of the heart rate. Subsequent activation of the non-myelinated fibers attenuated this negative chronotropic effect. Stimulation of adrenergic fibers was excluded, based on data from histochemical studies and the effects of blockade of beta- and muscarinic receptors. The lengthening of the RR interval during activation of the myelinated fibers alone was about 3.5 times larger than during activation of all fiber groups. When the conduction of the myelinated fibers was blocked (anodal block), activation of the nonmyelinated fibers did not show any effect on heart rate. It was concluded that the negative chronotropic effect is conducted via small myelinated fibers and that the nonmyelinated fibers modulate the effect of these cardiomotor fibers, without having an effect on heart rate on their own. This modulation is effected either via a presynaptic mechanism on preganglionic fibers or via an interaction with postganglionic neurons. The nonmyelinated fibers constitute a novel peripheral system to modulate heart rate.

The depolarization of feline ventral horn group Ia spinal afferent terminations by GABA.

The unmyelinated terminal regions of extensor muscle Ia afferent fibres were stimulated electrically near lumbar motoneurones in anaesthetised cats using 300 microseconds pulses of less than 1 microA passed through the central NaCl barrel of seven barrel micropipettes. Such terminations were identified by anodal blocking factors of less than four and the latency of the antidromic impulse recorded in the appropriate peripheral muscle nerve. Although the effects of microelectrophoretically administered GABA were occasionally complex, the most consistent finding was a reduction in termination threshold followed by an increase. Both this reduction in threshold by GABA, and that produced by tetanic stimulation of low threshold flexor afferents (PAD) were diminished by microelectrophoretic bicuculline methochloride. This GABA antagonist alone elevated the threshold of some terminations but did not reduce the depolarizing action of either potassium or L-glutamate. Furthermore, since reductions in threshold by GABA, but not by either potassium or L-glutamate, were associated with a decrease in PAD, GABA appears to increase terminal membrane conductance. Since neither GABA nor bicuculline methochloride influenced the threshold or afferent depolarization of non-terminal regions of Ia fibres, there results are consistent with the function of GABA as a depolarizing transmitter at gabergic axoaxonic synapses upon the terminals of Ia afferent fibres synapsing with motoneurones.

Peripheram origins of human evoked spinal cord potentials : H. Shimizu, Y. Maruyama, H. Endo, S. Urano and K. Shimoji (Niigata, Japan)

This study has combined physiological pressure stimulation of carotid baroreceptors via a vascularly isolated carotid sinus and anodal block of baroreceptor afferent fibers in the carotid sinus nerve to examine the medullary projections of type I vs. type II (large A- vs. small A- and C-fiber afferent) baroreceptors. The control distribution of cells in the nucleus tractus solitarius expressing c-fos in response to physiological activation of carotid baroreceptors in the isolated sinus was compared to that during anodal block of large A-fibers in the carotid sinus nerve. Carotid baroreceptor stimulation primarily activated neurons in the ipsilateral commissural and medial subnuclei of the caudal nucleus tractus solitarius and the dorsolateral, dorsomedial and medial subnuclei in the intermediate and rostral levels of the nucleus tractus solitarius. Elimination of large A-fiber carotid baroreceptor afferents, during similar carotid baroreceptor stimulation resulted in a decrease in the nmber of cells expressing c-fos in the dorsomedial subnucleus of the rostral nucleus tractus solitarius. These data indicate that projections of larger A-fiber (type I) carotid baroreceptors are localized primarily to the rostral dorsomedial subnucleus, while those of smallr A- and C-fiber baroreceptors are more widely distributed to the commissural, medial and dorsal subnuclei of the nucleus tractus solitarius.

Effect of anodal blockade of myelinated fibers on vagal C-fiber afferents.

The effects of monopolar cathodal, monopolar anodal, and bipolar anodal polarizing currents on vagal A- and C-fiber activity were studied in anesthetized dogs. Monopolar cathodal polarization consistently produced excitation of spontaneous and evoked A- and C-fibers. Monopolar anodal and bipolar anodal polarizations differentially blocked A-fibers as a function of fiber diameter. The specific purpose of this study was to compare the effects of monopolar anodal and bipolar anodal currents on C-fiber excitability. Small fiber preparations were dissected from the vagal trunk, cut centrally, and placed on recording electrodes. A constant-current stimulus pulse was applied to the nerve at various distances from the blocking electrodes. The stimulus current strength was increased until an isolated C-fiber spike was observed. This value was defined as 100% of threshold. The stimulus current was then reduced to zero, the blocking current was increased slowly to 100 microA, and the procedure repeated. Threshold data obtained in this manner for each set of stimulation electrodes was plotted as a function of distance from the blocking electrode(s) for both modes of anodal blockade. No significant change in C-fiber excitability was observed with bipolar anodal blockade, whereas excitability was significantly (P less than or equal to 0.05) decreased using the monopolar technique. Thus, monopolar anodal block may reduce the possibility of asynchronous C-fiber discharge, which has been associated with a bipolar block of A-fibers.

Binaural Interaction in the Cod

ABSTRACT Recordings were made from 93 single units in the acoustical lobes and the torus semicircularis of the cod during sound stimulation and anodal blocking of the posterior saccular nerves. Most medullary sound responses were phase locked to the stimulus to some degree. The phase locking was less pronounced in the torus semicircularis, and sound stimulation sometimes caused clear inhibition of activity in this area. A large fraction of the units in both recording loci was insensitive to our sound stimuli, which acted mainly via the swimbladder. Peripheral blocking caused decreased activity and inhibition of sound responses in the acoustic lobes, indicating excitatory ascending input to this region. Binaural interaction was found in 8 of 29 medullary units tested during both ipsi- and contralateral block. Single-sided blocking experiments revealed both inhibitory and excitatory input to the torus semicircularis region. Binaural interaction was found in 3 of the 5 units tested during both ipsi- and contralateral block in this area.

Demonstration of re-entry within the canine specialized conduction system

There is suggestive evidence that bundle branch re-entry occurs in man in response to premature right ventricular stimulation. Demonstration of the activation sequence during re-entrant excitation in the in vivo dog was accomplished by placing recording electrodes on the major portions of the specialized conduction system. A temporary right heart bypass was utilized to place two or more electrodes on both right and left bundle branches and place electrodes on the His bundle and on the left and right ventricular endocardium. Premature excitation of the right ventricle was found not to retrogradely activate the right bundle but was able to cause slow right to left myocardial activation that resulted in retrograde activation within the left bundle branch. Retrograde conduction in the left bundle caused activation of the His bundle and the proximal right bundle. Activation of the right bundle resulted in antegrade conduction of the impulse across the site of previous conduction block and re-excitation of the right ventricle, to complete the re-entrant circuit. This type of re-entry, utilizing the bundle branches, was demonstrated in 19 dogs. This re-entry circuit was found to be facilitated by shortening of the right ventricular refractory period by local epicardial warming and was abolished by interruption of conduction in the right bundle by anodal blocking current applied to the right bundle. The sites of slow conduction, site of unidirectional block, and pathways of conduction were demonstrated. The validity of the concept of re-entry occurring within the specialized conduction system is substantiated.

Vagus cardioinhibitory fibers in rats.

In chloralose-urethane anesthetized rats, compound spike potentials provoked in the cervical vagus nerve with electrical stimulation of the central cut end were found to consist of three major groups, A, delta-B and C. A remarkable cardioinhibition was observed on repetitive stimulation of the vagus nerve with an intensity which gave rise to the delta-B spike potential group. However, when the stimulus intensity was increased further beyond the level where the delta-B potential group had reached the maximum, the potency of cardioinhibition continued to be reinforced with the development of the C potential group. Selective activation of C fibers by anodal block of conductions along A and delta-B fibers was still associated with a considerable degree of cardioinhibition. These findings indicate that activities of both C fibers and delta-B fibers contribute to vagal cardioinhibition in rats and the view that the vagal cardioinhibition is mediated by B fibers, although valid in cats, can not be held applicable to all species of mammalians.

The function of baroreceptor C fibres in the rabbit's aortic nerve.

The participation of aortic nerve C fibers in the baroreflex was investigated by recording changes in renal nerve activity in response to acute increases in arterial pressure, in two experimental situations: with the A and C fibres of the aortic nerve intact, and with the A fibres temporarily blocked by a hyperpolarizing current (anodal block). Pressure was increased by manual inflation of an intra-aortic balloon, and interference from other barorecptor areas was avoided by carotid occlusion and sectioning of the right aortic nerve. Rises in mean arterial pressure exceeding 20 mmHg, to levels above 110 mmHg, were needed to trigger sympathetic inhibition via C fibres. A rise of 45 mmHg caused 50% reduction in sympathetic activity, equal to that obtained by stimulation of aortic nerve C fibres at about 3 Hz. In contrast, a rise of 20-30 mmHg evoked more than 60% reduction in sympathetic activity when the A fibres were operative. Judged by these studies of peak reflex responses to brief pressure rises, baroreceptors with C fibres in the aortic nerve have a much higher threshold to pressure than their myelinated counterpart; the C fibres contribute to the baroreflex inhibition sympathetic discharge only when pressure is increased well above normal resting levels.

Anodal block of medullated cardiopulmonary vagal afferents in cats.

In anesthetized cats examination was made of the conditions under which the application of DC current to a mixed nerve permits determination of whether a reflex response is mediated by medullated or nonmedullated afferents. The following precautions should be observed. The nerve must be stripped of its sheath and bleeding avoided. The anode should be 6 mm distant from the cathode. The temperature of the mineral oil surrounding the nerve should be 30-32 degrees C to avoid activation of nonmedullated fibers during the block. The major limitation is a time- and frequency-dependent block of nonmedullated fibers which makes the technique suitable only for differentiating between medullated fibers and nonmedullated fibers with low frequency traffic. Observing these criteria, anodal block of the cervical vagus in sino-aortic denervated cats resulted in a mean rise in aortic pressure of 8 Torr; subsequent cold block caused a further mean rise of 30 Torr. Thus 80% of the total increase in aortic pressure could be ascribed to interruption of vagal C-fiber activity.

Role of heart and lung receptors with nonmedullated vagal afferents in circulatory control.

Vagal afferents from the cardiopulmonary region exert a tonic inhibition on the vasomotor center. This is demonstrated by constriction of systemic resistance and splanchnic capacitance vessels and by increased output of renin when the vagi are cut or blocked. In dogs, removal or selective denervation of organs showed that receptors in the lungs, the atria, and the ventricles each are responsible for the vasomotor inhibition. That this inhibition is due to nonmedullated vagal afferents (C fibers) was demonstrated by selective cooling of the vagi, anodal block of medullated afferents, and selective electrical stimulation of medullated and nonmedullated fibers. In the open-chest cat the discharge frequency of individual C fibers is sparse and irregular (mean, 1.4 impulses/sec) but increases to 10 or more impulses/sec with moderate increases in cardiac filling pressure and exhibits cardiac rhythmicity or is continuous throughout the cardiac cycle. The inhibition of sympathetic vasomotor outflow effected through the cardiopulmonary receptors is inversely related to that exerted by the arterial baroreceptors. The former receptors have less influence on the muscle circulation than the latter, but have an equal or greater effect on the renal circulation. In summary, receptors in the heart and lungs with nonmedullated vagal afferents are an important component of the integrated neural control of the circulation.