J. Phy.~iol. (1975), 247, pp. 1-241 With 9 text -ft gurem Printed in Great Britain

SPONTANEOUS AND SYNAPTIC EXCITATION OF PARAMEDIAN RETICULAR NEURONES IN THE DECEREBRATE CAT

By A. W. DUGGAN* AND C. J. A. GAME From the Department of Pharmacology, Australian National University, Canberra, Australia

(Received 18 February 1974) SUMMARY

1. In decerebrate cats neurones in the region of the paramedian reticular nucleus were identified by responses to stimulation of implanted cerebellar electrodes. Approximately one half were antidromically acti-

vated and one half orthodromically. 2. Somatic stimulation and electrical stimulation of both hind limb and cranial nerves activated many of these cells. There was no correlation between the effects of these stimuli on cell firing and on blood pressure. 3. A number of rhythms in spontaneous firing were observed. One fifth of cells fired with the rhythm of efferent activity in the recurrent laryngeal nerve. In animals showing cycles of stability and instability in blood pressure corresponding phases of activity and inactivity in the firing of paramedian reticular neurones were observed. 4. Most paramedian reticular cells showed bursts of firing preceding abrupt rises in blood pressure but this was also observed with cells lateral to this area. 5. One third of cells studied showed changes in firing rate correlated with the changes in blood pressure which followed i.v. acetylcholine and noradrenaline. 6. Bilateral carotid artery occlusion, which always produced a rise in blood pressure, had little effect on cell firing. INTRODUCTION

The paramedian reticular nucleus (Brodal, 1953) lies between the hypoglossal nucleus and the inferior olive and is bounded laterally by the emerging hypoglossal rootlets. It merges with the periphypoglossal nuclei and both nuclei send efferents to and receive afferents from the cerebellum * C. J. Martin Fellow of the National Health and Medical Research Council of Australia. 1-2

A. W. DUGGAN AND C. J. A. GAME (Brodal, 1953; Brodal & Torvik, 1954; Torvik & Brodal, 1954; Brodal & Gogstad, 1957). Physiological studies of areas of the brain stem which include this nucleus have been from two view points: as a site which on stimulation produces electroencephalographic arousal (Moruzzi & Magoun, 1949) and inhibition of extensor rigidity (Magoun & Rhines, 1946) and secondly as an area which, when stimulated or ablated, modifies heart rate and blood pressure (Wang & Ranson, 1939; Alexander, 1946; Miura & Reis, 1969, 1972; Calaresu & Henry, 1970; Calaresu & Thomas, 1971). The possible relevance of some of these studies to the function of the paramedian reticular nucleus has been reviewed by Brodal (1953). Neurones of the paramedian reticular nucleus have been reported to differ from other reticular neurones in that nearly all are excited by electrophoretically administered acetylcholine and 5-hydroxytryptamine and inhibited by noradrenaline (Avanzino, Bradley & Wolstencroft, 1966). The present study was undertaken to attempt to assign some functional significance to the excitation of these cells by acetylcholine. Since little is known about afferent inputs to these neurones (Spyer & Wolstencroft, 1971), and there is controversy in the literature regarding possible connexions of the sinus nerve to the area which includes this nucleus (Crill & Reis, 1968; Miura & Reis, 1969; Biscoe & Sampson, 1970; Spyer & Wolstencroft, 1971; Lipski, McAllen & Spyer, 1972), this first paper describes spontaneous firing patterns of these neurones and their activation from a number of sources. A second communication reports findings on the pharmacology of excitation of these cells by acetylcholine and an inability to relate this to synaptic excitation. 2

METHODS Experiments were performed on thirty-five cats decerebrated under halothane anaesthesia by mid-brain coagulation (Crawford & Curtis, 1966). The external carotid arteries were ligated in six animals. Bilateral lateral ventricular drains were used to minimize the effects of obstructive hydrocephalus. The brain stem was approached from the ventral side. During the dissection the right hypoglossal (XII), recurrent laryngeal (RLN), superior laryngeal (SLN), glossopharyngeal (IX) and, in a few experiments, sinus nerves were prepared for stimulation. A pool of liquid paraffin made by the raising of skin flaps minimized stimulus spread between these nerves. Three fine needles insulated except for 1-2 mm at the tips were implanted in the cerebellum at Horsley-Clarke co-ordinates F -10 and V + 1, the central needle being in the mid line and the others 3 mm on either side. At this position a pair of electrodes was above and at either side of each fastigial nucleus. In some experiments, the ipsi- and/or contralateral saphenous and sciatic nerves were stimulated with implanted electrodes. Blood pressure was recorded with a polyethylene cannula introduced into the femoral artery with the tip probably in the lower aorta in most animals. Extracellular recordings were obtained with the 4 M-NaCl filled centre barrel of

PA RAMEDIAN RETICULAR NEURONES

3

seven barrel micropipettes, the outer barrels of which contained the drugs to be ejected electrophoretically. These, together with their concentration and pH, if adjusted, were: acetylcholine bromide (0.5 M), L-glutamate Na (0.5 M, pH 6.8) acetyl-,f-methylcholine Cl (0-5 M), nicotine HCl (0-2 M, pH 7 5), atropine S04 (10 mir in 165 mM-NaCl or 041 M), dihydro-fi-erythroidine HBr (10 mm in 165 mM-NaCl or 0.1 M) and gallamine triethiodide (0.2 M). Action potentials were converted into pulses using a continuously monitored level discriminator and the firing rate, measured with a ratemeter, displayed on a pen recorder. In some experiments the total efferent discharge in the recurrent laryngeal nerve was also displayed on a pen recorder.

Activation of cells from the cerebellum In distinguishing antidromic from orthodromic activation the following criteria were used. (i) Collision between action potentials initiated at the soma and those from the stimulating electrodes. This was tested by using spontaneous action potentials, or those produced by the excitant L-glutamate, to trigger the oscilloscope sweep and by timing the stimulus to the cerebellum appropriately. Examples are shown in Fig. 2A and B. (ii) Frequency following. When testing for collision obliteration of the cerebellar evoked action potential will occur when the interval between spontaneous and evoked action potentials is less than the refractory period of the soma. Frequency following was mainly used to exclude this possibility. (iii) Constanry of latency. Analysis of data showed that cells which showed collision had variation in latency of cerebellar activation usually less than 0-1 msec and always less than 0-2 msec. Hence when collision was not tested a latency variation of less than 0.1 msec was regarded as indicating antidromic activation. Cell locations Neurones were identified by the responses to stimulation of the implanted cerebellar electrodes. During an experiment the position of the tip of the micropipette was determined approximately by micromanipulator readings in relation to the ventral surface of the medulla and by reference to the field potentials produced by stimulation of the hypoglossal nerve and by antidromic activation of inferior olivary neurones from the cerebellar electrodes. For tracks lateral in the medulla, the field potentials from stimulation of afferents in the glossopharyngeal and superior laryngeal nerves served as reference markers. At the conclusion of nearly all experiments pontamine sky blue was ejected electrophoretically and the resultant spot located subsequently in frozen sections. Tissue sections were 50 am in thickness and distance from the obex of the site of dye deposition was measured in eleven cases. The mean distance was 2-4 mm rostral (range 1-75--5 mm). With a further ten animals tissue sections at the site of dye deposition indicated a similar distance from the obex. At approximately this level, Brodal (1953) observed that nearly all cells within the triangle bounded by the inferior olive, mid line raph6 and emerging hypoglossal rootlets underwent degeneration following removal of the cerebellum. Rostrally, however, degenerating, and a few non-degenerating cells, were intermingled and the latter were still described as part of the nucleus. Such a definition cannot be complied with when studying cells in the intact animal and for this reason all cells activated from the cerebellum and in the area outlined have been classified as belonging to the paramedian reticular nucleus. As pial openings were necessary for electrode penetrations and the vertebral artery prevented such openings

4

A. W. DUGGAN AND C. J. A. GAME

being made within 0-5 mm from the mid line it is unlikely that any mid line raph6 cells were studied. Cells of the perihypoglossal and paramedian reticular nuclei have been grouped together as the frozen sections were not of sufficient quality to allow accurate separation of cells at the junction of the two groups. Cells of the lateral reticular nucleus have been separated on the basis of the emerging hypoglossal nerve rootlets. Cells within these rootlets (N. interfascicularis of Jacobsohn; Brodal, 1957) have been included with paramedian reticular neurones. Brain 8tem movement and extracellular recording Movement between tissue components and the recording micro-electrode can be a problem when recording in the brain stem. Respiratory pulsations were minimized by making a bilateral pneumothorax after paralyzing the animal with i.v. gallamine and artificially ventilating at a rapid rate (50/min) with a small tidal volume. End-tidal C02 was maintained between 3-5 and 4%. To minimize cardiac pulsations, a small Perspex ring was applied to the brain stem surface using a small micromanipulator but this was an imperfect device because of failure of accurate apposition to the irregular ventral surface of the medulla. Since these experiments sought to correlate changes in blood pressure with cell firing it was important to distinguish direct (synaptic) effects from those arising indirectly: alterations in blood pressure are accompanied by changes in the volume of exposed nervous tissue and hence in the distance between the recording microelectrode and neurones. The most common source of error from this movement is that the action potential of the cell being studied can gradually decrease in size and not be accurately counted, or worse, that another cell be counted in its place. This occurrence was guarded against by continually monitoring action potentials and generated pulses on the same oscilloscope and, when specifically correlating cell firing with changing blood pressure, by studying only spontaneously active cells with relatively large action potentials. It should be noted that because of this problem the number of cells from which technically acceptable records were obtained during conditions of changing blood pressure was less than one half of those studied. Changes in the firing rate of a particular cell could result from contact between the electrode and cell membrane, from alterations in the tissue distribution of excitants ejected from adjacent micropipettes and even from variations in the effects on cell activity of electrophoretic currents used to retain pharmacologically active substances within micropipettes (Spehlmann, 1964). The phase relationships of the artifactual changes associated with ejecting an excitant, however, differ from those reported in this paper and, as the distinction is important, are illustrated in Fig. 1. The paramedian neurone, the responses of which are shown in Fig. 1A was fired by the continuous ejection of L-glutamate 150 nA, and as the blood pressure rose spontaneously, the firing rate decreased and was accomplished by a change in action potential amplitude (not illustrated). The important relationship is that firing rate and blood pressure do not have a phase lag. This neurone did not show these fluctuations in firing rate in the absence of the ejection of glutamate and, whilst it is possible that this cell did receive a synaptic input in phase with changes in blood pressure, it is more likely, in view of the changing action potential amplitude, that these fluctuations in firing rate were produced by varying concentrations of L-glutamate being delivered to the cell surface. Hence it is probable that movement of tissue relative to the electrode tip was in phase with blood pressure changes measured in the femoral artery and as displayed by the quite slow chart speeds used for recording. In attempting to distinguish real from artifactual changes emphasis will be placed on such phase relationships. Fig. 1 B illustrates the firing rate of another paramedian reticular neurone which increased

PA-4RAMEDIAN RETIGULAR1 NEUJRONES

5

before a rise in blood pressure, a phase relationship which almost certainly excludes an artifactual basis for the change in firing rate. Carotid occlusion was produced by snares of linen thread passed through polyethylene tubing which was sutured to adjacent muscle and taped to the cat frame. One end of the polythene tubing rested lightly on the arterial wall but some slight stretch of the artery still occurred before its occlusion. Initially, snares were placed

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through the fore brain. By tying the external carotid arteries just above the carotid sinus relatively stable recording conditions following occlusion of the common carotids were obtained. The concentrations of drugs given ixv. were: acetylcholine bromide 2 #g ml-' and noradrenaline bitartrate 40 gg ml-'.

66

~~A. W. DUGGAN AND C. J. A. GAME RESULTS

Cerebellar activation of paramedian reticular neurones As electrodes passed between the inferior olive and the hypoglossal nucleus neurones activated by stimulation of the cerebellum occurred in clusters. Commonly, a group of cells was found just dorsal to the inferior olive and another just ventral to the hypoglossal nucleus. Usually field potentials from both the ipsi- and the contralateral cerebellum were observed at each site but as one needle was common to each pair of stimulating electrodes no emphasis will be placed on whether a cell was activated from the ipsi- or contralateral cerebellum. Using the criteria outlined in Methods, forty-two of 103 cells were antidromically activated (seven of these were also fired orthodromically), forty-six were excited orthodromically. The data were inadequate for a decision upon the remaining fifteen. The ability of paramedian cells to respond to two closely spaced stimuli varied greatly. The antidromically activated cell in Fig. 2A (collision is shown) responded to two stimuli with an interval of 0-7 msec. At this high frequency the interval between action potentials was greater than the interval between stimuli. By contrast the antidromically activated cell of Fig. 2 B did not follow a stimulus interval of P-0 msec but the orthodromically activated cell of Fig. 20 (a wide latency variation is shown) fired to each of two stimuli with an interval of 0-7 msec. Although antidromically and orthodromically activated cells were not equally sampled in all experiments and in two experiments no antidromically activated cells were encountered, it was usual for these two types of cell to be intermingled throughout the area of the paramedian reticular nucleus and not to occur in separated clusters. The distribution of latencies of activation for antidromic and orthodromic cells is shown in Fig. 2 D and the locations, when determined, of these two types of cell are mapped in Fig. 2 E. A cell activated both antidromically and orthodromically from the cerebellum is shown in Fig. 3 B. Inhibition of spontaneous firing of a paramedian neurone following a stimulus to the cerebellum was observed once. Activation from the glossopharyngjeal, superior laryngeal and sinus nerves A previous study in the rat (Duggan, Headley & Lodge, 1975), indicated that neurones in the paramedian reticular area could be activated by stimulation of the glossopharyngeal nerve. This area contains many fibre tracts as well as cells and it is important to distinguish between action potentials from these two sources when studying activation from the

7i PA RAMEDIAN RETICULAR NEURONES periphery. This is readily done in experiments employing microelectrophoresis as ejection of an excitant (such as L-glutamate) will cause cells but not fibres to fire. Ejection of an excitant also gives information on A

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A. W. DUGGAN AND C. J. A. GAME

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Fig. 3. Somatic activation of paramedian reticular neurones. A, the responses of a paramedian reticular neurone to stimulation of the ipsilateral cerebellum, ipsilateral glossopharyngeal nerve, contra and ipsilateral saphenous nerves. Each stimulus artifact is marked with an arrow. The record of the response to stimulation of the cerebellum shows collision between action potentials produced by ejection of L-glutamate and those evoked from the cerebellum. A 500 ,uV calibration pulse is the first deflexion in each of the other records. The increased positivity of the action potential which followed ipsilateral saphenous stimulation was due to changing electrode-cell distances. B, activation of a paramedian reticular neurone from the cerebellum and the glossopharyngeal nerve. The cerebellar record shows both antidromic and orthodromic activation. Two stimuli, marked by arrows, were delivered to the cerebellum at an interval of 2 msec. This record is not a collision test. The time of stimulation of the glosso-pharyngeal nerve is marked by an arrow. A 500 /W calibration pulse is the first deflexion in each record. This cell was not activated from the ipsilateral superior laryngeal nerve. C, the firing of a paramedian reticular neurone in response to stroking the leg and abdomen and the effects on blood pressure. The upper trace is cell firing measured by a rate meter (time constant 3.3 sec), the lower trace is blood pressure. At the time of the first arrow the left leg was stroked, at all others the abdomen was stroked.

PARAMEDIAN RETICULAR NEURONES 9 the number of cells in the vicinity of the electrode tip. This was particularly important in ensuring that an action potential distorted by superimposition on a cerebellar field was derived from the same cell responding to peripheral stimulation, and that small changes in action potential configuration were due to changing electrode-cell distances and not to the presence of another neurone. Collision between an L-glutamate evoked action potential and that from the cerebellum of course leaves no doubt about a similar origin. In twelve cats the effects of stimulation of the ipsilateral glossopharyngeal and superior laryngeal nerves (both a single stimulus and two stimuli at an interval of 3-3 msec were used) were observed with twentyone neurones. Of these, five were activated from the glossopharyngeal nerve alone, two from the superior laryngeal nerve, three from both nerves and eleven could not be activated from either. The latency of activation from these nerves was relatively long (5-15 msec) as shown in Fig. 3A and B. There was no correlation between type of activation from the cerebellum and the response to stimulation of cranial nerves. The ipsilateral sinus nerve was stimulated in four experiments. In two animals small depressor effects on blood pressure were consistently observed, in one small pressor responses resulted and in one a large pressor response changed to a consistent depressor response following the intravenous administration of pentobarbitone sodium, 3 mg kg-1. No paramedian neurone was excited by stimulation of the sinus nerve nor was any field potential observed. Activation by somatic stimulation Paramedian reticular neurones were commonly excited by gentle stroking of, or blowing on, the integument of limbs and abdomen. Of forty-six neurones tested in this way thirty-two were excited. A systematic study of receptive fields was not made but in most cases responses were obtained from all parts of the body tested. With a further two cells light stimuli were ineffective but a noxious stimulus (squeezing of limbs with forceps) resulted in excitation. With twenty neurones responses to stimulation of the ipsi- and/or contralateral sciatic and saphenous nerves were studied. Thirteen cells were excited with latencies of activation of 15-20 msec (Fig. 3A). No differences in responses to somatic stimulation were observed between cells antidromically and orthodromically activated from the cerebellum. Stroking or blowing on the integument was often followed by a change in blood pressure. There was, however, a lack of correlation between effects on blood pressure and on cell firing as shown in Fig. 3 B.

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Spontaneous firing patterns of paramedian reticular neurones A number of variations of spontaneous firing was observed. (1) Firing with the frequency of efferent activity recorded in the recurrent laryngeal nerve. The animals were paralysed and ventilated with air at a rapid rate (50 min-'). Monitoring of the efferent discharge in the recurrent laryngeal nerve (RLN) usually showed bursts of firing at a frequency approximately half that of the respiratory pump frequency. This frequency, which could also be recorded in the nucleus of the tractus solitarius was the animal's 'endogenous' breathing rate and in some experiments appeared in the blood pressure tracing with or without the respiratory pump frequency (Fig. 4 C). This 'RLN rhythm' was observed with sixteen of eighty-two cells. These sixteen cells came from nine animals of the total thirty-five used, and whilst in two experiments nearly all PRN cells showed this rhythm, in others its presence on cell firing was unpredictable. The appearance of the RLN rhythm on the blood pressure tracing did not necessarily mean that it would be present in the firing of PRN cells and conversely cells were observed with this firing pattern without its presence on blood pressure tracings. This rhythm did not necessarily remain with cell firing throughout the period of observation. Fig. 4A illustrates the accentuation of this rhythm on cell firing just prior to its appearance in the blood pressure record. The fluctuations in blood pressure with the RLN rhythm were not marked in this case but they are present on the record following the drop in blood pressure produced by i.v. acetylcholine 0-3 #ug. The factors determining the appearance of this rhythm on blood pressure and/or cell firing were not defined. In particular arterial oxygen was not monitored. With the animal referred to in Fig. 4 the RLN rhythm disappeared from the blood pressure record following the injection of 4 jug noradrenaline i.v., an observation made with other animals. Pentobarbitone sodium (2-3 mg kg-1) was given i.v. to four animals during the course of an experiment and with the two in which this rhythm Fig. 4. Paramedian reticular neurones firing with the rhythm of the efferent discharge in the recurrent laryngeal nerve. A, the upper trace is cell firing rate, the lower is blood pressure. B, cell firing rate and blood pressure were recorded on different pen recorders at different chart speeds. The records have been positioned around the time of administration of pentobarbitone Na, 5 mg kg-'. The cell was excited by the electrophoretic administration of L-glutamate 100 nA and acetylcholine 150 nA at times indicated by the bars above the record. C, the upper record is total efferent activity in the recurrent laryngeal nerve measured with a rate-meter, the lower trace is blood pressure.

12 A. W. DUGGAN AND C. J. A. GAME was present on the blood pressure tracing, it was abolished. In one of these animals a cell showing this rhythm was being observed whilst barbiturate was given and the rhythm was abolished both from the blood pressure tracing and from cell firing. This is illustrated in Fig. 4 B. As ejection of the excitants L-glutamate and acetylcholine did not affect the fluctuations in firing frequency with the RLN rhythm an artifactual basis can be excluded. With the two cells illustrated the RLN discharge was monitored on a loudspeaker but Fig. 4C illustrates results from another experiment showing the correlation between total efferent RLN discharge and blood U

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pressure. The presence of this rhythm was not significantly correlated with any other means of activating each cell. (2) Firing with a cardiac rhythm. Only one neurone which fired with a cardiac rhythm was found. (3) Firing with the respiratory pump frequency. Such cells were observed in the nucleus of the tractus solitarius. No paramedian reticular neurone fired with this frequency. (4) Long duration cycles of activity-inactivity associated with blood pressure instability-stability. In three animals, the blood pressure showed periods of instability with rapid fluctuations, alternating with periods of relative stability. The periods of stability lasted from 3 to 10 min and were usually

PARAMEDIAN RETICULAR NEURONES 13 of shorter duration than the preceding periods of instability. In each animal the firing of one paramedian cell was observed throughout these cycles and with all three cells little activity was observed when the blood pressure was stable and irregular increased firing occurred throughout the periods of instability. A

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Records obtained from one animal are illustrated in Fig. 5. The relationship of the firing of this neurone to abrupt rises in blood pressure is less obvious than that shown in Fig. 6 but there is a clear relationship between overall stability of the blood pressure record and cell firing rate. The period of cell inactivity illustrated was 8 min and the periods of activity preceding and succeeding were 10 and 19 min respectively. This

A. W. DUGGAN AND C. J. A. GAME cell was antidromically activated from the cerebellum, was excited following i.v. acetylcholine and was excited by electrophoretically administered acetylcholine. Periodic non-responsiveness of reticular neurones to peripheral stimuli has been described (Scheibel & Scheibel, 1965) with periods much longer than those observed here with blood pressure instability. No quantitative study of responsiveness of these neurones during periods of inactivity was performed but it was observed that peripheral stimuli (stroking or blowing on limbs or abdomen) could still excite. These peripheral stimuli were not observed to terminate periods of inactivity. (5) Bursts of firing preceding abrupt changes in blood pressure. The stability of arterial blood pressure varied with different animals. The following rhythms were present to varying extents. (a) Small fluctuations with the frequency of the ventilating pump. (b) Fluctuations with the frequency of the animals endogenous respiratory rhythm. (c) Abrupt, rapidly rising and relatively large amplitude fluctuations. If the animals were incompletely paralysed, movements of limbs sometimes were associated with these rises. (d) Periods of stability alternating with periods of instability. (e) Gross fluctuations of a slower rise than those of c and which were observed in deteriorated preparations. This section is concerned with firing of paramedian reticular neurones in relation to abrupt blood pressure changes. Such changes were observed in ten of eighteen animals decerebrated by mid-brain coagulation alone and in which blood pressure recordings were made. They were not observed in the six animals in which the external carotids were also tied. Associated with these changes many paramedian reticular neurones were observed to fire but the changes in action potential amplitude associated with varying blood pressure limited to fifteen the number of cells from which accurate recordings could be made. Of these, thirteen showed a short burst of firing immediately preceding the change in blood pressure, one was inhibited and one showed no change. An example of these bursts is shown in Fig. 6A. This cell was antidromically activated from the cerebellum. A sharp burst of firing preceded each abrupt rise in blood pressure and three such rises are shown. The phase relationships of firing and blood pressure from another neurone are shown in Fig. 1 B and the increase clearly precedes any change in blood pressure. Such phase relationships practically exclude movement of electrode tip relative to cell as a cause of the change in firing rate. Inhibition of a paramedian reticular neurone preceding an abrupt blood pressure change is shown in Fig. 6 B. The arrows mark the start of each 14

PA RAMEDIA N RETICULAR NEURONES 15 decrease in firing rate and this preceded the rise in blood pressure in each case. Because many paramedian neurones showed these sharp bursts of firing and the correlation with blood pressure changes was so definite, -cells lateral to the paramedian reticular nucleus were also studied in relation to abrupt blood pressure change. As an example of a cell far removed from the paramedian reticular nucleus showing this firing pattern Fig. 6 C contains results obtained from a cell of the nucleus of the tractus solitarius. This neurone was activated with a short latency by a stimulus to the superior laryngeal nerve, and, when excited by L-glutamate, fired with the animals endogenous respiratory rhythm. Small bursts of spontaneous firing preceded each abrupt blood pressure change but with excitation by L-glutamate these resembled the sharp bursts of Fig. 6A. Recordings from either sciatic nerve also showed gross activity preceding such abrupt rises in blood pressure. Hence whilst paramedian reticular neurones commonly showed this firing pattern it was by no means peculiar to them. The responses of paramedian reticular neurones to induced changes in blood pressure (1) I.v. acetylcholine and noradrenaline. Noradrenaline and histamine given i.v. have been used to characterize 'cardiovascular neurones' (Salmoiraghi, 1962). A change in blood pressure and heart rate is not the sole consequence of injecting these compounds, but, nevertheless, neurones concerned with cardiovascular regulation might be expected to respond to blood pressure changes induced in this way. In the present experiments acetylcholine bromide and noradrenaline bitartrate were injected intravenously and effects on firing of paramedian reticular neurones were sought. Only spontaneously active cells were studied because of the need to monitor action potential amplitude during changing electrode-cell distances with changing blood pressure. Technically acceptable results were obtained from thirty-five cells (twenty-one antidromically activated from the cerebellum, ten orthodromically and four unassessed). The firing of twelve was affected by these substances, that of twenty-three unaffected. Of the twelve responsive neurones, the firing rate of ten was increased following i.v. acetylcholine (0.03-0.2,ug kg-1) and of these, four were inhibited following noradrenaline (1-4 /zg kg-'). One neurone was excited following i.v. noradrenaline and inhibited by i.v. acetylcholine and one cell showed slight excitation following both substances. The phase relationships of these responses are best described by reference to an illustration.

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A. W. DUGGAN AND C. J. A. GAME The results illustrated in Fig. 7 A were obtained from a cell orthodromically activated from the cerebellum and insensitive to electrophoretically ejected acetylcholine. It had a low level of spontaneous firing. Acetylcholine 0.5 gug i.v. produced a fall in blood pressure of approximately 50 mmHg and towards the end of this falling phase a large increase in firing rate occurred. Whilst there was considerable variation between cells in the duration and magnitude of this response, there was less scatter in 16

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its time of onset. The record illustrating the effect of ixv. noradrenaline (Fig. 7A) initially shows a spontaneous rise in blood pressure which is preceded by a burst of eel] firing. Because of the low spontaneous firing of this cell the onset of inhibition was hard to define but with this, and the three other neurones inhibited in this way, the inhibition outlasted the period of raised blood pressure. Similar effects of i.v. acetylcholine and noradrenaline on the firing of another cell are shown in Fig. 9.

PARAMEDIAN RETICULAR NEURONES 17 Fig. 7 B illustrates the firing of the one cell showing opposite response to those of Fig. 7A. The phase relationships of the inhibition of this cell following i.v. acetylcholine are approximate mirror images of those of cells excited by this procedure. No correlation was observed between responsiveness of cells to I.v. acetylcholine and noradrenaline and type of activation from the cerebellum. Cell firing pattern following i.v. ACh or NA * Change following ACh e Change following ACh and NA A

No change

And/~~~

Fig. 8. The distribution of cells in relation to cell firing following i.v. acetylcholine and noradrenaline. The outline of the medulla was drawn from a tissue section and is representative of the area studied. The hypoglossal and inferior olivary nuclei are outlined.

Of cells showing bursts of firing preceding abrupt blood pressure changes (see the preceding section), results with i.v. acetylcholine and noradrenaline were satisfactory with only four and of these, two showed a change in firing following these agents. The distribution of cells responsive and unresponsive to intravenous acetylcholine and noradrenaline is shown in Fig. 8. There was a concentration of responsive cells in the region just dorsal to the inferior olivary nucleus and two of the three cells which were excited by carotid occlusion are also in this group. (2) Firing patterns of paramedian neurones following carotid occlusion. The firing pattern of twenty-six paramedian reticular neurones (six animals) was observed following carotid occlusion. With sixteen of these, the response to intravenous acetylcholine and noradrenaline was also assessed and Table 1 summarizes these results. Firing in response to carotid occlusion was a rare event. The firing of all three neurones excited by carotid occlusion was affected following intravenous acetylcholine and/or noradrenaline. These three cells were half of those tested which showed responses to these substances. All twenty-six

A. W. DUGGAN AND C. J. A. GAME

18

Occlude common carotids

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.

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Acetylcholine Noradrenaline i.V 0-6 ug L.V. 6 ag

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Fig. 9. The effects of occlusion of both common carotid arteries and I.v. noradrenaline and acetylcholine on the firing of a paramedian reticular neurone. A, filmed records of action potentials and blood pressure. The duration of carotid occlusion is marked by the interrupted bars. B and a, the upper trace is cell firing measured with a rate- meter, the lower is blood pressure. In B, the time of common carotid occlusion (0.0.0.) is marked by bars above the cell firing record. TABLE 1. The responses of paramedian reticular neurones to carotid occlusion and to I.v. acetylcholine Response to I.v. acetylcholine Excitation No effect Unassessed Totals

Response to carotid occlusion A No effect Excitation 3 3 0 10 0 10 3 23

Totals 6 10 10 26

cells were spontaneously active but inhibition was not observed with any following carotid occlusion. The six animals used for these experiments did not show abrupt changes in blood pressure. The cell, the firing of which is shown in Fig. 9, was excited soon after the onset of carotid occlusion and just before any change in blood pressure occurred. As sinus nerve activity was not monitored, accurate phase relationships between the stimulus and cell firing cannot be described. This neurone was inhibited following I.v. noradrenaline (8 lug) and was

PA 1?RAMEDIAN RETICULAR NEURONES 19 excited following I.v. acetylcholine (0-6 4ug). The phase relationships of these responses are shown in Fig. 9 C. Two of the neurones activated by carotid occlusion were located just dorsal of the inferior olive. The position of the third was not plotted with any accuracy but was within the paramedian reticular area. Of these three neurones, two were orthodromically activated from the cerebellum and one antidromically. DISCUSSION

As there are many reports on firing patterns of reticular neurones in general but comparatively few on cells of the paramedian reticular nucleus, the limits of this nucleus need to be defined anatomically. Three cell groupings have been described; ventral, dorsal and accessory (Brodal, 1953) all lying within the confines of the hypoglossal nucleus, inferior olive, emerging hypoglossal rootlets and mid line raphe cells. These three groupings were recognized on the basis of cell degeneration following removal of the cerebellum and, since Brodal (1953) describes variations in the nucleus between different cats, when studying the intact animal it is perhaps better to speak of the 'paramedian reticular area'. In this study approximately half of the cells in the paramedian area were considered to be antidromically activated from the cerebellum. This is a higher proportion of cells than that reported by Spyer & Wolstencroft (1971) who stimulated the ipsilateral cerebellar peduncle (it is not clear which peduncle was stimulated) and found twenty-nine of eighty-eight cells, to be antidromically activated. The difference may be explained by differing proportions of cerebellar projecting axons being stimulated particularly as the paramedian reticular projection to the cerebellum is crossed and uncrossed (Brodal, 1953). In addition, electrode tracks in the rostral part of the nucleus will tend to sample cells of the nucleus gigantocellularis and thus lower the proportion of cells antidromically activated from the cerebellum (but see Avanzino, Hdsli & Wolstencroft, 1966). This probably explains the very low proportion of antidromically responding cells found in a few of the present experiments. The findings of both Spyer & Wolstencroft (1971), and the present study that many cells in this area cannot be activated antidromically from the cerebellum is at variance with the anatomical findings (Brodal, 1953) but in the ensuing discussion both cell types have been included in the paramedian reticular nucleus as the many stimuli used did not differentiate between them. Spyer & Wolstencroft (1971) were unable to activate paramedian cells projecting to the cerebellum by electrical stimulation of the sinus or vagus nerves, the hypothalamic 'depressor area' or peripheral somatic

A. W. DUGGAN AND C. J. A. GAME 20 stimulation. In the present study both orthodromic and antidromic cerebellar activated cells were activated both by peripheral somatic stimuli and by stimuli to the glossopharyngeal and superior laryngeal nerves. The published maps of Biscoe & Sampson (1970) indicate that paramedian reticular cells were activated by stimuli to these cranial nerves but cerebellar stimulation was not used in their experiments. The present failure to activate cells by sinus nerve stimulation confirms the results of Spyer & Wolstencroft (1971), Biscoe & Sampson (1970) and' McAllen & Spyer (1972). One-fifth of paramedian cells fired spontaneously with the rhythm of the efferent discharge in the recurrent laryngeal nerve. The associated fluctuations when present in the blood pressure tracing do not quite fit the definitions for either Traube-Hering or Mayer Waves. Traube-Hering waves are fluctuations in blood pressure in phase with breathing whilst Mayer waves are fluctuations slower than the breathing rate and said to be commonest in animals in poor experimental condition (Andersson, Kenny & Neil, 1950). An in-phase relationship between efferent discharges in the inferior cardiac and phrenic nerves in decerebrate cats artificially ventilated has been reported (Przybla & Wang, 1967). Salmoiraghi (1962) interpreted a correlation between Mayer waves and the firing of medullary neurones as evidence for a cardiovascular function for the cell being studied but the phrenic or recurrent laryngeal nerve discharge was not monitored in these experiments. Whether the appearance of the RLN rhythm on the firing of paramedian neurones was commoner in deteriorated preparations cannot be stated definitely but is considered probable. The paucity of cells firing with a cardiac rhythm in this area has been reported by others (Biscoe & Sampson, 1970; Miura & Reis, 1972; Middleton, Woolsey, Burton & Rose, 1973). The significance of the long lasting activity-inactivity cycles in cell firing cannot be assessed but it is possible that they represent states of 'arousal' and inactivity of the animal and not specifically related to the corresponding phases in blood pressure stability. In their reports of periodic non-responsiveness of reticular neurones, the Scheibels (1965) found such cycles were not related to other manifestations of sleep. It is of interest that a role for blood pressure and activity of the baroreceptors in the regulation of sleep and wakefulness has been proposed (Baust & Heinemann, 1967). The sharp bursts of firing preceding abrupt rises in blood pressure were observed in medullary areas apart from the paramedian reticular nucleus. Because of associated limb movements and that such bursts were seldom seen in animals in which the external carotid arteries had been ligated, it is probable that such bursts were part of a generalized 'arousal' of the animal,

PA RAMEDIAN RETICULAR NEURONES 21 and a rise in blood pressure but one component part. The pathway for the cardiovascular accompaniment of the 'defence reaction' produced by hypothalamic stimulation is ventrally situated in the mid-brain (Abrahams, Hilton & Zbrozyna, 1960) and may have escaped destruction in some animals decerebrated by mid-brain coagulation alone. The 'type C' bursts described by Moruzzi (1954) in decerebrate and encephale isole cats may be identical to the sharp bursts of firing described here. The activity recorded in the parahypoglossal area by Alanis, Mascher & Miyamoto (1966) following hypothalamic stimulation and correlated with activity in cardiac nerves may also have been part of a generalized 'arousal' as the medulla was not systemically probed. The present experiments found no evidence that the paramedian reticular nucleus was of importance in the cardiovascular response to somatic stimulation. The effects on blood pressure and cell firing were frequently unrelated. The firing of one third of the neurones studied was influenced by intravenous noradrenaline and acetylcholine, a finding which is at least consistent with a role in cardiovascular regulation. Fig. 9 shows that cells responsive to these agents were observed on the fringes of the paramedian reticular area and hence it is doubtful if they belong to the paramedian reticular nucleus. Neurones showing responses following agents which change blood pressure on intravenous administration have, however, been reported scattered throughout the medulla (Bradley & Mollica, 1958; Salmoiraghi, 1962; Przybla & Wang, 1967) in the mesencephalic reticular formation (Baust & Niemczyk, 1963) and in the hypothalamus (Frazier, Taquini, Boyarsky & Wilson, 1965). The firing patterns can be interpreted in terms of compensatory reactions to restore blood pressure to normal but other explanations are possible. The possibility that changes in cell firing following systemically vasoactive compounds are artifactual cannot be entirely excluded (discussed by Baust & Niemczyk, 1963), but the phase relationships between cell response and blood pressure make this unlikely. Noradrenaline and acetylcholine given systemically affect the activity of many viscera and afferent impulses from these could affect the firing of reticular neurones. A direct effect by intravenous acetylcholine can be excluded as some neurones affected by this substance, given systemically, were insensitive to electrophoretically administered acetylcholine. There is evidence that a rise in blood pressure alone causes EEG arousal (Baust & Niemczyk, 1963; Baust, Niemczyk & Vieth, 1963; Capon & Castiau, 1973), a mechanism of change in the firing of reticular neurones which is probably not specifically related to control of circulation. If the sinus nerve does project to the paramedian reticular nucleus (Crill & Reis, 1968; Homma, Miura & Reis, 1970), then it is reasonable to

A. W. DUGGAN AND C. J. A. GAME 22 expect that the firing of neurones of this nucleus will be affected by bilateral carotid artery occlusion, as this procedure was always followed by a large rise in blood pressure. The finding that only three of twenty-six paramedian reticular cells were excited by bilateral carotid occlusion does not lend much support to the proposal that the cardiovascular response to this procedure is organized in this nucleus (Miura & Reis, 1972). Whilst this stimulus is relatively crude (blood flow through the carotid body is also abnormal, Langren & Neil, 1951), the lack of effect on paramedian neurones made it unnecessary to attempt a more specific stimulation of carotid baroreceptors. The cardiovascular responses to fastigial nucleus stimulation have been shown to be abolished by lesions in the region of the paramedian reticular nucleus (Miura & Reis, 1970) but recently stimulation of this nucleus in the conscious cat has been shown to be accompanied by arousal and behavioural changes (Reis, Doba & Nathan, 1973). In the present experiments, the change in blood pressure following carotid occlusion was probably the stimulus most devoid of effect on arousal of the animal. It is probably significant, in assessing the role of the paramedian reticular nucleus in cardiovascular regulation, that this was the least effective stimulus in altering the firing of neurones of this nucleus. The authors wish to thank Mrs H. Rath for technical assistance and Professor D. R. Curtis for criticism of the manuscript.

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