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Renal afferent nerves affect discharge rate of medullary and hypothalamic single units in the cat
Journal of the Autonomic Nervous System, 3 (1981) 311--320
311
© Elsevier/North-Holland Biomedical Press
R E N A L AFFERENT NERVES AFFECT DISCHARGE RATE OF M E D U L L A R Y AND HYPOTHALAMIC SINGLE UNITS IN THE CAT
F.R. CALARESU and J. CIRIELLO
Department of Physiology, University of Western Ontario, London, Ont. N6A 5C1 (Canada)
Keywords: afferent renal nerves -- carotid sinus nerve - - h y p o t h a l a m i c neurons -- medullary neurons -- visceral afferent pathways
ABSTRACT
Electrical activity of spontaneously firing single units in the medulla and hypothalamus of 22 cats anesthetized with chloralose was monitored for changes in firing frequency during electrical stimulation of afferent renal (ARN) and carotid sinus (CSN) nerves. Stimulation of A R N altered the firing frequency of 214 out of 540 units studied in the ipsi- and contralateral medulla; the majority of the responses were excitatory b u t a few units (8%) were inhibited b y stimulation. Of the units responding to A R N stimulation, 57% were found to respond in the same manner to stimulation o f the CSN. Responsive units were found primarily in 3 regions: the lateral tegmental field, the area o f the paramedian reticular nucleus and the region o f the dorsal vagal complex around the obex. In the hypothalamus stimulation of A R N affected the activity of 197 o f the 407 units studied ipsi- and contralaterally; the majority o f the units were excited b u t 8% were found to be inhibited. Of the units responding to A R N 75% also responded to stimulation o f the CSN. Responsive units were found in most areas b u t were concentrated in 3 anterior regions: lateral preoptic area, lateral hypothalamic area and the region of the paraventricular nucleus. This is the first demonstration that stimulation of afferent renal nerves can influence the electrical activity of medullary and hypothalamic neurons bilaterally. Because of the demonstrated physiological role o f the structures where these responsive units were found these results suggest that sensory receptors in the kidney convey important information to central sites involved in physiological responses related to cardiovascular adjustments and fluid balance. Furthermore it has been demonstrated that the majority o f medullary and hypothalamic neurons responding to stimulation of ARN also receive an input from the CSN suggesting that certain regions of b o t h medulla and hypothalamus can integrate peripheral information from the kidney and from cardiovascular receptors to bring a b o u t appropriate homeostatic responses.
312
INTRODUCTION
Although a variety of sensory receptors signalling important physiological information to the central nervous system undoubtedly exist in the mammalian kidney [1] the search for the physiological function of these receptors has proven an arduous task. Since 1960 when renal mechanoreceptors were first described [17] only two more types of receptors have been classified, the R1 receptors responding to renal ischaemia [18], and the R2 receptors presumably monitoring ionic concentration in the renal interstitium [19]. Renal receptors conveying sensory information of physiological significance to integrative circuits in the central nervous system would give rise to command signals controlling the function of effector organs involved in homeostatic responses. Although it has already been established that afferent information from the kidney can affect the electrical activity of efferent fibers to both the ipsi- and the contralateral kidney, the type of physiological information relayed from the kidney and the specific renal responses controlled by the firing of efferent nerves are still a matter of speculation [1,5]. These reno-renal reflexes can be evoked by stimulation of renal afferent fibers with latencies of a magnitude suggesting long central pathways involving supraspinal structures. Of these supraspinal structures, because of their well known role in the central control of visceral homeostatic mechanisms, the medulla and the hypothalamus would be t w o likely sites of integration of information originating in the kidney. For these reasons experiments were done to explore systematically the medulla and the hypothalamus of cats for single units responding to stimulation of afferent renal nerves to identify the precise location of structures involved in the handling of renal afferent information. Furthermore the central units responding to stimulation of afferent renal nerves were subsequently tested for their responsiveness to stimulation of the carotid sinus nerve to investigate the possibility of convergence of signals from carotid and renal receptors on central neurons. Some of the results of this study have been presented elsewhere in preliminary form [9 ]. MATERIALS AND METHODS
Results were obtained in 22 adult cats (2.4--4.5 kg) of either sex anesthetized with alpha~hloratose (60 mg/kg i.v. initially, supplemented by additional doses of 30 mg/kg at 8--10 h intervals) after ethyl chloride and ether induction. The animals were paralyzed with decamethonium bromide (0.5 mg/kg i.v. initially and additional doses when necessary) and artificially ventilated through a tracheal cannuta. Arterial pressure and heart rate monitored from a cannula in the femoral artery were recorded on a polygraph. A catheter in the femoral vein was used for administration of drugs. Rectal temperature was maintained at 37 -+ 0.2°C by a heating pad connected to a Yellow Springs 73 temperature controller. The left renal nerves were exposed as previously described [5] and the central ends of the crushed nerves were placed on bipolar stainless-steel electrodes and covered with cotton pellets soaked
313
in warm medical fluid (Dow Coming 360, Midland, Mich.). The left carotid sinus nerve (CSN) was exposed [7] and a segment of the hypoglossal nerve overlying the sinus region was removed. The head of the animal was fixed in a K o p f stereotaxic frame. The medulla was exposed b y removing the occipital bone and part of the cerebellum; the hypothalamus was approached through a bilateral parietal craniotomy. Exposed nervous tissue was covered with warm medical fluid to prevent drying.
Recording and stimulation Electrical activity was recorded from spontaneously firing single units using stainless4teel microelectrodes and a conventional system for recording and analyzing single unit activity [8]. The medulla was explored bilaterally on a grid with points 0.5 mm apart, from 2 mm caudal to 5 mm rostral to the obex, from 0.5 mm lateral to the edge, and from the dorsal to the ventral surface of the brain stem. The rostrocaudal extent o f the hypothalamus was explored bilaterally on a grid with points 0.5 mm apart, from 8 to 16 mm rostral to the interaural line, from 0.3 to 4.3 mm lateral to the midline and from the horizontal stereotaxic zero line to the ventral surface. Single units were tested for their responses to electrical stimulation of afferent renal nerves (ARN) with trains of 2--5 rectangular waves at 200 Hz, 0.5 msec duration at 1--5 times the threshold current required to elicit an increase in mean arterial pressure of approximately 10 mm Hg using at 15 sec train at 60 Hz, 0.5 msec [6]. Units whose activity was modified by stimulation of A R N were also tested for their response to stimulation of the carotid sinus nerve (CSN) at parameters used previously [2]. The latency was calculated from peristimulus time histograms from the time o f occurrence of the shock artifact corresponding to the last pulse delivered to the peripheral nerves to the time of appearance o f a change in firing frequency of at least 50% from control. Localization of sites of recording and statistical comparison of results were done using methods previously described [2]. RESULTS
The effect of electrical stimulation of ARN on the electrical activity of 540 spontaneously firing (0.1--30 spikes/sec) medullary units was studied in 10 cats. In the ipsilateral medulla 37% (106 of 289 units tested) altered their rate of discharge during stimulation o f ARN: 95 were excited (mean latency 16.5 ± 0.9 msec) and 11 were inhibited (mean latency 17.5 + 1.9 msec). Of the units excited, 56% (53 of 95) were also excited b y stimulation of the CSN (mean latency 13.7 + 1.0, significantly shorter than latency to A R N stimulation) and of the 11 units inhibited 4 were also inhibited b y CSN stimulation (mean latency 11.8 ± 3.1 msec). In the contralateral medulla 43% of the units tested (108 o f 251) altered their rate of discharge. Of the responsive units 101 were excited (mean latency 15.5 ± 0.7 msec) and 7 were inhibited (mean latency 23.1 ± 4.8 msec). Of the units excited 60% (60 of 101) were also excited b y stimulation
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Fig. 1. Excitatory responses of a medullary (A) and of a hypothalamic unit (B) to stimulation of afferent renal (ARN) and carotid sinus (CSN) nerves. Five superimposed sweeps for each record. Arrows indicate time of stimulation. For locus see Fig. 2. Fig. 2. Anatomical drawings of representative transverse sections of the medulla (from 2 mm caudal to 4 mm rostral to the obex) showing the location of single units responding to stimulation of the ipsilateral ARN and CSN. For convenience of presentation each section includes sites of recording nearest to the section shown. *, units excited by ARN and CSN; e, units excited by ARN only; v, units inh~bited by ARN and CSN; ©, units inhibited by ARN only. AMB, nucleus ambiguus; AP, area postrema; CI, inferior central nucleus; DMV, dorsal motor nucleus o f the vagus; ECN, external cuneate nucleus; FTL, lateral tegmental field; IFT, infratrigeminal nucleus; ION, inferior olivary nucleus; LRN, lateral reticular nucleus; NTS, nucleus of the solitary tract; P, pyramidal tract; PH, nucleus praepositus hypoglossi; PPR, postpyramidal nucleus of the raphe; PRN, paramedian reticular nucleus; RB, restiform body; RFN, retrofacial nucleus; S, solitary tract; VIN, inferior vestibular nucleus; VMN, medial vestibular nucleus; 5SP, spinal trigeminal nucleus; 5ST, spinal trigeminal tract; 12M, hypoglossal nucleus.
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of the CSN (mean latency 22.1 -+ 1.6 msec), significantly greater than latencies to stimulation of ipsilateral CSN and of ARN), and of the 7 units 5 were also inhibited by the CSN (mean latency 23.4 + 7.4 msec). The excitatory response to stimulation of the ARN could be evoked by short trains of 2--5 pulses at 200 Hz and consisted of 1--3 spikes. On the other hand, similar excitatory responses could be evoked by single stimuli to the CSN. Fig. 1A shows typical excitatory responses of a medullary unit to stimulation of the ARN and CSN. The anatomical location in the medulla of units responding to electrical stimulation of either the ARN or the ARN and CSN is shown in Figs. 2 and 3. Responsive units were found primarily in 3 regions: the lateral tegmental
316
field, the area of the paramedian reticular nucleus and the region of the dorsal vagal complex around the level of the obex.
Hypothalamic units In 12 cats, 407 spontaneously firing (0.1--35 spikes/sec) units were tested during stimulation of ARN. In the ipsilateral hypothalamus 101/203 {50%) altered their rate of discharge; 98 were excited (mean latency 20.9 + 1.2 msec) and 3 were inhibited (12.5 -+ 3.2 msec). Of the excited units 80 (80%) also responded with excitation to stimulation of the CSN (mean latency 25.4 -+ 1.6 msec, significantly greater than latency to ARN stimulation), and 2 of the 3 inhibited units were similarlyaffected during stimulation o f the CSN (mean latency 8.5 -+ 3.5 msec).
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Fig. 4. Location of single units responding to stimulation of ARN and CSN plotted on transverse sections of the hypothalamus (8 ram--16 mm rostral to the interaural line). Refer to legend to Fig. 2 for identification of symbols. ACB, nucleus accumbens; AH, anterior hypothalamic nucleus; BST, bed nucleus of the stria terminalis; CP, pes pedunculi; DBB, diagonal band of Broca; DMH, dorsomedial hypothalamic area; FX, fornix; IC, internal capsule; LH, lateral hypothalamus; MB, mammillary body; MM, medial mammillary nucleus; MT, mammiliothalamic tract; OC, optic chiasm; OT, optic tract; PAH, paraventricular nucleus; PH, posterior hypothalamic area; POA, preoptic area; PVH, periventricular hypothalamic area; RN, nucleus reuniens thalami; SON, supraoptic nucleus; VMH, ventromedial hypothalamic nucleus; V3, third ventricle.
317 Of 204 units tested in the contralateral hypothalamus 47% (96/204) changed their firing frequency to stimulation of ARN; 84 units responded with excitation (mean latency 18.7 -+ 1.1 msec), and 12 responded with inhibition (mean latency 20.8 -+ 3.5 msec). The majority (69%) of units excited by ARN (58/84) responded similarly to stimulation of the CSN with a mean latency of 22.6 -+ 1.4 msec (significantly greater than latency to ARN stimulation} and 7 of the 12 units inhibited by ARN were similarly affected during CSN stimulation (mean latency 24.3 -+ 4.4 msec). The excitatory responses evoked in hypothalamic single units during stimulation of ARN and CSN were qualitatively and quantitatively similar to those described for medullary units as shown in Fig. lB. Responsive units were found primarily in 3 anterior hypothalamic regions: lateral preoptic area, lateral hypothalamic area and the region of the paraventricular nucleus. However, a small number of units were found scattered throughout the hypothalamus. The anatomical distribution of responsive units is shown in Fig. 4.
Comparisons of hypothalamic and medullary units Comparisons were not made for units inhibited because of their small number. Analysis of the units excited by stimulation of ARN revealed 3 major differences. Firstly, hypothalamic units responded to stimulation of both ARN and CSN with a significantly longer latency than medullary units. Secondly, approximately three
318
responded with excitation and only a few with inhibition during stimulation of ARN. This great preponderance of excitatory responses in medullary and hypothalamic neurons to stimulation of A R N has also been found to occur in hypothalamic neurons during stimulation of the CSN and aortic depressor nerve [2,3]. The few inhibited units were found exclusively in the lateral tegmental fields o f the medulla and in the preoptic region {medial preoptic and lateral preoptic nuclei) of the hypothalamus, whereas units excited were found to be distributed broadly. Although no effort was made to study the responses of these central units to activation of specific renal receptors [ 1,18,1 9 ] and therefore it is not possible to assign functional significance to these responses, the high proportion o f units responding to stimulation of afferent renal nerves u n d o u b t e d l y indicates that a variety of physiological signals originate in the kidney and that a large number of medullary and hypothalamic neurons are involved in integrating this information a b o u t renal function. This study has also shown that the majority of neurons responding to stimulation of A R N also responded to activation of the CSN, although proportionately more hypothalamic than medullary units responded to both inputs, suggesting that a larger number o f hypothalamic neurons are involved in integrating information from cardiovascular and renal receptors. This convergence o f inputs on central neurons can be interpreted to indicate either that the physiological information carried b y these t w o nerves is similar, an unlikely possibility in view o f the nature of the stimuli required to activate renal receptors [15,17,18,19], or that these neurons integrate a number of physiological signals of dissimilar nature necessary for the homeostatic control of circulation and fluid balance. Another interesting finding is that the medullary units responding either to only A R N or both inputs were evenly distributed in the 3 medullary projection areas, whereas hypothalamic units responding only to stimulation of A R N were located primarily in the preoptic region. Finally, two of the electrophysiological findings in this study deserve comment. First, the relatively short mean latency and small standard error of the latencies of the responses of single units to stimulation o f A R N indicate that the pathway carrying renal information is mostly made up of a homogeneous population of relatively fast conducting fibers. Comparison of latencies of units responding to stimulation of A R N and CSN showed significantly different latencies of responses to CSN stimulation, which prompts the suggestion that medullary and hypothalamic units were activated by different afferent pathways. Whether this difference was due to activation of different populations of renal receptors and renal afferent fibers or to different routing in the central nervous system cannot be ascertained from the information available. It should also be noted that the latencies o f the responses to stimulation of the CSN of hypothalamic neurons reported in this study are comparable to those previously described [2]. The second electrophysiological observation is that central units responded only if a minimum o f two pulses were delivered to ARN, in contrast to the CSN pathway which could be acti-
319
rated by single pulses. The different physiological properties of ARN and CSN pathways may be related to the longer and possibly more complicated route through the spinal cord followed by ARN compared to the more direct routes followed by the CSN. The observation that ARN project to several distinct medullary and hypothalamic regions merits discussion. The area of the dorsal vagal complex is well established as a site of integration of afferent information from and efferent signals to the cardiovascular system [4], and presumably physiological signals from the kidney are just one of the many inputs necessary for the homeostatic control of the circulation. In addition, although no precise localization is available, this area may correspond to an area described in the dog to elicit an increase in plasma renin activity on electrical stimulation [16]. The area of the medial reticular formation corresponding to the region of the paramedian nucleus has been implicated in the cardiovascular adjustments related to changes of posture [14] and it may be suggested that the paramedian nucleus is also involved in the adjustments in renal function known to occur during postural changes, although these adjustments have usually been attributed to activation of arterial and cardiopulmonary baroreceptors [10]. Finally, the projection to the lateral tegmental field cannot be assigned any specific significance, as this area is part of the poorly defined vasomotor centre [4]. It is worth noting that in the hypothalamus CSN and ARN share projections to the lateral hypothalamus and to the paraventricular nucleus, but only ARN project to the preoptic area. This latter finding is interesting in view of the demonstration of adipsia elicited by ablation of this region [13]. It is therefore reasonable to postulate that afferent information from the kidney is essential to the preoptic region in the coordination of neural activity concerned with body fluid balance. Furthermore the two other areas which receive ARN and CSN projections have also been implicated in body fluid control as it is known that the paraventricular nucleus is involved in the production and release of ADH [12] and ACTH [11] and that the lateral hypothalamus can control the release of renin [20]. In summary, we have provided the first electrophysiological demonstration of discrete locations of medullary and hypothaiamic single units responding to stimulation of afferent renal nerves. These results provide useful information on medullary and hypothalamic sites probably concerned with body fluid balance, although this suggestion is somewhat tempered by the demonstration that the majority of the units responding to stimulation of ARN also responded to stimulation of the CSN. ACKNOWLEDGEMENTS This work was supported by a grant to F.R.C. from the Medical Research Council of Canada. J.C. was holder of a Fellowship from the Canadian Heart Foundation.
320 REFERENCES 1 Calaresu, F.R., Studies on renal nerves. In R. Barcelo et al. (Eds.), Proc. VII Int. Congr. Nephrology, Montreal, Karger, Basel, 1978, pp. 565--571. 2 Calaresu, F.R. and Ciriello, J., Projections to the hypothalamus from buffer nerves and nucleus tractus solitarius in the cat, Amer. J. Physiol., 239 (1980) R130--R136. 3 Calaresu, F.R. and Ciriello, J., Projections of buffer nerves to medulla and hypothalamus. In P. Sleight (Ed.), Baroreceptors and Hypertension, Oxford Univ. Press, Oxford, 1980, pp. 252--260. 4 Calaresu, F.R., Faiers, A.A. and Mogenson, G.J., Central neural regulation of heart and blood vessels in mammals, Progr. Neurobiol., 5 (1975) 1--35. 5 Calaresu, F.R., Kim, P., Nakamura, H. and Sato, A., Electrophysiological characteristics of reno-renal reflexes in the cat, J. Physiol. (Load.), 238 (1978) 141--154. 6 Calaresu, F.R., Stella, A. and Zanchetti, A., Haemodynamic responses and renin release during stimulation of afferent renal nerves in the cat, J. Physiol. (Lond.), 255 (1975) 687--700. 7 Ciriello, J. and Calaresu, F.R., Lateral reticular nucleus: a site of somatic and cardiovascular integration in the cat, Amer. J. Physiol., 233 (1977) 100--109. 8 Ciriello, J. and Calaresu, F.R., Separate medullary pathways mediating reflex vagal bradycardia to stimulation of buffer nerves in the cat, J. auton. Nerv. Syst., 1 (1979) 13--32. 9 Ciriello, J. and Calaresu, F.R., Hypothalamic projections of renal afferent nerves in the cat, Canad. J. Physiol. Pharmacol., 58 (1980) 574--576. 10 DiBona, G.F. and Johns, E.J., A study of the role of renal nerves in the renal responses to 60 ° head-up tilt in the anaesthetized dog, J. Physiol. (Lond.), 299 (1980) 117--126. 11 Grizzle, W.E., Johnson, R.N., Schramm, L.P. and Gann, D.S., Hypothalamic cells in an area mediating ACTH release respond to right atrial stretch, Amer. J. Physiol., 228 (1975) 1039--1045. 12 Hayward, J.N., Functional and morphological aspects of hypothalamic neurons, Physiol. Rev., 57 (1977) 574--657. 13 Johnson, A.K. and Buggy, J., Periventricular preoptic-hypothalamus is vital for thirst and normal water economy, Amer. J. Physiol., 234 (1978) R122--R129. 14 Miura, M. and Reis, D.J., The paramedian reticular nucleus: a site of inhibitory interaction between projections from fastigial nucleus and carotid sinus nerve acting on blood pressure, J. Physiol. (Lond,), 216 (1971) 441--460. 15 Niijima, A., Studies on the blood-pressure sensitive receptors in the rabbit kidney in vivo, Jap. J. Physiol., 22 (1972) 433---440. 16 Passo, S.S., Assaykeen, K., Otsuka, B., Wise, L., Goidfien, A. and Ganong, W.F., Effect of stimulation of the medulla oblongata on renin secretion in dogs, Neuroendocrinology, 7 (1971) 1--10. 17 Pines, Iu.L., The electrophysiological characteristics of the afferent connexions of the kidney with the central nervous system, Fiziol. Zh. (Leningr.), 46 (1960) 1622-1630. 18 Recordati, G.M., Moss, N.G., Genovesi, S. and Rogenes, P.R., Renal receptors in the rat sensitive to chemical alterations of their environment, Circulat. Res., 46 (1980) 395--400. 19 Recordati, G.M., Moss, N.G. and Waselkov, L., Renal chemoreceptors in the rat, Circulat. Res., 43 (1978) 534--543. 20 Zanchetti, A. and Stella, A., Neural control of renin release, Clin. Sci. molec. Med., 48 (1975) 215--223.
311
© Elsevier/North-Holland Biomedical Press
R E N A L AFFERENT NERVES AFFECT DISCHARGE RATE OF M E D U L L A R Y AND HYPOTHALAMIC SINGLE UNITS IN THE CAT
F.R. CALARESU and J. CIRIELLO
Department of Physiology, University of Western Ontario, London, Ont. N6A 5C1 (Canada)
Keywords: afferent renal nerves -- carotid sinus nerve - - h y p o t h a l a m i c neurons -- medullary neurons -- visceral afferent pathways
ABSTRACT
Electrical activity of spontaneously firing single units in the medulla and hypothalamus of 22 cats anesthetized with chloralose was monitored for changes in firing frequency during electrical stimulation of afferent renal (ARN) and carotid sinus (CSN) nerves. Stimulation of A R N altered the firing frequency of 214 out of 540 units studied in the ipsi- and contralateral medulla; the majority of the responses were excitatory b u t a few units (8%) were inhibited b y stimulation. Of the units responding to A R N stimulation, 57% were found to respond in the same manner to stimulation o f the CSN. Responsive units were found primarily in 3 regions: the lateral tegmental field, the area o f the paramedian reticular nucleus and the region o f the dorsal vagal complex around the obex. In the hypothalamus stimulation of A R N affected the activity of 197 o f the 407 units studied ipsi- and contralaterally; the majority o f the units were excited b u t 8% were found to be inhibited. Of the units responding to A R N 75% also responded to stimulation o f the CSN. Responsive units were found in most areas b u t were concentrated in 3 anterior regions: lateral preoptic area, lateral hypothalamic area and the region of the paraventricular nucleus. This is the first demonstration that stimulation of afferent renal nerves can influence the electrical activity of medullary and hypothalamic neurons bilaterally. Because of the demonstrated physiological role o f the structures where these responsive units were found these results suggest that sensory receptors in the kidney convey important information to central sites involved in physiological responses related to cardiovascular adjustments and fluid balance. Furthermore it has been demonstrated that the majority o f medullary and hypothalamic neurons responding to stimulation of ARN also receive an input from the CSN suggesting that certain regions of b o t h medulla and hypothalamus can integrate peripheral information from the kidney and from cardiovascular receptors to bring a b o u t appropriate homeostatic responses.
312
INTRODUCTION
Although a variety of sensory receptors signalling important physiological information to the central nervous system undoubtedly exist in the mammalian kidney [1] the search for the physiological function of these receptors has proven an arduous task. Since 1960 when renal mechanoreceptors were first described [17] only two more types of receptors have been classified, the R1 receptors responding to renal ischaemia [18], and the R2 receptors presumably monitoring ionic concentration in the renal interstitium [19]. Renal receptors conveying sensory information of physiological significance to integrative circuits in the central nervous system would give rise to command signals controlling the function of effector organs involved in homeostatic responses. Although it has already been established that afferent information from the kidney can affect the electrical activity of efferent fibers to both the ipsi- and the contralateral kidney, the type of physiological information relayed from the kidney and the specific renal responses controlled by the firing of efferent nerves are still a matter of speculation [1,5]. These reno-renal reflexes can be evoked by stimulation of renal afferent fibers with latencies of a magnitude suggesting long central pathways involving supraspinal structures. Of these supraspinal structures, because of their well known role in the central control of visceral homeostatic mechanisms, the medulla and the hypothalamus would be t w o likely sites of integration of information originating in the kidney. For these reasons experiments were done to explore systematically the medulla and the hypothalamus of cats for single units responding to stimulation of afferent renal nerves to identify the precise location of structures involved in the handling of renal afferent information. Furthermore the central units responding to stimulation of afferent renal nerves were subsequently tested for their responsiveness to stimulation of the carotid sinus nerve to investigate the possibility of convergence of signals from carotid and renal receptors on central neurons. Some of the results of this study have been presented elsewhere in preliminary form [9 ]. MATERIALS AND METHODS
Results were obtained in 22 adult cats (2.4--4.5 kg) of either sex anesthetized with alpha~hloratose (60 mg/kg i.v. initially, supplemented by additional doses of 30 mg/kg at 8--10 h intervals) after ethyl chloride and ether induction. The animals were paralyzed with decamethonium bromide (0.5 mg/kg i.v. initially and additional doses when necessary) and artificially ventilated through a tracheal cannuta. Arterial pressure and heart rate monitored from a cannula in the femoral artery were recorded on a polygraph. A catheter in the femoral vein was used for administration of drugs. Rectal temperature was maintained at 37 -+ 0.2°C by a heating pad connected to a Yellow Springs 73 temperature controller. The left renal nerves were exposed as previously described [5] and the central ends of the crushed nerves were placed on bipolar stainless-steel electrodes and covered with cotton pellets soaked
313
in warm medical fluid (Dow Coming 360, Midland, Mich.). The left carotid sinus nerve (CSN) was exposed [7] and a segment of the hypoglossal nerve overlying the sinus region was removed. The head of the animal was fixed in a K o p f stereotaxic frame. The medulla was exposed b y removing the occipital bone and part of the cerebellum; the hypothalamus was approached through a bilateral parietal craniotomy. Exposed nervous tissue was covered with warm medical fluid to prevent drying.
Recording and stimulation Electrical activity was recorded from spontaneously firing single units using stainless4teel microelectrodes and a conventional system for recording and analyzing single unit activity [8]. The medulla was explored bilaterally on a grid with points 0.5 mm apart, from 2 mm caudal to 5 mm rostral to the obex, from 0.5 mm lateral to the edge, and from the dorsal to the ventral surface of the brain stem. The rostrocaudal extent o f the hypothalamus was explored bilaterally on a grid with points 0.5 mm apart, from 8 to 16 mm rostral to the interaural line, from 0.3 to 4.3 mm lateral to the midline and from the horizontal stereotaxic zero line to the ventral surface. Single units were tested for their responses to electrical stimulation of afferent renal nerves (ARN) with trains of 2--5 rectangular waves at 200 Hz, 0.5 msec duration at 1--5 times the threshold current required to elicit an increase in mean arterial pressure of approximately 10 mm Hg using at 15 sec train at 60 Hz, 0.5 msec [6]. Units whose activity was modified by stimulation of A R N were also tested for their response to stimulation of the carotid sinus nerve (CSN) at parameters used previously [2]. The latency was calculated from peristimulus time histograms from the time o f occurrence of the shock artifact corresponding to the last pulse delivered to the peripheral nerves to the time of appearance o f a change in firing frequency of at least 50% from control. Localization of sites of recording and statistical comparison of results were done using methods previously described [2]. RESULTS
The effect of electrical stimulation of ARN on the electrical activity of 540 spontaneously firing (0.1--30 spikes/sec) medullary units was studied in 10 cats. In the ipsilateral medulla 37% (106 of 289 units tested) altered their rate of discharge during stimulation o f ARN: 95 were excited (mean latency 16.5 ± 0.9 msec) and 11 were inhibited (mean latency 17.5 + 1.9 msec). Of the units excited, 56% (53 of 95) were also excited b y stimulation of the CSN (mean latency 13.7 + 1.0, significantly shorter than latency to A R N stimulation) and of the 11 units inhibited 4 were also inhibited b y CSN stimulation (mean latency 11.8 ± 3.1 msec). In the contralateral medulla 43% of the units tested (108 o f 251) altered their rate of discharge. Of the responsive units 101 were excited (mean latency 15.5 ± 0.7 msec) and 7 were inhibited (mean latency 23.1 ± 4.8 msec). Of the units excited 60% (60 of 101) were also excited b y stimulation
314
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Fig. 1. Excitatory responses of a medullary (A) and of a hypothalamic unit (B) to stimulation of afferent renal (ARN) and carotid sinus (CSN) nerves. Five superimposed sweeps for each record. Arrows indicate time of stimulation. For locus see Fig. 2. Fig. 2. Anatomical drawings of representative transverse sections of the medulla (from 2 mm caudal to 4 mm rostral to the obex) showing the location of single units responding to stimulation of the ipsilateral ARN and CSN. For convenience of presentation each section includes sites of recording nearest to the section shown. *, units excited by ARN and CSN; e, units excited by ARN only; v, units inh~bited by ARN and CSN; ©, units inhibited by ARN only. AMB, nucleus ambiguus; AP, area postrema; CI, inferior central nucleus; DMV, dorsal motor nucleus o f the vagus; ECN, external cuneate nucleus; FTL, lateral tegmental field; IFT, infratrigeminal nucleus; ION, inferior olivary nucleus; LRN, lateral reticular nucleus; NTS, nucleus of the solitary tract; P, pyramidal tract; PH, nucleus praepositus hypoglossi; PPR, postpyramidal nucleus of the raphe; PRN, paramedian reticular nucleus; RB, restiform body; RFN, retrofacial nucleus; S, solitary tract; VIN, inferior vestibular nucleus; VMN, medial vestibular nucleus; 5SP, spinal trigeminal nucleus; 5ST, spinal trigeminal tract; 12M, hypoglossal nucleus.
315
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of the CSN (mean latency 22.1 -+ 1.6 msec), significantly greater than latencies to stimulation of ipsilateral CSN and of ARN), and of the 7 units 5 were also inhibited by the CSN (mean latency 23.4 + 7.4 msec). The excitatory response to stimulation of the ARN could be evoked by short trains of 2--5 pulses at 200 Hz and consisted of 1--3 spikes. On the other hand, similar excitatory responses could be evoked by single stimuli to the CSN. Fig. 1A shows typical excitatory responses of a medullary unit to stimulation of the ARN and CSN. The anatomical location in the medulla of units responding to electrical stimulation of either the ARN or the ARN and CSN is shown in Figs. 2 and 3. Responsive units were found primarily in 3 regions: the lateral tegmental
316
field, the area of the paramedian reticular nucleus and the region of the dorsal vagal complex around the level of the obex.
Hypothalamic units In 12 cats, 407 spontaneously firing (0.1--35 spikes/sec) units were tested during stimulation of ARN. In the ipsilateral hypothalamus 101/203 {50%) altered their rate of discharge; 98 were excited (mean latency 20.9 + 1.2 msec) and 3 were inhibited (12.5 -+ 3.2 msec). Of the excited units 80 (80%) also responded with excitation to stimulation of the CSN (mean latency 25.4 -+ 1.6 msec, significantly greater than latency to ARN stimulation), and 2 of the 3 inhibited units were similarlyaffected during stimulation o f the CSN (mean latency 8.5 -+ 3.5 msec).
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Fig. 4. Location of single units responding to stimulation of ARN and CSN plotted on transverse sections of the hypothalamus (8 ram--16 mm rostral to the interaural line). Refer to legend to Fig. 2 for identification of symbols. ACB, nucleus accumbens; AH, anterior hypothalamic nucleus; BST, bed nucleus of the stria terminalis; CP, pes pedunculi; DBB, diagonal band of Broca; DMH, dorsomedial hypothalamic area; FX, fornix; IC, internal capsule; LH, lateral hypothalamus; MB, mammillary body; MM, medial mammillary nucleus; MT, mammiliothalamic tract; OC, optic chiasm; OT, optic tract; PAH, paraventricular nucleus; PH, posterior hypothalamic area; POA, preoptic area; PVH, periventricular hypothalamic area; RN, nucleus reuniens thalami; SON, supraoptic nucleus; VMH, ventromedial hypothalamic nucleus; V3, third ventricle.
317 Of 204 units tested in the contralateral hypothalamus 47% (96/204) changed their firing frequency to stimulation of ARN; 84 units responded with excitation (mean latency 18.7 -+ 1.1 msec), and 12 responded with inhibition (mean latency 20.8 -+ 3.5 msec). The majority (69%) of units excited by ARN (58/84) responded similarly to stimulation of the CSN with a mean latency of 22.6 -+ 1.4 msec (significantly greater than latency to ARN stimulation} and 7 of the 12 units inhibited by ARN were similarly affected during CSN stimulation (mean latency 24.3 -+ 4.4 msec). The excitatory responses evoked in hypothalamic single units during stimulation of ARN and CSN were qualitatively and quantitatively similar to those described for medullary units as shown in Fig. lB. Responsive units were found primarily in 3 anterior hypothalamic regions: lateral preoptic area, lateral hypothalamic area and the region of the paraventricular nucleus. However, a small number of units were found scattered throughout the hypothalamus. The anatomical distribution of responsive units is shown in Fig. 4.
Comparisons of hypothalamic and medullary units Comparisons were not made for units inhibited because of their small number. Analysis of the units excited by stimulation of ARN revealed 3 major differences. Firstly, hypothalamic units responded to stimulation of both ARN and CSN with a significantly longer latency than medullary units. Secondly, approximately three
318
responded with excitation and only a few with inhibition during stimulation of ARN. This great preponderance of excitatory responses in medullary and hypothalamic neurons to stimulation of A R N has also been found to occur in hypothalamic neurons during stimulation of the CSN and aortic depressor nerve [2,3]. The few inhibited units were found exclusively in the lateral tegmental fields o f the medulla and in the preoptic region {medial preoptic and lateral preoptic nuclei) of the hypothalamus, whereas units excited were found to be distributed broadly. Although no effort was made to study the responses of these central units to activation of specific renal receptors [ 1,18,1 9 ] and therefore it is not possible to assign functional significance to these responses, the high proportion o f units responding to stimulation of afferent renal nerves u n d o u b t e d l y indicates that a variety of physiological signals originate in the kidney and that a large number of medullary and hypothalamic neurons are involved in integrating this information a b o u t renal function. This study has also shown that the majority of neurons responding to stimulation of A R N also responded to activation of the CSN, although proportionately more hypothalamic than medullary units responded to both inputs, suggesting that a larger number o f hypothalamic neurons are involved in integrating information from cardiovascular and renal receptors. This convergence o f inputs on central neurons can be interpreted to indicate either that the physiological information carried b y these t w o nerves is similar, an unlikely possibility in view o f the nature of the stimuli required to activate renal receptors [15,17,18,19], or that these neurons integrate a number of physiological signals of dissimilar nature necessary for the homeostatic control of circulation and fluid balance. Another interesting finding is that the medullary units responding either to only A R N or both inputs were evenly distributed in the 3 medullary projection areas, whereas hypothalamic units responding only to stimulation of A R N were located primarily in the preoptic region. Finally, two of the electrophysiological findings in this study deserve comment. First, the relatively short mean latency and small standard error of the latencies of the responses of single units to stimulation o f A R N indicate that the pathway carrying renal information is mostly made up of a homogeneous population of relatively fast conducting fibers. Comparison of latencies of units responding to stimulation of A R N and CSN showed significantly different latencies of responses to CSN stimulation, which prompts the suggestion that medullary and hypothalamic units were activated by different afferent pathways. Whether this difference was due to activation of different populations of renal receptors and renal afferent fibers or to different routing in the central nervous system cannot be ascertained from the information available. It should also be noted that the latencies o f the responses to stimulation of the CSN of hypothalamic neurons reported in this study are comparable to those previously described [2]. The second electrophysiological observation is that central units responded only if a minimum o f two pulses were delivered to ARN, in contrast to the CSN pathway which could be acti-
319
rated by single pulses. The different physiological properties of ARN and CSN pathways may be related to the longer and possibly more complicated route through the spinal cord followed by ARN compared to the more direct routes followed by the CSN. The observation that ARN project to several distinct medullary and hypothalamic regions merits discussion. The area of the dorsal vagal complex is well established as a site of integration of afferent information from and efferent signals to the cardiovascular system [4], and presumably physiological signals from the kidney are just one of the many inputs necessary for the homeostatic control of the circulation. In addition, although no precise localization is available, this area may correspond to an area described in the dog to elicit an increase in plasma renin activity on electrical stimulation [16]. The area of the medial reticular formation corresponding to the region of the paramedian nucleus has been implicated in the cardiovascular adjustments related to changes of posture [14] and it may be suggested that the paramedian nucleus is also involved in the adjustments in renal function known to occur during postural changes, although these adjustments have usually been attributed to activation of arterial and cardiopulmonary baroreceptors [10]. Finally, the projection to the lateral tegmental field cannot be assigned any specific significance, as this area is part of the poorly defined vasomotor centre [4]. It is worth noting that in the hypothalamus CSN and ARN share projections to the lateral hypothalamus and to the paraventricular nucleus, but only ARN project to the preoptic area. This latter finding is interesting in view of the demonstration of adipsia elicited by ablation of this region [13]. It is therefore reasonable to postulate that afferent information from the kidney is essential to the preoptic region in the coordination of neural activity concerned with body fluid balance. Furthermore the two other areas which receive ARN and CSN projections have also been implicated in body fluid control as it is known that the paraventricular nucleus is involved in the production and release of ADH [12] and ACTH [11] and that the lateral hypothalamus can control the release of renin [20]. In summary, we have provided the first electrophysiological demonstration of discrete locations of medullary and hypothaiamic single units responding to stimulation of afferent renal nerves. These results provide useful information on medullary and hypothalamic sites probably concerned with body fluid balance, although this suggestion is somewhat tempered by the demonstration that the majority of the units responding to stimulation of ARN also responded to stimulation of the CSN. ACKNOWLEDGEMENTS This work was supported by a grant to F.R.C. from the Medical Research Council of Canada. J.C. was holder of a Fellowship from the Canadian Heart Foundation.
320 REFERENCES 1 Calaresu, F.R., Studies on renal nerves. In R. Barcelo et al. (Eds.), Proc. VII Int. Congr. Nephrology, Montreal, Karger, Basel, 1978, pp. 565--571. 2 Calaresu, F.R. and Ciriello, J., Projections to the hypothalamus from buffer nerves and nucleus tractus solitarius in the cat, Amer. J. Physiol., 239 (1980) R130--R136. 3 Calaresu, F.R. and Ciriello, J., Projections of buffer nerves to medulla and hypothalamus. In P. Sleight (Ed.), Baroreceptors and Hypertension, Oxford Univ. Press, Oxford, 1980, pp. 252--260. 4 Calaresu, F.R., Faiers, A.A. and Mogenson, G.J., Central neural regulation of heart and blood vessels in mammals, Progr. Neurobiol., 5 (1975) 1--35. 5 Calaresu, F.R., Kim, P., Nakamura, H. and Sato, A., Electrophysiological characteristics of reno-renal reflexes in the cat, J. Physiol. (Load.), 238 (1978) 141--154. 6 Calaresu, F.R., Stella, A. and Zanchetti, A., Haemodynamic responses and renin release during stimulation of afferent renal nerves in the cat, J. Physiol. (Lond.), 255 (1975) 687--700. 7 Ciriello, J. and Calaresu, F.R., Lateral reticular nucleus: a site of somatic and cardiovascular integration in the cat, Amer. J. Physiol., 233 (1977) 100--109. 8 Ciriello, J. and Calaresu, F.R., Separate medullary pathways mediating reflex vagal bradycardia to stimulation of buffer nerves in the cat, J. auton. Nerv. Syst., 1 (1979) 13--32. 9 Ciriello, J. and Calaresu, F.R., Hypothalamic projections of renal afferent nerves in the cat, Canad. J. Physiol. Pharmacol., 58 (1980) 574--576. 10 DiBona, G.F. and Johns, E.J., A study of the role of renal nerves in the renal responses to 60 ° head-up tilt in the anaesthetized dog, J. Physiol. (Lond.), 299 (1980) 117--126. 11 Grizzle, W.E., Johnson, R.N., Schramm, L.P. and Gann, D.S., Hypothalamic cells in an area mediating ACTH release respond to right atrial stretch, Amer. J. Physiol., 228 (1975) 1039--1045. 12 Hayward, J.N., Functional and morphological aspects of hypothalamic neurons, Physiol. Rev., 57 (1977) 574--657. 13 Johnson, A.K. and Buggy, J., Periventricular preoptic-hypothalamus is vital for thirst and normal water economy, Amer. J. Physiol., 234 (1978) R122--R129. 14 Miura, M. and Reis, D.J., The paramedian reticular nucleus: a site of inhibitory interaction between projections from fastigial nucleus and carotid sinus nerve acting on blood pressure, J. Physiol. (Lond,), 216 (1971) 441--460. 15 Niijima, A., Studies on the blood-pressure sensitive receptors in the rabbit kidney in vivo, Jap. J. Physiol., 22 (1972) 433---440. 16 Passo, S.S., Assaykeen, K., Otsuka, B., Wise, L., Goidfien, A. and Ganong, W.F., Effect of stimulation of the medulla oblongata on renin secretion in dogs, Neuroendocrinology, 7 (1971) 1--10. 17 Pines, Iu.L., The electrophysiological characteristics of the afferent connexions of the kidney with the central nervous system, Fiziol. Zh. (Leningr.), 46 (1960) 1622-1630. 18 Recordati, G.M., Moss, N.G., Genovesi, S. and Rogenes, P.R., Renal receptors in the rat sensitive to chemical alterations of their environment, Circulat. Res., 46 (1980) 395--400. 19 Recordati, G.M., Moss, N.G. and Waselkov, L., Renal chemoreceptors in the rat, Circulat. Res., 43 (1978) 534--543. 20 Zanchetti, A. and Stella, A., Neural control of renin release, Clin. Sci. molec. Med., 48 (1975) 215--223.