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Electrophysiological identification of neurons in ventrolateral medulla sending collateral axons to paraventricular and supraoptic nuclei in the cat
Brain Research, 305 (1984) 375 - 379 Elsevier
375
BRE 20268
Electrophysiological identification of neurons in ventrolateral medulla sending collateral axons to paraventricular and supraoptic nuclei in the cat MONICA M. CAVERSON and JOHN CIRIELLO*
Departmentof Physiology, Health Sciences Centre, Universityof Western Ontario, London, Ontario N6A 5C1 (Canada) (Accepted March 6th, 1984)
Key words: ventrolateral medulla - - paraventricular nucleus - - supraoptic nucleus - - carotid sinus nerve - - aortic depressor nerve - medullo-hypothalamic pathways - - cardiovascular reflex pathways
Experiments were done in chloralosed, paralyzed and artificiallyventilated cats to identify single units in the ventrolateral medulla (VLM) that send collateral axons directly to the region of the paraventricular (PVH) and supraoptic (SON) nuclei, and responding to peripheral inputs carrying cardiovascular afferent information. Twenty-six single units were antidromically activated in the VLM to stimulation of both the PVH and SON, and in each case the antidromic potential evoked by stimulation of one site was cancelled by stimulation of the other site. These units responded with latencies corresponding to conduction velocities of 5.1 _+0.4 m/s. Of these 26 units, 10 responded orthodromically to stimulation of either the carotid sinus or aortic depressor nerves. These data have demonstrated the existence of VLM neurons which send collateral axons to the PVH and SON and have provided evidence for their role in mediating cardiovascular afferent information directly to hypothalamic regions involved in autonomic and neuroendocrine regulation. In recent years there has b e e n an increasing amount of e x p e r i m e n t a l evidence suggesting that ascending pathways from the ventrolateral m e d u l l a (VLM) to the h y p o t h a l a m u s are i m p o r t a n t components of neural mechanisms controlling autonomic and n e u r o e n d o c r i n e functions (for a review see ref. 13). In particular, it has been suggested that the V L M exerts an influence on the release of vasopressin by magnocellular n e u r o s e c r e t o r y neurons in the paraventricular ( P V H ) and supraoptic (SON) nucleP 3. This suggestion is based on the following observations. First, application of nicotine to the surface of the V L M 1 or destruction of V L M neurons by either electrolytic lesions or kainic acid injections 3 have been shown to result in a m a r k e d elevation in the plasma level of vasopressin. O n the o t h e r hand, application of the putative inhibitory neurotransmitters glycine and y-aminobutyric acid to the surface of the V L M has been shown to inhibit the release of vasopressin in response to carotid occlusion 10. In support of this suggestion n e u r o a n a t o m i c a l studies using the t r a n s p o r t of a n t e r o g r a d e and retro-
grade tracers have d e m o n s t r a t e d direct projections from the V L M to magnocellular n e u r o s e c r e t o r y portions of the P V H and S O N 12,13. In addition, single units in the V L M antidromically activated by stimulation of either the P V H or S O N and o r t h o d r o m i c a l l y excited by stimulation of cardiovascular afferent fibers have been recently identified7,S. In the course of these latter electrophysiological studies by Ciriello and Caverson7, 8 it was o b s e r v e d that the anatomical distribution of neurons projecting directly to the P V H and S O N overlapped. This observation suggested the possibility that some. of these V L M neurons m a y have sent collateral axons to both hypothalamic structures. The present study was done to provide electrophysiological evidence of divergent axon collaterals of neurons in the V L M to the P V H and SON. The V L M was systematically explored for single units antidromically excited by electrical stimulation of both the P V H and SON. These units were then tested for their o r t h o d r o m i c response to stimulation of the carotid sinus (CSN) and aortic depressor ( A D N ) nerves. A preliminary r e p o r t of
Correspondence: J. Ciriello, Department of Physiology, Health Sciences Centre, University of Western Ontario, London, Ontario N6A 5C1 Canada. 0006-8993/84/$03.00 (~ 1984 Elsevier Science Publishers B.V.
376 these data has been p r e s e n t e d elsewhere 6. Experiments were done in 8 adult cats of either sex anesthetized with a-chloralose (60 mg/kg, i.v. initially, s u p p l e m e n t e d by additional doses of 30 mg/kg at 8- to 10-h intervals) after ethyl chloride and ether induction. The trachea was cannulated, the animals were paralyzed with d e c a m e t h o n i u m b r o m i d e (0.5 mg/kg, i.v., initially and additional doses when necessary) and then artificially ventilated. The femoral artery and vein were cannulated to record arterial pressure and heart rate and for the administration of drugs, respectively. The CSN and A D N were exposed as previously described 7 and the central ends of the isolated crushed nerves were placed on bipolar stainless steel electrodes for stimulation. Access to the P V H and SON was gained through a parietal craniotomy 7,8. The procedures for stimulation of the CSN, A D N and hypothalamus, for recording of electrical activity extracellularly from single units in the V L M , and for histological identification of recording and stimulation sites have been described in detail elsewhere 7. Spontaneously active and silent units in the V L M e n c o u n t e r e d during an electrode penetration were assessed for antidromic activation to electrical stimulation of the P V H and SON according to previously established criteria 7,11. Units were first tested for their antidromic response to stimulation of either the P V H or S O N on the ipsilateral side with 0 . 1 - 0 , 2 ms rectangular pulses at 0.5 Hz and at current intensities of 0 . 2 - 1 . 0 m A . A n t i d r o m i c a l l y identified units were then tested for their antidromic response to stimulation of the other structure on the same side using similar stimulation parameters. In addition, antidromic potentials e v o k e d by stimulation of one structure were tested for cancellation by stimulation of the other structure by applying the stimuli at intervals that were less than the sum of the latencies to the onset of the two e v o k e d antidromic action potentials. Units antidromically activated by stimulation of both the P V H and S O N were further tested for their o r t h o d r o m i c responses to electrical stimulation of the CSN and A D N with pulses of 0.3 ms duration at 0.5 Hz at current intensities up to 5 times the current required to elicit a cardiac slowing of 10-15 beats/min when using a 15 s stimulus train at 25 Hz and pulse duration of 0.3 ms. Currents at this intensity have previously been shown to elicit maximal reflex bradycardia during stimulation of
A
B
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SON D
I_
Fig. 1. VLM neuron antidromically activated by electrical stimulation of both the PVH and SON. Each record is 5 superimposed sweeps. Stimuli delivered at arrows. A: response of VLM neuron to stimulation of PVH at 0.5 Hz (0.1 ms and 0.6 mA). B: response of VLM neuron to stimulation of SON at 0.5 Hz (0.1 ms and 0.8 mA). C: when two stimuli are applied 10 ms apart both antidromic potentials are present. D: when the interval between stimuli is reduced to 8.8 ms, the SON potential is cancelled. E: orthodromic response of unit to electrical stimulation of the CSN. Calibration, 2 ms and 200/~v.
either the CSN or A D N 5 Stimulation and recording sites were m a p p e d on transverse sections of the hypothalamus modified from a stereotaxic atlas 2 and from projection drawings of the medulla. The conduction distance for each unit was calculated from these maps and ranged between 23.9-26.6 mm to the P V H and between 22.1-31.9 mm to the SON. Statistical analysis of means + S.E. of latencies and conduction velocities was done by using Student's t-test. P < 0.01 was considered to be statistically significant. A total of 160 electrode penetrations were made through the region of the V L M in the 8 cats. Although a large number of single units were antidrom-
377
A i
Si
SON
SON
PVH
PVH
L
SON
Fig. 2. Response of VLM unit antidromically activated by electrical stimulation of both the SON (A; 0.1 ms and 0.4 mA) and PVH (B; 0.1 ms and 0.4 mA) at 0.5 Hz. When two stimuli are delivered at 4.5 ms apart both antidromic potentials are present, but when the stimulus interval is reduced to 2.7 ms the PVH potential is cancelled. Each record is 5 superimposed sweeps. Stimuli delivered at arrows. Calibration, 2 ms and 50 ~v.
ically identified in the V L M by electrical stimulation of either the P V H or S O N (>150), these units will not be r e p o r t e d as similar results have previously been published 7.8. Twenty-six histologically verified single units in the V L M were antidromically activated by electrical stimulation of both the P V H and S O N (Figs. 1 and 2). Of these units, 5 (19%) were spontaneously active ( 2 - 1 7 spikes/s) and 21 were silent. A l l units responded with a single spike to stimulation of the P V H (mean duration, 1.6 + 0.1 ms) and S O N (mean duration, 1.6 + 0.1 ms) at threshold and suprathreshold stimulus intensities. Units r e s p o n d e d to stimulation of the P V H with a m e a n latency of 7.0 + 0.9 ms and to stimulation of the S O N with a m e a n latency of 6.6 + 0.8 ms, corresponding to conduction velocities of 5.0 ___ 0.5 and 5.3 + 0.6 m/s, respectively. The conduction velocities of these collateral axons were not significantly different and therefore have a c o m b i n e d mean conduction velocity of 5.1 + 0.4 m/s. The latencies of the antidromic responses were constant for
any one unit and followed rates of stimulation of 269 + 22 and 272 + 18 H z during stimulation of the P V H and SON, respectively. The following frequency of one collateral axon was not significantly different from the other. The antidromic action potential e v o k e d by stimulation of one structure was always cancelled by stimulation of the other structure (Figs. 1 and 2). This occurred for all units regardless of which structure was stimulated first. The timing of cancellation did not correlate well with the conduction velocities of the axons. A s shown in Fig. 1D cancellation occurred when the stimuli were applied at an interval (8.8 ms) that was less than the sum of the latencies of the two antidromic potentials (9.6 ms). This suggests that these neurons (Fig. 1) likely possessed two long axons with the branching point for the two axons occurring close to the soma. H o w e v e r , as shown in Fig. 2C this was not always the case as application of the two stimuli at an interval of 4.5 ms did not result in cancellation of the second antidromic spike. Instead, in the majority of units (22/26), as r e p r e s e n t e d in Fig. 2D, a very short time interval was n e e d e d between the two stimuli (2.7 ms) for cancellation of the P V H e v o k e d antidromic spike. This suggests that this type of n e u r o n (Fig. 2) likely possessed two short axon collaterals with the branching point for the axons close to their terminal sites. Of the 26 antidromically identified units, 10 (38%) were excited orthodromically by stimulation of either the CSN (n = 5; m e a n latency, 9.0 + 2.1 ms) or A D N (n = 5; m e a n latency; 11.2 + 2.8 ms). The remaining 16 units were not responsive to stimulation of the two buffer nerves. Fig. 1E shows the o r t h o d r o m i c response of an antidromically activated unit to stimulation of the CSN. Units excited by stimulation of the buffer nerves r e s p o n d e d antidromically with latencies corresponding to a m e a n conduction velocity of 4.6 + 0.2 m/s, whereas the m e a n conduction velocity of units non-responsive to the buffer nerves was 5.6 _+ 0.6 m/s. The anatomical distribution of units activated antidromically and either o r t h o d r o m i c a l l y excited or unresponsive to stimulation of the buffer nerves is shown in Fig. 3. A n t i d r o m i c a l l y activated units were found in an area that e x t e n d e d from the obex to 3 m m rostral to the obex. These units were located primarily in a region dorsal and lateral to the lateral
378
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Fig. 3. Series of representative transverse sections of the VLM of the cat (obex to 3 mm rostral to obex) showing the location of units antidromically activated by electrical stimulation of both the PVH and SON. O, units excited orthodromically only by stimulation of the CSN; A, units excited orthodromically only by stimulation of the ADN; ©, units not responding to stimulation of the buffer nerves. AMB, nucleus ambiguus; ION, inferior olivary nucleus; LRN, lateral reticular nucleus; 5ST, spinal trigeminal tract; 12N, hypoglossal nerve. Calibration, 2 mm.
reticular nucleus at caudal levels and lateral to the intramedullary rootlets of the hypoglossal nerve near the ventral surface of the medulla at more rostral levels. Responsive and non-responsive units to stimulation of the buffer nerves were found throughout this area. These data provide the first demonstration of neurons in the VLM that send divergent collateral axons to the P V H and SON and which mediate cardiovascular afferent information to these hypothalamic structures. The finding of neurons in the VLM with
branching axons was not unexpected as previous studies from this laboratory have demonstrated a considerable overlap of the anatomical distribution of single units antidromically activated by stimulation of either the SON or PVH 7~8. These findings are also supported by the recent anatomical demonstration in the cat of retrogradely double labeled neurons in the VLM after injections of two different fluorescent tracers in the regions of the P V H and SON (Ciriello and Caverson, 1984, unpublished observations). However, an unexpected finding was the difference in the timing of cancellation of an antidromic potential after the delivery of the two stimuli to the P V H and SON. This difference suggests the existence of at least two morphologically different VLM neurons; those with two long collateral axons branching near the soma and those with short collateral axons branching near the termination site. The functional significance of these two types of branching VLM neurons is unknown, but may be related to chemical transmitters associated with these neurons (for a review see ref. 13). The finding that VLM units antidromically activated by stimulation of the P V H and SON responded orthodromically to stimulation of the CSN and A D N is in agreement with previous studies showing that VLM units projecting directly to either hypothalamic structure alter their rate of discharge during stimulation of the buffer nerves 7.8 and that single units in the PVH and SON respond to stimulation of the buffer nerves 4. The latencies of the antidromic responses correspond to conduction velocities of these ascending fibers of 5.1 _+ 0.4 m/s, which is in agreement with earlier estimates 7& The functional significance of this ascending pathway may be related to the demonstration that activation of baroreceptors have been shown to alter the release of vasopressin ]4 and not oxytocin 9, to the finding that application of inhibitory neurotransmitters to the surface of the VLM inhibits the release of vasopressin during carotid occlusion 10, and to the demonstration that lesions of the VLM result in an increase in the plasma levels of vasopressin3. This evidence, taken together with the finding that neurons in the VLM projecting directly to the PVH and SON receive cardiovascular afferent inputs suggests that baroreceptor information involved in the regulation of vasopressin release is relayed to VLM neurons
379 which m a i n t a i n a tonic inhibition on h y p o t h a l a m i c
m a t i o n to these h y p o t h a l a m i c structures, c a n n o t be
vasopressinergic neurons.
excluded.
However,
as s u g g e s t e d
p r e v i o u s l y 7~8 the possibilities that the n o n - r e s p o n s i v e units to s t i m u l a t i o n of t h e b u f f e r n e r v e s are i n v o l v e d
This w o r k was s u p p o r t e d by the O n t a r i o H e a r t
in the c o n t r o l of o x y t o c i n r e l e a s e , and that b o t h re-
F o u n d a t i o n . J . C . is a C a n a d i a n H e a r t F o u n d a t i o n
sponsive and n o n - r e s p o n s i v e units m a y be i n v o l v e d in
Scholar and M . M . C .
relaying p e r i p h e r a l and c e n t r a l c h e m o r e c e p t o r infor-
G r a d u a t e Scholarship.
1 Bisset, G. W., Feldberg, W., Guertzenstein, P. G. and Rocha E Silva, M., Vasopressin release by nicotine: the site of action, Brit. J. Pharraacol. Chemother., 54 (1975) 463-474. 2 Bleier, R., The Hypothalamus of the Cat, The Johns Hopkins Press, Baltimore, MD, 1961. 3 Blessing, W. W., Sved, A. F. and Reis, D. J., Destruction of noradrenergic neurons in rabbit brainstem elevates plasma vasopressin causing hypertension, Science, 217 (1982) 661-663. 4 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. 5 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. 6 Ciriello, J. and Caverson, M. M., Electrophysiological identification of neurons in the ventrolateral medulla that send collateral axons to the paraventricular and supraoptic nuclei and receive cardiovascular afferent inputs in the cat, Soc. Neurosci. Abstr., 9 (1983) 181. 7 Ciriello, J. and Caverson, M. M., Direct pathway from neurons in the ventrolateral medulla relaying cardiovascular afferent information to the supraoptic nucleus in the cat, Brain Research, 292 (1984) 221-228.
is the h o l d e r of an O n t a r i o
8 Ciriello, J. and Caverson, M. M., Ventrolateral medullary neurons relay cardiovascular inputs to the paraventricular nucleus, Amer. J. Physiol., in press. 9 Clark, B. J. M. and Rocha E Silva, M., An afferent pathway for the selective release of vasopressin in response to carotid occlusion and hemorrhage in the cat, J. Physiol. (Lond.), 191 (1967) 529-542. 10 Feldberg, W. and Rocha E Silva, M., Inhibition of vasopressin release to carotid occlusion by gamma-aminobutyric acid and glycine, Brit. J. PharmacoL, 72 (1981) 17-24. 11 Lipski, J., Antidromic activation of neurones as an analytic tool in the study of the central nervous system, J. Neurosci. Meth., 4 (1981) 1-32. 12 Loewy, A. D., WaUach, J. H. and McKellar, S., Efferent connections of the ventral medulla oblongata in the rat, Brain Res. Rev., 3 (1981) 63-80. 13 Sawchenko, P. E. and Swanson, L. W., The organization of noradrenergic pathways from the brainstem to the paraventricular and supraoptic nuclei in the rat, Brain Res. Rev., 4 (1982) 275-325. 14 Share, L. and Levy, M. N., Cardiovascular receptors and blood titer of antidiuretic hormone, Amer. J. Physiol., 203 (1962) 425-428.
375
BRE 20268
Electrophysiological identification of neurons in ventrolateral medulla sending collateral axons to paraventricular and supraoptic nuclei in the cat MONICA M. CAVERSON and JOHN CIRIELLO*
Departmentof Physiology, Health Sciences Centre, Universityof Western Ontario, London, Ontario N6A 5C1 (Canada) (Accepted March 6th, 1984)
Key words: ventrolateral medulla - - paraventricular nucleus - - supraoptic nucleus - - carotid sinus nerve - - aortic depressor nerve - medullo-hypothalamic pathways - - cardiovascular reflex pathways
Experiments were done in chloralosed, paralyzed and artificiallyventilated cats to identify single units in the ventrolateral medulla (VLM) that send collateral axons directly to the region of the paraventricular (PVH) and supraoptic (SON) nuclei, and responding to peripheral inputs carrying cardiovascular afferent information. Twenty-six single units were antidromically activated in the VLM to stimulation of both the PVH and SON, and in each case the antidromic potential evoked by stimulation of one site was cancelled by stimulation of the other site. These units responded with latencies corresponding to conduction velocities of 5.1 _+0.4 m/s. Of these 26 units, 10 responded orthodromically to stimulation of either the carotid sinus or aortic depressor nerves. These data have demonstrated the existence of VLM neurons which send collateral axons to the PVH and SON and have provided evidence for their role in mediating cardiovascular afferent information directly to hypothalamic regions involved in autonomic and neuroendocrine regulation. In recent years there has b e e n an increasing amount of e x p e r i m e n t a l evidence suggesting that ascending pathways from the ventrolateral m e d u l l a (VLM) to the h y p o t h a l a m u s are i m p o r t a n t components of neural mechanisms controlling autonomic and n e u r o e n d o c r i n e functions (for a review see ref. 13). In particular, it has been suggested that the V L M exerts an influence on the release of vasopressin by magnocellular n e u r o s e c r e t o r y neurons in the paraventricular ( P V H ) and supraoptic (SON) nucleP 3. This suggestion is based on the following observations. First, application of nicotine to the surface of the V L M 1 or destruction of V L M neurons by either electrolytic lesions or kainic acid injections 3 have been shown to result in a m a r k e d elevation in the plasma level of vasopressin. O n the o t h e r hand, application of the putative inhibitory neurotransmitters glycine and y-aminobutyric acid to the surface of the V L M has been shown to inhibit the release of vasopressin in response to carotid occlusion 10. In support of this suggestion n e u r o a n a t o m i c a l studies using the t r a n s p o r t of a n t e r o g r a d e and retro-
grade tracers have d e m o n s t r a t e d direct projections from the V L M to magnocellular n e u r o s e c r e t o r y portions of the P V H and S O N 12,13. In addition, single units in the V L M antidromically activated by stimulation of either the P V H or S O N and o r t h o d r o m i c a l l y excited by stimulation of cardiovascular afferent fibers have been recently identified7,S. In the course of these latter electrophysiological studies by Ciriello and Caverson7, 8 it was o b s e r v e d that the anatomical distribution of neurons projecting directly to the P V H and S O N overlapped. This observation suggested the possibility that some. of these V L M neurons m a y have sent collateral axons to both hypothalamic structures. The present study was done to provide electrophysiological evidence of divergent axon collaterals of neurons in the V L M to the P V H and SON. The V L M was systematically explored for single units antidromically excited by electrical stimulation of both the P V H and SON. These units were then tested for their o r t h o d r o m i c response to stimulation of the carotid sinus (CSN) and aortic depressor ( A D N ) nerves. A preliminary r e p o r t of
Correspondence: J. Ciriello, Department of Physiology, Health Sciences Centre, University of Western Ontario, London, Ontario N6A 5C1 Canada. 0006-8993/84/$03.00 (~ 1984 Elsevier Science Publishers B.V.
376 these data has been p r e s e n t e d elsewhere 6. Experiments were done in 8 adult cats of either sex anesthetized with a-chloralose (60 mg/kg, i.v. initially, s u p p l e m e n t e d by additional doses of 30 mg/kg at 8- to 10-h intervals) after ethyl chloride and ether induction. The trachea was cannulated, the animals were paralyzed with d e c a m e t h o n i u m b r o m i d e (0.5 mg/kg, i.v., initially and additional doses when necessary) and then artificially ventilated. The femoral artery and vein were cannulated to record arterial pressure and heart rate and for the administration of drugs, respectively. The CSN and A D N were exposed as previously described 7 and the central ends of the isolated crushed nerves were placed on bipolar stainless steel electrodes for stimulation. Access to the P V H and SON was gained through a parietal craniotomy 7,8. The procedures for stimulation of the CSN, A D N and hypothalamus, for recording of electrical activity extracellularly from single units in the V L M , and for histological identification of recording and stimulation sites have been described in detail elsewhere 7. Spontaneously active and silent units in the V L M e n c o u n t e r e d during an electrode penetration were assessed for antidromic activation to electrical stimulation of the P V H and SON according to previously established criteria 7,11. Units were first tested for their antidromic response to stimulation of either the P V H or S O N on the ipsilateral side with 0 . 1 - 0 , 2 ms rectangular pulses at 0.5 Hz and at current intensities of 0 . 2 - 1 . 0 m A . A n t i d r o m i c a l l y identified units were then tested for their antidromic response to stimulation of the other structure on the same side using similar stimulation parameters. In addition, antidromic potentials e v o k e d by stimulation of one structure were tested for cancellation by stimulation of the other structure by applying the stimuli at intervals that were less than the sum of the latencies to the onset of the two e v o k e d antidromic action potentials. Units antidromically activated by stimulation of both the P V H and S O N were further tested for their o r t h o d r o m i c responses to electrical stimulation of the CSN and A D N with pulses of 0.3 ms duration at 0.5 Hz at current intensities up to 5 times the current required to elicit a cardiac slowing of 10-15 beats/min when using a 15 s stimulus train at 25 Hz and pulse duration of 0.3 ms. Currents at this intensity have previously been shown to elicit maximal reflex bradycardia during stimulation of
A
B
C~p~!
SON D
I_
Fig. 1. VLM neuron antidromically activated by electrical stimulation of both the PVH and SON. Each record is 5 superimposed sweeps. Stimuli delivered at arrows. A: response of VLM neuron to stimulation of PVH at 0.5 Hz (0.1 ms and 0.6 mA). B: response of VLM neuron to stimulation of SON at 0.5 Hz (0.1 ms and 0.8 mA). C: when two stimuli are applied 10 ms apart both antidromic potentials are present. D: when the interval between stimuli is reduced to 8.8 ms, the SON potential is cancelled. E: orthodromic response of unit to electrical stimulation of the CSN. Calibration, 2 ms and 200/~v.
either the CSN or A D N 5 Stimulation and recording sites were m a p p e d on transverse sections of the hypothalamus modified from a stereotaxic atlas 2 and from projection drawings of the medulla. The conduction distance for each unit was calculated from these maps and ranged between 23.9-26.6 mm to the P V H and between 22.1-31.9 mm to the SON. Statistical analysis of means + S.E. of latencies and conduction velocities was done by using Student's t-test. P < 0.01 was considered to be statistically significant. A total of 160 electrode penetrations were made through the region of the V L M in the 8 cats. Although a large number of single units were antidrom-
377
A i
Si
SON
SON
PVH
PVH
L
SON
Fig. 2. Response of VLM unit antidromically activated by electrical stimulation of both the SON (A; 0.1 ms and 0.4 mA) and PVH (B; 0.1 ms and 0.4 mA) at 0.5 Hz. When two stimuli are delivered at 4.5 ms apart both antidromic potentials are present, but when the stimulus interval is reduced to 2.7 ms the PVH potential is cancelled. Each record is 5 superimposed sweeps. Stimuli delivered at arrows. Calibration, 2 ms and 50 ~v.
ically identified in the V L M by electrical stimulation of either the P V H or S O N (>150), these units will not be r e p o r t e d as similar results have previously been published 7.8. Twenty-six histologically verified single units in the V L M were antidromically activated by electrical stimulation of both the P V H and S O N (Figs. 1 and 2). Of these units, 5 (19%) were spontaneously active ( 2 - 1 7 spikes/s) and 21 were silent. A l l units responded with a single spike to stimulation of the P V H (mean duration, 1.6 + 0.1 ms) and S O N (mean duration, 1.6 + 0.1 ms) at threshold and suprathreshold stimulus intensities. Units r e s p o n d e d to stimulation of the P V H with a m e a n latency of 7.0 + 0.9 ms and to stimulation of the S O N with a m e a n latency of 6.6 + 0.8 ms, corresponding to conduction velocities of 5.0 ___ 0.5 and 5.3 + 0.6 m/s, respectively. The conduction velocities of these collateral axons were not significantly different and therefore have a c o m b i n e d mean conduction velocity of 5.1 + 0.4 m/s. The latencies of the antidromic responses were constant for
any one unit and followed rates of stimulation of 269 + 22 and 272 + 18 H z during stimulation of the P V H and SON, respectively. The following frequency of one collateral axon was not significantly different from the other. The antidromic action potential e v o k e d by stimulation of one structure was always cancelled by stimulation of the other structure (Figs. 1 and 2). This occurred for all units regardless of which structure was stimulated first. The timing of cancellation did not correlate well with the conduction velocities of the axons. A s shown in Fig. 1D cancellation occurred when the stimuli were applied at an interval (8.8 ms) that was less than the sum of the latencies of the two antidromic potentials (9.6 ms). This suggests that these neurons (Fig. 1) likely possessed two long axons with the branching point for the two axons occurring close to the soma. H o w e v e r , as shown in Fig. 2C this was not always the case as application of the two stimuli at an interval of 4.5 ms did not result in cancellation of the second antidromic spike. Instead, in the majority of units (22/26), as r e p r e s e n t e d in Fig. 2D, a very short time interval was n e e d e d between the two stimuli (2.7 ms) for cancellation of the P V H e v o k e d antidromic spike. This suggests that this type of n e u r o n (Fig. 2) likely possessed two short axon collaterals with the branching point for the axons close to their terminal sites. Of the 26 antidromically identified units, 10 (38%) were excited orthodromically by stimulation of either the CSN (n = 5; m e a n latency, 9.0 + 2.1 ms) or A D N (n = 5; m e a n latency; 11.2 + 2.8 ms). The remaining 16 units were not responsive to stimulation of the two buffer nerves. Fig. 1E shows the o r t h o d r o m i c response of an antidromically activated unit to stimulation of the CSN. Units excited by stimulation of the buffer nerves r e s p o n d e d antidromically with latencies corresponding to a m e a n conduction velocity of 4.6 + 0.2 m/s, whereas the m e a n conduction velocity of units non-responsive to the buffer nerves was 5.6 _+ 0.6 m/s. The anatomical distribution of units activated antidromically and either o r t h o d r o m i c a l l y excited or unresponsive to stimulation of the buffer nerves is shown in Fig. 3. A n t i d r o m i c a l l y activated units were found in an area that e x t e n d e d from the obex to 3 m m rostral to the obex. These units were located primarily in a region dorsal and lateral to the lateral
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Fig. 3. Series of representative transverse sections of the VLM of the cat (obex to 3 mm rostral to obex) showing the location of units antidromically activated by electrical stimulation of both the PVH and SON. O, units excited orthodromically only by stimulation of the CSN; A, units excited orthodromically only by stimulation of the ADN; ©, units not responding to stimulation of the buffer nerves. AMB, nucleus ambiguus; ION, inferior olivary nucleus; LRN, lateral reticular nucleus; 5ST, spinal trigeminal tract; 12N, hypoglossal nerve. Calibration, 2 mm.
reticular nucleus at caudal levels and lateral to the intramedullary rootlets of the hypoglossal nerve near the ventral surface of the medulla at more rostral levels. Responsive and non-responsive units to stimulation of the buffer nerves were found throughout this area. These data provide the first demonstration of neurons in the VLM that send divergent collateral axons to the P V H and SON and which mediate cardiovascular afferent information to these hypothalamic structures. The finding of neurons in the VLM with
branching axons was not unexpected as previous studies from this laboratory have demonstrated a considerable overlap of the anatomical distribution of single units antidromically activated by stimulation of either the SON or PVH 7~8. These findings are also supported by the recent anatomical demonstration in the cat of retrogradely double labeled neurons in the VLM after injections of two different fluorescent tracers in the regions of the P V H and SON (Ciriello and Caverson, 1984, unpublished observations). However, an unexpected finding was the difference in the timing of cancellation of an antidromic potential after the delivery of the two stimuli to the P V H and SON. This difference suggests the existence of at least two morphologically different VLM neurons; those with two long collateral axons branching near the soma and those with short collateral axons branching near the termination site. The functional significance of these two types of branching VLM neurons is unknown, but may be related to chemical transmitters associated with these neurons (for a review see ref. 13). The finding that VLM units antidromically activated by stimulation of the P V H and SON responded orthodromically to stimulation of the CSN and A D N is in agreement with previous studies showing that VLM units projecting directly to either hypothalamic structure alter their rate of discharge during stimulation of the buffer nerves 7.8 and that single units in the PVH and SON respond to stimulation of the buffer nerves 4. The latencies of the antidromic responses correspond to conduction velocities of these ascending fibers of 5.1 _+ 0.4 m/s, which is in agreement with earlier estimates 7& The functional significance of this ascending pathway may be related to the demonstration that activation of baroreceptors have been shown to alter the release of vasopressin ]4 and not oxytocin 9, to the finding that application of inhibitory neurotransmitters to the surface of the VLM inhibits the release of vasopressin during carotid occlusion 10, and to the demonstration that lesions of the VLM result in an increase in the plasma levels of vasopressin3. This evidence, taken together with the finding that neurons in the VLM projecting directly to the PVH and SON receive cardiovascular afferent inputs suggests that baroreceptor information involved in the regulation of vasopressin release is relayed to VLM neurons
379 which m a i n t a i n a tonic inhibition on h y p o t h a l a m i c
m a t i o n to these h y p o t h a l a m i c structures, c a n n o t be
vasopressinergic neurons.
excluded.
However,
as s u g g e s t e d
p r e v i o u s l y 7~8 the possibilities that the n o n - r e s p o n s i v e units to s t i m u l a t i o n of t h e b u f f e r n e r v e s are i n v o l v e d
This w o r k was s u p p o r t e d by the O n t a r i o H e a r t
in the c o n t r o l of o x y t o c i n r e l e a s e , and that b o t h re-
F o u n d a t i o n . J . C . is a C a n a d i a n H e a r t F o u n d a t i o n
sponsive and n o n - r e s p o n s i v e units m a y be i n v o l v e d in
Scholar and M . M . C .
relaying p e r i p h e r a l and c e n t r a l c h e m o r e c e p t o r infor-
G r a d u a t e Scholarship.
1 Bisset, G. W., Feldberg, W., Guertzenstein, P. G. and Rocha E Silva, M., Vasopressin release by nicotine: the site of action, Brit. J. Pharraacol. Chemother., 54 (1975) 463-474. 2 Bleier, R., The Hypothalamus of the Cat, The Johns Hopkins Press, Baltimore, MD, 1961. 3 Blessing, W. W., Sved, A. F. and Reis, D. J., Destruction of noradrenergic neurons in rabbit brainstem elevates plasma vasopressin causing hypertension, Science, 217 (1982) 661-663. 4 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. 5 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. 6 Ciriello, J. and Caverson, M. M., Electrophysiological identification of neurons in the ventrolateral medulla that send collateral axons to the paraventricular and supraoptic nuclei and receive cardiovascular afferent inputs in the cat, Soc. Neurosci. Abstr., 9 (1983) 181. 7 Ciriello, J. and Caverson, M. M., Direct pathway from neurons in the ventrolateral medulla relaying cardiovascular afferent information to the supraoptic nucleus in the cat, Brain Research, 292 (1984) 221-228.
is the h o l d e r of an O n t a r i o
8 Ciriello, J. and Caverson, M. M., Ventrolateral medullary neurons relay cardiovascular inputs to the paraventricular nucleus, Amer. J. Physiol., in press. 9 Clark, B. J. M. and Rocha E Silva, M., An afferent pathway for the selective release of vasopressin in response to carotid occlusion and hemorrhage in the cat, J. Physiol. (Lond.), 191 (1967) 529-542. 10 Feldberg, W. and Rocha E Silva, M., Inhibition of vasopressin release to carotid occlusion by gamma-aminobutyric acid and glycine, Brit. J. PharmacoL, 72 (1981) 17-24. 11 Lipski, J., Antidromic activation of neurones as an analytic tool in the study of the central nervous system, J. Neurosci. Meth., 4 (1981) 1-32. 12 Loewy, A. D., WaUach, J. H. and McKellar, S., Efferent connections of the ventral medulla oblongata in the rat, Brain Res. Rev., 3 (1981) 63-80. 13 Sawchenko, P. E. and Swanson, L. W., The organization of noradrenergic pathways from the brainstem to the paraventricular and supraoptic nuclei in the rat, Brain Res. Rev., 4 (1982) 275-325. 14 Share, L. and Levy, M. N., Cardiovascular receptors and blood titer of antidiuretic hormone, Amer. J. Physiol., 203 (1962) 425-428.