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The axons of raphespinal sympathoinhibitory neurons branch in the cervical spinal cord
Brain Research, 441 (1988) 371-376 Elsevier
371
BRE 22733
The axons of raphespinal sympathoinhibitory neurons branch in the cervical spinal cord Susan M. Barman and Gerard L. Gebber Departmems of Pharmacology and Toxicology, and of Physiology, Michigan State University, East Lansing, M148824 (U.S.A.)
(Accepted 27 October 1987) Key words: Antidromic mapping: Axonal branching: Medullospinal sympathetic neuron: Spike-triggered averaging: Sympatheticnerve discharge
This study shows that the axons of some raphespinal sympathoinhibitory neurons projecting to the third thoracic spinal segment emit branches in the third-fourth cervical spinal segments of cats. This was demonstrated by using time-contro!led collision of neuronal action potenttals initiated by stimuli applied at these spinal levels. Antidromic mapping revealed that the cervical branches of these neurons likely terminated in Rexed's lamina VII. In contrast to these raphe neurons, the axons of ventrolateral medullospinal sympathoexcitatory neurons did not emit cervical branches.
Barman and Gebber I and Morrison and Gebber II characterized the electrophysiological properties of cat medullary raphe and rostral ventrolateral medullary (VLM) neurons whose axons innervate the intermediolateral nucleus (IML) of the thoracic spinal cord (as demonstrated with antidromic mapping). Spike-triggered averaging and post R-wave analysis revealed that their activity is synchronized to the card~ac-related rhythm in sympathetic nerve discharge ($ND). The raphespinal neurons likely subserve a sympathoinhibitory function since their firing rate increases during baroreceptor reflex activation. In contrast, the VLM spinal neurons likely are sympathoexcitatory since their activity is decreased during baroreceptor reflex activation. The current investigation tested the hypothesis that the axons of such rapheand VLM-spinal neurons emit branches in the cervical spinal cord. This possibility was considered since double-labeling studies 5"~'!° show that some raphe and VLM neurons that project to the thoracic or lumbar spinal cord also innervate the cervical gray matter. These studie,~ did not assess whether these neurons project to different nuclei at the two spinal lev-
els. Such would be the case if a neuron that innervates the thoracic IML branched at cervical levels, since there is no IML in these spinal segments. Twenty-three cats (2.0-4.0 kg) were anesthetized by an i.p. injection of sodium diallyibarbiturate (60 mg/kg) and urethan (240 mg/kg), paralyzed with gallamine triethiodide (4 mg/kg, i.v., initial dose), placed in a stereotaxic apparatus and spinal investigation unit, and artificially respired. End-tidal CO2 was kept at 4 + 0.5% and rectal temperature was kept at 37 + 1 °C. Standard procedures were used to record brachial arterial pressure. As detailed in earlier reports from this laboratory L~], multibarrel glass micropipettes were used to record extracellularly the action potentials of single medullary neurons and to iontophorese 1 M L-glutamate at pH 8. Recordings were made from the somadendritic region of raphe and VLM neurons since their firing rate was increased by iontophoresing Lglutamate (ejection c u r r e n t s , - 5 to -30 hA), an amino acid that selectively depolarizes neuronal cell bodies 4. Raphespinal sympathoinhibitory neurons were identified on the midline, 2-5 mm rostral to the
Correspondence: S.M. Barman, Department of Pharmacology and Toxicology, Michigan State University, East Lansing, M! 48824. U.S.A.
0006-8993/88/$03.50© 1988Elsevier Science Publishers B.V. (Biomedical Division)
372 obex, and 3-5.5 mm below the dorsal surface. VLMspinal sympathoexcitatory neurons were identified 4-6 mm rostral to the obex, 3.5-4.0 mm lateral to the midline, and 3.5-6 mm below the dorsal surface. These recording sites were similar to those described in other reports from this laboratory TM and were identified by using histological procedures detailed in these reports. Left postganglionic inferior cardiac SND was recorded monophasically with a platinum bipolar electrode by using a capacity-coupled preamplifier (bandpass of 1-1,000 Hz). As previously described TM spike-triggered averaging (RC Electronics Computerscope) was used to identify single medullary neurons whose activity was synchronized to SND. A Grass $8800 digital stimulator and Grass PSIU-6 constant-current units were used to deliver cathodal square-wave pulses (0.5 ms duration) through tungsten microelectrodes (tip impedance, 10-30 k•) positioned in the third-fourth cervical (C3-C4) and third thoracic (T3) spinal segments (exposed by laminectomy). Stimulus current was measured by monitoring the voltage drop across a 100-t2 resistor in series with the anode (an alligator clip on back muscle). The T3 microelectrode was positioned at a site below the dorsolateral sulcus that required low stimulus current (<15 gA) to elicit a short-latency (<20 ms) excitatory response in the ipsilateral inferior cardiac nerve. Histological analysis 1.11 revealed that these sites were in the IML. The medullary neurons studied were antidromically activated by stimuli (threshold current, 119 _+ 22 gA; mean + S.E.M.) applied to this region. Whereas the stimulating microelectrode in T 3 was stationary, the one in C3-C 4 was moved to search for sites from which these neurons could be antidromically activated. Initially an electrode track was made through the gray matter, just medial to the dorsolateral sulcus. Forty-nine raphe neurons with sympathetic nerverelated activity were antidromically activated by microstimulation in the T3 IML and in the C3-C4 spinal segments. Neuronal responses were considered antidromic when the minimum interval between a spontaneous (or L-glutamate-induced) action potential and the stimulus (1.5 x threshold) that always elicited a response was close to the sum of the onset latency of the stimulus-induced action potential and the axonal refractory period (i.e., the critical delay
for antidromic activation9). An estimate of the axonal refractory period was obtained by determining the minimum interval between paired stimuli producing two action potentials 100% of the time. The data in Fig. 1 are for a : aphe neuron that was activated by stimuli applied in the C3 white matter and in the T3 IML. The response elicited by C 3 stimulation had an onset latency of 28.5 ms and followed paired stimuli separated by 5.8 ms (Fig. 1AI). The response elicited by T 3 IML stimulation had an onset latency of 50 ms and followed paired stimuli separated by 4.2 ms (Fig. 1B1). Each response passed the time-controlled collision test for antidromic activation. Specifically, a response was recorded when the Ca stimulus was applied 34.6 ms, but not 34.0 ms, after the occurrence of a spontaneous action potential (Fig. 1A2,3). A response was recorded when the T3 stimulus was applied 54.5 ms, but not 53.5 ms, after the occurrence of a spontaneous action potential (Fig. 1B2.3). The spike-triggered average of SND (Fig. 1Cl) shows that the activity of this neuron was synchronized to a rhythm in inferior cardiac SND. Although not shown, post R-wave analysis revealed that this rhythm had the period of the cardiac cycle. The spike-triggered average shows SND that preceded (left of zero lag) and followed (right of zero lag) the action potential of the raphespinal neuron. Medullary neurons were considered to have sympathetic nerve-related activity if the amplitude of the first peak to the right of zero lag in the spike-triggered average exceeded by at least a factor of 3 that of the largest deflection in the dummy pulse-triggered average (Fig. 1C2) constructed by using a random pulse train with the same frequency as the neuronal spike train. Since the firing rate of these raphespinai neurons increased during the inhibition of SND produced by baroreceptor reflex activation (Fig. 1D), they likely subserved a sympathoinhibitory function. The baroreceptors were activated by inflating the balloon-tipped end of a Fogarty embollectomy catheter (size 4 Fr; American Edwards Labs.) that was positioned in the aorta near the level ef the diaphragm. Arterial pressure was increased proximal to the point of aortic obstruction, thus activating the baroreceptors. In the example shown in Fig. 1D, blood pressure fell below control level when the balloon was deflated. As expected, neuronal firing rate decreased during this time.
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Fig. 1. Discharge characteristics o~ a raphespinal sympathoinhibitory ~euron. A, B: antidromic responses initiated by stimuli (!.5 x threshold) applied in the white matter of the third cervical (C3) segment and the third thoracic (T3) intermediolaterai nucleus, respectively. Three superimposed traces in each panel. Dots mark spontaneous and stimulus-induced action potentials; arrows mark stimuli. Panel ]: estimate of axonal refractory period with paired stimuli. Panels 2 and 3: time-controlled collision test for antidromic activation (see text for details). Horizontal calibration is 20 ms; vertical calibration is 50 pV. C: normalized midsignal spike-triggered (trace 1) and 'dummy' (trace 2) averages of inferior cardiac sympathetic nerve discharge (SND), each based on 250 trials. Bin width is 1 ms and vertical calibration is 30 pV. D: baroreceptor reflex response produced by aortic obstruction. Traces show (top to bottom): blood pressure (BP; mm Hg), time base (1 s/division), standardized pulses derived from neuronal action potentials, and SND. Vertical calibration is 100#V for SND.
Time-controlled collision of the action potentials initiated by stimuli applied in the Ca-C4 spinal segments and in the T3 IML was used to determine whether the C3-C4 stimulus excited an axonal branch or the main axon. This test is described in other reports from this laboratory TM. Specifically, we determined the maximum interval (collision interval, CI) after a C3-C4 threshold stimulus at which a T3 IML stimulus failed to elicit an antidromic response at the recording site. If the C3-C4 stimulus activates the main axon, then Cl equals the difference between the latencies of the antidromic responses elicited by stimuli applied at the two spinal levels (LI-Lc) plus the thoracic axonal refractory period (Rt): Cl = Lt -Lc + Rt. If a C3-C4 axonal branch is activated, then CI > L t - L c + Rt. The conduction time in the axonal branch (ta) is calculated by using the formula: tB =
1/2(CI -Lt + Lc -Rt). The axons of 13 of 49 raphespinal sympathoinhibitory neurons were shown to branch in the Ca-C4 spinal segments. Fig. 2 shows two examples of the collision test for the raphe neuron whose discharges were illustrated in Fig. 1. In Fig. 2A the T3 IML stimulus Was paired with a stimulus applied in the Ca gray matter. The onset latency of the antidromic response elicited by C3 stimulation was 32 ms (Fig. 2Al). The maximum interval after the C3 stimulus at which the T3 stimulus failed to elicit an antidromic response at the recording site was 30.8 ms (Fig. 2A2). Since CI > L t - L c + Rt (50 ms-32 ms + 4.2 ms), the C 3 response monitored activation of an axonal branch (t 8 = 4.3 ms). The tB averaged 9.6 + 1.9 ms for the C3-C4 branches of 13 raphespinal sympathoinhibitory neurons. In Fig. 2B the T3 IML stimulus was paired with a stimulus applied in the C3
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Fig. 2. Axonal branching in the third cervical segment (Ca) of a raphespinal sympathoinhibitory neuron that projected to the third thoracic intermediolateral nucleus (T3 IML). Same neuron as in Fig. I. Three superimposed traces in each panel. Dots mark stimulus-induced action potentials: arrows mark stimuli. A: collision of action potentials initiated by stimulation of C3 gray matter and T.~ IML. The maximum collision interval indicated that an axonal branch was activated by C3 stimulation (see text for details). B: collision of action potentials initiated by stimulation of C~ white matter and T.~ IML. The maximum collision interval indicated that the main axon was activated by C~ stimulation (see text for details), Responses to Ca stimulation alone are shown in panels A~ and B~. These responses overlap with the T~ stimulus artifacts in the other panels, Horizontal calibration is 10 ms: vertical calibration is 5014V,
white matter. The onset latency of the antidromic response elicited by this Ca stimulus was 28.5 ms. CI was 26.9 ms and ta was 0.6 ms in this case, indicating that a site near the main axon in the C 3 white matter was activated. Antidromic mapping was used to locate the main axons and likely sites of termination of C3-C~ axonal branches of 4 raphespinal sympathoinhibitory neurons. The stimulating microelectrode was moved in 200-~m steps from the dorsal to the ventral surface of the spinal cord in tracks separated by 0.4-1 mm in the mediolateral and rostrocaudal directions. The threshold current for antidromic activation, the antidromic response latency, and the depth of the electrode tip below the dorsal surface were recorded at each site of stimulation. Depth-threshold curves
were constructed from these data. Spinal stimulation sites were referenced to the dorsal surface and to a lesion (0.5 mA direct anodal current for 5 s) placed at a site requiring low stimulus-current to antidromically activate the neuron. The axonal branch was assumed to terminate near the point that required the least stimulus current (27 + 8/tA) to elicit the longest latency antidromic response. Mapping was continued until it was established that this point was surrounded by sites requiring higher currents to elicit the longest latency response. The main axon was assumed to be near the site in the white matter that required the least stimulus current (34 + 13/~A) to elicit the shortest latency antidromic response. Collision with spontaneous or L-glutamate-induced action potentials indicated that each response with a different latency monitored the antidromic activation of the same neuron. The C3-C4 branches of two raphespinal neurons whose main axons were located in the ventral funiculus appeared to terminate in the medial portion of Rexed's 12 lamina VII. Fig. 31 shows the depththreshold curves for one of these neurons. The plane of stimulation illustrated includes the presumed site of termination of the axonal branch (track C). The longest latency (36 ms) antidromic response was elicited with the least stimulus current (15/~A) from this site. The intermediate onset latency (33 ms) response likely reflected activation of the branch at a point between the main axon (response onset latency, 29 ms) in the ventral funiculus (track B) and the presumed site of termination in lamina VII. Although the depth-threshold curve for track A is not shown, currents >500 # A were needed to antidromically activate the neuron from sites in this track. The main axons of two raphespina! sympathoinhibitory neurons were in the dorsolateral funiculus, and their branches appeared to terminate in the lateral portion of Rexed's lamina VII. Fig. 311 shows the results of antidromic mapping for one of these neurons, We were unable to demonstrate cervical axonal branching of 36 raphespinal sympathoinhibitory neurons. in these experiments the C3-C4 spinal segments were extensively searched bilaterally, and the collision test was performed using the longest onset latency antidromic response elicited by C3-C 4 stimulation. When the C3-C4 and T3 IML stimuli were paired, CI was close to Lz-Lc + Rt and ta = 0.1 + 0.1
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Fig. 3. Antidromic mapping of cervical branches of two raphespinal sympathoinhibitory neurons. I: ventral funicular axon with a branch projecting to the medial portion of Rexed's lamina VII in the fourth cervical segment (C4). C4 cross-section with electrode tracks (A-D) is on the left. Calibration is I ram. Depth-threshold curves for Iracks B-D are on the right. Dashed lines show extent of ordinate relative to the cross-section. Depth below the surface relates to track B. Unfilled and filled circles on cross.section show the location of the main axon and presumed site of termination of the axonal branch, respectively. II: dorsolateral funicular axon (unfilled circle) with a branch projecting to the lateral portion of Rexed's lamina VII in C~ (filled circle). Same format as in I.
ms. Thus the C3-C 4 stimulus activated the main axon of these 36 raphe-spinal neurons. Whereas all 36 neurons were antidromicaUy activated by microstimulation of the C3--C 4 white matter, only 14 of these neurons could be activated by stimuli applied in the C3-C4 gray matter. In these 14 cases the onset latency of the antidromic response was the same whether stimulating the gray or white matter. Higher currents were needed to activate these neurons from the gray matter than from the white matter, suggesting that current spread from the gray matter to the main axon in the white matter.
The axonal conduction velocity (2.2 + 0.3 m/s) of raphespinal sympathoinhibitory neurons that emitted branches in C3-C4 was not significantly different (P > 0.05, Student's t-test) from that (3.1 + 0.5 m/s) of neurons that did not appear to branch at this level. Conduction velocity was calculated by dividing the distance between the recording microelectrode in the medulla and the stimulating microelectrode in T3 by the onset latency of the antidromic response. These values we.~e similar to those reported by Morrison and Gebber il for raphespinal sympathoinhibitory neurons that innervated the IML.
376 Eighteen VLM neurons with sympathetic nerverelated activity were antidromically activated by microstimulation in both the C3-C4 spinal segments and the T 3 IML. Although the C3-C4 gray and white matter were extensively searched, in no case did the time-controlled collision test indicate that these neurons branched in this region. That is, CI was close to L t-L¢ + R t and ts = 0.0 + 0.2 ms. These neurons had properties similar to those of VLM-spinal sympathoexcitatory neurons studied by Barman and Gebber I. One, spike-triggered averaging demonstrated that their activity was synchronized to the cardiac-related rhythm in SND. Two, their firing rate decreased in parallel to SND during baroreceptor reflex activation. Three, their axonal conduction velocity was 3.4 + 0.3 m/s. The most significant finding of this study is that the axons of 27% of raphespinal sympathoinhibitory neurons projecting to the T3 spinal segment emit branches in the Ca-C4 spinal segments. These neurons likely innervated the thoracic IML since antidromic mapping experiments by Morrison and Gebber ~ showed this to be the case for over 90% of raphespinal sympathoinhibitory neurons with similar discharge patterns and axonai conduction velocities. Although no attempt was made to identify the neu-
ronal types in the cervical cord that were innervated by raphespinal sympathoinhibitory neurons, several possibilities are worth considering. First, the cervical branches of these neurons may have excited descending propriospinal neurons that inhibited SND. Evidence supporting the exist,:nce of such propriospinal neurons was provided by Schramm and Livingston ~3 and Kirchner et al. 7. Second, these raphespinal neurons may have innervated propriospinal neurons involved in the spinal component of the somatosympathetic reflex elicited by electrical stimulaton of cervical dorsal root afferents 2. Regarding this possibility, Coote and Macleod 3 postulated that a raphespinal pathway mediates baroreceptor-induced inhibition of somatosympathetic reflexes. Third, the cervical branches of raphespinal sympathoinhibitory neurons may have synapsed on lamina VII interneutons innervating motoneuronal pools in the ventral horn 8. In this case, some raphespinal neurons might mediate baroreceptor influences on both ventral horn neurons 3 and preganglionic sympathetic neurons in the IML.
1 Barma~l, S.M. an,.i Oc0ber, O,L., Axonal projection patterns of ventrolateral medullospinal sympathoexcitatory neurons, J. Neurophysiol., 53 (1985) 1551-1566. 2 Barman, S.M. and Wurster, R.D., Interaction of descending spinal sympathetic pathways and afferent nerves, Am. J. Physiol., 234 (1978) H223-H229. 3 Coote, J.H. and Macleod, V.H., Evidence for the involveL-~, ment in the baroreceptor reflex of a descending inhibitory pathway, J. Physiol. (Lond.), 241 (1974) 477-496. 4 Freis, W. and Zieglgfinsberger, W.A., A method to discriminate axonai from cell body activity and to analyze 'silent' cells, Exp. Brain Res., 21 (1974)441-445. 5 Huisn'lan, A.M., Kuypers, H.G.J.M. and Verbugh, C.A., Differences in collateralization of the descending spinal pathways from red nucleus and other brain stem cell groups in cat and monkey, In H.G.J.M. Kuypers and G.F. Martin (Eds.), Descending Pathways to the Spinal Cord, Progress in Brain Research, Vol. 57, Elsevier, Amsterdam, 1982, pp. 185-217. 6 Huisman, A.M., Ververs, B., Cavada, C. and Kuypers, H.G.J.M., Collateralization of brainstem pathw~,ys in the spinal ventral horn in rat as demonstrated with the retrograde fluorescent double-labeling technique, Brain Research, 300 (1984) 362-367. 7 Kirchner, F., Wyszogrodski, I. and Poiosa, C., Some prop-
erties of sympathetic neuron inhibition by depressor area and intraspinal stimulation, Pfliigers Arch., 357 (1975) 349-360. 8 Kuypers, H.G.J.M., Anatomy of the descending pathways. In V.B. Brooks (Ed.), Handbook of Physiology, Section 1, The Nervous System, Vol. ll, Motor Control, Part I, American Phyg|ological Society, Bethesda, MD, 1981, pp. 597-666. 9 Lipski, J., Antidromic activation of neurones as an analytic tool in the study of the central nervous system, J. Neurosci. Methods, 4 (1981) 1-32. 10 Martin, G.F., Canaba, T., Humbertson, A.O., Jr., Laxson, L.C. and Panneton, W.M., Spinal projections from the medullary reticular formation of the North American opossum: Evidence for connectional heterogeneity, J. Comp. Neurol., 196 (1981) 663-682. 11 Morrison, S.F. and Gebber, G.L., Axonal branching patterns and funicular trajectories of raphespinal sympathoinhibitory neurons, J. Neurophysiol., 53 (1985) 759-772. 12 Rexed, B., A cytc,architectonic atlas of the spinal cord in the cat, J. Cot~ p. Neurol., 100 (1954) 297-379. 13 Schramm, L.P. and Livingston, R.H., Inhibition of renal nerve sympathetic activity by spinal stimulation in the rat, Am. J. Physiol., 252 (1987) R514-R525.
The authors thank Ms. Diane Hummel for typing this manuscript. This study was supported by NIH Grants HL-13187 and HL-33266.
371
BRE 22733
The axons of raphespinal sympathoinhibitory neurons branch in the cervical spinal cord Susan M. Barman and Gerard L. Gebber Departmems of Pharmacology and Toxicology, and of Physiology, Michigan State University, East Lansing, M148824 (U.S.A.)
(Accepted 27 October 1987) Key words: Antidromic mapping: Axonal branching: Medullospinal sympathetic neuron: Spike-triggered averaging: Sympatheticnerve discharge
This study shows that the axons of some raphespinal sympathoinhibitory neurons projecting to the third thoracic spinal segment emit branches in the third-fourth cervical spinal segments of cats. This was demonstrated by using time-contro!led collision of neuronal action potenttals initiated by stimuli applied at these spinal levels. Antidromic mapping revealed that the cervical branches of these neurons likely terminated in Rexed's lamina VII. In contrast to these raphe neurons, the axons of ventrolateral medullospinal sympathoexcitatory neurons did not emit cervical branches.
Barman and Gebber I and Morrison and Gebber II characterized the electrophysiological properties of cat medullary raphe and rostral ventrolateral medullary (VLM) neurons whose axons innervate the intermediolateral nucleus (IML) of the thoracic spinal cord (as demonstrated with antidromic mapping). Spike-triggered averaging and post R-wave analysis revealed that their activity is synchronized to the card~ac-related rhythm in sympathetic nerve discharge ($ND). The raphespinal neurons likely subserve a sympathoinhibitory function since their firing rate increases during baroreceptor reflex activation. In contrast, the VLM spinal neurons likely are sympathoexcitatory since their activity is decreased during baroreceptor reflex activation. The current investigation tested the hypothesis that the axons of such rapheand VLM-spinal neurons emit branches in the cervical spinal cord. This possibility was considered since double-labeling studies 5"~'!° show that some raphe and VLM neurons that project to the thoracic or lumbar spinal cord also innervate the cervical gray matter. These studie,~ did not assess whether these neurons project to different nuclei at the two spinal lev-
els. Such would be the case if a neuron that innervates the thoracic IML branched at cervical levels, since there is no IML in these spinal segments. Twenty-three cats (2.0-4.0 kg) were anesthetized by an i.p. injection of sodium diallyibarbiturate (60 mg/kg) and urethan (240 mg/kg), paralyzed with gallamine triethiodide (4 mg/kg, i.v., initial dose), placed in a stereotaxic apparatus and spinal investigation unit, and artificially respired. End-tidal CO2 was kept at 4 + 0.5% and rectal temperature was kept at 37 + 1 °C. Standard procedures were used to record brachial arterial pressure. As detailed in earlier reports from this laboratory L~], multibarrel glass micropipettes were used to record extracellularly the action potentials of single medullary neurons and to iontophorese 1 M L-glutamate at pH 8. Recordings were made from the somadendritic region of raphe and VLM neurons since their firing rate was increased by iontophoresing Lglutamate (ejection c u r r e n t s , - 5 to -30 hA), an amino acid that selectively depolarizes neuronal cell bodies 4. Raphespinal sympathoinhibitory neurons were identified on the midline, 2-5 mm rostral to the
Correspondence: S.M. Barman, Department of Pharmacology and Toxicology, Michigan State University, East Lansing, M! 48824. U.S.A.
0006-8993/88/$03.50© 1988Elsevier Science Publishers B.V. (Biomedical Division)
372 obex, and 3-5.5 mm below the dorsal surface. VLMspinal sympathoexcitatory neurons were identified 4-6 mm rostral to the obex, 3.5-4.0 mm lateral to the midline, and 3.5-6 mm below the dorsal surface. These recording sites were similar to those described in other reports from this laboratory TM and were identified by using histological procedures detailed in these reports. Left postganglionic inferior cardiac SND was recorded monophasically with a platinum bipolar electrode by using a capacity-coupled preamplifier (bandpass of 1-1,000 Hz). As previously described TM spike-triggered averaging (RC Electronics Computerscope) was used to identify single medullary neurons whose activity was synchronized to SND. A Grass $8800 digital stimulator and Grass PSIU-6 constant-current units were used to deliver cathodal square-wave pulses (0.5 ms duration) through tungsten microelectrodes (tip impedance, 10-30 k•) positioned in the third-fourth cervical (C3-C4) and third thoracic (T3) spinal segments (exposed by laminectomy). Stimulus current was measured by monitoring the voltage drop across a 100-t2 resistor in series with the anode (an alligator clip on back muscle). The T3 microelectrode was positioned at a site below the dorsolateral sulcus that required low stimulus current (<15 gA) to elicit a short-latency (<20 ms) excitatory response in the ipsilateral inferior cardiac nerve. Histological analysis 1.11 revealed that these sites were in the IML. The medullary neurons studied were antidromically activated by stimuli (threshold current, 119 _+ 22 gA; mean + S.E.M.) applied to this region. Whereas the stimulating microelectrode in T 3 was stationary, the one in C3-C 4 was moved to search for sites from which these neurons could be antidromically activated. Initially an electrode track was made through the gray matter, just medial to the dorsolateral sulcus. Forty-nine raphe neurons with sympathetic nerverelated activity were antidromically activated by microstimulation in the T3 IML and in the C3-C4 spinal segments. Neuronal responses were considered antidromic when the minimum interval between a spontaneous (or L-glutamate-induced) action potential and the stimulus (1.5 x threshold) that always elicited a response was close to the sum of the onset latency of the stimulus-induced action potential and the axonal refractory period (i.e., the critical delay
for antidromic activation9). An estimate of the axonal refractory period was obtained by determining the minimum interval between paired stimuli producing two action potentials 100% of the time. The data in Fig. 1 are for a : aphe neuron that was activated by stimuli applied in the C3 white matter and in the T3 IML. The response elicited by C 3 stimulation had an onset latency of 28.5 ms and followed paired stimuli separated by 5.8 ms (Fig. 1AI). The response elicited by T 3 IML stimulation had an onset latency of 50 ms and followed paired stimuli separated by 4.2 ms (Fig. 1B1). Each response passed the time-controlled collision test for antidromic activation. Specifically, a response was recorded when the Ca stimulus was applied 34.6 ms, but not 34.0 ms, after the occurrence of a spontaneous action potential (Fig. 1A2,3). A response was recorded when the T3 stimulus was applied 54.5 ms, but not 53.5 ms, after the occurrence of a spontaneous action potential (Fig. 1B2.3). The spike-triggered average of SND (Fig. 1Cl) shows that the activity of this neuron was synchronized to a rhythm in inferior cardiac SND. Although not shown, post R-wave analysis revealed that this rhythm had the period of the cardiac cycle. The spike-triggered average shows SND that preceded (left of zero lag) and followed (right of zero lag) the action potential of the raphespinal neuron. Medullary neurons were considered to have sympathetic nerve-related activity if the amplitude of the first peak to the right of zero lag in the spike-triggered average exceeded by at least a factor of 3 that of the largest deflection in the dummy pulse-triggered average (Fig. 1C2) constructed by using a random pulse train with the same frequency as the neuronal spike train. Since the firing rate of these raphespinai neurons increased during the inhibition of SND produced by baroreceptor reflex activation (Fig. 1D), they likely subserved a sympathoinhibitory function. The baroreceptors were activated by inflating the balloon-tipped end of a Fogarty embollectomy catheter (size 4 Fr; American Edwards Labs.) that was positioned in the aorta near the level ef the diaphragm. Arterial pressure was increased proximal to the point of aortic obstruction, thus activating the baroreceptors. In the example shown in Fig. 1D, blood pressure fell below control level when the balloon was deflated. As expected, neuronal firing rate decreased during this time.
373
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Fig. 1. Discharge characteristics o~ a raphespinal sympathoinhibitory ~euron. A, B: antidromic responses initiated by stimuli (!.5 x threshold) applied in the white matter of the third cervical (C3) segment and the third thoracic (T3) intermediolaterai nucleus, respectively. Three superimposed traces in each panel. Dots mark spontaneous and stimulus-induced action potentials; arrows mark stimuli. Panel ]: estimate of axonal refractory period with paired stimuli. Panels 2 and 3: time-controlled collision test for antidromic activation (see text for details). Horizontal calibration is 20 ms; vertical calibration is 50 pV. C: normalized midsignal spike-triggered (trace 1) and 'dummy' (trace 2) averages of inferior cardiac sympathetic nerve discharge (SND), each based on 250 trials. Bin width is 1 ms and vertical calibration is 30 pV. D: baroreceptor reflex response produced by aortic obstruction. Traces show (top to bottom): blood pressure (BP; mm Hg), time base (1 s/division), standardized pulses derived from neuronal action potentials, and SND. Vertical calibration is 100#V for SND.
Time-controlled collision of the action potentials initiated by stimuli applied in the Ca-C4 spinal segments and in the T3 IML was used to determine whether the C3-C4 stimulus excited an axonal branch or the main axon. This test is described in other reports from this laboratory TM. Specifically, we determined the maximum interval (collision interval, CI) after a C3-C4 threshold stimulus at which a T3 IML stimulus failed to elicit an antidromic response at the recording site. If the C3-C4 stimulus activates the main axon, then Cl equals the difference between the latencies of the antidromic responses elicited by stimuli applied at the two spinal levels (LI-Lc) plus the thoracic axonal refractory period (Rt): Cl = Lt -Lc + Rt. If a C3-C4 axonal branch is activated, then CI > L t - L c + Rt. The conduction time in the axonal branch (ta) is calculated by using the formula: tB =
1/2(CI -Lt + Lc -Rt). The axons of 13 of 49 raphespinal sympathoinhibitory neurons were shown to branch in the Ca-C4 spinal segments. Fig. 2 shows two examples of the collision test for the raphe neuron whose discharges were illustrated in Fig. 1. In Fig. 2A the T3 IML stimulus Was paired with a stimulus applied in the Ca gray matter. The onset latency of the antidromic response elicited by C3 stimulation was 32 ms (Fig. 2Al). The maximum interval after the C3 stimulus at which the T3 stimulus failed to elicit an antidromic response at the recording site was 30.8 ms (Fig. 2A2). Since CI > L t - L c + Rt (50 ms-32 ms + 4.2 ms), the C 3 response monitored activation of an axonal branch (t 8 = 4.3 ms). The tB averaged 9.6 + 1.9 ms for the C3-C4 branches of 13 raphespinal sympathoinhibitory neurons. In Fig. 2B the T3 IML stimulus was paired with a stimulus applied in the C3
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Fig. 2. Axonal branching in the third cervical segment (Ca) of a raphespinal sympathoinhibitory neuron that projected to the third thoracic intermediolateral nucleus (T3 IML). Same neuron as in Fig. I. Three superimposed traces in each panel. Dots mark stimulus-induced action potentials: arrows mark stimuli. A: collision of action potentials initiated by stimulation of C3 gray matter and T.~ IML. The maximum collision interval indicated that an axonal branch was activated by C3 stimulation (see text for details). B: collision of action potentials initiated by stimulation of C~ white matter and T.~ IML. The maximum collision interval indicated that the main axon was activated by C~ stimulation (see text for details), Responses to Ca stimulation alone are shown in panels A~ and B~. These responses overlap with the T~ stimulus artifacts in the other panels, Horizontal calibration is 10 ms: vertical calibration is 5014V,
white matter. The onset latency of the antidromic response elicited by this Ca stimulus was 28.5 ms. CI was 26.9 ms and ta was 0.6 ms in this case, indicating that a site near the main axon in the C 3 white matter was activated. Antidromic mapping was used to locate the main axons and likely sites of termination of C3-C~ axonal branches of 4 raphespinal sympathoinhibitory neurons. The stimulating microelectrode was moved in 200-~m steps from the dorsal to the ventral surface of the spinal cord in tracks separated by 0.4-1 mm in the mediolateral and rostrocaudal directions. The threshold current for antidromic activation, the antidromic response latency, and the depth of the electrode tip below the dorsal surface were recorded at each site of stimulation. Depth-threshold curves
were constructed from these data. Spinal stimulation sites were referenced to the dorsal surface and to a lesion (0.5 mA direct anodal current for 5 s) placed at a site requiring low stimulus-current to antidromically activate the neuron. The axonal branch was assumed to terminate near the point that required the least stimulus current (27 + 8/tA) to elicit the longest latency antidromic response. Mapping was continued until it was established that this point was surrounded by sites requiring higher currents to elicit the longest latency response. The main axon was assumed to be near the site in the white matter that required the least stimulus current (34 + 13/~A) to elicit the shortest latency antidromic response. Collision with spontaneous or L-glutamate-induced action potentials indicated that each response with a different latency monitored the antidromic activation of the same neuron. The C3-C4 branches of two raphespinal neurons whose main axons were located in the ventral funiculus appeared to terminate in the medial portion of Rexed's 12 lamina VII. Fig. 31 shows the depththreshold curves for one of these neurons. The plane of stimulation illustrated includes the presumed site of termination of the axonal branch (track C). The longest latency (36 ms) antidromic response was elicited with the least stimulus current (15/~A) from this site. The intermediate onset latency (33 ms) response likely reflected activation of the branch at a point between the main axon (response onset latency, 29 ms) in the ventral funiculus (track B) and the presumed site of termination in lamina VII. Although the depth-threshold curve for track A is not shown, currents >500 # A were needed to antidromically activate the neuron from sites in this track. The main axons of two raphespina! sympathoinhibitory neurons were in the dorsolateral funiculus, and their branches appeared to terminate in the lateral portion of Rexed's lamina VII. Fig. 311 shows the results of antidromic mapping for one of these neurons, We were unable to demonstrate cervical axonal branching of 36 raphespinal sympathoinhibitory neurons. in these experiments the C3-C4 spinal segments were extensively searched bilaterally, and the collision test was performed using the longest onset latency antidromic response elicited by C3-C 4 stimulation. When the C3-C4 and T3 IML stimuli were paired, CI was close to Lz-Lc + Rt and ta = 0.1 + 0.1
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Fig. 3. Antidromic mapping of cervical branches of two raphespinal sympathoinhibitory neurons. I: ventral funicular axon with a branch projecting to the medial portion of Rexed's lamina VII in the fourth cervical segment (C4). C4 cross-section with electrode tracks (A-D) is on the left. Calibration is I ram. Depth-threshold curves for Iracks B-D are on the right. Dashed lines show extent of ordinate relative to the cross-section. Depth below the surface relates to track B. Unfilled and filled circles on cross.section show the location of the main axon and presumed site of termination of the axonal branch, respectively. II: dorsolateral funicular axon (unfilled circle) with a branch projecting to the lateral portion of Rexed's lamina VII in C~ (filled circle). Same format as in I.
ms. Thus the C3-C 4 stimulus activated the main axon of these 36 raphe-spinal neurons. Whereas all 36 neurons were antidromicaUy activated by microstimulation of the C3--C 4 white matter, only 14 of these neurons could be activated by stimuli applied in the C3-C4 gray matter. In these 14 cases the onset latency of the antidromic response was the same whether stimulating the gray or white matter. Higher currents were needed to activate these neurons from the gray matter than from the white matter, suggesting that current spread from the gray matter to the main axon in the white matter.
The axonal conduction velocity (2.2 + 0.3 m/s) of raphespinal sympathoinhibitory neurons that emitted branches in C3-C4 was not significantly different (P > 0.05, Student's t-test) from that (3.1 + 0.5 m/s) of neurons that did not appear to branch at this level. Conduction velocity was calculated by dividing the distance between the recording microelectrode in the medulla and the stimulating microelectrode in T3 by the onset latency of the antidromic response. These values we.~e similar to those reported by Morrison and Gebber il for raphespinal sympathoinhibitory neurons that innervated the IML.
376 Eighteen VLM neurons with sympathetic nerverelated activity were antidromically activated by microstimulation in both the C3-C4 spinal segments and the T 3 IML. Although the C3-C4 gray and white matter were extensively searched, in no case did the time-controlled collision test indicate that these neurons branched in this region. That is, CI was close to L t-L¢ + R t and ts = 0.0 + 0.2 ms. These neurons had properties similar to those of VLM-spinal sympathoexcitatory neurons studied by Barman and Gebber I. One, spike-triggered averaging demonstrated that their activity was synchronized to the cardiac-related rhythm in SND. Two, their firing rate decreased in parallel to SND during baroreceptor reflex activation. Three, their axonal conduction velocity was 3.4 + 0.3 m/s. The most significant finding of this study is that the axons of 27% of raphespinal sympathoinhibitory neurons projecting to the T3 spinal segment emit branches in the Ca-C4 spinal segments. These neurons likely innervated the thoracic IML since antidromic mapping experiments by Morrison and Gebber ~ showed this to be the case for over 90% of raphespinal sympathoinhibitory neurons with similar discharge patterns and axonai conduction velocities. Although no attempt was made to identify the neu-
ronal types in the cervical cord that were innervated by raphespinal sympathoinhibitory neurons, several possibilities are worth considering. First, the cervical branches of these neurons may have excited descending propriospinal neurons that inhibited SND. Evidence supporting the exist,:nce of such propriospinal neurons was provided by Schramm and Livingston ~3 and Kirchner et al. 7. Second, these raphespinal neurons may have innervated propriospinal neurons involved in the spinal component of the somatosympathetic reflex elicited by electrical stimulaton of cervical dorsal root afferents 2. Regarding this possibility, Coote and Macleod 3 postulated that a raphespinal pathway mediates baroreceptor-induced inhibition of somatosympathetic reflexes. Third, the cervical branches of raphespinal sympathoinhibitory neurons may have synapsed on lamina VII interneutons innervating motoneuronal pools in the ventral horn 8. In this case, some raphespinal neurons might mediate baroreceptor influences on both ventral horn neurons 3 and preganglionic sympathetic neurons in the IML.
1 Barma~l, S.M. an,.i Oc0ber, O,L., Axonal projection patterns of ventrolateral medullospinal sympathoexcitatory neurons, J. Neurophysiol., 53 (1985) 1551-1566. 2 Barman, S.M. and Wurster, R.D., Interaction of descending spinal sympathetic pathways and afferent nerves, Am. J. Physiol., 234 (1978) H223-H229. 3 Coote, J.H. and Macleod, V.H., Evidence for the involveL-~, ment in the baroreceptor reflex of a descending inhibitory pathway, J. Physiol. (Lond.), 241 (1974) 477-496. 4 Freis, W. and Zieglgfinsberger, W.A., A method to discriminate axonai from cell body activity and to analyze 'silent' cells, Exp. Brain Res., 21 (1974)441-445. 5 Huisn'lan, A.M., Kuypers, H.G.J.M. and Verbugh, C.A., Differences in collateralization of the descending spinal pathways from red nucleus and other brain stem cell groups in cat and monkey, In H.G.J.M. Kuypers and G.F. Martin (Eds.), Descending Pathways to the Spinal Cord, Progress in Brain Research, Vol. 57, Elsevier, Amsterdam, 1982, pp. 185-217. 6 Huisman, A.M., Ververs, B., Cavada, C. and Kuypers, H.G.J.M., Collateralization of brainstem pathw~,ys in the spinal ventral horn in rat as demonstrated with the retrograde fluorescent double-labeling technique, Brain Research, 300 (1984) 362-367. 7 Kirchner, F., Wyszogrodski, I. and Poiosa, C., Some prop-
erties of sympathetic neuron inhibition by depressor area and intraspinal stimulation, Pfliigers Arch., 357 (1975) 349-360. 8 Kuypers, H.G.J.M., Anatomy of the descending pathways. In V.B. Brooks (Ed.), Handbook of Physiology, Section 1, The Nervous System, Vol. ll, Motor Control, Part I, American Phyg|ological Society, Bethesda, MD, 1981, pp. 597-666. 9 Lipski, J., Antidromic activation of neurones as an analytic tool in the study of the central nervous system, J. Neurosci. Methods, 4 (1981) 1-32. 10 Martin, G.F., Canaba, T., Humbertson, A.O., Jr., Laxson, L.C. and Panneton, W.M., Spinal projections from the medullary reticular formation of the North American opossum: Evidence for connectional heterogeneity, J. Comp. Neurol., 196 (1981) 663-682. 11 Morrison, S.F. and Gebber, G.L., Axonal branching patterns and funicular trajectories of raphespinal sympathoinhibitory neurons, J. Neurophysiol., 53 (1985) 759-772. 12 Rexed, B., A cytc,architectonic atlas of the spinal cord in the cat, J. Cot~ p. Neurol., 100 (1954) 297-379. 13 Schramm, L.P. and Livingston, R.H., Inhibition of renal nerve sympathetic activity by spinal stimulation in the rat, Am. J. Physiol., 252 (1987) R514-R525.
The authors thank Ms. Diane Hummel for typing this manuscript. This study was supported by NIH Grants HL-13187 and HL-33266.