Journal of the Autonomic NerL'ous System, 40 (1992) 255-260
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1838/92/$05.00 JANS 01320
Responses of single units in the midline medulla to stimulation of the rostral ventrolateral medulla Kathryn A. King and Robert B. McCall Cardiot:ascular Diseases Research, The Upjohn Company, Kalamazoo, Michigan, USA (Received 2 April 1992) (Revision received 9 June 1992) (Accepted 6 July 1992)
Key words: Sympathetic nerve activity; Rostral ventrolateral medulla; Raphe; Serotonin; Cat Abstract Single units in the midline medulla were characterized as sympathoexcitatory (SE), sympathoinhibitory (SI) or serotonin (5-HT) neurons. Post-stimulatory changes in the firing patterns of sympathoexcitatory, sympathoinhibitory and 5-HT units were observed during single shock stimulation of the pressor area of the rostral ventrolateral medulla (RVLM). Excitation, inhibition, or no change in cell firing patterns were observed for each cell type, but each cell showed only one type of response to stimulation. No midline neurons were antidromically activated by stimulation of the rostral ventrolateral medulla. These results are discussed in relation to neuronal pathways between the RVLM and the midline medulla involved in the generation of sympathetic activity.
The cell bodies of sympathetic preganglionic neurons are located in the intermediolateral cell column (IML) of the spinal cord. This area receives supraspinal input from several sources, including the medulla. The rostral ventrolateral medulla (RVLM), which is believed to be the brainstem area responsible for maintaining basal vasomotor tone (see review, ), provides sympathoexcitatory information to the IML. Electrical stimulation of the RVLM results in excitation of sympathetic preganglionic neurons which is characterized by biphasic onset latency of excitation (i.e. early and late) in the rat . The early latency response is mediated via a direct medul-
Correspondence to: R.B. McCall, The Upjohn Company, 7243209-3, Kalamazoo, MI 49001, USA.
lospinal pathway while the latter response is thought to result from the antidromic activation of midline neurons which project to both the RVLM and the IML (Morrison, personal communication). Neurons in the midline medulla also project to the IML and, in addition, provide a tonic inhibition to sympathoexcitatory neurons in the RVLM [5,7,9,13]. These electrophysiological studies suggest that areas of the medulla which project to the IML may be reciprocally innervated. Recent anatomical studies have identified cells within the RVLM which project to the nucleus raphe pallidus and nucleus raphe obscurus of the midline medulla. Axons of RVLM neurons were identified in close proximity to serotonin (5-HT) containing cell bodies in these nuclei . Projections from the nucleus raphe obscurus to the
nucleus paragigantocellularis lateralis of the RVLM have also been identified . The studies described above suggest that neurons in both the RVLM and the midline medulla project to the I M L and that reciprocal connections may exist between these two groups of neurons. Three types of midline medullary neurons have been associated with central autonomic function. The discharge of two of these neuronal types are temporally related to sympathetic nerve activity [12,13]. Sympathetic-related neurons have been identified whose discharges increase during baroreceptor reflex activation. These neurons project to the I M L of the spinal cord and are thought to subserve a sympathoinhibitory function (i.e. sympathoinhibitory neurons). The discharge of other midline sympathetic neurons are inhibited during baroreceptor reflex activation and are thought to subserve a sympathoexcitatory function (i.e. sympathoexcitatory neurons). Finally, 5-HT neurons project to the I M L cell column to provide a tonic excitatory input to sympathetic preganglionic neurons . In the present study, we attempted to identify pathways between the R V L M and midline autonomic neurons by determining the effects of electrical stimulation of the pressor area of the R V L M on midline SE, S1 and putatively identified 5-HT cells. Adult cats (2,5-4.0 kg) were anesthetized by intramuscular injection of ketamine (11 mg/kg). This was followed by intravenous injection of chloralose (80 m g / k g ) or by intraperitoneal injection of a mixture of dial barbiturate (60 m g / k g ) , urethane (240 m g / k g ) and monoethylurea (240 mg/kg). These doses of anesthetic were sufficient to maintain an appropriate level of anesthesia for the duration of the experiments as assessed by the corneal reflex and hind-limb abduction tests. Each animal was placed in a stereotaxic apparatus and a femoral artery and vein were cannulated for recording blood pressure and for peripheral drug administration, respectively. A Fogarty occluding catheter was inserted into the descending aorta via the opposite femoral artery. Baroreceptor reflex activation was accomplished by inflation of the balloon portion of the catheter. A tracheal cannula was inserted, and, following surgery, the animals were artificially ventilated
and paralyzed with gallamine (4 m g / k g , i.v.). Rectal temperature was maintained between 37 ° and 38°C using a heating pad. Sympathetic nerve discharge (SND) was recorded from the central end of the sectioned left inferior cardiac nerve. The nerve was located distal to its exit from the steilate ganglion and was isolated outside the pleural cavity after removal of the vertebral portion of the first rib. Nerve activity was recorded under mineral oil using a bipolar platinum electrode with capacity coupled preamplification at low and high frequency half-amplitude responses of l and 1000 Hz, respectively. Portions of the occipital bone and cerebellum were removed to expose the dorsal aspect of the medulla. A stimulating electrode was placed in the RVLM as has been described previously [1,2,8]. Using the obex as a surface landmark, the electrode was inserted into the left medulla in the area containing sympathoexcitatory neurons [2,9] (i.e. 4.0 to 6.0 mm rostral to obex, 3.5 to 4.0 mm lateral to midline, and 4.0 to 6.0 m m from the dorsal surface of the medulla). The stimulating electrode was always located ipsilateral to the recorded inferior cardiac nerve. The stimulation site was confirmed to be in the pressor area by high frequency stimulation (100 g A , 0.5 ms, 20-50 Hz). Single shock stimulation of the RVLM elicited an evoked potential in the inferior cardiac nerve with a mean onset latency of 37.6 _+ 1.2 ms. This value is similar to that reported in the literature for the onset of activation of inferior cardiac sympathetic activity following RVLM stimulation and was an electrophysiological demonstration of an accurate electrode placement in the RVLM . The placement of the stimulating electrode was also confirmed histologically. Following each experiment, the brainstem was removed, fixed in 10% formalin, sliced in a cryostat (20 ~ m ) and stained with cresyl violet. Sections were examined under the light microscope in order to locate the electrode track. Single-unit recordings were made from the midline medulla, 2.0 to 5.(I mm rostral to obex and 2.0 to 5.0 mm from the dorsal surface of the medulla. Three cell types were identified: (1) putative serotonin cells; (2) sympathoexcitatory
257 (SE) units; and (3) sympathoinhibitory (SI) units. Cells were characterized using methods previously described [10,12,13]. Briefly, mid-signal spike-triggered averages of sympathetic activity were used to assess the relation of unitary discharge to sympathetic activity. Cells whose discharges were related to sympathetic activity were classified as sympathoexcitatory if inhibited during baroreceptor activation or sympathoinhibitory if excited during baroreceptor activation. Identification of 5-HT neurons was based on several criteria previously described . Putative 5-HT cells were initially identified by the regular manner in which they spontaneously fired. The regular nature of unit discharge was confirmed by spike-triggered post event time histogram analysis. In addition, putative 5-HT cells were tested for their response to baroreceptor reflex activation and for their relationship to sympathetic nerve activity. No putative 5 - H T cell fired with a temporal relation related to sympathetic discharge or responded to baroreceptor activation. Finally, the sensitivity of some 5 - H T neurons to the inhibitory effects of intravenous 8-OH D P A T were determined. In this regard, intravenous 8 - O H D P A T inhibits the firing of 5-HT neurons by acting on 5-HT~A autoreceptors . Following characterization of each midline unit, the R V L M was stimulated using single pulses at a frequency of 0.5 Hz (25-100 /xA). A threshold current required to alter the firing of neurons was determined. Cells were then tested at multiples of this threshold of current intensity (2-3 times threshold). In addition, the effects of three shock trains of stimuli were determined for most units. Blood pressure, SND, unit activity and the stimulus trigger were recorded on magnetic tape. Unit activity was subjected to window discrimination, the output of which was used to build spike-triggered averages of SND and poststimulatory time histograms (200 sweeps). Excitation was defined as a peak in the histogram greater than two times baseline (Fig. 1A), and inhibition as a decrease in firing to less than 70% of baseline (Fig. 1B). Ceils which were considered not affected by stimulation of the R V L M did not change their firing pattern from baseline. Figure 1C illustrates the response of a unit whose dis-
B 11E o
0 100msec Fig. 1. Post-stimulatory time histograms illustrating excitation (A), inhibition (B) and no change (C) in midline medullary cell firing following single-pulse stimulation of the RVLM. Each histogram illustrates the effect of stimulation (64 trials) of the RVLM on a different midline unit.
charges were not affected by stimulation of the RVLM. For each unit, the spontaneous firing rate was recorded. During stimulation of the RVLM, the onset of excitation or inhibition and the duration of the effect was determined, and, for excited cells, the time from the stimulus to the peak excitatory effect was also measured. A total of 36 units were recorded from 21 animals. O f the units recorded, 12 were identified as putative 5 - H T units, 17 as SI, and seven as SE. Stimulation of the R V L M produced either excitation, inhibition, or did not affect the unit firing pattern. Each individual cell responded to stimulation with only one of these patterns, but each type of response was observed for each cell type (SI, SE, putative 5-HT). Figure 2 illustrates the distribution of each type of response among all cells recorded. Increasing the intensity of the stimulus of single shocks increased the intensity but failed to affect the character of the response to stimulation. Similarly, the responses to trainstimulation were typically more obvious or pronounced than the responses to single pulses, but followed the same pattern. In no instance did we
observe midline neurons antidromically activated by stimulation of the rostral ventrolateral medulla. Table I summarizes the mean times to the onset and peak of the stimulation-induced effect, and the duration of the effect (excitation or inhibition) for each cell type following single-pulse stimulation at three times the threshold intensity. A consistent pattern of responses of midline units to stimulation of the R V L M was not observed. There were no significant differences in the onset or duration of excitatory or inhibitory responses to stimulation within or between cell types. Units excited by the stimulus responded with a single discharge to shocks applied to the RVLM. Unit type or spontaneous firing frequency did not allow prediction of the response to stimulation, nor did the location of either the recording or stimulating electrodes. In addition, stimulation of a pressor site which resulted in a 25 to 30 m m H g increase in mean blood pressure did not necessarily produce more obvious excitation or inhibition of unit firing than a stimulation site which produced smaller increases in blood pressure at the same stimulus intensity. Similarly, in four of the ten animals in which more than one unit was recorded, both excitatory and inhibitory responses were observed in units of the same type (i.e. SI units).
10 987 6 5 4 3 2 1 Excited
Inhibited No Effect Response to Stimulation SE
Fig. 2. Number of sympathoexcitatory (SE), sympathoinhibitory (SI) and putative serotonin (5-HT) cells in the midline medulla in which stimulation of the rostral ventrolateral medulla produced excitation, inhibition, or no effect.
Examination of cresyl violet stained sections of the medulla indicated that the stimulating electrode was consistently placed in the rostral ventrolateral medulla (Fig. 3). The current study was designed to determine the effects of electrical stimulation of the R V L M on the firing of SE, SI and putative 5-HT neurons located in the midline medulla. Anatomic studies indicate that neurons within the R V L M project
Responses of midline medullary cells to stimulation of the rostraI centrolateral medulla Frequency spike/s
5-HT cells excited inhibited no effect
1.2 + 0.4 2.3 _+ 0.5 4.3 + 1.3
36.9 + 1/I.4 36.7 ± 17.6
53.2 + 13.3 -
34.4 + 5.3 102.6 + 36.8
SE cells excited inhibited no effect
1.5 1.3 2.3 + 0.2
SI cells excited inhibited no effect
1.3 + 0.2 1.3 ± 0.2 0.5
16,2 + 32.9 + -
34.1 _+ 8.4
29.3 ± 6.8 153.8 ± 30.1 -
All values are mean +_ S.E., except where n is < 3 (see Fig 1). Frequency: spontaneous firing frequency; onset: latency to onset of effect; peak: latency to peak effect.
259 to the caudal raphe nuclei of the midline medulla . We found that stimulation of the pressor area of the R V L M produced variable effects on the firing of midline SE, SI and putative 5-HT neurons. Indeed, examples of excitation, inhibition or no effect were found for each of the three cell types following R V L M stimulation. The constellation of responses suggests a heterogenous population of pathways from the R V L M to the midline medulla. Part of the variability in response observed in this study likely results from the fact that we electrically stimulated cell bodies and fibers of passage in the RVLM. Stimulation of cell bodies alone with glutamate microinjection into the R V L M was not undertaken since the pressor response elicited by the microinjection would affect the firing of midline neurons independent of any direct activation of R V L M neurons projecting to the midline. In spite of this limitation, we can conclude that neuronal elements in the R V L M can affect the firing of sympathetic and putative 5-HT neurons in the midline medulla. These pathways may be involved in the generation and synchronization of sympathetic activity. Electrical stimulation of the R V L M results in the biphasic excitation of sympathetic preganglionic neurons in the I M L cell column of the rat spinal cord. Morrison  recently demonstrated
that the early onset excitation results from activation of neurons in the R V L M which project directly to the IML. The late onset of excitation involved antidromic activation of medullospinal neurons in the midline medulla of the rat (Morrison, personal communication). In the present study we could not antidromically activate midline neurons following stimulation of the R V L M in the cat. Interestingly, a biphasic excitation of sympathetic preganglionic n e u r o n s following R V L M stimulation has never been reported in the cat. Morrison's data , in combination with our own, suggest that this may be because a direct monosynaptic pathway from the midline to the rostral ventrolateral medulla is lacking in the cat. However, Lovick, using retrograde tracing with horseradish peroxidase, identified afferent inputs to the nucleus paragigantocellularis lateralis of the R V L M from the raphe obscurus . The apparent discrepancy in our findings and the anatomic data may indicate that the direct pathway identified anatomically is not made up of sympathetically-related or serotonin neurons, but of cells that serve another function. Alternatively, the apparent lack of direct projections may have been due to sampling bias based on the fact that we recorded only spontaneously firing midline neurons. Finally, if only a relatively few midline neurons project directly to the RVLM, then our
Fig. 3. Representative drawing of a section of the cat medulla illustrating the location of the stimulating electrodes in the rostral ventrolateral medulla as determined stereotaxically. Closed circles represent sites where high frequency stimulation elicited pressor responses of > 25 mmHG. Open circle represents a site where high frequency stimulation altered blood pressure < 10 mmHg. Drawing adapted from .
small sampling size might have been insufficient to identify these neurons. In any case, our data do not support a direct autonomic pathway from the midline to the rostral ventrolateral medulla. In a previous study we found that neuronal elements in the midline medulla provide a tonic inhibitory input to sympathoexcitatory neurons in the rostral ventrolateral medulla . Stimulation of the midline medulla produced a GABA-mediated inhibition of sympathoexcitatory neurons in the RVLM. This inhibition resulted from activation of presumed GABAergic interneurons which were found in close apposition to the sympathoexcitatory neurons. In this previous study we did not determine if the pathway from the midline to the GABAergic interneurons in the RVLM was mono- or poly-synaptic. Our inability to antidromically activate midline neurons from the RVLM suggests that the pathway from the midline to G A B A interneurons in the RVLM is polysynaptic involving a synapse outside the RVLM.
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