Download Different adrenal sympathetic preganglionic

Am J Physiol Regulatory Integrative Comp Physiol
279: R1763–R1775, 2000.
Different adrenal sympathetic preganglionic neurons
regulate epinephrine and norepinephrine secretion
SHAUN F. MORRISON AND WEI-HUA CAO
Department of Physiology, Northwestern University Medical School, Chicago, Illinois 60611
Received 24 April 2000; accepted in final form 10 July 2000
have provided histochemical differentiation of the populations of adrenal medullary chromaffin cells secreting epinephrine and those secreting
norepinephrine (13, 15, 23, 25). Several lines of evidence have suggested that the two populations of adrenal chromaffin cells are regulated by distinct neural
pathways to the adrenal medulla. First, nerve terminals on epinephrine-secreting chromaffin cells are morphologically different from those on norepinephrinesecreting cells (24). More direct evidence for the
existence of distinct populations of sympathetic
preganglionic neurons (SPNs) innervating cat epinephrine- and norepinephrine-synthesizing chromaffin cells
comes from an elegant anatomic study showing that
adrenal SPNs that contain the calcium-binding protein
calretinin terminate in the vicinity of noradrenergic
chromaffin cells and are located more caudally than
adrenal SPNs, which do not contain calretinin and
whose terminals are located preferentially among adrenergic chromaffin cells (18). Whether this is also the
case in the rat remains to be determined. Second,
stimulation at different hypothalamic sites in the cat
(21, 42) or stimulation of the splanchnic nerve at different frequencies (17, 29) can evoke selective or differential secretion of adrenal epinephrine or norepinephrine, suggesting that medullary chromaffin cells
secreting epinephrine and those that secrete norepinephrine are controlled by different descending and
preganglionic pathways. Similar data are available in
the rat. Third, certain stimuli in the rat, such as the
hypoglycemia induced by insulin or mimicked by 2-deoxyglucose (2-DG), produce a response consistent with
a selective activation of epinephrine-secreting adrenal
chromaffin cells (22, 34, 43, 48, 53, 54), while, in contrast, acute cold exposure in the rat results in a preferential secretion of norepinephrine (54).
In the present study, we sought to provide electrophysiological evidence for the independent regulation
of adrenergic and noradrenergic chromaffin cells by
characterizing the physiological and stimulus-evoked
responses of adrenal SPNs. Because the chromaffin
cells of the adrenal medulla are the sole sympathetic
target tissue directly innervated by SPNs, the adrenal
medulla provides a unique opportunity to study antidromically identified SPNs controlling a known target
tissue. We reasoned that adrenal SPNs regulating adrenergic chromaffin cells could be differentiated from
those regulating noradrenergic chromaffin cells by
their excitatory responses to the glucopenia after
2-DG, which produces a selective increase in adrenal
epinephrine secretion (31, 34, 48). In addition, we determined the responses of adrenal SPNs to stimulation
in the rostral ventrolateral medulla (RVLM), a location
of adrenal sympathetic premotor neurons (47, 57), and
to acute activation of the baroreceptor reflex, a potent,
Address for reprint requests and other correspondence: S. F. Morrison, Dept. of Physiology (M211), Northwestern Univ. Medical
School, 303 E. Chicago Ave., Chicago, IL 60611 (E-mail: s-morrison2
@northwestern.edu).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
adrenal medulla; sympathetic nerve activity; hypoglycemia;
rostral ventrolateral medulla; 2-deoxyglucose; vasoconstrictor neurons; intermediolateral nucleus; blood pressure;
splanchnic nerve activity
ANATOMIC STUDIES
http://www.ajpregu.org
0363-6119/00 $5.00 Copyright © 2000 the American Physiological Society
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Morrison, Shaun F., and Wei-Hua Cao. Different adrenal sympathetic preganglionic neurons regulate epinephrine
and norepinephrine secretion. Am J Physiol Regulatory Integrative Comp Physiol 279: R1763–R1775, 2000.—Brain stimulation or activation of certain reflexes can result in differential activation of the two populations of adrenal medullary
chromaffin cells: those secreting either epinephrine or norepinephrine, suggesting that they are controlled by different
central sympathetic networks. In urethan-chloralose-anesthetized rats, we found that antidromically identified adrenal
sympathetic preganglionic neurons (SPNs) were excited by
stimulation of the rostral ventrolateral medulla (RVLM) with
either a short (mean: 29 ms) or a long (mean: 129 ms) latency.
The latter group of adrenal SPNs were remarkably insensitive to baroreceptor reflex activation but strongly activated
by the glucopenic agent 2-deoxyglucose (2-DG), indicating
their role in regulation of adrenal epinephrine release. In
contrast, adrenal SPNs activated by RVLM stimulation at a
short latency were completely inhibited by increases in arterial pressure or stimulation of the aortic depressor nerve,
were unaffected by 2-DG administration, and are presumed
to govern the discharge of adrenal norepinephrine-secreting
chromaffin cells. These findings of a functionally distinct
preganglionic innervation of epinephrine- and norepinephrine-releasing adrenal chromaffin cells provide a foundation
for identifying the different sympathetic networks underlying the differential regulation of epinephrine and norepinephrine secretion from the adrenal medulla in response to
physiological challenges and experimental stimuli.
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DIFFERENTIATION OF ADRENAL SPNS
short-term regulator of cardiac and vasoconstrictor
sympathetic outflows that has also been shown to modulate adrenal nerve activity in the rat (26, 51). Our
results provide strong support for the existence of a
unique population of adrenal SPNs controlling the secretion of epinephrine and suggest that the adrenal
SPNs regulating noradrenergic chromaffin cells behave in a manner similar to that expected of SPNs
controlling vasoconstriction.
METHODS
RESULTS
Antidromic identification of adrenal SPNs. In 81
rats, 139 neurons in the IML region of thoracic segments T8-T10 (located beneath thoracic vertebrae T7T9) responded to stimulation of the adrenal nerve at a
constant latency and followed paired adrenal nerve
stimuli separated by ⬍5 ms. The antidromic nature of
the constant latency responses of these neurons was
confirmed with the collision test. In the example in Fig.
1, adrenal SPN action potentials were evoked at a
constant onset latency (51 ms) when adrenal nerve
stimuli were delivered at 53.5 ms after a spontaneous
discharge of the SPN (Fig. 1, trace 1) but collided when
adrenal nerve stimuli were applied at 52 ms after a
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General procedures. Male Sprague-Dawley rats (300–400
g) were anesthetized with intravenous injections of urethane
(800 mg/kg) and ␣-chloralose (70 mg/kg). The trachea, femoral vein, and femoral artery were cannulated for artificial
ventilation, drug administration, and measurement of arterial pressure, respectively. Rectal temperature was maintained at 37°C with a thermostatically controlled heating
lamp and table. Animals were placed in a stereotaxic apparatus and spinal investigation unit with the incisor bar at
⫺12 mm below the interaural line and the spinal clamp on
the T10 and T11 vertebrae. An occipital craniotomy and T7-T9
laminectomy were performed, and exposed nervous tissue
was covered with a warmed mixture of mineral oil and
silicone grease. After paralysis with d-tubocurarine (0.8
mg/kg iv), animals were respired with 100% oxygen at a
minute volume of 140–180 ml. Respiratory rate or tidal
volume was adjusted to maintain expired CO2 between 3.5
and 4.5%. A bilateral pneumothorax reduced respiratory
pump-related movements of tissue near the recording electrode. After 4 h, supplemental doses of ␣-chloralose (20 mg/kg
iv) were given every 2 h.
Recording and identification of adrenal SPNs. The extracellular action potentials of adrenal SPNs were recorded
(capacity coupled, 300–3,000 Hz) with glass microelectrodes
containing a carbon filament. The indifferent electrode was
inserted in muscle adjacent to the vertebral column. The
region between 700 and 1,100 ␮m ventral to the dorsal
surface of the spinal cord and along the dorsal root entry zone
was explored for adrenal SPNs that were antidromically
identified with stimuli (20–200 ␮A, 1 ms, 0.5 Hz) applied to
the left adrenal nerve just proximal to its entrance to the
adrenal capsule. Three criteria were used to establish the
antidromic nature (35, 37) of the responses of spinal neurons
to adrenal nerve stimulation: 1) constant onset latency, 2)
high following frequency, and 3) collision with spontaneous
action potentials or those evoked orthodromically by stimulation in the RVLM. To perform time-controlled collision
tests, individual unit action potentials were selected from
ongoing activity and used to trigger an adrenal nerve stimulus at a specified delay. At the end of some experiments,
current (15 ␮A, 1 min) was passed through the carbon filament to make a small lesion marking the SPN recording site.
These were consistently located within the principal nucleus
of the intermediolateral (IML) cell column.
RVLM stimulation. A tungsten microelectrode with a
50-␮m exposed tip was used to deliver paired (6-ms interpulse interval) stimuli (50–300 ␮A, 1 ms, 0.5 Hz) to the
region of the RVLM containing spinally projecting, sympathetic premotor neurons, including those projecting monosynaptically to adrenal SPNs (47, 57). The stimulating electrode was positioned 2.6 mm rostral, 1.9 mm lateral, and 2.4
mm ventral to the calamus scriptorius. In some experiments,
the stimulating microelectrode was replaced with a micropipette (20-␮m tip diameter) containing the excitatory amino
acid L-glutamate (10 mM), from which microinjections (60 nl)
were made to determine the effect on adrenal SPN discharge
of exciting only neurons in RVLM. At the conclusion of the
experiment, the position of the stimulating electrode and/or
microinjection pipette was marked with an electrophoretic
deposit of fast green dye from a micropipette stereotaxically
positioned at the same RVLM site. After perfusion with
fixative, the brain stem was sliced (50 ␮m), and the locations
of dye marks were plotted on drawings from a rat atlas (39).
Activation of the baroreceptor reflex. To determine the
effect of activation of baroreceptor afferent nerves on the
discharge of adrenal SPNs, the left aortic depressor nerve
(ADN) was dissected in the neck and separated from the
cervical vagus nerve, and the sectioned central end was
placed on platinum hook electrodes for stimulation with
square wave pulses. The following two stimulus paradigms
were used: 1) a short burst of three pulses, 6-ms interpulse
interval, 20–100 ␮A, 1 ms duration, 0.25 Hz and 2) highfrequency train of 20 Hz for 3 s, 1 ms duration, 20–100 ␮A.
Natural stimulation of the baroreceptors was produced by
the rise in arterial pressure after an intravenous bolus injection of phenylephrine (0.1 ml, 50–100 ␮g/ml).
Adrenal SPN response to glucopenia. To determine the
response of adrenal SPNs to simulated hypoglycemia [a stimulus that produces selective release of adrenal epinephrine
(22, 43, 53, 54)], spontaneous SPN activity was recorded
before and for at least 15 min after an intravenous bolus of
2-DG (250 mg/kg). Histograms (1-s bins) of unit activity were
computed, and differences between control activity levels and
those at various intervals after the 2-DG administration
were determined.
Data analysis. Action potentials of adrenal SPNs and the
arterial pressure were digitized at 22 kHz and recorded on
VCR tape along with 5-volt pulses coincident with stimuli
applied to the adrenal or aortic nerves or to the RVLM.
Computer-aided data analysis consisted of stimulus- or arterial pulse-triggered histograms of the discharges of adrenal
SPNs. To identify poststimulus periods of increased or decreased probability of discharge of adrenal SPNs, the mean
discharge rate during the 100-ms period before stimulus
delivery was determined from peristimulus time histograms
of adrenal SPN discharge. The onsets of excitatory or inhibitory responses of adrenal SPNs were identified as the first
bin in peristimulus time histograms that was followed by at
least three bins whose values were greater than (or less than,
for inhibitory responses) the mean control discharge rate and
whose values continued to increase (or decrease, for inhibitory responses). The results are expressed as means ⫾ SE.
Significant differences between groups were determined with
the Student’s t-test.
DIFFERENTIATION OF ADRENAL SPNS
spontaneous SPN action potential (Fig. 1, trace 2). As
previously described (35, 37), the SPN axonal refractory period was determined by delivering twin pulses
to the adrenal nerve at an interval (40 ms; Fig. 1, traces
3 and 4) after a spontaneous action potential, resulting
in collision of the first antidromic spike. The SPN
recording showed that a second antidromically evoked
action potential was generated when the interpulse
interval was 2.4 ms (Fig. 1, trace 3), i.e., equal to or
greater than the axonal refractory period, but not when
the interpulse interval was 2.2 ms (Fig. 1, trace 4).
RVLM-evoked discharge of adrenal SPNs. All of the
adrenal SPNs were excited by paired stimuli applied to
the region of the RVLM containing sympathetic premotor neurons. Most of the SPNs responded to each
RVLM stimulus with a single action potential. Threshold responses in which only 10–20% of the RVLM
stimuli evoked an SPN spike typically occurred at
stimulus currents between 40 and 80 ␮A. Maximal
responses in which an SPN action potential occurred
for nearly every RVLM stimulus were attained at a
mean current of 225 ␮A. Splanchnic SPNs, which
would include those with axons in the adrenal nerve,
were previously divided into four groups on the basis of
the patterns and latencies of their responses to RVLM
stimulation (37). The antidromically identified adrenal
SPNs in the present study exhibited RVLM stimulusevoked responses characteristic of the splanchnic
SPNs in either group I (short onset latency excitation)
or group IV (early inhibition and late excitation). The
adrenal SPNs with the characteristics of splanchnic
group IV SPNs were designated Epi Adr SPNs, and
adrenal SPNs with RVLM stimulus-evoked responses
similar to those of splanchnic group I SPNs were designated NE Adr SPNs.
Epi Adr SPN responses to RVLM stimulation. In 71
(51%) adrenal SPNs, RVLM stimulation produced a
biphasic response (Fig. 2A) consisting of an early reduction in unit action potential probability followed by
a period of increased discharge probability during
which the unit could fire more than one time to a
paired RVLM stimulus. This response, characteristic of
Epi Adr SPNs, is shown in the peristimulus time histogram in Fig. 2A, top. The probability of unit discharge fell below control levels for ⬃80 ms after the
RVLM stimulation (mean control discharge rate in the
100 ms before the stimulation: 4.9 Hz; mean discharge
rate between 15 and 80 ms after the stimulation: 1.8
Hz). This was followed by a period of increased discharge probability lasting from 110 to 180 ms after the
RVLM stimulus, during which the 100 RVLM stimuli
evoked 86 spikes with a mean latency of 139 ms and a
modal latency of 150 ms. These two phases of the
RVLM stimulus-evoked response of Epi Adr SPNs are
clearly seen in the mean peristimulus time histogram
(Fig. 2A, bottom) derived from the individual response
histograms of 71 Epi Adr SPNs. The early period of
inhibition lasted for 68 ms after the stimulus, and
during this time (i.e., from the end of the stimulus
blanking at 12–68 ms) the mean discharge rate averaged 64% of control. The late period of excitation of Epi
Adr SPNs lasted for 108 ms, between 76 and 184 ms,
during which the mean response latency was 129 ⫾ 2.8
ms and the modal response latency was 136 ms.
As shown in Fig. 3, top, the antidromic latencies of
Epi Adr SPNs ranged from 6 to 60 ms (mean: 35 ⫾ 1.5
ms), corresponding to approximate conduction velocities between 0.55 and 5.5 m/s, based on an average
distance from the spinal recording site to the adrenal
nerve stimulation site of 33 ⫾ 2 mm (n ⫽ 31 rats in
which this distance was measured).
Epi Adr SPNs were excited by activation of RVLM
neurons with microinjections of glutamate. In the example in Fig. 2B, the discharge of an adrenal SPN with
an RVLM stimulus-evoked response pattern characteristic of Epi Adr SPNs (see Fig. 2A) was increased from
a basal rate of 5.5 spikes/s to a peak rate of 12 spikes/s
after an ipsilateral microinjection of glutamate (10
mM, 60 nl) in the RVLM, which also resulted in an
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Fig. 1. Antidromic activation and collision test for an adrenal sympathetic preganglionic neuron (SPN). Trace 1: single pulse stimulation of the adrenal nerve (2 mA) delivered 53.5 ms after a spontaneous action potential results in a constant-onset latency response (51
ms) in the SPN; trace 2: adrenal nerve stimuli at 52 ms after a
spontaneous action potential fail to elicit an SPN response; trace 3: a
single constant-onset latency response is recorded from the SPN
after twin adrenal nerve stimuli 2.4 ms apart delivered 40 ms after
spontaneous SPN spikes; trace 4: no response is recorded from the
SPN after twin adrenal nerve stimuli 2.2 ms apart delivered 40 ms
after spontaneous SPN spikes. All traces are superpositions of 4
stimulation trials. Vertical calibration is 125 ␮V.
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DIFFERENTIATION OF ADRENAL SPNS
increase in mean arterial pressure of 60 mmHg. Similar stimulation of neuronal cell bodies in the RVLM
produced an increase in discharge rate of 89 ⫾ 5.6% in
three Epi Adr SPNs and an increase in mean arterial
pressure of 47 ⫾ 10.7 mmHg. In none of these cases
was an early inhibition (see the histograms of the
electrically evoked responses in Fig. 2A) observed, although the temporally diffuse nature of the glutamate
microinjection stimulus and the presence of RVLM
neurons with excitatory inputs to Epi Adr SPNs could
have masked such an effect.
NE Adr SPN responses to RVLM stimulation. Sixtyeight (49%) adrenal SPNs responded to RVLM stimulation with single action potentials that all had latencies ⬍50 ms. The peristimulus time histogram for a
representative NE Adr SPN (Fig. 2C, top) indicates a
dramatic, short-lasting increase in discharge probabil-
ity between 20 and 40 ms after the RVLM stimuli
(mean response latency: 28.8 ms). This excitation was
usually followed by a period of decreased discharge or
silence often lasting for 100–200 ms. The mean peristimulus time histogram derived from the responses of
68 NE Adr SPNs (Fig. 2C, bottom) shows the consistency of this early and remarkable excitation, which
occurred from 12 ms (the end of the stimulus-blanking
period) to 52 ms, with a mean response latency of 29 ⫾
0.9 ms (range: 16–47 ms). As shown in Fig. 3, bottom,
the antidromic latencies of NE Adr SPNs ranged from
7 to 70 ms (mean: 37 ⫾ 2.0 ms), corresponding to
approximate conduction velocities between 0.47 and
4.7 m/s. These did not differ from those for Epi Adr
SPNs.
NE Adr SPNs were also excited by activation of
RVLM neurons with microinjections of glutamate. In
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Fig. 2. Responses of adrenal SPNs to
activation of the rostral ventrolateral
medulla (RVLM). A: top, peristimulus
time histogram of the activity of adrenal
SPNs with early inhibition and late excitation (Epi Adr SPN) during 100 stimuli (120 ␮A, twin pulses) to the ipsilateral RVLM. Bin width was 5 ms.
Bottom, average peristimulus time histogram of the responses of 71 Epi Adr
SPNs. Each bin represents the mean ⫹
SE of the values (normalized for no. of
RVLM stimuli delivered) for that bin
from all of the individual peristimulus
time histograms. Bin width is 4 ms. B:
response of an Epi Adr SPN to a microinjection (arrowhead, 60 nl) of glutamate (10 mM) in the ipsilateral RVLM.
Top, extracellular action potentials of
the SPN; middle, rate histogram
(500-ms bin width) of the SPN activity;
bottom, arterial pressure (AP). C: top,
peristimulus time histogram of the activity of SPNs with short onset latency
excitation (NE Adr SPN) during 30
stimuli (80 ␮A, twin pulses) to the ipsilateral RVLM. Bin width was 5 ms. Bottom: average peristimulus time histogram of the responses of 68 NE Adr
SPNs. Bin width is 4 ms. D: response of
an NE Adr SPN to a microinjection (arrowhead, 60 nl) of glutamate (10 mM) in
the ipsilateral RVLM. Traces are as
in B.
DIFFERENTIATION OF ADRENAL SPNS
the example in Fig. 2D, the discharge of an adrenal
SPN with an RVLM stimulus-evoked response pattern
characteristic of NE Adr SPNs (see Fig. 2C) was increased from a basal rate of 2 spikes/s to a peak rate of
10 spikes/s after an ipsilateral microinjection of glutamate (10 mM, 60 nl) in the RVLM, which also resulted
in an increase in mean arterial pressure of 51 mmHg.
Similar stimulation of neuronal cell bodies in the
RVLM produced an increase in discharge rate of 171 ⫾
14.8% in four NE Adr SPNs and an increase in mean
arterial pressure of 54 ⫾ 2.2 mmHg.
Baroreceptor reflex influences on the discharge of Adr
SPNs. Adrenal SPNs with RVLM stimulus-evoked responses characteristic of Epi Adr SPNs (see Fig. 2A)
had a mean spontaneous discharge frequency of 5.6 ⫾
0.3 Hz at a mean arterial pressure of 99 ⫾ 3.0 mmHg.
Perisystolic averaging indicated that the spontaneous
discharge of Epi Adr SPNs was not synchronized to the
cardiac cycle, suggesting that the activity of these
neurons was not strongly influenced by the baroreceptor reflex. In the example in Fig. 4A, there is no significant alteration in the average spikes per bin for an Epi
Adr SPN over the course of 1.5 cardiac cycles, including
two systolic pressure increases of ⬃70 mmHg. The
average perisystolic time interval histogram (Fig. 4B)
for 15 Epi Adr SPNs indicates that, although there was
a tendency for a reduced discharge probability during
middiastole, this modulation represented an inhibition
of only 18% of the normalized maximum firing rate
over the course of the cardiac cycle (average mean
arterial pressure: 108 mmHg).
NE Adr SPNs had a mean discharge frequency of
3.5 ⫾ 0.3 Hz, which was significantly slower (P ⬍ 0.01)
than that of Epi Adr SPNs at an equivalent mean
arterial pressure of 99 ⫾ 2.4 mmHg. In contrast to
those of Epi Adr SPNs, the perisystolic time interval
histograms of the spontaneous discharge of all NE Adr
SPNs exhibited prominent peaks and troughs indicating a strong influence of the baroreceptor reflex on the
networks governing the activity of NE Adr SPNs. In
the example in Fig. 4D, the discharge probability of
this NE Adr SPN fell precipitously from 16 to 40 ms
after the onset of systole and then rose throughout
diastole. The mean perisystolic time interval histogram constructed for 10 NE Adr SPNs (Fig. 4E) indicated a similarly large reduction in mean discharge
probability in middiastole (average mean arterial pressure: 118 mmHg). This resulted in an inhibitory modulation representing 83% of the maximum discharge,
which was significantly (P ⬍ 0.01) greater than that for
Epi Adr SPNs, although the average mean arterial
pressures were not different between the groups.
The effect of natural activation of the baroreceptor
reflex indicated a similarly reduced sensitivity of the
discharge of Epi Adr SPNs to baroreceptor reflex-mediated sympathoinhibition. As shown in Fig. 4C, the
mean discharge rate of an Epi Adr SPN was changed
from 3.8 spikes/s at a mean arterial pressure of 90
mmHg to 3.6 spikes/s at a mean arterial pressure of
183 mmHg during the pressor response to a bolus
injection of phenylephrine (5 ␮g in 0.1 ml iv). In contrast, increasing arterial pressure to 186 mmHg resulted in a complete inhibition of the discharge of an
NE Adr SPN (Fig. 4F). In a group of eight Epi Adr
SPNs, an average phenylephrine-induced increase in
arterial pressure of 71 ⫾ 6.4 mmHg resulted in a mean
reduction in discharge frequency of 18 ⫾ 3.9%, whereas
a similar increase in arterial pressure (70 ⫾ 5.2
mmHg) evoked a complete inhibition (100% reduction)
of the discharge of 10 NE Adr SPNs.
The responses of adrenal SPNs to electrical stimulation of baroreceptor afferents in the ADN also indicated a differential sensitivity of the medullary networks governing the discharge of Epi Adr SPNs and of
NE Adr SPNs to baroreceptor reflex inputs. Averaged
responses to three pulse stimuli applied to the ADN
showed no consistent period of reduced discharge probability in Epi Adr SPNs but a dramatic reduction in
discharge probability in NE Adr SPNs. Figure 5A, top,
which shows superimposed records from 30 trials of
ADN stimulation for an Epi Adr SPN, shows no period
of unit discharge inhibition comparable to that in a
typical NE Adr SPN whose superimposed records of 30
trials of ADN stimulation (Fig. 5D) indicate a reduced
discharge probability between 65 and 190 ms after the
ADN stimulus onset. The mean peristimulus time interval histogram for 37 Epi Adr SPNs (Fig. 5A, bottom)
indicates that, on average, Epi Adr SPNs experienced a
reduced discharge probability (to 72% of control) between 230 and 390 ms after the onset of the ADN
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Fig. 3. Histograms of the antidromic onset latencies of adrenal SPNs
putatively regulating adrenal medullary epinephrine secretion (Epi
Adr SPN, top) and those regulating adrenal medullary norepinephrine secretion (NE Adr SPN, bottom).
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Fig. 4. Relationship of adrenal SPN discharge to activation of the baroreceptor reflex by arterial pressure. A: top,
systolic-triggered average of the arterial pressure. Bottom, perisystolic time interval histogram of the spontaneous
activity of an Epi Adr SPN during 1,077 cardiac cycles. Horizontal axis is expressed as % of the cardiac cycle length,
which was 149 ms. Vertical axis in the histogram is normalized to the maximum no. of counts in the modal bin. B:
top, mean (solid line) and mean ⫹ SE (dotted line) systolic-triggered average of the arterial pressure. Bottom,
average perisystolic time interval histogram derived from analysis of the spontaneous discharge of 15 Epi Adr
SPNs. In the latter, each bin contains the mean ⫹ SE of the values for that bin from all of the individual perisystolic
time interval histograms, after normalizing to the modal bin value. Horizontal axis is expressed as % of the cardiac
cycle length to normalize for differing cardiac cycle lengths among animals. Bin width is 5% of the cardiac cycle.
C: response of an Epi Adr SPN to the pressor response after iv bolus of phenylephrine (PE, 5 ␮g). Top, extracellular
action potentials of the SPN; middle, corresponding rate histogram (1-s bin width) of the SPN activity; bottom,
arterial pressure. D: top, systolic-triggered average of the arterial pressure. Bottom, perisystolic time interval
histogram of the spontaneous activity of an NE Adr SPN. Analysis is from 500 cardiac cycles, and cardiac cycle
length was 159 ms. E: top, mean (solid line) and mean ⫹ SE (dotted line) systolic-triggered average of the arterial
pressure. Bottom, average perisystolic time interval histogram derived from analysis of the spontaneous discharge
of 10 NE Adr SPNs. F: response of NE Adr SPN to the pressor response after iv bolus of phenylephrine (PE, 5 ␮g).
Top, extracellular action potentials of the SPN; middle, corresponding rate histogram (1-s bin width) of the SPN
activity; bottom, arterial pressure. Horizontal calibration bar is 8 s for C and 10 s for F. Vertical calibration is 40
␮V for C, top, and 30 ␮V for F, top.
DIFFERENTIATION OF ADRENAL SPNS
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stimuli. In contrast, the average discharge probability
of NE Adr SPNs (n ⫽ 32) was reduced to 16% of the
control discharge rate between 50 and 260 ms after the
ADN stimulation. This baroreceptor-mediated inhibitory effect on the discharge of NE Adr SPNs was
significantly greater (P ⬍ 0.001) than that on the
discharge of Epi Adr SPNs. The slower conduction
velocity in the pathway from the RVLM mediating the
excitation of Epi Adr SPNs (see Fig. 2A) may have
contributed to the longer onset latency and longer
period of reduced discharge probability in Epi Adr
SPNs.
High-frequency stimulation of baroreceptor afferents in the ADN that produced equivalent reductions
in arterial pressure (Fig. 5, B and E, bottom) resulted
in no perceptible change in the discharge of an Epi Adr
SPN (Fig. 5B) but was accompanied by a complete
inhibition of an NE Adr SPN (Fig. 5E). The mean
peristimulus histogram for the high-frequency, ADN
stimulus-evoked responses of 19 Epi Adr SPNs (Fig.
5C) indicated a reduction in discharge probability of
11% of the control firing frequency during the 3-s ADN
stimulation and a subsequent elevation in average
discharge frequency (⫹5% of control) that was maintained for at least the next 10 s. From the mean
peristimulus histogram (Fig. 5F) for the responses of
13 NE Adr SPNs to similar ADN stimulations (maximum reductions in mean arterial pressure of 23 and 20
mmHg for Epi and NE Adr SPNs, respectively; Fig. 5,
C and F, bottom), it is clear that the reduction in
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Fig. 5. Responses of adrenal SPNs to
stimulation of the aortic depressor
nerve (ADN). A: top, superposition of
30 records of the discharges of an Epi
Adr SPN during stimulation of the
ADN (200 ␮A, 3 pulses). Bottom, mean
peristimulus time interval histogram
for 37 Epi Adr SPNs in which each bin
contains the mean ⫹ SE of the values
for that bin from all of the individual
peristimulus time interval histograms.
Bin width is 10 ms. Counts from stimulus artifacts were eliminated by zeroing the two corresponding bins. B: top,
extracellular action potentials of an
Epi Adr SPN (and smaller stimulus
artifacts). Bottom, arterial pressure
during a high-frequency stimulation of
the ADN (20 ␮A, 20 Hz, 3 s). C: top,
mean peristimulus time interval histogram of the responses of 19 Epi Adr
SPNs. Bottom, mean peristimulus histogram of the arterial pressures. Each
bin of these histograms contains the
mean ⫹ SE of the values for that bin
from all of the individual peristimulus
histograms. Bin width is 1 s. D: top,
superposition of 30 records of the discharges of an NE Adr SPN during
stimulation of the ADN (200 ␮A, 3
pulses). Bottom: mean peristimulus
time interval histogram for 32 NE Adr
SPNs. E: top, extracellular action potentials of an NE Adr SPN (and
smaller stimulus artifacts). Bottom, arterial pressure during a high-frequency stimulation of the ADN (100
␮A). Horizontal calibration is 4 s for B
and E. Vertical calibration is 80 ␮V for
A and D, 40 ␮V for B, and 50 ␮V for E.
F: top, mean peristimulus time interval histogram of the responses of 13
NE Adr SPNs. Bottom, mean peristimulus histogram of the arterial
pressures. Each bin of these histograms contains the mean ⫹ SE of the
values for that bin from all of the individual peristimulus histograms. Bin
width is 1 s.
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DIFFERENTIATION OF ADRENAL SPNS
in the RVLM on a drawing (39) through the rat medulla 2.5 mm rostral to the calamus scriptorius. Each
of the recovered sites is within the vasomotor region of
the RVLM, which contains sympathetic premotor neurons regulating vasoconstrictor sympathetic outflow
(36) and adrenal medullary catecholamine secretion
(47, 57).
DISCUSSION
The principal new finding from these experiments is
that adrenal SPNs can be divided into two distinct
populations based of their evoked and reflex responses.
Epi Adr SPNs were markedly excited during the glucopenia evoked by intravenous 2-DG administration,
consistent with SPNs that regulate the secretion from
adrenal, epinephrine-producing chromaffin cells,
whereas NE Adr SPNs were unaffected by 2-DG, consistent with SPNs regulating the discharge of adrenal,
norepinephrine-secreting chromaffin cells. Epi Adr
SPNs consistently responded to electrical stimulation
of the RVLM with a biphasic change in discharge
probability: an early inhibition followed by a long latency excitation. In contrast, the response of NE Adr
SPNs to RVLM stimulation was always a dramatic,
short latency excitation. Epi Adr SPNs exhibited little
or no sensitivity to the baroreceptor reflex, as indicated
by the relative absence of a cardiac cycle-related modulation of their spontaneous discharge and only a minimal inhibition of their discharge during large, phenylephrine-evoked pressor responses or in response to
stimulation of baroreceptor afferents in the ADN. The
discharge of NE Adr SPNs, by comparison, was exquisitely sensitive to baroreceptor reflex activation: the
perisystolic time histograms of NE Adr SPNs contained bins with zero counts, and natural (increased
arterial pressure) or electrical activation of the baroreceptor reflex produced a prompt and complete inhibition of NE Adr SPN discharge. These data provide the
first functional differentiation among populations of
SPNs, thereby supporting the existence of functional
specificity at the level of the SPN (1, 18, 40). These
results also form a foundation for determining the
organization and properties of the networks selectively
controlling adrenal epinephrine secretion (21, 52, 54),
and they suggest that the central pathways determining adrenal norepinephrine release may be indistinguishable from those controlling sympathetic tone to
vasoconstrictor targets.
Several lines of evidence indicate that the SPNs from
which we recorded innervated the epinephrine- and
the norepinephrine-secreting adrenal chromaffin cells.
The SPNs reported in this study were found in thoracic
segments T8-T10, which are within the T5-T11 distribution containing the majority of adrenal SPNs retrogradely labeled after adrenal medullary injection of
tracer in the rat (2, 28, 44, 46, 47) and other species (18,
27). Jensen et al. (27) have shown that the population
of SPNs innervating the rabbit adrenal medulla is
separate from that innervating ganglion cells in the
aorticorenal ganglion, indicating that the axons of ad-
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discharge probability during the ADN stimulation
(84% of control frequency) is significantly (P ⬍ 0.001)
greater than that for Epi Adr SPNs and that the mean
discharge frequency after the inhibition (3.4 ⫾ 0.1
spikes/s) is not different from the control firing rate
(3.5 ⫾ 0.1 spikes/s).
Responses of Adr SPNs to 2-DG-induced glucopenia.
Because plasma levels and adrenal secretion rates of
epinephrine and norepinephrine have been shown to
be differentially sensitive to reductions in blood glucose
(53, 54), we reasoned that adrenal SPNs regulating the
discharge of epinephrine-secreting chromaffin cells
should be excited during the glucopenia produced by
2-DG, while little or no change in discharge frequency
would be expected of adrenal SPNs regulating the
activity of chromaffin cells secreting norepinephrine.
We tested the response of 20 adrenal SPNs with
RVLM stimulus-evoked responses characteristic of Epi
Adr SPNs (see Fig. 2A) and 17 adrenal SPNs with
RVLM stimulus-evoked responses characteristic of NE
Adr SPNs (see Fig. 2C) to an intravenous bolus of 2-DG
(250 mg/kg). In the example shown in Fig. 6A, the
discharge rate of an Epi Adr SPN increased from 5.2
spikes/s during the control preinjection and early
postinjection period (inset on left) to 8.6 spikes/s during
the sustained activation (inset on right) after the 2-DG
injection. The mean post-2-DG time interval histogram
for Epi Adr SPNs (Fig. 6B) shows that 2-DG produced
a significant (P ⬍ 0.05) increase in discharge frequency
beginning ⬃30 s after the 2-DG injection. This excitation reached a plateau (mean discharge frequency:
143 ⫾ 0.7% of control) between 1 and 2 min after the
2-DG injection and remained at this level for at least 5
min (mean discharge frequency: 144 ⫾ 12.4% of control
at 180 s; 149 ⫾ 10.3% of control at 240 s; 148 ⫾ 14.9%
of control at 300 s).
In contrast, the discharge frequency of the NE Adr
SPN shown in Fig. 6C was slightly reduced from 0.5
spikes/s during the control preinjection and early
postinjection period (inset on left) to 0.3 spikes/s during
the period ⬃90–110 s (inset on right) after the 2-DG
injection. The mean post-2-DG time interval histogram
for the NE Adr SPNs (Fig. 6D) suggests a slight,
short-lasting increase in SPN discharge between 5 and
15 s after the 2-DG injection; thereafter, mean SPN
discharge frequency was reduced, averaging 90 ⫾ 0.5%
of control in the period between 1 and 2 min after the
2-DG injection. This level of activity was maintained
for at least 5 min (mean firing frequency: 86 ⫾ 6.8% of
control at 180 s, 89 ⫾ 6.4% of control at 240 s, 88 ⫾
3.3% of control at 300 s).
Location of stimulation and microinjection sites in
the RVLM. Figure 7, top, shows two histological sections at the level of the RVLM containing fast green
dye spots indicating the positions of the stimulating
electrode in two experiments in which Epi and NE Adr
SPNs were recorded. These stimulation sites were located ventral to the compact division of the nucleus
ambiguus, close to the ventral surface of the medulla
and within 500 ␮m of the caudal end of the facial
nucleus. Figure 7, bottom, shows the stimulation sites
DIFFERENTIATION OF ADRENAL SPNS
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Fig. 6. Responses of adrenal SPNs to iv injection of 2-deoxyglucose (2-DG, 250 mg/kg, time 0). A: top, post-2-DG
injection time interval histogram of the discharge of an Epi Adr SPN. Bin width is 1 s. Bottom: corresponding
record of arterial pressure. Inset on left: extracellular action potentials of the Epi Adr SPN during the 5 s just after
the 2-DG injection (discharge rate: 5.2 Hz). Inset on right: similar record during 5 s of the peak activation of the
Epi Adr SPN (discharge rate: 8.6 Hz). B: top, mean ⫹ SE discharge frequency (expressed as %control) histogram
(bin width: 1 s) obtained by averaging the responses of 20 Epi Adr SPNs to administration of 2-DG. Bottom:
average ⫹ SE mean arterial pressure recorded during responses to 2-DG. C: top, post-2-DG injection time interval
histogram of the discharge of an NE Adr SPN. Bin width is 1 s. Bottom: corresponding record of arterial pressure.
Inset on left, extracellular action potentials of the NE Adr SPN during the 5 s just after the 2-DG injection
(discharge rate: 0.5 Hz). Inset on right, similar record during a 5-s interval at 110 s (discharge rate: 0.3 Hz). D: top,
mean ⫹ SE discharge frequency (expressed as %control) histogram (bin width: 1 s) obtained by averaging the
responses of 17 NE Adr SPNs to administration of 2-DG. Bottom: average ⫹ SE mean arterial pressure recorded
during responses to 2-DG.
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DIFFERENTIATION OF ADRENAL SPNS
renal SPNs project selectively to the adrenal medulla.
It seems unlikely that our antidromic stimulus would
have activated SPN axons other than those of adrenal
SPNs, since the adrenal nerve was always well isolated
and the stimulating electrode hooks were positioned
close to the entrance of the adrenal nerve in the adrenal capsule. Although adrenal SPNs may innervate
adrenal medullary ganglion cells and adrenal chromaffin cells (14), the former appear to be a minor cell
population in the adrenal medulla, and it is not known
whether or when they receive a unique preganglionic
input. In view of the conclusion, based on anatomic
data, that there are ⬃2.7 times as many epinephrinecontaining as norepinephrine-secreting chromaffin
cells in the adult rat medulla (50), we were initially
surprised to record from nearly equal numbers of Epi
Adr SPNs and NE Adr SPNs. However, given the
potential for different degrees of convergence of adrenal SPN inputs onto chromaffin cells (24) and our small
sample size in each rat, we might not expect a close
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Fig. 7. Localization of stimulation sites in the RVLM. Top: fast green
dye deposits (arrows) at the positions of the RVLM stimulating
electrode in 2 experiments. NA, compact division of the nucleus
ambiguus. Bottom: filled circles on a drawing through the medulla
⬃2.5 mm rostral to the calamus scriptorius indicate the positions of
the RVLM stimulating electrode in 15 representative experiments.
NTS, nucleus tractus solitarius; PrH, prepositus hypoglossal nucleus; MVe, medial vestibular nucleus; SpVe, spinal vestibular nucleus; SpV, spinal trigeminal nucleus; IO, inferior olive; PYR, pyramidal tract.
correlation between the relative numbers of each type
of adrenal SPN and chromaffin cells.
The simulated hypoglycemia induced by the glucopenic agent 2-DG produces a strong stimulus for the
release of adrenal epinephrine, with a relatively minor
increase in plasma norepinephrine, an indeterminate
fraction of which arose as “spillover” from sympathetic
postganglionic terminals (7). This result parallels that
seen with insulin-induced hypoglycemia (11, 16, 53)
and with chronic 2-DG administration (41). Because
only one of the populations of adrenal SPNs was excited by 2-DG, we conclude that 2-DG administration
was an effective stimulus for distinguishing adrenal
SPNs projecting to epinephrine-secreting chromaffin
cells from those regulating adrenal norepinephrine release. Our results extend those indicating a large increase in preganglionic adrenal nerve activity after
2-DG administration (7) to suggest that this increase
was comprised predominantly of the activation of adrenal SPNs innervating epinephrine-secreting chromaffin cells.
Both electrical and chemical stimulation of the population of sympathetic premotor neurons in the RVLM
activated Epi Adr SPNs and NE Adr SPNs, consistent
with the anatomic finding that the RVLM contains
adrenal sympathetic premotor neurons (47, 56, 57).
Although the characteristics of the excitatory responses of Epi Adr SPNs and NE Adr SPNs to chemical
excitation of RVLM neurons were not markedly different, there were significant differences in the latencies
of the action potentials evoked in Epi Adr SPNs and in
NE Adr SPNs after electrical stimulation of RVLM.
The mean response latency of NE Adr SPNs (29 ms,
Fig. 2C) to RVLM stimulation corresponds to an average conduction velocity of ⬃2.4 m/s, which falls within
the range of “rapidly conducting” RVLM spinal, sympathetic premotor neurons with strong baroreceptor
reflex sensitivity (6, 32, 36, 45). In combination with
the high degree of baroreceptor reflex sensitivity of NE
Adr SPNs, these results suggest that NE Adr SPNs
receive baroreceptor-modulated, premotor excitatory
inputs from RVLM neurons similar to those that provide the tonic excitation to vasoconstrictor SPNs.
In contrast, RVLM stimulation excited Epi Adr
SPNs with a mean latency to peak of 129 ms (Fig. 2A),
indicating an average conduction velocity of RVLM
premotor neurons to Epi Adr SPNs of ⬃0.5 m/s, which
is within the range of “slowly conducting” sympathetic
premotor neurons (6, 36, 45). This observation raises
several points. The absence of baroreceptor modulation
of the discharge of Epi Adr SPNs suggests that their
excitatory premotor inputs from RVLM would arise
from an as-yet-undescribed population of RVLM neurons with slowly conducting spinal axons, but without
the strong inhibitory responses to increases in arterial
pressure that constitute a hallmark of sympathetic
premotor neurons. The recent finding that all slowly
conducting sympathetic premotor neurons (albeit
barosensitive) in the RVLM are C1 cells (45) leads to
the interesting speculation that a subpopulation of the
phenylethanolamine-N-methyltransferase-containing
DIFFERENTIATION OF ADRENAL SPNS
micity, whereas 50% of the latter had a weak cardiac
rhythmicity (9). Curiously, the same group indicated
subsequently (8) that preganglionic adrenal nerve activity was decreased (although not as much as renal or
postganglionic adrenal nerve activity) during a phenylephrine-evoked increase in arterial pressure. Because the adrenal nerves contain not only the axons of
SPNs innervating epinephrine-synthesizing chromaffin cells but also the preganglionic axons of SPNs
innervating the norepinephrine-synthesizing chromaffin cells and postganglionic axons innervating cortical
cells and arteriolar smooth muscle, it is not possible to
determine from these data whether such responses are
indicative of baroreceptor control of the discharge of
SPNs specifically controlling adrenal epinephrine secretion. Although determinations of catecholamine levels in adrenal venous blood could provide more specific
information on the baroreflex regulation of the secretion of the two adrenal catecholamines, the sensitivity
of the adrenal chromaffin cells to blood-borne agents,
including ANG II (4, 12, 20, 33, 55), could produce
significant changes in catecholamine secretion (e.g.,
during severe hypotension) independent of changes in
neural input to chromaffin cells. In this regard, the
responses of Epi Adr SPNs to hypotensive stimuli were
not tested in this study. Finally, the differences between our results and those indicating a reduction in
epinephrine in adrenal venous blood during a phenylephrine-evoked pressor response (26) may have
arisen from a marked reduction in adrenal blood flow
(not reported) leading to a compromised recovery of
epinephrine during the pressor response.
In summary, these results provide a characterization
of adrenal SPNs that has allowed us to distinguish
those regulating adrenal chromaffin cells secreting epinephrine from those controlling adrenal norepinephrine release. The dramatic differences between these
two populations of adrenal SPNs in their reflex- and
centrally evoked responses suggest that their discharge is controlled by different central neural circuits,
including unique populations of adrenal sympathetic
premotor neurons in the RVLM. Our findings support
the distinct functional organization of the networks
governing the activity of Epi Adr SPNs and NE Adr
SPNs that has been proposed (18, 52) to account for the
differential regulation of epinephrine and norepinephrine secretion from the adrenal medulla in response to
physiological challenges and experimental stimuli.
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