Innervation of the cat's adrenal medulla

17 Pages·2005·2.15 MB·English

J. Anat. (1968), 102,2, pp. 257-273 257
With 6figures
Printedin GreatBritain
Innervation of the cat's adrenal medulla
E. MARLEY AND GWENDA I. PROUT
Institute ofPsychiatry, Maudsley Hospital,
Denmark Hill, London, S.E. 5
INTRODUCTION
The mode offunctioning ofthe adrenalmedulla can only be understood in terms
of the properties of the nerves responsible for the activation and recruitment ofits
secretory cells. A detailed knowledge of the innervation of the gland is therefore
essential. The complex origin and course of the nerve supply to the gland can be
revealed by gross dissection, but Langley's excellent accounts (Langley, 1894, 1896;
Langley & Anderson, 1896) ofthe cat's autonomic nervous system do not consider
the innervation of the adrenal medullae in detail. Moreover, this method fails to
distinguish between secretomotor nerves and those unrelated to secretory function.
Alternative techniques available for investigating adrenal innervation include the
study of degeneration within the gland after denervation (Hollinshead, 1936;
Swinyard, 1937; Young, 1939) and the relation between the effects of stimulation
ofthe splanchnic nerves and the secretion ofsympathin (Maycock & Heslop, 1939).
Provided that themethodsusedfordetectionofcatecholamines are sensitiveenough
and extra-medullary sources of sympathin are excluded, the latter method should
provide the most reliable criterion of functional innervation but the method of
assay used by Maycock & Heslop, besides being relatively insensitiveincomparison
to methods now available, was only semi-quantitative and did not permit identifi-
cation ofthe particular amine secreted.
Inthepresentexperimentsacombinationofanatomicalandphysiologicalmethods
have been used involving acarefuldisplayandanalysis ofthesplanchnic nerves and
the determination ofadrenal medullary secretory function by stimulation of these
nerves.
METHODS
Macroscopic dissections ofthe sympathetic chain, splanchnicnerves, prevertebral
ganglia and connexions to the adrenal gland were made in fifteen cats preserved in
5% formol saline. The anatomy ofthe nerves oneach side was recorded. The para-
vertebral ganglia were designated by giving them the number of the spinal nerve
with which they were connected by the grey ramus (Langley, 1891; Ranson &
Billingsley, 1918).
Secretion by the adrenal medulla on exciting the splanchnic nerves was tested in
cats anaesthetized with chloralose (80mg/kg i.v) after induction withethyl chloride
and ether. The vagi and cervical sympathetic nerves were divided in the neck and
the cats artificially ventilated. The splanchnic nerves were approached through
a lumbar incision, identified and then divided. In order to mobilize the splanchnic
nerves for stimulation the intervening adrenolumbar vessels lateral to the gland
were divided between ligatures. The sympathetic trunk was removed from the first
258 E. MARLEY AND GWENDA I. PROUT
to fourth lumbar sympathetic ganglia to exclude the possibility of activating the
adrenal medulla through aberrant nerve pathways. In some cats the contralateral
splanchnic nerves were also identified and divided and the sympathetic trunk
excised. Theabdominalvisceraincluding thestomach, the smallandlarge intestines,
pancreas, spleen and the adrenal gland not under test were removed; the renal
vessels were then tied. By these means, sympathin release or secretion from sources
other thanthe adrenal medulla under test would be unlikely. Heparin (10mg/kgi.v)
was injected. The splanchnic nerves distal to the division were excited through
platinum electrodes (cathode distal) with supramaximal rectangular (05 ms) pulses
of 20V at varying frequencies.
The optimum rate of electrical stimulation for eliciting maximum secretion of
sympathin was 30-60/s for the first nerve but was usually slower for the smaller
nerves (Marley & Prout, 1963); secretion was much smaller with faster or slower
thanwithoptimalexcitation. Consequently, secretiononexcitingthesmallsplanchnic
nerves might be so trivialas to bemissed ifexcitationwasnotnear the optimal rate.
For these reasons, output was tested with briefbursts ofsupramaximal stimuli over
a range offrequencies to determine that which gave maximal spatial and temporal
recruitment. Stimuli were counted on a 'Racal' high-impedance digital frequency
meter in parallel with the stimulating electrodes. Blood pressure was recorded via
acannulatiedintoacarotidarteryandwritingonasmokeddrum.Thebloodpressure
ofthe cats prepared by the various methods lay between 75 and 125 mm Hg.
Adrenal medullary secretion was measured in two ways. In the first (assay by
superfusion) blood from the carotid artery or the adrenolumbar vein, or both, was
allowed to superfuse an isolated stomach strip; in the second (assay of plasma
samples) blood from the adrenolumbar vein was collected and assayed on the blood
pressure and uterus ofthe rat. The number ofcats tested by the two techniques are
given in the text. In the superfusion method (Vane, 1958, 1964) blood was pumped
from a carotid artery into an extracorporeal circuit of silicone rubber tubing by
a roller-pump, (Saxby, Siddiqi & Walker, 1960) driven by a Servomex motor
controller. Thebloodwaswarmedinawater-jacket at40 °Cbefore superfusingarat
isolated stomach strip (Vane, 1957) and then returned to a jugular vein. The tone
ofthe stomach strip was recorded with an auxotonic lever (Paton, 1957) writing on
smoked paper. The strip was normally contracted when superfused with blood but
relaxed when sympathin was secreted into the circulation on splanchnic nerve
stimulation. The amount of sympathin reaching the stomach strip was assayed
against adrenaline or noradrenaline injected into the systemic circulation. To assay
secretion ofverysmall quantities ofsympathinfrom theadrenalmedullathemethod
was modified (Marley, 1961). In this case, adrenal venous as well as carotid adrenal
blood was pumped in separate extracorporeal circuits and either mixed before
superfusing a single rat stomach strip or the blood streams superfused strips in
series, the upper strip receiving carotid arterial, and the lower adrenal venous as
well as carotid arterial blood. Sympathin secreted from the adrenal medulla was
estimated from the response of the strip superfused by adrenal and carotid arterial
blood and which would detect as little as 1-5 ng adrenaline. Sympathin in the
systemic circuit was estimated from the response of the strip superfused by carotid
arterial blood and this would detect 50-100ng or more ofadrenaline secreted into
Innervation of adrenal medulla 259
the general circulation. The arrangement of the extracorporeal circuits for assay of
sympathin is described more fully in Marley & Prout (1965).
When plasma samples were to be collected from the adrenolumbar venous blood
for assay on the rat blood pressure and rat uterus, a siliconed polyethylene cannula
described by Marley & Paton (1961) was tied into the adrenolumbar vein with the
tipjustlateral toandpointingtowards theadrenalgland. Theopeningoftheadreno-
lumbar vein into the vena cava was then closed and consequently the venous blood
flow through the adrenal gland was diverted into the cannula. The blood was
collected and centrifuged with precautions to prevent the development of vaso-
pressoractivityunrelated to sympathin (Gaddum, Peart&Vogt, 1949).Theamounts
of the two catechol amines in the plasma was calculated from the adrenaline and
noradrenaline equivalents determined with two test organs by the graphic solution
of simultaneous equations (Marley & Paton, 1961). Noradrenaline in the plasma
was assayed by its pressor effect in pithed rats (Shipley & Tilden, 1947); adrenaline
was assayed with the electrically stimulated uterus from a rat pretreated with
stilboestrol 100 ,tgi.m. (Harvey & Pennefather, 1962).
The present experiments were mostly concerned in quantitative comparisons of
secretion obtained by exciting the different splanchnic nerves. They were also
concerned in ascertaining which splanchnic nerves influenced adrenal medullary
secretion. Since this was a mensural problem, data from previous studies (Marley &
Paton, 1961; Marley & Prout, 1965) were used to supplement the present results.
The amounts ofadrenaline ornoradrenaline found byassay orgivenbyinjection are
given in terms of base.
RESULTS
Anatomical studies
The splanchnic nerves
In the 30 sides of 15 cats dissected, the upper splanchnic nerves varied from 3 to
4 in number on each side. There were 4 in 7 cats on the left side, and in 8 on the
right, and 3 on the left in 8 cats and in7 onthe right. The highest root contributing
to a splanchnicnerve was the 13th thoracic, and the lowest the 4th lumbar. Details
aregiveninTable 1. Thesplanchnicnervespassed to theupperprevertebral ganglia,
the renalplexus and intermesenteric nerves (Figs. 1, 2). Asimilarnumberofinferior
splanchnicnervesarisingfromthelowerlumbargangliarantotheinferiormesenteric
ganglia andintermesenteric nerves. The upper splanchnic nerves did not in any two
cases exactly correspond, nor did the nerves ofone side mirror those of the other,
theleftsideusuallybeingthemorecomplicated(Fig. 1).Astheterms'greater','lesser'
or 'least splanchnicnerves' have little tocommend them, thenerves have beennum-
bered 1-4from above downwards.
Thefirst, or major, splanchnic nerve
Thefirst splanchnic nerve left the sympathetic trunk at, or between the thirteenth
thoracic and the second lumbar sympathetic ganglion, and in seventeen sides from
or near the first lumbar ganglion (Table 1). The left and right nerves left the sympa-
thetic trunk at the same level with the exception of one cat in which the origin
differed by one segment. The first splanchnic nerve was the largest in diameter and
260 E. MARLEY AND GWENDA I. PROUT
always joined the superior lateral pole of the ipsilateral coeliac ganglion, although
in five sides direct branches to the adrenal gland left the nervejust proximal to its
junction with the ganglion. On 11 sides the first splanchnic nerve was a single trunk
throughout its course but in 4 sides it divided into two at the diaphragm and in
another4sidesjust distal to a ganglion onthenerve, thebranchesjoiningthecoeliac
ganglion. In 6 sides it united with the second splanchnic nerve (Fig. 1) proximal to
the coeliac ganglion. Variations in the anatomy of the upper splanchnic nerves are
summarized in Table 1.
Table 1. Roots, distribution and variations in anatomy ofthe left and
right upper splanchnic nerves infifteen cats (macroscopic analysis)
(Abbreviations asinLegend toFig. 1. Roots originating from interganglionic segments
allocated tonearest ganglion.)
Splanchnic
nerve Roots Distribution Variation
1st T XIII 11 Coeliacganglion Single nerve 11
LI 17 Divides at diaphragm 4
LII 2 Joins 2nd Spl.n. before 6
coeliac ganglion
Ganglion onnerveafter 4
whichnerve divides
Nervesto adrenal gland 5
proximal tocoeliac
ganglion
2nd T XIII Coeliac ganglion 27 Singlenerve 13
1
LI 8 Aortico-renal ganglion 4 Splits intotwo 5
LII 20 Renal plexus 4 Splitsinto three 1
LIII Tworoots 5
1
Loop from adjacent 2
ganglion
Branch frominter- 6
ganglionic segment
Branchto 3rd Spl.n. 3
Joins4th Spl.n. to form
ganglion 1
3rd LII 13 Adrenal gland 2 Singlenerve 11
LIII 17 Coeliacganglion 7 Splits into two 13
Aortico-renal ganglion 16 Splits into three 2
Coeliac-aortico-renal band 3 Splitsinto four 2
Renalplexus 11 Two roots 7
Kidney 4 Branch frominter- 1
Lateral intermesenteric 2 ganglionic segment
nerve Branchto 4th Spl.n. 3
Joins4th Spl.n. 3
4th Lll Coeliac ganglion 3 Singlenerve 6
1
LIII 15 Aortico-renal ganglion 12 Splitsinto two 14
LIV 9 Renal plexus 11 Splits into four 1
LIII and IV Kidney 3 Tworoots 5
1
Lateralintermesenteric 8 Branch to lateral inter- 5
nerve mesentericnerve
Medianintermesenteric 5
nerve
Inferiormesenteric 4
ganglion
Innervation ofadrenal medulla 261
The secondsplanchnic nerve
In 28 sides, the second splanchnic nerve left the sympathetic trunk from or near
the first or second lumbar ganglion. It joined the coeliac ganglion on 27 sides but
on 6 sides branched to the aortico-renal ganglion or renal plexus (Table 1). It was
smaller than, but could be almost as large as, the major splanchnic nerve. In 13
sides it ran as a single trunk; the commonest variations (Table 1) were branching
rootlets, especially from the interganglionic segments, and connexions to the first
and third nerves.
Right Left S
TXIIlt \ 1 Spin. LI
Lk::' CG CN SMN CN \, Si.
/SMN CG ~ LII
ARG0
Ik~~~~~~R'NARG A ~~~~~~~~~~~~~3Spi.n.
L;; II wAR ~~~~~K~KRNL SI I
L 1II-'
w L~~~~~~~~~~~~~~LIV
IV?/--
L I'l
LIMNL
LIMN
IMG YFG
Fig. 1. Arrangement ofthe splanchnic nerves and innervation ofthe adrenal glands in a cat
(ventral aspect). The kidneys (K) and adrenal glands (A) have been reflected medially and
ventrally. Abbreviations: LG, CG, ARG, IMG, lumbar sympathetic, coeliac, aortico-renal and
inferior mesenteric ganglia. ST, Sympathetic trunk. W, White rami. Dotted lines, grey rami.
LI-V, Firsttofifth lumbarnerves. Spl.n., Splanchnicnerves. CN, SMN, Coeliacandsuperior
mesentericnerves.LIMN,MIMN,Lateralandmedianintermesentericnerves.RN,Renalnerves.
The thirdsplanchnic nerve
Thisleftthesympathetic trunkfromthesecond orthirdlumbarganglion (Table 1).
In 11 sides it was a single trunk and in 13 formed two branches. It terminated in the
aortico-renal ganglion (17 sides), the renal plexus (11 sides), but also joined the
coeliac ganglion (7 sides), kidney (4 sides) and intermesenteric nerves. In 2 sides it
innervated the adrenal gland directly.
Thefourth splanchnic nerve
This left the sympathetic trunk from the third or fourth lumbar ganglion. On 14
sides the fourth nerve divided into two and on one side divided into four. A single
trunk from origin to distribution was found in only 6 sides. The categorization of
the splanchnic nerves into upper and lower was made between the third and fourth
lumbar ganglia. In 14 sides the nerves from the third lumbar ganglion joined the
I7 Anat. 102
262 E. MARLEY AND GWENDA I. PROUT
coeliac and aortico-renal ganglia whereas those nerves which rose from the fourth
ganglion passed to the intermesenteric nerves and inferior mesenteric ganglia.
On 12 sides branches from the fourth lumbar ganglion passed forward to join the
aortico-renal ganglia orbranchesfrom the third lumbar ganglion passed posteriorly
to the intermesenteric nerves (Fig. 1, Table 1); this division into upper and lower
splanchnic nerves was therefore not absolute. Branches from both ganglia passed
to the renal plexus.
Right Left Right Left
ISi
Fig. 2.Arrangementoftheupperprevertebral gangliaandconnexionsinthreecats
seenfromtheventral aspect. Abbreviations asinFig. 1.
Coeliacplexus andprevertebralganglia
Thenerves to theadrenalglandarosemainlyfromtheupperprevertebral ganglia,
which consists ofthe coeliac and aortico-renal ganglia interconnecting through the
coeliac plexus. These ganglia lay ventral to the aorta and inferior vena cava close
to the origins of the coeliac and superior mesenteric arteries. The ganglia were
joined through the intermesenteric nerves with the lower prevertebral group, the
inferior mesenteric ganglia (Figs. 1, 2).
The coeliac andaortico-renalganglia
The right coeliac and aortico-renal ganglia were fused in 9 cats in a semilunar
or comma-shaped mass but were connected by strands in 4. The left ganglia were
Innervation ofadrenal medulla 263
less closely associated, being linked only by strands in 12 cats and fused in 3. The
ganglia were irregularly shaped and either fenestrated or dense and homogeneous.
The aortico-renal ganglia were linked in all specimens; in 6, the ganglia were united
just totherightofthemid-lineandgaveconnectingbranches tobothadrenalglands;
in 2, all the upper prevertebral ganglia were fused in a horse-shoe-shaped mass
(Fig. 2c); in the remainder, the aortico-renal ganglia were connected by a stout
band below and a smaller strand above the superior mesenteric artery. A caudal
aortico-renal ganglion was found in two cats (Fig. 2b). The coeliac ganglia were
directly interconnected in only one cat.
The anterior extremity of the coeliac or of the fused ganglionic masses were
prolonged into the coeliac nerves. The right nerve accompanied the coeliac and
hepatic arteries and branches from it joined the left nerve which accompanied the
splenic artery. The superior mesenteric nerves rose from the medial border of the
aortico-renal ganglia and gave branches to circle the superior mesenteric artery.
Two lateral and a median intermesenteric nerve originated from the lower poles
of the aortico-renal and coeliac ganglia or their interconnexions (Fig. 2a). On the
median nerve opposite the kidneys there was in sixcats anelongated ganglion giving
renal, adrenal or testicular, ovarian and ureteric branches. Branches to the lateral
intermesenteric nerves came from the renal plexus, the fourth and fifth splanchnic
nerves, and in two sides from the third splanchnic nerve.
Innervation ofthe adrenalglandandkidney
Apart from the direct branches to the adrenal gland from the first splanchnic
nerve in five cats and in two from the third splanchnic nerve, 4-6 adrenal nerves
left the lateral border of the ipsilateral coeliac and aortico-renal ganglia (Fig. 2)
just caudal to the junction of the first splanchnic nerve with the ganglia. These
nerves overlapped the smaller splanchnic nerves joining the aortico-renal ganglia
and entered the adrenal gland separately and postero-medially or coalesced in
a longitudinal trunk on the posterior surface ofthe gland. Occasionally branches to
theglandcamefromthecontralateralaortico-renalganglion(Fig. 2a,c)theipsilateral
renal plexus or from the intermesenteric nerves.
The renal nerves left the lateral border of the ganglia and ran into a plexus and
in four cats a ganglion (Fig. 2c) between the renal artery and vein. The adrenal
nerves in twenty-five sides and the third and fourth splanchnic nerves each oneleven
sides gave branches direct to the kidney.
As judged by gross dissection, the innervation of the adrenal medulla was pre-
dominantly ipsilateral, the two largest splanchnic nerves passing to the coeliac
ganglion, the smaller nerves to the aortico-renal ganglia. Inaddition, there appeared
to be a number ofpossible pathways forcrossed innervation ofthe adrenal medulla.
Fibres could readily pass within the macroscopically demonstrable nerve network
found in all our dissections from the ipsilateral splanchnic nerves and aortico-renal
ganglion to the opposite aortico-renal ganglion and adrenal gland or from the
coeliac ganglion to anaortico-renal ganglion giving branches to both adrenal glands
(Fig. 2a, b, c). In one cat branches passed from one coeliac ganglion to the other.
No connexion between the splanchnic nerves above the diaphragm was found.
17-2
264 E. MARLEY AND GWENDA I. PROUT
Functional studies
Ipsilateral innervation
Stimulationoftheuppersplanchnicnervesofeithersideinvariablyelicitedsecretion
of sympathin from the ipsilateral adrenal medulla (233 left or right first splanchnic
nerves in 184 cats, 15 second left or right splanchnic nerves in 8 cats and 5 left third
splanchnic nerves in 5 cats) whereas secretion on exciting the fourth splanchnic
nerve was detected in only 2 of 6 cats.
Secretion resulting from briefbursts ofstimuli (180 or 256 shocks) was greater on
exciting the first than the smaller splanchnic nerves, and dwindled progressively
asthesizeofnerveexciteddiminished.Therelativesizeofandtime-courseofsecretion
were best shown in the same cat by using the superfusion method. As shown in
Fig. 3A, B, D, secretion was greater with excitation at 10, 40, 80 or 160/s to the
first splanchnic nerve than to the second orthird nerves. Secretion was mostmarked
for all nerves with excitation at 40/s. The delay between stimulating the nerve
and the beginning ofrelaxation of the rat stomach strip was due to the time taken
for the blood to pass from the adrenal medulla to the carotid arterial blood super-
fusing the rat stomach strip. The amount of sympathin secreted by the adrenal
medulla could be gauged from the responses of the superfused rat stomach strip
(Fig. 3C) to different doses ofadrenaline injected intravenously into the cat.
Secretion on exciting the fourth splanchnic nerve was insufficient to be detected
by a rat stomach strip superfused by carotid arterial blood (upper trace, Fig. 4.)
If, inaddition, blood fromtheadrenolumbarveinwas superfused overarat stomach
strip then secretion as shown by relaxation of the stomach strip was detected with
excitation at 4, 32, 16 and 8/s (lower trace, Fig. 4).
Although it was not possible to estimate the resting adrenal medullary secretion
with the superfusion method, this could be estimated by assay of plasma samples
obtained from adrenolumbar venous blood. The control secretion of combined
amine after acute division of the splanchnic nerves and before stimulation ranged
from 12-5 to 25 ng/min in the experiment ofFig. 5 and 35-47 ng/min in the experi-
ment of Fig. 6. These values are similar to those reported by Vogt (1952) for the
secretion from the chronically denervated feline adrenal medulla and indicated
that secretion had not been adversely influenced by the operative procedures.
Secretion was increased on exciting the splanchnic nerves. While it was possible
with assay on the rat uterus and blood pressure to compare in the same cat the
secretion obtained by stimulating the different splanchnic nerves at a certain fre-
quency, to do this over a range of frequencies required comparison in different
cats. Theconsiderably greatersecretion onexcitingthefirstthanthethirdsplanchnic
nerve is shown in the two experiments of Fig. 5A, B. Thus 215 ng/min total amine
was secreted on 32/s excitation ofthe first splanchnic nerve compared to 30 ng/min
on excitation ofthe third nerve, and this calculation does not take into account the
overflow ofamine in the subsequent 10 min collection period after exciting the first
splanchnic nerve. The ratio of adrenaline to noradrenaline secreted varied, being
predominantly noradrenaline in the cat from which Fig. 5A was obtained and
predominantly adrenaline in the case of Fig. 5C. The relative amounts of amines
secreted on stimulation of the splanchnic nerves at the different frequencies corre-
Innervation ofadrenal medulla 265
sponded well with the results obtained from the superfusion experiments. The
apparently longer time-course of secretion in experiments in which blood from the
adrenolumbar vein was assayed on the rat uterus and blood pressure was due to
r-MIi
I-I--
l_
A
1st
splanchnic
nerve
0 0 * 0
10 80 40 160
2nd
splanchnic
nerve
U , U . * 5 a
10 80 160 40 Ad Ad Ad
0.5 02 01
/pg pig lpig
D
3rd
splanchnic
nerve
V0 0 0
160 40 80 10
Fig. 3. Rat stomach strip; response to superfused blood from a carotid artery on electrical
stimulation ofthe ipsilateral splanchnic nerves. Cat, 3-4kg; chloralose anaesthesia A, B, D.
Sympathin secretion on exciting the left first, second and third splanchnic nerves respectively
with 160shocksatfrequencies/s indicated. C, Calibration responses dueto adrenalineinjected
intravenously. Time-marker, minutes.
266 E. MARLEY AND GWENDA I. PROUT
therelatively slow bloodflow (0-5-2 0 ml/min) incomparison to that of5-10 ml/min
for experiments with the superfusion method of assay.
The greater secretion on sustained excitation at 32/s of the first splanchnic as
compared tothat onstimulatingthesecondsplanchnicnerveisillustratedin Fig. SC.
Excitation was for 8 min in each instance, the secretion ofadrenal medullary amines
on exciting the first splanchnic nerve being 960ng/min compared to 445 ng/min on
stimulating the second nerve.
A B
a a0 u a
Ad S S S S
50 ng 4s 32s 16s 8/s
160 160 160 160
I I I--n
Min
Fig. 4. Adrenal medullary secretion on exciting the ipsilateral fourth splanchnic nerve. Cat,
2-4kg; chloralose. Upper rat stomach strip superfused by carotid arterial and lower strip by
carotid arterial and adrenal venous blood. A, Calibration response toadrenaline injected into
theadrenalcircuit.B,Sympathinsecretiononexciting(S)fourthsplanchnicnerveanddetected
with strip inadrenal circuit butnot bystrip insystemiccircuit. Time-marker, minutes.
Secretion was not detected on exciting the peripheral ends of the divided vagi
(4catsinwhichtheabdominalviscerawereleftinsitu)thefirstofthelowersplanchnic
nerves (3 cats), nor on splanchnic nerve excitation after removing the adrenal
glands (2 cats).