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In Vivo Analysis of Sympathetic Activity in Rat Tail, Study notes of Physiology

The first analysis of single unit sympathetic activity recorded in vivo from sympathetic fibres innervating an identified blood vessel, specifically the caudal ventral artery of the rat tail. The study examines the importance of tonic baroreceptor activity and respiratory-related activities in determining the patterning and frequency of activity in the sympathetic supply to this vessel. Methods include anesthetizing rats with sodium pentobarbitone and a-chloralose, recording phrenic nerve activity, and using a focal extracellular recording technique to record sympathetic activity from the caudal ventral artery.

What you will learn

  • How does tonic baroreceptor activity influence sympathetic activity in the caudal ventral artery?
  • What is the significance of recording single unit sympathetic activity from an identified blood vessel?
  • What role does respiratory-related activity play in sympathetic activity in the caudal ventral artery?

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Journal
of
Physiology
(1994),
476.3
Sympathetic
activity
recorded
from
the
rat
caudal
ventral
artery
in
vivo
Christopher
D.
Johnson
and
Michael
P.
Gilbey
Department
of
Physiology,
Royal
Free
Hospital
School
of
Medicine,
Rowland
Hill
Street,
London
NW3
2PF
1.
In
twenty-five
sodium
pentobarbitone
(a-chloralose
supplemented)-anaesthetized,
artificially
ventilated
and
paralysed
rats,
postganglionic
sympathetic
single
unit
activity
was
recorded
at
the
level
of
the
adventitia
of
the
caudal
ventral
artery
of
the
tail
using
a
focal
recording
technique.
2.
Ten
units
were
identified
as
being
sympathetic
in
nature,
as
they
were
activated
following
electrical
stimulation
of
the
lumbar
sympathetic
chain.
The
on-going
activity
of
seven
of
these
was
blocked
by
hexamethonium
(6-12
mg
kg').
3.
The
units
were
not
under
tonic
baroreceptor
modulation,
as
indicated
by
the
lack
of
pulse
modulation
of
discharge.
Respiratory
modulation
was
apparent,
with
neurones
firing
mainly
during
expiration
(phrenic
silence),
and
activity
was
influenced
also
by
the
lung
inflation
cycle.
Whole-body
warming
decreased
unit
activity.
4.
Interspike
interval
and
autocorrelation
analysis
showed
that
unit
discharge
was
dominated
by
the
respiratory
rhythm
and
that
units
tended
to
discharge
in
bursts
(often
duplets).
It
is
suggested
that
the
intraburst
interval
may
be
determined
by
a
hypothetical
sympathetic
oscillator.
5.
This
study
presents
the
first
analysis
of
single
unit
activity
recorded
in
vivo
from
sympathetic
fibres
innervating
an
identified
blood
vessel.
Complex
patterns
of
sympathetic
nerve
activity
are
generated
by
the
central
nervous
system
in
response
to
reflex
inputs
or
as
components
of
complex
behavioural
activity,
e.g.
those
produced
by
stimulation
of
the
upper
airways
with
smoke
(Peterson,
Coote,
Gilbey
&
Futuro-Neto,
1983),
desynchronized
sleep-like
periods
in
the
decerebrate
cat
(Futuro-Neto
&
Coote,
1982),
somatic
stimuli
(Jiinig,
1985)
and
central
respiratory
drive
(Zhou
&
Gilbey,
1992).
The
techniques
used
in
these
studies
have
failed
to
identify
the
precise
target(s)
to
which
the
various
activities
are
directed.
For
example,
even
if
activity
is
correctly
identified
as
being
skin
vasoconstrictor
(see
Janig,
1985),
the
vessel
innervated
has
not
been
identified.
This
has
been
a
substantial
hinderance
to
understanding
further
the
physiological
organization
of
the
sympathetic
nervous
system.
In
this
report,
data
are
presented
that
represent
the
first
analysis
of
single
unit
sympathetic
activity
supplying
an
identified
blood
vessel.
This
has
been
achieved
by
the
in
vivo
application
of
an
adaptation
of
the
technique
of
focal
extra-
cellular
recording
used
by
Cunnane
and
co-workers
in
vitro
(see
Brock
&
Cunnane,
1987;
Astrand,
Brock
&
Cunnane,
1988;
Evans,
1990;
Cunnane
&
Moss,
1993).
In
this
way,
the
nature
of
unit
activity
directed
specifically
at
the
caudal
ventral
artery
of
the
rat
tail
has
been
examined.
The
importance
of
tonic
baroreceptor
activity
and
respiratory-
related
activities
in
determining
the
patterning
and
frequency
of
activity
in
the
sympathetic
supply
to
this
vessel
has
been
examined.
In
addition,
the
effect
of
raising
whole-body
temperature
on
unit
activity
has
been
investigated
because
of
the
involvement
of
the
rat
tail
circulation
in
thermoregulation
(for
references
see
O'Leary,
Johnson
&
Taylor,
1985).
Some
results
obtained
using
more
conventional
recording
techniques
are
also
reported,
as
they
provide
information
relevant
to
the
interpretation
of
data
obtained
with
the
focal
recording
technique.
Preliminary
accounts
of
this
work
have
been
published
as
abstracts
(Johnson
&
Gilbey,
1993a,
b,
c).
METHODS
Experiments
were
carried
out
on
twenty-five
male
Sprague-
Dawley
rats
(200-350
g)
anaesthetized
with
sodium
pento-
barbitone
(60
mg
kg-')
and
supplemented
with
a-chloralose
(5-10
mg)
when
required,
as
judged
from
recordings
of
heart
rate,
blood
pressure,
phrenic
nerve
activity,
size
of
pupils,
and
palpebral
and
paw-pinch
reflexes.
The
muscle
relaxant
gallamine
triethiodide
(16
mg
kg')
was
administered
during
data
collection.
Within
this
period
animals
were
allowed
to
recover
from
neuromuscular
block
and
the
depth
of
anaesthesia
checked.
A
carotid
artery
and
a
jugular
vein
were
cannulated
to
monitor
arterial
pressure
and
administer
drugs,
respectively.
Artificial
ventilation
(rate,
90-120
min1)
was
performed
using
MS
3114,
pp.
437-442
437
pf3
pf4
pf5

Partial preview of the text

Download In Vivo Analysis of Sympathetic Activity in Rat Tail and more Study notes Physiology in PDF only on Docsity!

Journal of Physiology (1994), 476.

Sympathetic activity recorded from the rat caudal ventral

artery in vivo

Christopher D. Johnson and Michael P. Gilbey

Department of Physiology, Royal Free Hospital School of Medicine, Rowland Hill Street,

London NW3 2PF

  1. In twenty-five sodium pentobarbitone (a-chloralose supplemented)-anaesthetized, artificially ventilated^ and^ paralysed rats, postganglionic sympathetic single unit activity was recorded at the level of the adventitia of the caudal ventral artery of the tail using a focal recording technique.
  2. Ten units were identified as being sympathetic in nature, as they were activated following electrical stimulation of the lumbar (^) sympathetic chain. The on-going activity of seven of

these was blocked by hexamethonium (6-12 mg kg').

  1. The units were not under tonic baroreceptor modulation, as indicated by the lack of pulse
modulation of discharge. Respiratory modulation was apparent, with neurones firing
mainly during expiration (phrenic silence), and activity was influenced also by the lung

inflation cycle. Whole-body warming decreased unit activity.

  1. Interspike interval and autocorrelation (^) analysis showed that unit discharge was
dominated by the respiratory rhythm and that units tended to discharge in bursts (often
duplets). It is suggested that the intraburst interval may be determined by a hypothetical
sympathetic oscillator.
5. This study presents the first analysis of single unit activity recorded in vivo from
sympathetic fibres innervating an identified blood vessel.
Complex patterns of sympathetic nerve activity are

generated by the central nervous system in response to reflex

inputs or as components of complex behavioural activity,

e.g. those^ produced by stimulation^ of^ the^ upper airways

with smoke (Peterson, Coote, Gilbey & Futuro-Neto, 1983),
desynchronized sleep-like periods in the decerebrate cat
(Futuro-Neto & Coote, 1982), somatic stimuli (Jiinig, 1985)

and central respiratory drive (^) (Zhou & Gilbey, 1992). The

techniques used in these studies have failed to identify the

precise target(s) to which the various activities are directed. For example, even if activity is correctly identified as being

skin vasoconstrictor (see Janig, 1985), the vessel innervated

has not been identified. This has been a substantial

hinderance to understanding further the physiological

organization of the sympathetic nervous system. In this (^) report, data are presented that represent the first analysis of single unit sympathetic activity supplying an identified blood vessel. This has been achieved by the in vivo application of an adaptation of the technique of focal extra-

cellular recording used by Cunnane and co-workers in vitro
(see Brock & Cunnane, 1987; Astrand, Brock & Cunnane,
1988; Evans, 1990; Cunnane & Moss, 1993). In this way, the
nature of unit activity directed specifically at the caudal

ventral artery of the rat tail has been examined. The

importance of tonic baroreceptor activity and respiratory-

related activities in determining the patterning and

frequency of activity in the sympathetic supply to this

vessel has been examined. In addition, the effect of raising
whole-body temperature on^ unit activity has^ been

investigated because of the involvement of the rat tail

circulation in thermoregulation (for references see O'Leary,
Johnson & Taylor, 1985). Some results obtained using more
conventional recording techniques are also reported, as

they provide information relevant to the interpretation of

data obtained with the focal recording technique.
Preliminary accounts of this work have been published
as abstracts (Johnson & Gilbey, 1993a, b, c).

METHODS

Experiments were carried out on twenty-five male Sprague- Dawley rats (200-350 g) anaesthetized with sodium pento-

barbitone (60 mg kg-') and supplemented with a-chloralose

(5-10 mg) when required, as judged from (^) recordings of heart rate, blood pressure, phrenic nerve activity, size of pupils, and palpebral and paw-pinch reflexes. The muscle relaxant

gallamine triethiodide (16 mg kg') was administered during

data collection. Within this period animals were allowed to recover from neuromuscular block and the (^) depth of anaesthesia checked. A carotid artery and a jugular vein were cannulated to monitor arterial pressure and administer drugs, respectively. Artificial ventilation (^) (rate, 90-120 (^) min1) was (^) performed using

MS 3114, pp. 437-442 437

C. D. Johnson and M. P. Gilbey

02-enriched room air. Tracheal pressure (5-10 mmHg) and end-tidal CO2 were monitored continuously. Arterial blood samples were taken periodically and arterial pH and gas tensions kept within the following ranges: pH, 7 3-7 45;

arterial PCo2, 35-48 mmHg; arterial P02 > 100 mmHg. Animals

were given a pneumothorax and an end-expiratory pressure of 2-3 cmH2O was applied to the expiratory line to prevent atelectasis. Following a ventral (^) laparotomy, a thermocouple was placed over the abdominal aorta to monitor and regulate core (^) temperature (37 5 + 0 5 °C in the control state and up to 40 °C during whole-body (^) warming) by a heating blanket wrapped around the animal.

Preparation of nerves Following the laparotomy, the lumbar sympathetic chains were exposed and a silver wire bipolar electrode wrapped around them (^) between the third and fifth lumbar ganglia. Both sympathetic chains and electrodes were embedded in insulating material (Provil, Bayer Dental, Germany) and the laparotomy repaired. These (^) electrodes were used to stimulate the chain and thereby evoke activity in sympathetic fibres projecting into the tail. Phrenic nerve activity was recorded so that phrenic-related sympathetic activity could be assessed. A ventral incision was made above the left clavicle, which was removed, and the left phrenic nerve dissected free. The distal end of (^) the nerve was crushed and a silver wire bipolar electrode wrapped around it and the whole embedded in insulating material as described above. The tail was positioned in a Perspex bath for dissection. The fluid in the bath was at room temperature (20-23 °C). In one set of (^) experiments, a ventral collector nerve was exposed, cut

A

50 ms

B

5 ms C

distally, desheathed and covered in paraffin. Activity was either recorded from the whole nerve (^) (6 rats) or from dissected fibres in order to record single or multi-unit activity (9 rats). Conventional (^) bipolar platinum wire electrodes were used to record from the nerve; one pole of the electrode was placed on the cut central end and the other on the crushed distal end; a ground electrode was placed in close proximity. In another set of experiments (10 rats), where sympathetic activity was recorded from the (^) caudal ventral artery using a focal extracellular recording technique, the artery was exposed and the superficial connective tissue removed, but the adventitia left intact. The Perspex bath was filled with a standard Krebs solution (kstrand et al. 1988). Krebs solution-filled (^) glass electrodes (tip diameter < 80 uM) pulled from capillary tubing were placed on the vessel. To produce a 'seal' between the tip of the electrode and the blood (^) vessel, gentle suction was applied to the electrode via the side-arm of the electrode holder. A ground electrode was placed in close proximity.

Drugs

Hexamethonium bromide (Sigma, UK; 6-12 mg kg' in saline)

was administered i.v. and aq,/-methylene adenosine 5'-

triphosphate (Sigma; 10- M) was added to the stock Krebs solution.

Data collection and analysis All (^) neuronal discharges were recorded through high impedance headstages (NL 100, Neurolog, Digitimer Ltd, (^) UK), amplified and filtered. Nerve activities were monitored on an oscilloscope and VDU linked to an IBM computer. Single unit sympathetic activity was discriminated using a (^) spike processor (D130, Digitimer). Nerve discharges, ECG, arterial blood

Figure 1. A a, activity evoked in response to sympathetic chain stimulation (^) (indicated by arrow; 100 trials, 1 Hz, 1 ms pulse, supramaximal stimulus) recorded from the central end of a cut ventral collector nerve. Ab, response was^ reduced by hexamethonium (6 mg kg', i.v.). B, activity evoked (1 trial; sweep delay, 300 ms from stimulus) in response to sympathetic chain stimulation (stimulus parameters as above) recorded from caudal ventral (^) artery using focal (^) recording technique. C, the^ unit^ shown^ in^ B^ was^ discriminated^ so that its on-going activity could be analysed. Five superimposed sweeps are^ shown. Each^ sweep was triggered (^) by a transistor-to-transistor (^) logic (TTL)- pulse generated from the discriminated unit.

438 J.^ Physiol.^ 476.
C. D. Johnson and M. P. Gilbey

blocks excitatory junction currents; see Astrand et al. 1988). Under these conditions, unitary events were recorded which had durations in the range 2-4 ms, were typically triphasic (Fig. IC), and followed 1 Hz sympathetic chain stimulation (not seen with excitatory junction currents). The unitary events were thus confirmed as being action potentials.

Cardiac-related modulation of discharges

Systolic blood pressures in all experiments were in the range

100-140 mmHg. Sympathetic discharge was examined for cardiac-related activity by constructing ECG- or arterial pulse-triggered histograms. Neither the on-going activity recorded from fibre preparations (n =3) nor that recorded using the^ focal recording technique (n =^ 6) showed any clear cardiac-related activity. Figure 2A shows a typical example.

Phrenic-related activity

The activity of the five focally recorded units analysed ( hexamethonium sensitive, 2 hexamethonium resistant) had phrenic-related discharges. As there is a long delay in the sympathetic pathway (peripheral (see above) plus

central, approximately 100 ms; see Guyenet & Brown,
1986), an allowance was made for the phase shift between

phrenic and sympathetic nerve activities. Allowing for this

phase shift, peak firing of units was during phrenic silence,

with the period of depression of activity during the phrenic

discharge (Fig. 2C).

Lung inflation-related activity

The tracheal pressure recording (peak pressure,

5-10 mmHg; rate, 90-120 cycles min') was used as the

A (^) 20-

(^0 10) l

E :3i .

a)20-

E 0 z (^01) 40 (^0) c

E 38

o

s 36 ,,

o 10 1i& z n

i

4 0 s

trigger to generate (^) histograms to examine modulation of sympathetic activity related to the lung inflation cycle. Modulation was seen in three focally recorded units (1 of 4 hexamethonium-sensitive and 2 of 3 hexamethonium- resistant units; Fig. 2B).

Interspike interval and autocorrelation

analysis of unit activity

This analysis was carried out to examine the firing 'frequency' distribution and the possible presence of rhythmic discharges. The focally recorded on-going activity of all eight units analysed in this manner showed early peaks (median of modal intervals, 0-1-015 s; range, 0 05-0 20 s) in their interspike interval histograms. However, in five out of eight cases, although these intervals were similar to the pulse interval they were not coincident with it (see Fig. 3A). Recordings from five of these units were

made simultaneously with recordings of phrenic nerve

activity. These had a peak in their interspike interval

histograms coincident with the interphrenic burst interval
(see Fig. 3A). Autocorrelation analysis showed the discharge
of the units to have a rhythm dominated by that of the
respiratory cycle (as indicated^ by phrenic nerve^ discharge;
Fig. 3B). Units tended to discharge in 'bursts' of duplets at
the respiratory frequency, which is indicated by the similar

early peaks in both interspike interval and autocorrelation histograms.

Effect of whole-body warming on activity

Activity (2 of 3 fibres and 4 of 4 hexamethonium-sensitive
units, and 1 of 1 hexamethonium-insensitive units) was

Figure 3. A and B, interspike interval and autocorrelation histograms of a focally recorded unit. A, interspike interval histogram (50 ms^ bins, 400 intervals). The arrow on the left indicates modal (^) pulse interval and that on the right modal phrenic burst interval. B, autocorrelation (^) histogram (50 ms (^) bins, 400 sweeps). Arrows as above. The rhythm of unit discharge is dominated by that of the phrenic bursts. C, rate histogram (20 s^ bins) illustrating the^ influence of^ an increase in whole-body temperature on the discharge of a focally recorded unit. When abdominal temperature (top trace) reached^39 °C, activity declined to zero. Activity could still be evoked from the chain (indicated by bar) and returned to control levels as temperature fell. Activity was blocked by

hexamethonium (6 mg kg', i.v., given at arrow).

(^2000 3000) s

440 J.^ Physiol.^ 476.

0 1 000

J. Physiol. 476.3 Caudal ventral artery sympathetic activity 441

unaffected until a 'critical' core temperature was reached, which varied between 38 and 39 'C. At this temperature there was an abrupt 'switch-off' in (^) activity, which (^) was

maintained (Fig. 3C). As temperature began to fall, after
the blanket was switched off, the activity returned to control

levels. Blood pressure remained constant throughout the heating procedure.

DISCUSSION

Stimulation of the lumbar sympathetic chain evoked

responses in whole ventral collector nerves, teased fibres and units recorded from the ventral caudal artery with

latencies which furnished 'conduction velocities' which
were not significantly different from one another.

Hexamethonium could block these (^) evoked responses, which is (^) consistent with lumbar chain stimulation activating the preganglionic supply to postganglionic sympathetic neurones innervating the tail whose cell

bodies are located primarily in sacral (S) and coccygeal (Co)
ganglia (Sittiracha, McLachlan & Bell, 1987). On the basis of

the above (^) and a report showing that all afferents from the

tail pass into spinal segments S2-Co3 (Sittiracha et al.

1987), all units focally recorded from the ventral caudal artery can be considered (^) sympathetic. Not all activity,

evoked or on-going, was susceptible to 12 mg kg' hexa-

methonium given I.v. This is not surprising, as it has been
observed that doses of up to 36 mg kg1 hexamethonium

can be required to abolish the potential evoked in the ventral

collector nerve following sympathetic chain stimulation
(authors' unpublished observations). Furthermore, some of

the (^) hexamethonium resistance may be explained by non-

nicotinic transmission.
The unit activities recorded using the focal recording

system represented neuronal action potentials rather than

excitatory junction currents, as they had durations of

2-4 ms and were present when a,fi-methylene adenosine

5'-triphosphate was added to the Krebs solution; in the

presence of this (^) drug, excitatory junction currents are

blocked (Astrand et al. 1988). They also followed faithfully

(^1) Hz stimulation of the chain which is not seen with

excitatory junction currents (Astrand & Stjiirne, 1989).

As sympathetic fibres run (^) along the (^) caudal ventral

artery for a few millimetres before innervating it
(Sittiracha et al. 1987), the recorded activity was destined
for this target. Thus this study presents the first analysis of

single unit (^) activity recorded in vivo from sympathetic

fibres innervating an identified blood vessel, in this case the

caudal ventral artery. The principal rhythm in the

discharge of units was that of the frequency of phrenic
bursts. The additional peak in the interspike interval

histograms in the range 005-0O20 s relates to the intraburst

interval which, as it was not necessarily coincident with

the pulse interval, may be determined (^) by a (^) hypothetical sympathetic oscillator (Gebber, Barman & Zviman, 1989).

These 'bursts' of action potentials probably lead to more effective neuroeffector transmission than that which would occur with single action potentials (^) (Brock & Cunnane, 1992). In (^) the rat, the thermoregulatory control of tail blood flow is an important homoeothermic mechanism. O'Leary

et al. (1985) concluded that the increase in tail vascular

conductance during body heating was purely via (^) withdrawal of vasoconstrictor drive. The data from these experiments show unequivocally that there is withdrawal of sympathetic drive to the caudal ventral artery in response to hyperthermia. The activity recorded was not under tonic baroreceptor modulation, as indicated by its lack of clear pulse-related modulation. In all animals, artificial ventilation (^) was effected at rates higher than the frequency of phrenic bursts, so phrenic discharge primarily reflects

central respiratory (inspiratory) drive (see Gilbey, Numao
& Spyer, 1986; Hiibler, Jiinig, Krummel & Peters, 1993).
Therefore, the focally recorded units received an excitatory

drive which was greatest during expiration. These 'caudal

ventral artery' units therefore have similar discharge

characteristics in these two respects (i.e. respiratory modulation but no tonic baroreceptor input) to activity recorded from the saphenous nerve supplying hairy skin

(Hiibler et al. 1993) and some sympathetic preganglionic

neurones recorded from the lower thoracic and upper segments of the spinal cord projecting into the lower

lumbar chain (Zhou & Gilbey, 1992). Habler et al. (1993)
postulated that the non-respiratory-modulated units
recorded by Zhou & Gilbey (1992) might project to the tail.

This study shows that non-modulated units are unlikely to innervate the caudal ventral artery, but non-respiratory- modulated (^) activity may be directed at other (^) parts of the tail (^) circulation. Another possibility which cannot be excluded is that non-respiratory-modulated and

respiratory-modulated sympathetic preganglionic neurones

converge onto^ the^ same^ postganglionic neurone.

In conclusion, this study has defined some of the
characteristics of activity in sympathetic fibres innervating

the caudal ventral artery of the rat tail. It remains to be determined how these activities compare to those in the sympathetic (^) supply to other blood vessels of (^) the tail and other circulations.

REFERENCES

ASTRAND, P., BROCK, J. A. & CUNNANE, T. C. (1988). Time course of transmitter action at the (^) sympathetic neuroeffector (^) junction in rodent vascular and non-vascular smooth muscle. Journal of Physiology 401, 657-670.

ASTRAND, P. & STJARNE, L. (1989). On the secretory activity of

single varicosities in sympathetic nerves innervating the tail artery. Journal of Physiology 409, 207-220. BROCK, J. A. & CUNNANE, T. C. (1987). Relationship between the nerve action potential and transmitter release from sympathetic postganglionic nerve^ terminals.^ Nature^ 326, 605-607.