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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
- 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.
- 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').
- 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.
- 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.
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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.
