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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.
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Journal of Physiology (1994), 476.
inflation cycle. Whole-body warming decreased unit activity.
generated by the central nervous system in response to reflex
e.g. those^ produced by stimulation^ of^ the^ upper airways
and central respiratory drive (^) (Zhou & Gilbey, 1992). The
precise target(s) to which the various activities are directed. For example, even if activity is correctly identified as being
has not been identified. This has been a substantial
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-
ventral artery of the rat tail has been examined. The
related activities in determining the patterning and
frequency of activity in the sympathetic supply to this
investigated because of the involvement of the rat tail
they provide information relevant to the interpretation of
Experiments were carried out on twenty-five male Sprague- Dawley rats (200-350 g) anaesthetized with sodium pento-
(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
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
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;
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
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.
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.
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.
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
phrenic and sympathetic nerve activities. Allowing for this
with the period of depression of activity during the phrenic
The tracheal pressure recording (peak pressure,
A (^) 20-
(^0 10) l
E :3i .
a)20-
E 0 z (^01) 40 (^0) c
E 38
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).
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
activity. These had a peak in their interspike interval
early peaks in both interspike interval and autocorrelation histograms.
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
(^2000 3000) s
440 J.^ Physiol.^ 476.
0 1 000
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
levels. Blood pressure remained constant throughout the heating procedure.
responses in whole ventral collector nerves, teased fibres and units recorded from the ventral caudal artery with
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
the above (^) and a report showing that all afferents from the
1987), all units focally recorded from the ventral caudal artery can be considered (^) sympathetic. Not all activity,
can be required to abolish the potential evoked in the ventral
the (^) hexamethonium resistance may be explained by non-
system represented neuronal action potentials rather than
presence of this (^) drug, excitatory junction currents are
(^1) Hz stimulation of the chain which is not seen with
As sympathetic fibres run (^) along the (^) caudal ventral
single unit (^) activity recorded in vivo from sympathetic
caudal ventral artery. The principal rhythm in the
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
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
drive which was greatest during expiration. These 'caudal
characteristics in these two respects (i.e. respiratory modulation but no tonic baroreceptor input) to activity recorded from the saphenous nerve supplying hairy skin
neurones recorded from the lower thoracic and upper segments of the spinal cord projecting into the lower
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
converge onto^ the^ same^ postganglionic neurone.
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.
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.
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.