204 G
expression of c-fos, a marker for neuronal activation, in the supramaximal square-wave shocks 150 V, 250 ms
trigeminal nucleus caudalis and several other brain nuclei duration, 0.3 Hz with a Grass S88 stimulator.
[31]. This activation was suppressed by the intraperitoneal The central tungsten wire of a glass-coated tungsten
administration of the non-steroidal anti-inflammatory drug electrode [18] was used to record single unit activity in the
indomethacin [33], which is effective in migraine [2]. trigeminal nucleus caudalis. The electrode was placed on
GTN-induced expression of c-fos in the caudal trigemi- the dorsal surface of the brainstem, 1.5–3 mm caudal to
nal nucleus does not however demonstrate unequivocally the obex and 2.5–4.5 mm lateral to the midline, and
whether GTN specifically activates those neurons respon- advanced to a depth of up to 2500 mm below the surface
sible for dural or cerebrovascular pain processing, synaptic by means of a piezoelectric microdrive. Single unit activity
activation is only inferred. The experiments we describe was amplified, filtered and displayed on an oscilloscope.
here were carried out to determine whether GTN adminis- Peri-stimulus and post-stimulus histograms were compiled
tered into the carotid artery of cats can produce activation and used for post-experiment analysis of the latency and
of neurons which also process sensory information from firing frequency of single units.
the dura. Neurons were located first by the presence of a response
to the search stimulus-electrical stimulation of skin or superior sagittal sinus. Some units were tested for the
presence of a cutaneous receptive field. The skin and hair
2. Materials and methods of the face were examined systematically with a variety of
stimuli brush, light touch, heavy pressure and pinch, and Seven
male or
female cats
mass 2.760.5
kg; the cells classified according to response. Low threshold
mean6standard deviation were used in these experiments. mechanoreceptive LTM units responded to light touch or
They were anesthetised with halothane 1.5, and then brush, and did not increase firing rate with noxious stimuli.
with intraperitoneal injections of a-chloralose 60 mg Nociceptive specific NS units responded only to heavy
21
kg . The femoral artery and vein were cannulated to
pressure or pinch, and wide dynamic range WDR units measure blood pressure and heart rate and to administer
responded to non-noxious stimuli, but had an increased intravenous drugs and fluids respectively. Animals were
firing rate in response to noxious stimuli [9]. Congruence intubated and ventilated with 30 oxygen in air to keep
of action potential shape between SSS-induced responses end-expiratory CO in the range 3.5–4.0. Throughout the
and RF-induced responses was assessed visually or via an
2
experiment, the animal was immobilised with intermittent averaging program to ensure that both modes of stimula-
21
intravenous gallamine triethiodide, 20 mg kg . The depth
tion were activating the same neuron. of anesthesia during immobilisation was assessed period-
One to three post-stimulus histograms, each consisting ically during the experiment by testing for sympathetic
of 50 successive recordings of discharges in response to responses pupillary dilatation, tachycardia, raised blood
stimulation, were recorded for each neuron under control pressure to noxious stimulation, and regularly by allowing
conditions. Histograms were acquired in pairs, with sagittal the effects of gallamine to wear off and testing for
sinus and receptive field stimuli alternating in each acquisi- withdrawal reflexes to pinching a hindpaw. Supplementary
tion run. The spontaneous discharge rate of the neurons doses of either chloralose or gallamine were given when
was monitored by recording peri-event histograms in necessary. Rectal temperature was monitored throughout
which each bin recorded the number of discharges re- the experiment with a thermistor, and was maintained at
corded over a sample time of 4 s. Control discharge rates 37–388C by means of a servo-controlled heating blanket.
were recorded for 15–20 min. Glyceryl trinitrate David
21
The lingual artery on one side was catheterised re- Bull Laboratories 5 mg ml
in 30 v v ethanol, 30 trogradely with a small diameter catheter SV8 polyvinyl
v v propylene glycol was diluted 1:100 in normal saline. tubing, Critchley Electrics, advanced until its tip lay in the
Drug-free vehicle was made from ethanol 30 and common carotid artery. Heparinised normal saline 50 I.U.
propylene glycol 30 in distilled water, similarly diluted
21
ml was continuously infused through the catheter at 5
and infused as a control substance. Drug or vehicle was
21 21
21
ml h to maintain its patency.
infused at a rate of 100 mg kg min
or equivalent
21 21
The cat was mounted in a David Kopf stereotaxic frame volumetric rate 2 ml kg
min from a glass syringe,
and the lower brainstem was exposed through a C1 with a Braun Perfusor. Infusions were carried out for 15
laminectomy and occipital craniotomy. The superior sagit- min or until neuron discharge rates peaked and began to
tal sinus was exposed by making parallel incisions in the decline again, whichever came first.
dura and the falx; a plastic sheet was then passed through Selected recording sites were marked by producing an
the incision in the falx [16]. Current spread to the cortex electrolytic lesion with a cathodal DC current 5 mA
was further prevented by constructing a paraffin-filled well passed through the tungsten wire for 5–10 s. At the end of
around the craniotomy site. The superior sagittal sinus was the experiment, the cat was deeply anesthetised with
21
draped over a bipolar hook electrode, insulated except for sodium pentobarbitone 20 mg kg
and perfused via the the tips. The superior sagittal sinus was stimulated with
aorta with 0.9 saline followed by 10 phosphate-buf-
G .A. Lambert et al. Brain Research 887 2000 203 –210
205
fered formalin. The lower brainstem upper cord was 2, division not decidable and were classified as WDR
removed and stored in phosphate-buffered formalin. The 7 8 or LTM 1 8. The locations of 10 lesioned record-
brainstem was later sectioned on a freezing microtome 50 ing sites out of 12 could be found on sections of the
m sections and stained with cresyl violet. Recording sites brainstems of 6 cats. Three sites were in laminae I II of
were reconstructed from a combination of electrolytic the trigeminal nucleus caudalis mean depth 1.4 mm and 2
lesions or track marks and microdrive readings. were in laminae III IV mean depth 2.2 mm; all of these
All group data are presented as mean6standard error neurons responded to GTN with accelerated discharge
S.E.M., except where indicated. Neurons were classified rates following GTN infusion. Two sites were in the neck
as displaying an altered discharge rate by means of the of the nucleus caudalis mean depth 1.7 mm; neither of
critical ratio test [24]. A variance ratio test and a two-tailed the neurons recorded from these sites responded to GTN
Kolmogorov–Smirnov test [28] were used to test whether with acceleration. The other three sites were in the adjacent
the pre-GTN and post-GTN discharge rate frequency ventrolateral medulla or nucleus retroambigualis mean
histograms could have been drawn from the same popula- depth 2.5 mm; 2 of the neurons recorded from these sites
tion. The variance ratio test was used to compare more responded to GTN infusions with an increase in discharge
than two populations of neuronal discharge rates. rate.
All experiments described in this report were approved Fig. 1 shows a post-stimulus histogram obtained from a
by this university’s Animal Care Ethics Committee and neuron in the trigeminal nucleus caudalis activated with
conformed to its guidelines. A2d fibre latency by electrical stimulation of the superior
sagittal sinus. Infusion of GTN through the indwelling lingual artery
3. Results catheter resulted in a rapid increase in the discharge rate of
