Recordings From Brain Stem Neurons Responding to Chemical
Stimulation of the Subarachnoid Space
A. EBERSBERGER, M. RINGKAMP, P. W. REEH, AND H. O. HANDWERKER
Department of Physiology and Experimental Pathophysiology, University of Erlangen, 91054 Erlangen, Germany
INTRODUCTION
Since 1940, sensory nerve fibers supplying the dura mater
encephali have been thought to be involved in the generation
of headache (Penfield and McNaughton 1940; Ray and
Wolff 1940). In the last two decades, it has been shown
that the dura is innervated densely by thin myelinated and
unmyelinated nerve fibers (Edvinsson and Uddman 1981;
Keller and Marfurt 1991). Most of these fibers are probably
afferent because they contain substance P (SP) and calcitonin gene related peptide (CGRP) known to occur exclusively
in primary afferents (Edvinsson et al. 1989; Fang 1961;
Keller and Marfurt 1991; Matsuyama et al. 1986; Yamamoto
et al. 1983). The majority of the ganglion cells that innervate
the dura of cat and rat are located in the ophthalmic division
of the trigeminal ganglion (Keller et al. 1985; Mayberg et
al. 1984; O’Connor and Van Der Kooy 1986; Steiger et al.
1982).
Though these meningeal afferents are thought to provide
input for the generation of headaches, little is known about
the mode of their activation. The dreadful headaches associated with subarachnoid hemorrhage probably are caused by
bloodborne substances that come into contact with the meninges. In the case of headaches typical for meningitis, there
are presumably inflammatory mediators involved (part of
which, again, are bloodborne). According to a new hypothesis, the migraine, a more common form of headache, is
related to a neurogenic inflammation in the meninges
(Goadsby 1993; Moskowitz and MacFarlane 1993). Indeed,
it has been demonstrated that neuropeptides are released in
the meninges when trigeminal nerve fibers are stimulated
electrically (Goadsby et al. 1988; Zagami et al. 1989);
depletion of neuropeptides can be observed by the application of capsaicin to cerebral vessels (Duckels and Buck
1982; Janssen et al. 1989). Furthermore, SP, Neuropeptide
Y and serotonin-like immunostaining were reduced substantially after stimulating the dura by application of autologous
arterial blood (Keller et al. 1993). After release, neuropeptides may activate or sensitize meningeal nociceptive afferents (Holzer 1988). During migraine attacks, release of
CGRP and SP could be measured in the jugular vein
(Goadsby et al. 1988, 1990), adding evidence to the neurogenic inflammation hypothesis. A link between peripheral
neuronal activity and central excitation is indicated by the
observation that SP is released in the trigeminal brain stem
nuclear complex after chemical stimulation of the meninges
(Schaible et al. 1996) and by the measurement of an enhanced level of excitatory amino acids, such as glutamate,
in the cerebrospinal fluid of migraine patients during the
attack (Martı́nez et al. 1993).
Thus all lines of evidence lead to the conclusion that
stimulation of the meninges with chemical mediators provides an important mechanism in the generation of various
forms of headaches. Substances with algogenic activity may
be released either in the course of the pathophysiological
process itself or indirectly as a consequence of the release
of neuropeptides that induce plasma extravasation.
It is known from clinical observations that subarachnoidal
rather than epidural stimuli cause intense headache (Dalessio
1989) and that the meninges at the base of the skull are
particularly sensitive. However, the few studies on sensitivity of meningeal afferents applied chemical stimuli either
into the sagital sinus or to the epidural surface (Dostrovsky
et al. 1991; Lambert et al. 1991; Zagami and Lambert 1990,
1991). Therefore the aim of the study was to describe an
experimental method for applying chemical stimuli to the
subarachnoidal space by superfusion of the basal meninges
in the anesthetized rat. We assessed input from the dura in
response to application of inflammatory mediators by single
unit recordings of secondary neurons in the subnucleus caudalis, the principle relay of orofacial pain and headache (Ses-
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Ebersberger, A., M. Ringkamp, P. W. Reeh, and H. O. Handwerker. Recordings from brain stem neurons responding to chemical stimulation of the subarachnoid space. J. Neurophysiol. 77:
3122–3133, 1997. The subarachnoid space at the base of the skull
was perfused continuously with artificial cerebrospinal fluid in anesthetized rats. A combination of inflammatory mediators consisting of histamine, bradykinin, serotonin, and prostaglandin E2
(10 05 M) at pH of 6.1 was introduced into the flow for defined
periods to stimulate meningeal primary afferents. Secondary neurons in the caudal nucleus of the trigeminal brain stem were
searched by electrical stimulation of the cornea. Of the units receiving oligosynaptic input from the cornea, 44% were excited by
stimulation of the meninges with inflammatory mediators. Most of
these units had small receptive fields including cornea and the
periorbital region, and their responsiveness was restricted to stimuli
of noxiuos intensity. Three types of responses to stimulation of the
meninges with algogenic agents were encountered: responses that
did not outlast the stimulus period, responses outlasting the stimulus period for several minutes, and oscillating response patterns
containing periods of enhanced and suppressed activity. The response pattern of a unit was reproducible, however, upon repetitive
stimulation at 20-min intervals; the response magnitude showed
tachyphylaxis upon stimulus repetition. The preparation presented
mimics pathophysiolocial states normally accompanied by headache, e.g., subarachnoidal bleeding. Responsiveness of neurons in
the caudal nucleus of the trigeminal brain stem to inflammatory
mediators may play a role in the generation and maintenance of
headache, e.g., migraine.
NEURONS RESPONDING TO CHEMICAL MENINGEAL STIMULATION
sle 1990) to give a first description of the characteristics of
these cells and their responses to chemical stimulation.
METHODS
Animal preparation
Recording
Extracellular recordings from neurons of the trigeminal subnucleus caudalis were performed with carbon fiber microelectrodes
(impedance Ç1–2 MV ). The amplified signals were recorded online with a PC-AT (interface card DAB 1200, Microstar) using
the SPIKE/SPIDI software package (Forster and Handwerker
1990). The obex was used as a landmark for the microdrive positioning of the electrode. Because of the relative high convergence
of dural and corneal afferents onto brain stem neurons (Davis and
Dostrovsky 1986, 1988; Dostrovsky et al. 1991; Strassman et al.
1986), we used corneal stimulation to search for neurons. As a
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microelectrode was advanced through the brain stem, corneal electrical search stimuli (8 V, 0.4 ms, 0.5 Hz) were delivered via a
small agar-filled metal cup softly attached to the left cornea. Units
excited by this electrical stimulation, showing action potentials in
a latency range of 5–50 ms, were characterized further and tested
for responsiveness to a chemical stimulus to the meninges. For
technical reasons, no lesions at the recording sites were performed
after the experiment.
Characterization of neurons
Low- and high-intensity mechanical stimuli were applied to the
face following a standard protocol to characterize the receptive
fields. Blowing to the animal’s face (B) and striking the hairs and
skin with a fine hair brush (H) were employed as stimuli for lowthreshold mechanoreceptors. Pricking the skin (PR) and the eyelid
(E) with a pointed watchmaker forceps and pinching skin folds
with an anatomic forceps (PI) served as noxious stimuli. The receptive field of each neuron was drawn on a schematic figure of
the rat’s face. Convergent maxillary input was assessed by noxious
cold stimuli applied to the upper incisor teeth. To test for neuronal
activity due to mechanical stimulation alone, a foam rubber pellet
was at first slightly pressed to the incisors (O). This was repeated
after cooling the pellet with dichlorodifluoromethane gas (common
vitality test in dentistry; P: noxious cold).
Chemical stimulation of the subarachnoid space
As a control, a bolus of 100 ml ACSF was channeled into the
continuous perfusion flow of ACSF 10 min before the chemical
stimulation. To chemically stimulate the meninges, a mixture of
inflammatory mediators (IM) consisting of histamine, bradykinin,
serotonin, and prostaglandin E2 each 10 05 M, pH 6.1 in ACSF was
used (see Kessler et al. 1992). To visualize the in- and outflow
and to measure the duration of the chemical stimulation, Evans
Blue was added to the control (ACSF) and to the stimulus. With
the volume (100 ml) and the flow rate (4.5 ml/h) used, the passage
through the skull lasted Ç120 s. Twenty minutes later (in 3 cases,
15 min later), the stimulus was repeated. In some cases, an identical stimulus was applied 20 min after the second one. At maximum,
five chemical stimuli were applied to one animal at intervals of
¢20 min. In each animal, only one chemically excitable cell was
recorded. To control the extension of the chemically stimulated
area (stained area) at the base of the skull, the brain was removed
in 20 animals at the end of the experiment. Additionally, the stained
meninges were peeled from the ventral part of the brain and as far
as possible from the inside of the skull.
Data analysis
Action potentials were discriminated off-line by their shape and
size using the SPIKE/SPIDI software package (Forster and
Handwerker 1990). Responses to the stimuli of the standard protocol for characterization of the neuron (see above) were qualitatively analyzed in neurons that also responded to chemical stimulation. Neuronal responses to chemical stimulation of the meninges
were treated as follows: the 2-min period before stimulation to 12
min after stimulus application was subdivided in 10-s bins. The
spontaneous activity of a neuron was quantified as the mean number of action potentials { SD from the 10-s bins of the prestimulus
period. A response to a stimulus was defined as positive when the
activity of the unit exceeded the spontaneous activity at least two
times the standard deviation during the first 4 min after stimulus
application. The first 10-s bin meeting this criterion was taken as
start of the response. The same criterion was used to define the
end of a response. In figures showing averages from several units
(Fig. 5, A and B), the spontaneous activity has been subtracted.
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Experiments were performed on adult male Wistar rats (280–
450 g). The animals were anesthetized with thiopental (120
mg/kg body wt ip). After scoploamine injection (50 mg/kg sc),
the trachea as well as the femoral vein and artery were cannulated.
Subsequently, the animals were mounted in a stereotactic frame
with the head fixed in horizontal position. The animals were ventilated artificially. The end-tidal CO2 was adjusted between 4.5 and
5.5%. The rats were paralyzed with pancuronium (initial dose 3.7
mg/kg iv; supplemental doses 1 mgrkg 01rh 01 iv). This dosage
of pancuronium was sufficient to prevent movements due to spontaneous respiration but allowed control of blink and withdrawal reflexes. The intra-arterial blood pressure and the heart rate (ECG)
were monitored continuously. Anesthesia was monitored by regularly testing the corneal blink reflex and the responses to paw
pinch. Supplemental doses of thiopental were given to maintain
areflexia (25 mg/kg ip). The body temperature was kept at a
constant level of Ç387C by a rectal thermometer and a feedbackcontrolled infrared lamp. The eyes were covered with an ointment
(Bepanthen, Roche) to prevent noxious input due to drying of the
cornea.
The skullpan was exposed, and a hole (0.8 mm diam) was drilled
that led into the cleft between the underlying frontal lobe and the
left olfactory bulb (4 mm rostral to Bregma, 2.5 mm lateral from
midline). To get access to the subarachnoid space, a catheter
(0.8 mm diam) was inserted and pushed cautiously forward (4
mm) to the base of the skull. Finally, the catheter was glued with
acrylic to the surface of the skull. Thereafter the head of the animal
was ventroflexed (907 ), and, using standard surgical procedures,
the cerebellum and the brain stem were exposed. Before starting
the perfusion, Ç80% of the cerebellum was aspirated to ensure an
unimpeded outflow of the perfusion fluid. The lesion gave sight to
the bottom of the fourth ventricle. The subarachnoid space then
was perfused with artificial cerebrospinal fluid (ACSF) consisting
of (in mEql01 ) 150 Na / , 3 K / , 2.5 Ca 2/ , 1.2 Mg 2/ , 132 Cl 0 ,
and 25 HCO3 0 plus 3.7 mM glucose and 6 mM urea, pH 7.4
(Nozaki et al. 1992) via the frontal catheter. A continuous flow of
4.5 ml/h was maintained by a syringe pump (Fig. 1A). Chemical
stimuli were injected as a bolus into the continuous flow of ACSF.
After forming a pool with the surrounding skin, the brain stem was
prevented from contacting the stimulus substances in the perfusion
fluid by a sheet of parafilm vertically placed over the fourth ventricle. A cotton pad between two sheets of parafilm guided the fluid
out of the skull where it was collected. Finally, the brain stem
was covered with agarose (1.5%). The agarose layer covering the
recording site was removed carefully from the surface of the brain
stem. At the end of the experiment, the animal was killed by an
overdose of a local anesthetic (2 ml lidocaine 2%, iv).
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A. EBERSBERGER, M. RINGKAMP, P. W. REEH, AND H. O. HANDWERKER
FIG . 1. A: rat brain is shown in position it was fixed in
experimental setup. F, passage of perfusion fluid through
skull; black bar, catheter; arrows with broken line, way of
fluid at base of skull, see also Fig. 1B where superfused
area is shown (hatched). B: minimal extension of superfused area (stained with Evans Blue) at base of skull. Fat
lines show circle of Willis; F, direction of perfusion fluid.
RESULTS
Neurons were searched throughout the whole rostro-caudal extension of the subnucleus caudalis of the trigeminal
brain stem nuclear complex in a depth of 0–2,800 mm from
the surface of the brain. Recordings from 117 units responding to the electrical search stimulus at the cornea were
stable enough for characterization with regard to the type of
input they received, to their mechanoreceptive fields in the
face, and to responsiveness upon cold stimulation of the
incisor teeth. Those units were tested subsequently by stimulation of the meninges with inflammatory mediators (see
METHODS ). Fifty-one of the neurons (43.6%) responded to
chemical stimulation of the meninges.
intensity (noxious) mechanical stimuli (PR Å prick; PI Å
pinch; E Å pinch at the eyelid).
Most cutaneous RFs were small and entirely restricted to
the innervation territory of the ophthalmic division of the
trigeminal nerve, typically including the upper and/or lower
eyelid. Because electrical stimulation of the cornea was used
as a search stimulus, RFs often included the cornea when
tested mechanically; RFs of 31 (out of 51) neurons were
confined to this region. In some cases, however, additional
RFs were found in the innervation area of the maxillary
trigeminal division, including the upper incisor teeth (in 13
of 51 units), the nostril (8 of 51 units), or the snout (6 of
51 units).
Table 1 shows a summary of these data. For each of the
three most common combinations of receptive properties,
an example of the complete characterization protocol is provided in Fig. 3. Figure 3A shows a unit with a mechanical
receptive field at the eyelid and the cornea that responded
Characterization of the neurons
LATENCIES OF THE RESPONSES TO ELECTRICAL STIMULATION
OF THE CORNEA. In neurons receiving convergent input
from meninges and cornea, the latencies between the stimulus and the first action potential ranged from 7 to 22 ms (in
1 case 47 ms; Fig. 2). Assuming mono- or oligosynaptic
input, these latencies suggest Ad- and/or C-fiber input from
the cornea because the estimated conduction distance was
Ç2 cm (mean conduction velocity 1.74 m/s). To ensure a
mono- or oligosynaptic input, only units with constant latencies to repeated stimulation were included in the sample.
Examples of spikes evoked by electrical corneal stimulation
are shown in several specimen figures (Figs. 3B and 4A).
RECEPTIVE
FIELDS
OF
CHEMICALLY
EXCITABLE
NEURONS.
Only 12 of 51 units (23,5%) driven by chemical stimulation
of the subarachnoid space responded to weak mechanical
stimuli like air puffs (in Figs. 3 and 4, B Å blowing) or
stroking the hairs in the receptive field (RF) with a soft
brush (H Å hair). In contrast, all 51 units responded to high
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FIG . 2. Distribution of latencies (mean 11.5 ms) of responses of units
to electrical stimulation of cornea resulting in a mean conduction velocity
of 1.74 m/s (n Å 46).
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A period of 12 min after the start of a response was used to analyze
the time course of the response.
NEURONS RESPONDING TO CHEMICAL MENINGEAL STIMULATION
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FIG . 3. Examples for 3 most common response patterns of the neurones in nc caudalis submitted to a standard characterization protocol. A: neuron with a mechanical receptive field at eyelid and cornea (a), action potential after electrical stimulation
of cornea (b). In c, responses to mechanical stimulation of face and to tooth pulp stimulation are shown (see METHODS ).
Unit responded only to high intensity stimuli at eyelid (E, PE) and cornea (C). F, begining of stimulus applications. B: unit
with a receptive field at eyelid and skin above eye (a), activity after electrical stimulation of cornea (b). Unit showed a
low frequent spontaneous activity throughout characterization procedure. It responded to low-intensity stimuli like striking
hairs of face with a fine brush (H) but also to high-intensity stimuli like pricking (PR) skin above eye and pricking and
pinching at eyelid (E, PE). C: neuron with a receptive field only at upper eyelid ( a), activity after electrical stimulation of
cornea (b). Unit was excited only by high-thresold stimulus at eyelid (E, PE). Abbreviations: B, blowing to face; H, striking
hairs of face with a fine brush; PR, pricking to facial skin; PI, pinching to facial skin; E, pinching to eyelid; PE, pricking
to eyelid; C, tapping to cornea; O, applying a foam pellet (room temperature) to upper incisor teeth; P, noxious cold (applying
a cooled foam pellet to upper incisor teeth).
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FIG . 4. Specimen of a neuron responding to chemical stimulation of meninges with a mixture of inflammatory mediators.
A: response to electrical stimulation of cornea (7 V, 0.4 ms). B: mechanical receptive field at upper eyelid. C: responses
to stimulation of facial receptive field. D: response to chemical stimulation of meninges with inflammatory mediators (IM,
pH 6.1). Response shows a brief onset and a relatively fast decline. ACSF, application of artificial cerebrospinal fluid as a
control; bars, period of stimulus application (Ç120 s).
to pinching and pricking of the eyelid (E, PE) and to lightly
tapping at the stimulation electrode attached to the cornea
(C). There was no response to weak mechanical stimuli
(B, H) and to noxious cold at the toothpulp (P). In contrast,
the unit shown in Fig. 3B was also excited by low-intensity
mechanical stimulation of the skin above the eye. In Fig.
3C, a unit only responding to pricking and pinching of the
eyelid is shown. Six units were of this type.
Only 1 out of 13 neurons with a facial receptive field
restricted to the maxillary region responded to chemical
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stimulation of the meninges, whereas 50 out of 100 neurons
including the ophthalmic region were activated by stimulation of the meninges.
Thirty seven of the units with input from the chemically
stimulated meninges (72%) were spontaneously active ( ú0.5
s 01 ). Twenty two of them had larger than average RFs, including skin areas outside the periorbital region in the ophthalmic
(n Å 5) or maxillary region (n Å 17). In contrast, eight meningeal units with very small receptive fields (cornea, eyelid, or
eyelid and cornea) showed no spontaneous activity ( õ0.5 s 01 ).
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NEURONS RESPONDING TO CHEMICAL MENINGEAL STIMULATION
TABLE
1.
Summary of receptive field properties encountered
Cornea
/
/
/
0
/
/
0
/
/
/
/
/
/
/
/
/
/
/
/
0
/
0
0
0
0
/
/
/
0
/
0
0
/
/
0
0
0
/
0
0
0
0
0
0
/
/
0
0
/
/
0
/
/
/
0
/
0
0
0
/
0
/
0
0
0
0
0
/
/
0
/
0
0
/
0
0
0
/
0
0
0
0
0
0
/
0
0
0
0
/
0
0
0
/
0
0
/
0
/
/
0
0
0
0
0
0
0
0
0
0
/
/
0
/
/
0
0
/
/
0
11
8
6
4
3
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
44
21
13
15
8
6
51
Skin Above
the Eye
Nostril
Snout
No. of
Cells
The last column shows the numbers of neurons that showed a specific
combination of receptive areas as indicated in the line left to the number.
Numbers in the last line of the table show how many neurons responded
to stimulation of a specific area. /, response to the applied stimulus; 0, no
response to the applied stimulus in the indicated area (stimulation procedure
described in METHODS).
Localization of the neurons
Two clusters of neurons receiving convergent input from
the meninges and the cornea were encountered, one in the
very rostral and the other one in the caudal part of the subnucleus caudalis. The rostral cluster was found in a region
extending in the rostro-caudal direction between 0–300 mm
from obex and 2,000–2,800 mm lateral from the midline.
The caudal cluster was located between 2,000–2,300 mm
caudal from the obex and 1,600–2,500 mm mediolateral
from midline. The units in both clusters were found at a
mean depth of 1,245 { 532 mm from the dorsal surface of
the brain stem, indicating a localization primarily in the
ventral part of the subnucleus caudalis.
Chemical stimulation of the subarachnoid space
LOCALIZATION OF THE AREA STIMULATED. In all experiments, Evans Blue was added either to the solutions containing the stimulating agents or to the bolus of ACSF (used
for control in this study) The extension of the stained part
of the basal meninges was examined in 20 animals postmortem. In all of these animals, at least the ipsilateral and medial
parts of the dura overlying the circle of Willis were stained
(Fig. 1B). In some cases (n Å 9), the perfusion fluid was
spreading to the contralateral site. The frontal meninges at
the olfactory bulbs and the parts of the dura covering the
convexities of the brain remained unstained. No staining was
found in the tissue underlying the meninges.
RESPONSES TO PERFUSION OF THE SUBARACHNOID SPACE. Superfusion of the meninges with ACSF did not excite any of
the units within an observation period of 10 min. In four
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neurons, slightly enhanced activity exceeding spontaneous
activity by one standard deviation was observed. However,
44% of the 117 neurons tested showed a clear response
to superfusion of the meninges with ACSF containing a
combination of IM consisting of histamine, bradykinin, serotonin, and prostaglandin E2 at pH 6.1. Figure 4 shows a
neuronal response with a quick onset and a relatively fast
decline upon passage of inflammatory mediators within
Ç120 s. In this neuron, a second application of the stimulus
at an interval of 20 min led to a similar response.
RESPONSE PATTERNS. According to their different response
patterns, the neurons were categorized in three groups: units
in which firing did not outlast the stimulus (120 s), units
showing responses that considerably outlasted the stimulus
duration of 120 s, with a slow recovery to their initial level
of spontaneous activity before the next stimulus was applied
20 min later, and units exhibiting bi- or polyphasic responses
(definition see METHODS ). Figure 5 shows histograms of the
mean responses of the units of the first and second group.
In this figure, spontaneous activity has been subtracted. In
mean longer lasting responses showed higher response rates
when shorter lasting ones.
The units belonging to the third class with long lasting
bi- or polyphasic response pattern are displayed in Fig. 6.
The responses of most of these units were biphasic with an
initial excitation followed by a depression of spike activity.
Both parts of the response were apparently not related to the
duration of the stimulus. Four out of the 16 neurons of this
class showed oscillating excitation after chemical stimulation and were therefore regarded to respond polyphasically.
Duration and frequency of the oscillating activity varied
from cell to cell.
Neurons with different chemical response patterns also
could be distinguished by their different spontaneous activity. In the first class, only 22% of the neurons (showing a
response not outlasting the stimulus duration) were spontaneously active ( ú0.5 s 01 ) in the interval 2 min before the
stimulus. More frequently, neurons of the other two classes
with the outlasting response patterns showed spontaneous
discharges exceeding one spike/2 s before the application
of inflammatory mediators (49 and 44% of class 2 and 3
units, respectively).
TACHYPHYLAXIS. Independent of the response pattern, the
units usually were excited less by repetition of the chemical
stimulus. Although the first stimulus elicited, on average,
15.43 { 6.93 impulses/10 s during the following 10 min,
the second stimulus evoked only 4.49 { 9.18 impulses/10
s. In some cases, a third stimulus was applied that yielded
similar responses as the second one. Figure 7 demonstrates
the tachyphylaxis in a unit to which three stimuli were applied at intervals of 15 min.
DISCUSSION
This study demonstrates that a proportion of neurons of
the trigeminal subnucleus caudalis responds to chemical
stimulation of meningeal structures with a combination of
inflammatory mediators. Dural afferents were stimulated by
chemicals that had direct access to the inner site of the
dura. This form of application resulted in a rapid onset of
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Eyelid
Upper
Incisor
Teeth
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responses of brain stem neurons comparable with that observed in primary nociceptive afferents studied by superfusion with a similar cocktail of mediators in a skin-nerve
preparation (Kessler et al. 1992).
Functional characteristics of neurons receiving input from
the meninges
For searching, electrical stimulation of
the cornea was used rather than chemical stimulation of
the meninges, because meningeal nerve terminals may have
become sensitized during the search procedure or, alternatively, tachyphylaxis after repeated chemical stimulation
might have concealed responses. Electrical stimulation of
the subarachnoid space was avoided to prevent the trigeminal
ganglia and nerve bundles from direct activation by current
spread. By the chosen searching procedure (electrical cornea
stimulation), a subpopulation of neurons has been selected
that has convergent input from the eye and from the meninges. The total proportion of brain stem neurons excited by
chemical stimulation of the meninges may therefore be underestimated. However, similar populations of neurons were
found when electrical search stimuli were applied to the dura
(see below, RECEPTIVE FIELDS ).
LATENCIES. The long activation latencies after electrical
stimulation of the cornea indicate input from slowly conducting myelinated or unmyelinated afferents. This was exSEARCH STRATEGY.
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pected, because the cornea mainly is innervated by thin afferent nerve fibers (Belmonte et al. 1991). Presumably the
input from the meninges also is transmitted by thin nerve
fibers, because electrical stimulation of the dura in cat revealed an exclusive Ad input from the dura to the caudal
trigeminal nucleus (Davis and Dostrovsky 1986, 1988; Dostrovsky et al. 1991; Strassman et al. 1986). These data also
are supported by recordings in the subnucleus caudalis and
in the trigeminal ganglion in rat where the response latencies
to electrical stimuli reflect input from Ad-fibers (Messlinger
et al. 1995; Strassman et al. 1995).
RECEPTIVE FIELDS. Ninety percent (46/51) of the neurons
activated by chemical stimulation of the meninges had facial
receptive fields restricted to the innervation territory of the
ophthalmic division of the trigeminal nerve. This observation
is consistent with receptive field locations reported for neurons searched by direct stimulation of the dura after craniotomy (cat: Davis and Dostrovsky 1986, 1988; Dostrovsky et
al. 1991; Lambert et al. 1991; Messlinger et al. 1995; Strassman et al. 1986; human: Wirth and Van Buren 1971). Clinical experience also shows that headache often is localized
to some extend behind or above the eye and, vice versa,
ophthalmic diseases like glaucoma often are accompanied
by severe headache. Additional receptive areas were observed in the maxillary trigeminal division in 20 neurons.
As described by Sessle et al. (1986), caudalis neurons may
receive input from up to six different areas of receptive fields
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FIG . 5. Averaged responses (minus spontaneous activity) to inflammatory mediators; 2 populations of units with
different response patterns were found. Each column represents a 10-s bin width. Bar indicates application period of
stimulus ( Ç120 s). A: mean response of neurons that were
activated for longer than stimulus duration. B: mean response of neurons, activity of which did not outlast stimulus
duration.
NEURONS RESPONDING TO CHEMICAL MENINGEAL STIMULATION
3129
including ophthalmic, maxillar and mandibular cutaneous
regions, intranasal and intraoral structures, and deep tissue
like the masseter muscle.
Because extracranial and intracranial structures such as
meninges are not likely to be innervated via collaterals of
branching primary afferents (Borges and Moskowitz 1983;
McMahon et al. 1985), the complex receptive fields of the
neurons result from convergent inputs onto brain stem neurons from different primary afferents innervating extra- and
intracranial structures.
The notion that units in the nucleus caudalis mediating
meningeal input are getting mainly nociceptor input from the
body surface is corroborated by the finding that the observed
receptive fields had a high activation threshold and were
relatively small, suggesting that they had not been much
altered in their size by sensitizing processes (compare with
Hu et al. 1992; Sessle et al. 1986). The activation threshold
and the receptive field size of the neurons were not tested
at the end of the experimental procedure. Units with larger
receptive fields and additional input from low-threshold
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mechanoreceptors often exhibited spontaneous activity as
also described for unit populations with input from deep
somatic tissue and/or skin in brain stem and spinal cord
(Gilette et al. 1993; Hu 1990).
Recording sites
In this study, neurons with convergent inflow from the
basal meninges, the cornea and/or the ophthalmic region
were found in the very rostral and caudal part of the subnucleus caudalis of the trigeminal brain stem complex. Because the trigeminal subnuclei are spindle shaped and overlap in their transition zones, it cannot be excluded that some
of the recorded neurons belonged to subnucleus interpolaris
or the cervical spinal cord (C1). There are no data from
electrophysiological studies in the rat concerning the location of brain stem neurons with meningeal input. In accordance with studies in the cat, we found neurons with meningeal input in the deeper laminae of the caudal trigeminal
nucleus (Davis and Dostrovsky 1986, 1988; Strassman et
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FIG . 6. Activities of neurons (minus spontaneous activity) in nc caudalis that responded bi- or
polyphasically to stimulation with IM. rrr, beginning and end of application (duration 120 s). Each
column represents a 10-s bin.
3130
A. EBERSBERGER, M. RINGKAMP, P. W. REEH, AND H. O. HANDWERKER
FIG . 7. Repeated responses of a neuron that
exemplifies tachyphylaxis to chemical stimulation
of meninges with IM. Different line plots show
response of unit to first, second, and third stimulus
given to animal at a 15-min interval for a duration
of Ç2 min.
1989b; Steiger and Meakin 1984). Very few of them project
to the brain stem via the maxillary and mandibular division
of the trigeminal nerve.
It needs to be considered that the chemical stimulation
procedure could have activated the brain stem neurons rather
directly than synaptically by the activation of meningeal
afferent fibers. This is unlikely for the following reasons.
First, no staining could be seen on the surface of the brain
stem after peeling off the stained meninges from the underlying tissue. Short stimulus duration and continuous flow of
the fluid may have prevented a passing of the algogenic
molecules through the meninges to the underlying tissue.
Second, only 1 out of 13 neurons with a facial receptive
field restricted to the maxillary region responded to chemical
stimulation of the meninges whereas, 50 out of 100 neurons
including the ophthalmic region were activated by stimulation of the meninges. Thus the neurons were activated by
the chemical stimulus only in a region with substantial input
from the meninges (via the ophthalmic branch). It is most
likely, therefore, that the responses of the brain stem neurons
were evoked synaptically by activation of meningeal afferents rather than by direct stimulation.
Sites of chemical stimulation
The staining of the passage way of the test solutions
through the subarachnoid space showed that at least the ipsilateral part of the circle of Willis was exposed to the chemical stimulus. The innervation of the meninges is particularly
dense around the large vessels at the base of the skull (Edvinsson et al. 1981, 1987; Suzuki et al. 1989). Presumed
sensory fibers containing SP and CGRP form a dense network close to dural and pial vessels (Edvinsson et al. 1989;
Fang 1989; Keller and Marfurt 1991; Matsuyama et al. 1986;
Yamamoto et al. 1983). Single beaded fibers run along the
vessel wall or terminate in the connective tissue between
blood vessels (Messlinger et al. 1993). Most of these fibers
run within the ophthalmic division of the trigeminal nerve
to the trigeminal ganglion (Keller et al. 1985; Mayberg et
al. 1984; O’Connor and Van Der Kooy 1986; Suzuki et al.
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Chemical stimulus
Inflammatory mediators as used in the present study are
presumably involved in the headache accompanying meningitis or subarachnoid hemorrhage where they can be assumed
to be bloodborne. Additionally, in the last decade, migraine
headaches often have been considered to be due to a neurogenic inflammation of the meninges (Goadsby 1993; Lauritzen 1994; Moskowitz and MacFarlane 1993) by plasmaextravasation. This again would lead to exposure of meningeal
nociceptors to inflammatory mediators. The present experiments have shown an intense activation of second order
neurons in the subnucleus caudalis by inflammatory mediators, indicating a dominant role of these nociceptive brain
stem neurons in headache. The inflammatory mediators applied in this study to the meninges are substances that indeed
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al. 1986). There is some controversy in these previous studies upon the rostro-caudal extension of the neurons. Immunohistochemical studies (HRP, c-Fos) favor a distribution
of neurons with meningeal input throughout the caudal trigeminal nucleus (Arbab et al. 1988; Nozaki et al. 1992;
Ruiz-Pesini et al. 1995; Strassman et al, 1994) whereas we,
like Strassman et al. (1986), found units with meningeal
input especially in the transition zone to subnucleus interpolaris and the spinal dorsal horn. These differences in the
location may be due to the selection of units with meningeal
input using electrical stimulation of the cornea for search.
Morphological data in the rat and cat show that corneal
afferents project to the ventrolateral caudal nucleus, the transition between the interpolar and caudal nucleus and the C1/
2 area (Lu et al. 1993; Marfurt and Del Toro 1987; Nagano
et al. 1975; Shigenaga et al. 1986). However, this distribution was not confirmed in electrophysiological studies in the
rat (Nagano et al. 1975; Pozo and Cervero 1993). Combined
morphological and physiological evidence indicate that the
existence of a single narrow projection site for dural afferents
is not very likely.
NEURONS RESPONDING TO CHEMICAL MENINGEAL STIMULATION
can be found under pathophysiological conditions as during
neurogenic inflammation. Serotonin, for example, can be
released from perivascular sympathetic nerve endings (Stanley et al. 1993), from platelets and mast cells after release
of neuropeptides by activation of sensory afferents (Dimitriadou et al. 1992; Kraeuter Kops et al. 1990; Öhlèn et al.
1989). All of the substances are able to excite or sensitize
primary afferents (Kessler et al. 1992; Lang et al. 1990) as
well as postsynaptic neurons, resulting in lowered stimulus
threshold, expansion of the receptive fields, and increased
spontaneous activity (Woolf 1994). Thus inflammatory mediators released in meningeal structures (see Goadsby et al.
1990; Juul et al. 1995; Moskowitz et al. 1983; Saito et al.
1987; Zagami et al. 1989) should be able to cause headache
by activation of trigeminal brain stem neurons.
Excitation of neurons in the nucleus caudalis of the cat
was observed by Dostrovsky et al. (1991) after application
of bradykinin into the sagittal sinus. Epidural application of
capsaicin and bradykinin also caused activation of thalamic
neurons (Lambert et al. 1991; Zagami and Lambert 1990,
1991). In accordance, the present data show that 44% of
the nucleus caudalis neurons recruited by electrical stimulation of the cornea also responded vigorously to stimulation
of the subarachnoid space with a mixture of inflammatory
mediators.
In an in vitro skin-nerve preparation, a similar mixture
activated 85% of the polymodal primary nociceptive afferents (Kessler et al. 1992). In this test solution, the excitatory
action seemed to be mainly due to the presence of bradykinin
(Lang et al. 1990) and a low pH (Steen et al. 1995). In
contrast, histamine, and serotonin are described to play a
minor role in excitation. At present similar studies on primary meningeal afferents are lacking.
RESPONSE PATTERNS. After stimulation with acidic inflammatory mediators, three different response types were
found: responses outlasting or not outlasting the stimulus
duration and responses of bi- or polyphasic character. It is
unclear to what extent these response patterns reflect the
peripheral input and/or central modifications. Single polymodal receptors in the skin, in vitro, showed a large variability in the response patterns after stimulation with bradykinin
(Lang et al. 1990), whereas responses to pH 6.1 tended to
last as long as the stimulus was present, showing little or
no adaptation (Steen et al. 1992). If meningeal afferents
behave similarly, a test solution with low pH should cause
sustained responses as observed in all of the three response
patterns. However, the perfusion of the subarachnoid space
has the disadvantage that the duration and temporal position
of a stimulus depended on the exact location of the receptive
field of a neuron. In spite of this, most of the neurons with
the stimulus-outlasting response pattern definitely fired for
longer than algesic substances could have been present in
the subarachnoid space ( ú10 min). A similar time course
has rarely been found in primary afferents of the skin. Although nothing is known about afferents of the subarachnoid
space in this respect, it may be that the long duration of the
response probably was generated, at least in part, by the
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second order neurons. Such a phenomenon of a much slower
decline of the discharges in secondary dorsal horn neurons
as compared with primary afferent fibers has been observed
after heat stimuli (Handwerker et al. 1975).
TACHYPHYLAXIS. Induction of tachyphylaxis was observed
regularly upon repeated application of inflammatory mediators. In the skin, a lack of tachyphylaxis to inflammatory
mediators was described when the pH of the solution was
lowered to 6.1 (Steen et al. 1995). However, tachyphylaxis
was well present in an in vitro preparation of visceral afferents from the rabbit pleura upon repeated stimulation with
the mixture of inflammatory mediators at pH 6.1 (Weidner
et al. 1996), which might be a better model for dural afferents. In addition, low pH was probably not safely maintained
in our preparation throughout the stimulus duration due to
neutralization by the buffering capacity of the subarachnoid
space. When bradykinin alone or a mixture of inflammatory
mediators were applied to primary cutaneous afferents at
neutral pH pronounced tachyphylaxis was observed regularly (Kessler et al. 1992; Lang et al. 1990). Therefore it is
very likely, that the tachyphylaxis observed in the present
study was due to altered primary afferent activity. As shown
in Figs. 7 and 4D, the shape of a response was not much
altered by repeating the stimulus. Therefore it is not likely
that cells with a rapid decrease in response (not outlasting
the stimulus duration) can be explained by tachyphylaxis.
In conclusion, an experimental model is described that
allows to study neuronal mechanisms involved in nociceptive processes in the meninges. In the present experiments,
we have demonstrated that the application of inflammatory
mediators is a powerful stimulation to excite neurons in the
trigeminal nuclear complex. The receptive fields of these
neurons are located mainly in regions supplied by the ophthalmic branch of the trigeminal nerve and reflect locations
of pain often observed in headache patients. Because headaches have been associated with the occurrence of pathophysiological processes in the meninges (e.g., neurogenic
inflammation), we believe that the experimental model described here is suitable to investigate mechanisms of certain
forms of headaches.
We thank Prof. H.-G. Schaible for critically reading the manuscript, A.
Linhardt and R. Beuscher for technical assistance, and K. Burian for expert
help with the art work.
This project was supported by the Deutsche Forschungsgemeinschaft,
SFB 353.
Address for reprint requests: A. Ebersberger, Dept. of Physiology II,
University of Wuerzburg, Roentgenring 9, 97070 Wuerzburg, Germany.
Received 23 July 1996; accepted in final form 11 February 1997.
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