3041 Journal of Physiology (1995), 485. 1, pp.

203-212 203

Localization of chemosensitive structures in the isolated

brainstem of adult guinea-pig
M. P. Morin-Surun, E. Boudinot, T. Schafer and M. Denavit-Saubie
Institut Alfred Fessard, Biologie Fonctionnelle du Neurone, CNRS,
91198 Gf sur Yvette Cedex, France
1. Central respiratory chemosensitivity has been intensively examined but some questions
remain unsolved; namely, what is the nature of the stimulus (fixed acid and/or C02) and
where is the site of brainstem chemosensitivity (near the ventral medullary surface or
structures deeper within the brainstem)? To examine these questions, we used the in vitro
isolated brainstem of adult guinea-pig perfused independently through the basilar artery
and the bath.
2. Respiratory motor output was recorded with a suction electrode from cranial hypoglossal
(XII) roots. Changes in pH and CO2 in the Krebs perfusate were made by changing either
the bicarbonate concentration or the Pco, saturating the Krebs solution.
3. Changes in basilar artery perfusate consisting of (i) an acidifying increase in PCO, (hyper-
capnic acidic Krebs solution), (ii) an increase in Pco, with no change in pH (hypercapnic
Krebs solution), or (iii) a decrease in pH with no change in Pco, (acidic Krebs solution)
evoked increases in respiratory frequency and a concomitant decrease in inspiratory burst
amplitude.
4. Bath superfusion with hypercapnic acidic Krebs solution increased the inspiratory burst
amplitude with no effect on respiratory burst frequency.
5. Bath superfusion with hypercapnic non-acidic Krebs solution increased the inspiratory
burst amplitude and decreased the respiratory frequency, while normocapnic acidic Krebs
solution increased the respiratory frequency with no change in burst amplitude.
6. These results show that respiratory responses to changes in CO2 and pH depend upon the
sites of action. While a CO2 increase or a pH decrease affected the respiratory frequency in
the deep brainstem structures (perfused through the basilar artery), CO2 respiratory
chemosensitivity at the ventral surface could be differentiated from the hydrogen ion
chemosensitivity. This suggests that different mechanisms mediated respiratory responses
when deep versus superficial brainstem structures were stimulated.

It has been well established that after deafferentation of
peripheral chemoreceptors, central respiratory chemo-
sensitivity persists in the brainstem. However, questions
regarding the location and the stimuli involved in central
respiratory acid-base homeostasis have not yet been
resolved.
The central chemosensitive mechanism has been ascribed
to defined areas on the ventral medullary surface
(Mitchell, Loeschcke, Massion & Severinghaus, 1963;
Schlaefke, See & Loeschcke, 1970). However, recent
studies have shown that other areas within the brainstem
could also be responsible for central respiratory responses
to CO2 (Arita, Kogo & Ichikawa, 1988; Konig & Seller,
1991; Sato, Severinghaus & Basbaum, 1992). Several
attempts have been made to record from cells involved in
this respiratory regulation within the brainstem and to
relate changes in their firing rate to changes in the acidity
of the brainstem induced by CO2 inhalation (Cohen, 1968;
Mitchell & Herbert, 1974; Trouth, Patrickson, Holloway &
Wright, 1982; Arita, Kuwana, Ichikawa & Kogo, 1989;
Kogo & Arita, 1990). One particular stimulus may either
activate, inhibit or have no effect on the cells (Arita et al.
1989). However, it is unknown whether or not the
activation or inhibition of neuronal firing rate is due to a
direct effect on chemosensitive neurones or is dependent
upon synaptic connections. Recently Takeda & Haji
(1991) have shown that most respiratory neurones located
in the ventral respiratory group (nucleus ambiguus) have
a postsynaptic response to a hypercapnic ventilatory
stimulation. On the other hand, in the brainstem slice
preparation, Dean and collaborators (Dean, Bayliss,
Erickson, Lawing & Millhorn, 1990) have described a
depolarization of cells recorded in the nucleus tractus
solitarius after bath superfusion of a hypercapnic Krebs
204 M. P. Morin-Surun and others
solution, even after synaptic blockade. These different
results reopen the question about the location of
brainstem chemosensitive sites.
Regarding the nature of the central chemosensitive
stimulus, it has long been thought that the hydrogen ion
concentration of the medullary extracellular fluid is of
primary importance rather than the content of carbon
dioxide in the extracellular space (Loeschcke, 1982;
Millhorn & Eldridge, 1986). Recent evidence, however,
indicates that C02 plays an independent role. Using the
isolated brainstem preparation of the newborn rat,
Harada, Kuno & Wang (1985) have differentiated
between the respiratory effects of hydrogen ions and
molecular C02. Similarly, in tissue culture, Neubauer,
Chou, Gonzales, Sterbenz, Geller & Edelman (1991) have
demonstrated that ventral brainstem neurones respond
directly to C02.
Recently, we have developed a perfused isolated brain-
stem preparation of the adult guinea-pig which maintains
a functional respiratory network (Morin-Surun, Boudinot,
Sarraseca, Fortin & Denavit-Saubie, 1992). In this
preparation it is possible to separate the ventral surface
superfusion (accessed by the bath) from the extracellular
fluid perfusion (accessed by the basilar artery) (see
Methods). Furthermore this preparation allows us to
control the metabolite concentration of the perfusion
medium.
The aim of the present study was to separate the
respiratory effects of CO2 and pH in different brainstem
areas. Preliminary reports of this work have been
presented in symposia (Denavit-Saubie, Morin-Surun,
Jacquin, Schiifer & Boudinot, 1993; Morin-Surun, Schiifer
& Denavit-Saubie, 1994)
METHODS
In vitro perfused isolated brainstem preparation of adult
guinea-pig
Data were obtained from twenty-five adult guinea-pigs
(200-320 g) anaesthetized with sodium pentobarbitone
(30 mg kg' i.P.), decapitated at the first cervical vertebra and
decerebrated at the intercollicular level. The brainstem with the
cerebellum was rapidly removed from the cranium (within
2-3 min). During this surgical procedure, cold (4 °C), oxygenated
Krebs solution was dripped onto the exposed brain surfaces. The
brainstem was held in place by pins, ventral surface upward, in a
recording chamber filled with oxygenated Krebs solution at room
temperature. The basilar artery was rapidly (within 2-3 min)
cannulated with a catheter inserted at the level of the pons, and
the vascular system was perfused (1X6-2 ml min-) with
oxygenated Krebs solution at room temperature using a
peristaltic pump (Minipuls 2, Gilson Medical Electronics SA,
France). Caudal ends of arteries at the first cervical segment of
the spinal cord were ligated (within 2-3 min). The pia mater was
completely removed from the ventral surface. The entire surface
of the brainstem was immersed and continuously superfused at a
constant rate (2-2-5 ml min-). The control Krebs solution
J. Physiol. 485.1
consisted of (mM): NaCl, 124; KCl, 5; MgSO4, 2-2; KH2PO4,112;
NaHCO3, 26; CaCl2, 2; glucose, 10; and was saturated with 95%
02 and 5% C02 (pH 7.38 + 0 06). Respiratory-related activity
was monitored from the hypoglossal roots through a glass suction
electrode. Nerve activity was amplified and filtered (10-100 Hz)
with a preamplifier. The half-wave activity of the hypoglossal
roots was integrated through a passive RC integrating circuit
(time constant 20 ms). All signals were displayed on an
oscilloscope and simultaneously recorded on digital tape and a
thermal paper recorder.
Changes in Po, and H+ concentration of the Krebs
perfusate
The hydrogen ion concentration of the perfusate was adjusted by
changing the bicarbonate (NaHCO3) concentration to 10 mm for
acidic Krebs solution (pH 6 9-7 2) and to 60 mm for alkaline
Krebs solution (pH 7 6-7 8). The osmolarity of the Krebs
solution was maintained by adjusting the NaCl concentration.
Increases in Pco2 were obtained by increasing the percentage of
C02 (FCO2) bubbled through the Krebs solution. Hypercapnic
acidic Krebs solution was obtained by increasing Fco2 (from 5%
to 9-13%) giving an acidic pH (pH 6 9-7 2). Hypercapnic Krebs
solution without change in pH (hypercapnic non-acidic Krebs
solution) was obtained by increasing FCO2 (from 5% to 9-13%)
and increasing [NaHCO3] to 60 mm. The FCO2 in the gas mixture
equilibrating the Krebs solution was measured with an infrared
C02 gas analyser.
Durations of different perfusate applications through the basilar
artery were estimated after obtaining a respiratory effect that
was reversible within -15 min. Basilar artery applications of
acid or alkaline Krebs solution were effective after 1 min.
Hypercapnic acid and hypercapnic non-acidic Krebs solution
applications were effective after 3 min. Bath superfusion was
effective after 15 min. The long delay of the response in bath
superfusion is due to the experimental conditions. When we
measured the bath concentrations of H+, we observed that the
superfusion had to be continued for 15 min before the entire
volume of the bath was completely replaced (see Fig. 5). Control
ionic composition reappeared within 35 min of superfusion with
control Krebs solution.
Data analysis
The phasic neural activity recorded from hypoglossal roots
consisted of a single burst followed by low-amplitude tonic
activity. The burst of activity represents the inspiratory-related
phase and the low tonic activity the expiratory-related phase
(Fukuda & Honda, 1982; Hwang, St-John & Bartlett, 1983). One
cycle was considered to begin at the onset of the inspiratory
burst and to end at the onset of the next inspiratory burst. The
integrated hypoglossal nerve activity was digitized with an
A/D-converter CED 1401 (Cambridge Electronic Design Limited,
Cambridge, UK) to calculate the following respiratory variables:
respiratory burst frequency, duration of the inspiratory and
expiratory phases, and amplitude of the integrated neural
activity. For each experiment, respiratory variables were
averaged over ten consecutive cycles during control conditions
and at the time of the maximal effect. The maximal effect for
each protocol was expressed as a percentage of the control value.
All data are given as means + standard error of the mean.
Statistical analysis was performed using Student's t test for
related measures and data were judged to be significant when
P< 0*05.
J. Phy8iol. 485.1 Central chemosensitivity in isolated brainstem 205

RESULTS Figure 2 illustrates the mean values of the maximal

Respiratory effects induced by changes in pH
and/or Pco, of the vascular perfusion
Figure 1 illustrates the pattern of the inspiratory activity
recorded from the hypoglossal roots of the brainstem
preparation. Under control conditions, the respiratory
variables showed slight burst-to-burst variations. When
the control Krebs solution perfusing the basilar artery
was replaced (for 3 min) by a hypercapnic acidic Krebs
solution, the respiratory burst frequency increased
significantly with a concomitant decrease in the
respiratory integrated neural activity.
pH 6-9
Fco'13%
respiratory effects after the various modifications to the
perfusion solutions. Hypercapnic acidic Krebs solution
(FCO2 11P3 + 0-6%, pH 6-97 + 0-06, n = 5) increased the
maximal respiratory burst frequency by 118 + 16%
(P< 0-01, n = 5,) within 3 min of the arrival of the test
solution to the brainstem, whereas the maximal decrease
in the burst amplitude (284 + 45%, P< 001, n = 5)
appeared within 5 min. The effect on respiratory burst
frequency was essentially due to a decrease in the
expiratory duration (59X8 + 3-8%, P< 0 01) together
with inconsistent change in the inspiratory duration
(1P7 + 6-2%). The control values recovered within
7-10 min of return to the control medium.
3 min

Int XII uJ%

pH 7-4
Fc°2 1 1 %

Int XII L"

Figure 1. Effects of intrabasilar artery perfusion on integrated respiratory activity recorded
from hypoglossal nerve activity (nt XII)
Horizontal bars indicate the duration of the application of the medium into the perfusion. The values of
the FCO, and the pH perfused are indicated over the bars. Control values were FCO, 5% and pH 7'4.
Upper trace, respiratory variation during hypercapnic acidic Krebs solution application. Lower trace,
respiratory variation during hypercapnic non-acidic Krebs solution application.
206 M. P. Morin-Surun and others J. Physiol. 485.1

Vascular perfusion Bath superfusion

** ***
120 120 -

o 80 80 -
-0.-
C.)
40 -
0)
cmc
C
cr
-
0)
40-

0-
I*
0 LJi

-40 -40-

o 40- 40 -
0..a

a) C 0 0

cCX
E
-s -40 - U
-U
-40 J

Figure 2. Effects of perfusate on respiratory variables

Maximal variations in respiratory frequency and amplitude induced by the different Krebs solutions in
the vascular system perfusion and the bath superfusion. O, hypercapnic + acidic pH; 0, hypercapnic
+ normal pH; U, normocapnic + acidic pH. Mean values (+ S.E.M.) are given as a percentage of the
control values. ***P< 0 01, **P< 0-02.

Int XII
, -. MI I 1 ;
pH 6-9 1 min

Int XII IIL- LLL

pH 7-7

Figure 3. Respiratory effects of an acidic and alkaline solution perfused through the basilar
artery
Recordings are effected on the same preparations. Upper trace, increase of respiratory frequency,
recorded as integrated hypoglossal nerve activity (Int XII), after intrabasilar artery perfusion of acidic
solution for 1 min (horizontal bar). Lower trace, decrease of respiratory frequency after alkaline
perfusion for 1 min (horizontal bar). Thirty minutes elapsed between both applications; the pH of the
control medium was 7-3.
J Physiol. 485.1 Central chemosensitivity in isolated brainstem 207

When the isolated brainstem was perfused through the burst frequency (53 9 + 17-8%, P< 0 01, n = 4) with a
basilar artery with hypercapnic non-acidic solution (Fco, slight and inconsistent increase in the amplitude of the
10 3 + 0 5%, pH 7-32 + 0-02, n = 5), the respiratory burst inspiratory burst (7 1 + 5-8%; Fig. 3). This decrease of
frequency increased significantly (115-3 + 15-7 %, the respiratory burst frequency was due to an increase in
P< 0. 01, n = 5; Fig. 2, left-hand histogram). As with a the expiratory duration (88'1 + 36'0%, P< 0 01) and a
hypercapnic acidic medium, this effect was rapidly slight but significant increase in the inspiratory duration
followed by a significant decrease in the amplitude (i1O0 + 6'1 %, P < 0'02). Respiratory variables recovered
(23f7 + 5-2 %, P< 0 01, n = 5) of the inspiratory burst to control values within 9 0 + 09 min.
(Fig. 1). The increased respiratory frequency was due to a
decrease in the expiratory duration (55'2 + 7 9%, Respiratory effects induced by changes in pH
P< 0 01) together with little change in the inspiratory and/or Pco of the bath superfusion
duration (-5-48 + 6-4%). Recovery to control values Bath superfusion of hypercapnic acidic medium (Fco.
appeared within 8 min of return to the control medium 10'36 + 0-67%I pH 7 07 + 0 03, n = 5) for 15 min
(8-1 + 1P3 min). increased the inspiratory burst amplitude (Fig. 2). The
maximal increase (17-8 + 6-0%, P< 0-02, n = 5)
A 1 min application of acidic Krebs solution perfused appeared within the first 10 min (9'9 + 1-3 min) of
through the basilar artery (Fco, 5%, pH 6-99 + 0-02, superfusion of the test solution. During this time, the
n = 4) increased the frequency with a decrease in the burst frequency was unaffected (457 + 417%, n = 5).
amplitude of the inspiratory burst (Figs 2 and 3). This After 25 min of wash, the burst amplitude had nearly
increase in the respiratory frequency was due to a recovered to control values.
simultaneous decrease in both the expiratory duration
(38'6 + 9-6%, P< 0 01) and the inspiratory duration To dissociate the effects of FCO, and H+ on the ventral
(19-9 + 4-2%, P< 0 01). Recovery to control values was surface, we superfused a hypercapnic non-acidic medium
observed within 8 min (7-2 + 2 min) of return to the (FcO2 9'4 + 0 07 %, pH 7'34 + 0'03, n = 5) for 15 min. The
control medium. respiratory effects were different from those observed
after superfusion with the hypercapnic acidic medium
Alkalinization of the medium perfusing the basilar artery and are illustrated in Fig. 4A.
(FcO2 5%, pH 7-8 + 0 03, n = 4) decreased the respiratory

A FCO2 9-6%, pH 7-3

LLLI 11-d LIIIIIUIJNI.-IJA LM..Li.J L

.1-.& Llk-J.A IL-LILAU J.-I -1 -L LLj m . I -1

Int XII -, -.- -r -r

00000

B Fc0025%, pH 68
t = 30 min t = 60 min

Int XII .-I I L L-1- I I I II ILL-it I-JLL-,] I I I-LLA- I I-A Lit L-1 iljltl 1,11 000000
II
"Alu"AllnuJIMSMAIII 0 0 00 0 0

5 min

Figure 4. Respiratory effects of bath superfusion

Differential respiratory effects during bath superfusions of a hypercapnic non-acidic (A) and a
normocapnic acidic solution (B). Horizontal bars indicate the duration of the superfusion (15 min). Fco2
and pH values of the superfused medium are presented over the bars; Fco2 of the control medium was
5%.
208 M. P. Morin-Surun and others J. Physiol. 485.1

On average, we observed a significant increase in the DISCUSSION

burst amplitude (33 9 + 11P3%, P< 0-02, n = 5) together
with a significant decrease in the respiratory burst
frequency (31i6 + 5 5%, P< 0-01, n = 5) within the first
15 min (Fig. 2) with no significant change in the
inspiratory duration. After 60 min of wash, the
respiratory activity returned to control values.
Figure 4B illustrates the effects of pH on the hypoglossal
respiratory burst activity. On average, a 15 min
superfusion of normocapnic acidic medium (Fco2 5 %,
pH 6-99 + 0-02, n = 4) induced a long-lasting (10-40 min)
increase in the respiratory frequency (27-1 + 06 %,
P< 0.01, n = 4) without a significant change in the burst
amplitude (Fig. 2). This increase resulted from a decrease
in the expiratory duration (42-0 + 341 %, P< 0.01, n = 4).
Conversely, the respiratory frequency was decreased
(28 2 + 1P9%, P< 0 01, n = 4 ) after a 15 min superfusion
of alkaline normocapnic medium (Fco, 5%, pH 7-80 + 0 03,
n = 4) (Fig. 5). The maximal decrease appeared at the
same time as the plateau pH reached the steady-state pH
in the bath solution. This decrease was due to an increase
in the expiratory duration (45 4 + 2 7 %, P < 0 01, n = 4)
with little change in the inspiratory duration. The
decrease in burst frequency was accompanied by an
increase in the amplitude of the inspiratory burst
(15-3 + 1P2%, P< 0.01, n = 4).
In this study, we show that the isolated perfused adult
brainstem retains a central respiratory chemosensitivity.
This chemosensitivity may regulate the respiratory
rhythmicity by different mechanisms, depending upon
the site of action and the nature of the stimuli.
Oxygen diffusion measurements in this preparation have
shown that bath superfusion irrigates only the first
300 /sm of superficial tissue while the basilar perfusion
irrigates all brainstem structures (Schiifer, Morin-Surun &
Denavit-Saubie, 1993). When the brainstem was perfused
via the basilar artery, C02 and pH produced the same
changes in respiratory burst activity. This indicates first
that central chemosensitivity exists within the brainstem
and second that the nature of the chemostimuli within the
brainstem structures are not discernable by their specific
effects on the respiratory related variables, suggesting
that both stimuli act on the same target to achieve
chemosensitive regulation. On the other hand,
superfusion of the ventral surface with Krebs solution
equilibrated to different PCO2 and pH levels produced
distinct effects on the respiratory variables: acidic pH
stimulated respiratory burst frequency whereas increased
C02 levels increased the amplitude of the inspiratory
burst, concomitant with a decrease in the burst frequency.
This suggests that C02 and pH act as distinct stimuli on

A
7-8 -

pH

7-4 -

0 10 20 30 40 50 min

B
FC2 5% pH 7-8 5 m

1 _-

Int XII
C°2i '..L
5LLIL iLflhLn
1. -

Figure 5. Time course of the diffusion of a Krebs solution alkalinization in the bath
superfusion and the induced respiratory effect
A, time course of the pH values in the bath during 15 min of an alkaline Krebs solution application,
indicated by the horizontal bar. B, integrated hypoglossal nerve activity (Int XII) during the same
time. Control values were Fco, 5 % and pH 7.4.
J. Phy-siol. 485.1 Central chemosensitivity in isolated brainstem
the ventral surface to modulate respiratory burst
activity. This difference between respiratory responses
evoked by C02 acting within the brainstem and on the
ventral surface suggests the existence of different
chemosensitive mechanisms in deep and superficial
structures.
In vivo, perfusion of subarachnoid spaces with solutions
of low pH and high C02 increased ventilation and
contributed to proof of the existence of central respiratory
chemosensitivity (Schlaefke, 1981; Loeschcke, 1982;
Millhorn & Eldridge, 1986). CO2 (9-13%) and pH (7-7 8)
levels used in this study represent the extreme of values
observed in vivo. Similar pH fluctuations in vivo produced
respiratory responses many times larger than those
documented here. Several factors could account for the
quantitative differences in responsiveness observed in
vivo and in vitro. The extreme values of pH required to
evoke responses in the in vitro preparations (Kaila,
Voipio, Paalasmaa, Pasternack & Deisz, 1993) may be
related to the complete isolation of the brainstem
structures from any peripheral and central inputs.
Furthermore, the in vitro preparation is maintained in
survival conditions at 27 0C to reduce the metabolic rate
of the brain tissue, rendering the preparation stable for
a long period of time. It is well known that the
chemosensitive respiratory response is temperature
dependant. Decreases in the ventral surface temperature
decreased the ventilatory response to hypercapnia
inhalation (Cherniack, von Euler, Homma & Kao, 1979).
Respiratory responses to vascular and ventral
surface perfusions
In this study, superfusion of the different test solutions
over the ventral surface modified respiratory parameters.
These results confirm that respiratory chemosensitivity is
also present on the ventral medullary surface in the
isolated brainstem of the adult guinea-pig. As in vivo
(Schlaefke, 1981; Loeschcke, 1982; Millhorn & Eldridge,
1986), a hypercapnic acidic medium applied to the ventral
surface increased the respiratory burst amplitude which
corresponds to either an increased recruitment or a
depolarisation of neurones in the respiratory network.
Three different chemosensitive areas have been described
on the ventral medullary surface. Organized as a rostro-
caudal column, they are the rostral, the intermediate and
the caudal chemosensitive fields, named M, S and L,
respectively (Schlaefke, 1981; Loeschcke, 1982; Millhorn
& Eldridge, 1986). Bilateral cold block of the intermediate
area (S) causes apnoea when aortic and sinus nerves are
cut (Schlaefke et al. 1970). This effect has led to the
working hypothesis that the information from both
chemosensitive fields (rostral (M) and caudal (L) areas)
converges within the intermediate area (S), from where it
is relayed to the respiratory integrative network located
deeper within the brainstem. In the present study, the
time course of the respiratory response is related to the
209
time course of the changes in the bath concentrations of
C02 and pH (see Fig. 5). Since superfusion of a hyper-
capnic acidic medium can only activate cells located
within 300 ,um of the ventral surface, the effect on
respiratory neurones in the ventral respiratory group
could be due either to synaptic inputs coming from specific
chemosensitive cells located at the ventral surface, or to a
direct effect of C02 on the rostrally projecting dendritic
arborization of neurones comprising the Botzinger
complex (Grelot, Bianchi, Iscoe & Remmers, 1988; Nattie
& Li, 1990).
A change in Pco, and pH of the Krebs solution perfusing
the basilar artery consistently evoked changes in the
respiratory burst frequency. In vivo, intravertebral artery
injection of C02-saturated saline associated with an acidic
shift of extracellular fluid pH evokes a transient increase
in the respiratory frequency (Ichikawa, Kuwana & Arita,
1989). Furthermore, modification of the respiratory
frequency is observed in response to C02 inhalation in
chronically instrumented cats when the ventral surface is
destroyed (Schlaefke, See, Herker-See & Loeschcke, 1979).
Our results demonstrate that these effects described in
vivo on the respiratory frequency are induced in deep
structures of the brainstem.
An increase in respiratory burst frequency after basilar
artery perfusion or bath superfusion of an acidic Krebs
solution is also observed in the isolated newborn
brainstem preparation (Harada et al. 1985; Tarasiuk &
Grossman, 1991; Issa & Remmers, 1992). The main effect
of this stimulus is on the expiratory duration. We have
shown that in this in vitro preparation, as in vivo, there is
a relationship between the inspiratory and expiratory
duration (Morin-Surun et al. 1992). However, in the
present study as in other in vitro studies (Murakoshi,
Suzue & Tamai, 1985; Greer, Smith & Feldman, 1991; Di
Pascale, Morin, Monteau & Hilaire, 1992), this
relationship is easily disturbed by modifying the
composition of the extracellular fluid, i.e. the expiratory
duration is more sensitive than the inspiratory duration.
Changes in the inspiratory-expiratory relationship could
be the result of changes in the synaptic inputs to the
respiratory neurones. For example, the expiratory
duration might be affected by any non-specific excitatory
input such as (CO2) which triggers the 'on switch'
inspiratory mechanism.
In this study, only stimulation of external structures by
hypercapnic acidic Krebs solution produces a response
close to that seen in vivo in decerebrate vagotomized cats
after C02 inhalation (Bradley, von Euler, Marttila & Roos,
1974). This suggests that in vivo, ventral surface
stimulation plays the major role in producing the
respiratory response. Furthermore, this should lead to the
conclusion that both pH and C02 are important
respiratory stimuli since the in vivo response is only
observed in vitro when both stimuli are present.
210 M. P. Morin-Surun and others
Nature of the stimuli implicated in the different
respiratory effects
Basilar artery perfusion with either hypercapnic non-
acidic or normocapnic acidic Krebs solution evoked
qualitatively the same responses as perfusion with hyper-
capnic acidic Krebs solution. This shows that the same
respiratory response can be effected by either hydrogen
ions, bicarbonate ions or carboxylic acid within deeper
structures. There are at least two explanations for these
observations. One is that each stimulus alone is able to
affect all the respiratory neurones implicated in the
regulation of the respiratory burst frequency. The other
possibility is that changes in any one of these stimuli leads
to only one stimulus for respiratory cells because of the
chemical interdependence of these stimuli. C02 dissociates
freely in the extracellular space and, as such,
chemotransduction mechanisms may very well occur
here. The other key factor in this interdependence is the
enzyme carbonic anhydrase. This enzyme is present only
in a small number of neurones (Neubauer et al. 1991) but
exists to a large degree in glia and in the endothelium of
blood vessels (Pan, Nauss, Bernard, Douglas & Trouth,
1991), confirming the extra neuronal space for the
chemotransduction mechanisms.
The classical view is that the chemosensitive stimulus is
the hydrogen ion (Loeschke, 1982). In the present study
we differentiate between the role of pH (i.e. hydrogen
ions) and C02 in the ventral chemosensitive response. We
show that acidic pH stimulates respiratory activity by an
increase of the respiratory burst frequency while
elevation of CO2 affects the respiratory burst amplitude.
This implies that in the cerebrospinal fluid, CO2 acts
independently from hydrogen ions on a specific target to
increase the amplitude of the neural burst. Respiratory
effects induced by a change in pH on the ventral surface
and within the brainstem were similar while C02 had
different effects on the respiratory activity, depending on
the brainstem structures stimulated. This suggests that
C02 preferentially stimulated chemosensitive cells on the
ventral surface while hydrogen acted on the same target
both within the brainstem and on the ventral surface. Use
of a novel fast CO2/H+-sensitive microelectrode (Voipio &
Kaila, 1993) within the different chemosensitive areas
located at the ventral surface and within the brainstem
would help to reveal any correlation between effects on
respiratory motor output and variations in Fcoj/pH
levels.
Possible neuronal mechanisms implicated in the
respiratory chemosensitive responses
In vivo, C02 evokes changes in postsynaptic activity of
respiratory neurones located in the ventral respiratory
area (Takeda & Haji, 1991) which could result from a
modulation of neurotransmission. Such modulation has
been described for bicarbonate ions which act as charge
carriers of GABA-gated currents (Kaila et al. 1993). Thus,
J. Physiol. 485.1
there is a link between the neuronal excitability and the
regulation of the neuronal intracellular pH. It is well
established that respiratory rhythmicity is dependent
upon synaptic connections between different types of
respiratory neurones (Ballantyne & Richter, 1984;
Richter, Ballantyne & Remmers, 1986; Ezure, 1990;
Onimaru & Homma, 1992) and particularly important is
GABAergic reciprocal inhibition (Champagnat, Denavit-
Saubie, Moyanova & Rondouin, 1982; Pierrefiche, Foutz &
Denavit-Saubie, 1993). The modulation of GABAergic
transmission by bicarbonate or consequently by hydrogen
ion concentration in the extracellular space may explain
the change in the respiratory frequency that we observed
with changes in the bicarbonate concentration of the
perfusate solution.
Recent results of hypoxic acidification of the ventral
medullary chemosensitive areas in vivo (Xu, Sato,
Spellman, Mitchell & Severinghaus, 1992) contradict the
hypothesis that the normal stimulus of medullary ventral
respiratory chemoreceptors is an acidification of the
extracellular fluid. The authors speculate that the chemo-
receptor neurones respond to changes in their
transmembrane H+ gradient. In this model, an
electrogenic inward flux of H+ would depolarize the
chemoreceptor cells, whereas outward H+ flux would
hyperpolarize them. In the present study, such a
transmembrane H+ gradient could explain the effect of
high Fco, solution when applied superficially, as CO2
easily penetrates the extracellular space at the ventral
surface (followed by the intracellular compartment) where
the buffering capacity is much higher than in the
extracellular fluid. Thus, this mechanism could explain
the different respiratory effects observed after changes of
C02 in the perfusate solution between the ventral surface
and the brainstem deep structures.
Conclusion
The isolated perfused brainstem preparation is
particularly convenient for studying brainstem chemo-
sensitivity. Respiratory chemoresponses are evoked by
different brainstem areas. On the ventral surface, this
regulation seems to be functionally specific for the C02
content of the superfusing medium.
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Acknowledgements
This work was supported by CNRS and DRET 92.075. T. Schiafer
was supported by an European Neuroscience Programme Short-
Term Fellowship of the European Science Foundation. The
authors wish to thank Dr Schlaefke for her comments on the
discussion and Dr H. A. McLean for English revisions and
scientific comments.
Author's present address
T. Schiifer: Abteilung fur angewandte Physiologie, Ruhr-
Universitiat Bochum, UniversitUtstraB3e 150, D-44780 Bochum,
Germany.

Received 25 January 1994; accepted 2 November 1994.