Induction of c-los immunoreactivity in tyrosine hydroxylase and phenylethanolamine-N-methyltransferase immunoreactive neurons
of the medulla oblongata of the rat after phosphate-buffered saline load in the urethane-anaesthetized rat
J.A. Narvfiez a ,b R. Covefias b M. de Ledn b, J.A. Aguirre a ,b A. Cintra b
M. Goldstein c and K. Fuxe b
" Department of Physiology, University of Mdlaga, Mdlaga (Spain), h Department of Histology and Neurobiology, Karolinska Institute, Stockholm (Sweden) and " Department of Psychiatry, New York University Medical Center, New York (USA)
We have studied the induction of c-los immunoreactivity (c-los IR) in catecholaminergic and vasopressinergic immunoreactive neurons after repeated phosphate-buffered saline (PBS) loading or after repeated elicitation of the baroreceptor reflex via repeated infusion of the vasoconstrictor agent L-phenylephrine. About 75% and 30%, respectively, of the tyrosine-hydroxylase immunoreactive (IR) cell bodies of the ventral noradrenaline (NA) A1/adrenaline (A) C1 and dorsal NA A2/A C2 areas and 60% and 30%, respectively, of the phenylethanolamine N-methyltransferase IR nerve cells of the adrenaline CI and C2 areas and 25% of the vasopressin (VP) IR neurons of the supraoptic (SO) nucleus developed nuclear c-los IR after repeated PBS loading. This phenomenon remained unaltered by the repeated elicitation of the baroreceptor reflex. These results suggest that the activation of volume receptors promotes homeostatic responses via activation of early genes in subsets of central medullary noradrenaline and adrenaline neurons and SO VP neurons of the urethane-anaesthetized rat.
Central noradrenaline (NA) and adrenaline (A) neurons of the medulla oblongata and pons are known to importantly participate in central cardiovascular reg- ulation involving a participation also in the barorecep- tor reflex arch 831'19"21'3°'31. However, little is known
about how the individual NA and A nerve cell groups of the medulla oblongata and pons are influenced by activation of low-pressure receptors located in the atria and high-pressure receptors, that is the baroreceptors, present within the aortic arch and the carotid sinus TM. In the case of locus coeruleus it was, however, early demonstrated that they can become activated by vis- ceral afferents and inhibited by a blood volume load through vagal afferents 6'33. Recently, it has been demonstrated that peripheral nerve stimulation and changes in physiological states produced e.g. by water deficits, osmotic stress, adrenalectomy, or the central administration of angiotensin II, catecholamines, and
systemic cholecystokinin can induce c-fos immunoreac- tivity (IR) in distinct nerve cell populations of the mammalian brain t'3,9,t233'lS'23"32, c-fos protein has also
been demonstrated in neurons of the nucleus tractus solitarius and the ventrolateral medulla after electrical stimulation of the carotid sinus nerve or after hypoxic stimulation, opening up the possibility to trace neurons of the baroreceptor and chemoreceptor pathways 7.
In the present work we have,therefore, studied if activation of low-pressure 3~ and high-pressure recep- tors by use of repeated i.v. injections of phosphate- buffered saline (PBS) (volume load) or of the periph- eral vasoconstrictor agent e-phenylephrine (elicitation of the baroreceptor reflex) in the anaesthetized rat, can induce c-fos IR within tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) IR neurons in cardiovascular areas of the caudal and rostral medulla 836 as analyzed by double immunofluo-
Correspondence: K. Fuxe, Department of Histology and Neurobiology, Karolinska Institute, Box 60400, S-104 01 Stockholm, Sweden. Fax: (46) (8) 33-7941.
rescence procedures 35. In view of the role of low- and high-pressure receptors in the neural regulation of vasopressin secretion, the induction of c-los IR in the vasopressinergic neurons of the supraoptic (SO) and paraventricular hypothalamic nuclei have also been evaluated 14.
Twenty-four male specific pathogen free Sprague- Dawley rats (250 g b.wt. ALAB, Stockholm) were used in this study. The animals were kept under standarized lighting conditions (lights on at 6.00 and off at 20.00) with a room temperature of 26 + I°C and had free access to food pellets and tap water. All experiments were performed between 8.00 and 12.00 a.m.
The animals were deeply anaesthetized with ure- thane (1.2 g/kg i.p.). A plastic catheter (PE-50, Clay Adams, USA) with heparin (50 IE/ml 0.9% NaC1, w/v) was inserted aproximately 4 mm into the common carotid artery. This catheter was connected to a Statham PC 23 SC transducer (Statham Company, Hato Ray, Puerto Rico) linked to a polygraph (model 7, Grass Instruments, Quincy, MA, USA) to record pul- satile blood pressure. A second catheter (PE-10 linked to PE-50, Clay Adams, USA) was inserted in the femoral vein. At the end of the experiments animals were perfused via the ascending aorta with cold physio- logical saline solution followed by a cold formalin-picric acid mixture (4% paraformaldehyde and 0.2% picric acid in 0.16 M phosphate buffer, pH 6.9). The perfu- sion was continued for 5 min and the brains were removed and postfixed in the same fixative for 2 h. The brains were transferred into phosphate-buffered saline (PBS, pH 7.2) containing sucrose (10%) for 2 days.
Six groups (four rats per group) were studied. In the first one (group A, controls), rats were perfused imme- diately after anaesthesia. In the second group (group B, sham-operated), animals were cannulated with the catheters remaining for 1 h. A third group received repeated injections (10 /zl) of PBS i.v. with a total volume of 0.5 ml during 1 h (group C). The fourth group was repeatedly injected i.v. with 10 /xg of L- phenylephrine in 10 /xl PBS to elicit the baroreceptor reflex (BRR) (group D). During the whole period of one hour the BRR was elicited each 70 seconds, in such a way that each animal received 50 injections of L-phenylephrine and a total volume of 0.50 ml. Finally, the fifth and sixth groups were treated in a way similar to the C and D groups, respectively, but the total volume injected was 5 ml of PBS during 1 h in both cases (groups E and F, respectively).
Cryostat sections (14/zm thick) were made using a Leitz cryostat (Heidelberg, FRG) and were processed according to the indirect immunofluorescence tech- nique 4. Thus, the sections were incubated at 4°C in a
343
humid chamber overnight using a mixture of rabbit polyclonal antisera to rat TH (final dilution 1:200 to 1 : 400; characterized by Markey et al.22), to rat PNMT (final dilution 1:800; characterized by Goldstein et al. 1°) or to vasopressin (VP) (final dilution 1 : 500; char- acterized by Rascher et al. 27) and a sheep polyclonal antiserum against the c-los protein (final dilution 1 : 400 to 1 : 200; purchased from CRB, Cambridge, UK; char- acterized by Le Gal La Salle2°). The c-fos antibody was raised against the N terminal amino acids residues (2-16) of a synthetic peptide (Met-Phe-Ser-Gly-Phe- Asn-Ala-Asp-Tyr-Glu-Ala-Ser-Ser-Ser-Arg-[Cys]) de- rived from a conserved amino acid sequence of c-los common to both mouse and human 34 and was affinity- purified using the immobilized synthetic peptides as is specified by the manufactures (CRB, Cambridge), such antibodies tend to be selective for c-fos and do not recognize Fos-related antigens. The antibodies were diluted in 0.1 M PBS containing 0.3% Triton X-100. After rinsing in PBS, the sections were incubated for 1 h at 37°C with a mixture of fluorescein isothiocyanate (FITC)-conjugated donkey antisheep immunoglobulins (final dilution 1:40; Amersham Ltd., UK) and Texas- red conjugated donkey antirabbit immunoglobulins (fi- nal dilution 1 : 40; Amersham Ltd., UK) to demonstrate c-fos protein and TH, PNMT or VP, respectively. The sections were rinsed in PBS and mounted in an an- tifading medium 17. The sections were examined in a Nikon Microphot-FX epifluorescence microscope equipped with the appropiate filter combinations. Fi- nally, the stereotaxic atlas of Paxinos and Watson 25 was used. The proportions of TH, PNMT and VP IR neurons displaying c-los IR under the present experi- mental conditions was established from micropho- tographs. A total of 30 sections were analyzed per rat involving 3-4 sections for each coexistence evaluation made.
As seen in Table I, 10-14% increases in mean arterial pressure are observed after 1 h following either L-phenylephrine or PBS loading. The BRR was repeat- edly elicited by L-phenylephrine and a maximal 20% reduction in heart rate was observed in both groups treated with the vasoconstrictor agent e-phenylephrine. Similar cardiovascular responses were found whether the volume load involved 0.5 or 5 ml of PBS during 1 h.
No c-fos IR was observed in the control and shame-operated groups in any area studied. After the injections of L-phenylephrine or PBS volume loading alone, c-los IR neuronal profiles were evenly dis- tributed in the catecholaminergic (CA) A1, A2, C1 and C2 areas as revealed by a rostrocaudal analysis of coronal sections of the medulla oblongata from bregma level -12.30 to -14.30. c-fos IR nerve cell bodies
344
TABLE I
Effects of a repeated phosphate-buffered saline load with or without repeated elicitation of the baroreceptor reflex with L-phenylephrine on cardiovascular parameters in the urethane anaesthetized rat
Means-+ S.E.M. (n = 4 rats). Final mean arterial pressure (MAP) is the MAP value at the end of the experiment and the percentage increase of the MAP at the end of the experiment is obtained by comparison with the basal value at the onset of the experiment. The bradycardic response is the peak reduction of heart rate after the injection of L-phenylephrine (PE) (10 p,g). No significant changes were found between groups in terms of MAP change or with regard to the bradycardic response to PE. bpm = beats per minute.
Treatment Basal Final % change Maximal MAP MAP of MAP bradycardic mm Hg mm Hg response (%)
were also demons t ra ted at various levels of the SO
nucleus. In doub le - immunolabe l l ing experiments , nu-
clear c-los IR was observed in many neu rons contain-
ing cytoplasmatic T H or P N M T IR in the above CA
areas after PBS loading with or without L-phenyl-
ephr ine t rea tment . No differences were found among
these groups with regard to deve lopment of c-los IR
(Table II). In the ventrola tera l medul lary NA A1 (Fig.
2 D - F ) and A C1 groups (Fig. 1 A - C ) 75% (overall
mean value from bregma levels - 1 4 . 3 0 to - 1 2 . 3 0 ) of
the TH- immunoreac t ive neurons developed nuclear c-
los IR (Table II), whereas in the dorsal medul la (NA
A2 (Fig. 2 A - C ) and A C2 groups) the coexistence of
T H and c-los 1R amoun ted to 33% (overall m e a n value
from bregma levels - 14.30 to - 12.30) of the TH-im-
munoreac t ive nerve cell popula t ions (Table II). A simi-
lar pa t te rn of coexistence appeared within the PNMT-
immunoreact ive neurons . Thus, 60% (overall mean
value) of the PNMT- immunoreac t ive nerve cells devel-
oped nuc lear c-fos IR in the ventrola tera l medul la (C1)
from bregma - 12.30 to - 13.80 after PBS loading with
or without L-phenylephrine t rea tment , while in the
dorsal medul la (C2) the percent of coexistence
amoun ted to 30% from bregma - 1 3 . 2 4 to -13 .80 .
(Figs. 1 D - F ; 3 A - D ; Table II). Few c-los IR cell nuclei
were observed which did not be long to TH- or PNMT-
positive nerve cells in these CA areas. None of the
P N M T IR neurons of the C3 group of the dorsomedial
medul la showed c-los IR in any of the exper imental
groups.
Finally, after L-phenylephrine or PBS volume load-
ing c-los IR also appeared in the SO and parvocel lular
paravent r icular hypothalamic (PV) nuclei. About 25%
of the VP immunoreac t ive neurons of the SO also
conta ined nuclear c-los IR (Fig. 3 E - F ) . No c-los IR
was found in the VP IR neurons of the PV nucleus.
The present f indings give evidence that activation of
low-pressure receptors, mainly located within the atria,
can induce bi lateral c-los IR mainly within NA and A
neurons of the ventral ( A 1 / C 1 ) and dorsal ( A 2 / C 2 )
medul la oblongata of the u re thane-anaes the t i zed rat.
These observat ions were made after repeated injec-
t ions over 1 h with a total volume of 0.5 ml of PBS.
Apparen t ly a maximal activation of the volume recep-
tors was already present with 0.5 ml, since 5 ml of PBS
TABLE II
Effects of a repeated phosphate-buffered saline load with or without repeated elicitation of the baroreceptor reflex on the appearance of c-fos IR in CA cell groups and in the SO vasopressin cells in the urethane anaesthetized male rat
Means_+ S.E.M. (n = 4 rats) are given for coexistence shown in percentage of the mean number of TH, PNMT or VP IR nerve cells found per section at various rostrocaudal levels of the different regions. The results values are based on the overall mean value (3-4 observations per rat) for each rat obtained from bregma levels -14.30 to -12.3 (c-Fos/TH); -13.80 to -13.24 (c-Fos/PNMT); -1.40 to -0.80 (c-Fos/VP).
Types of CA cell Mean percentage of coexisting nerve cell bodies ((c-los/TH, c-fos / PNMT or c-los/VP) × 100)
coexistence group PBS (0.5 ml) PBS (0.5 ml) + PE PBS (5 ml) PBS (5 ml) + PE
c-vos/TH IR cells
c-Fos/PNMT IR cells
c-Fos/VP Supraoptic IR cells
AI 75+4 73+5 78_+4 73_+5 A2 33_+4 35+2 35_+3 30_+4
Fig. 1. Double- immunofluorescence procedures. TH, P NM T (Texas red)/c-fos (FITC) IR nerve cells are found in the C1 adrenergic cell group. Bregma--13.30: the animal was repeatedly injected with PBS with a total volume of 0.5 ml of PBS (see text). TH-immunoreact ive neurons (A,B). D, dorsal; M, medial. In B a high magnification of the region delimited in A can be observed. The arrows show the colocalization of nuclear c-los IR (C) in some of the T H IR cell bodies. Gi, gigantocellular reticular nucleus. Bregma--12.80: the animal was repeatedly injected for 1 h with L-phenylephrine ( 1 0 / x g / 10 g.l) (a total volume of 0.5 ml) (see text). The region delimited in D can be observed at a high magnification in E. Coexistence (arrows) of cytoplasmatic P NM T (E) and nuclear c-los (F) IR can be found in some of the C1 neurons. D, dorsal; M, medial.
Bar = 50/xm.
L
I
L _ - ,
FO
S
B
FO
S
i ,/
D
Fig.
3.
Dou
ble-
imm
unof
luor
esce
nce
proc
edur
es.
PN
MT
(T
exas
red
)/c-
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(FIT
C)
and
VP
(T
exas
red
)/c-
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(FIT
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IR n
erve
cel
ls a
re f
ound
in
the
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area
and
in
the
SO
nuc
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, re
spec
tive
ly.
Bre
gma-
-13.
30:
the
anim
al w
as r
epea
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ject
ed w
ith
PB
S w
ith
a to
tal
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me
of 0
.5 m
l P
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(se
e te
xt).
Arr
ow
poin
ts t
o a
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MT
-im
mu
no
reac
tiv
e ne
uron
(A
) w
ith
nucl
ear
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(B
).
Bre
gma-
-13.
30:
the
anim
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was
re
peat
edly
in
ject
ed
wit
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-phe
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phri
ne (
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e P
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mm
unor
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ron
(a
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) (C
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ns
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ear
c-lo
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(D
).
Bre
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: th
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imal
was
rep
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dly
inje
cted
wit
h L
-phe
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ne (
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Coe
xist
ence
(ar
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f V
P (
E)
and
c-lo
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) IR
is
dem
on
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, do
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, m
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ar =
50
~m
.
",--.I
348
appear to produce any additional increases in the
number of c-los IR neurons nor in the proportion of
TH or PNMT IR neurons showing c-los IR. Stress
stimuli are probably not involved in the demonstrated
induction of c-fos IR in the NA and A neurons of the
medulla, since no c-los IR was observed in control and
sham-operated groups. It should be noticed that con-
siderably fewer nerve cells showed c-los IR in our
study using PBS load compared with the large numbers
found in the same areas after electrical stimulation of
the carotid sinus nerve in the a-chloralose-anaesthe- tized rat 7.
The findings of a selective induction of c-los IR in
25% of the VP IR neurons of the SO nucleus are in
line with the idea that atrial receptors, representing
volume receptors, importantly participate in the regu-
lation of VP release 2. Thus, it seems possible that this
regulation may mainly be exerted at the level of the SO
nucleus, since c-los induction could not be demon-
strated within the VP IR neurons of the PV nucleus. In
contrast, osmotic stress such as water deficits leads to
the expression of c-fos IR in the PV nucleus 9.
The above results suggest that volume loading leads
to selective homeostatic responses in distinct NA and
A cell groups of the medulla oblongata, involving the
induction of c-los IR, leading to changes in gene ex- pression and long-term cellular responses 23'24. It must
be emphasized, however that it is unknown to which
degree the induction of c-los IR within distinct cardio-
vascular medullary NA and A neurons is related to the
neurophysiological response of these nerve cells. Their
metabolic activation, however, probably reflects their
presence in the central pathways mediating the synap-
tic responses to activation of volume receptors.
The NA cell group A1 and the A cell group C1 in the caudal and rostral ventrolateral medulla 5'16, respec-
tively, were the most affected nerve cell groups with 60-75% of these cells being altered by the PBS load. It
must also be underscored that the c-los IR was almost
exclusively present within CA cell groups of the ventral
medulla, underlining the importance of the A1 and C1
NA and A neurons, respectively, in control of blood
volume. In contrast, only around 30% of the NA A2 neurons of the nucleus of the tractus solitarius (nTS) as
well as of the C2 adrenaline cell group of the rostral nTS responded with the appearance of c-los IR upon
the PBS load. These results suggest a greater involve- ment of the ventrolateral NA and A cell groups in the volume receptor reflex pathways in the medulla. In
support of this view, it has been reported that the SO vasopressinergic neuron inputs from medulla oblongata are catecholaminergic in nature with an origin mainly in the ventrolateral medulla, whilst A 2 / C 2 efferents
reach almost exclusively SO oxytocinergic neurons 2~''2s.
Also electrical stimulation of A2 neurons produce an activation of PV neurons 29. Nevertheless, in the dorsal
medulla the CA cell groups appear to be a major target
to the actions induced by volume receptor activation in
view of the predominant localization of the c-fos IR
nerve cell nuclei within the NA A2 and A C2 cell
groups. In contrast, the A cell group C3 within the
dorsomedial reticular formation of the rostral medulla
did not show any detectable induction of c-fos IR upon
the PBS load suggesting a lack of involvement of C3
adrenalin neurons in the volume receptor reflex path- ways.
In conclusion, activation of volume receptors via a
PBS load appears to induce homeostatic responses in
subsets of NA and A neurons of the dorsomedial and
especially the ventrolateral medulla as well as within
subsets of SO VP neurons as seen from the selective
induction of c-fos IR within a proportion of these CA
cell groups together with the selective appearance of
c-fos IR in some SO VP nerve cells. It seems possible
that volume receptors present in the atria may induce
coordinated responses in sympathetic tone and VP
release via activation of subsets of neurons within the cardiovascular NA and A cell groups of the medulla
oblongata.
This work has been supported by a grant (04X-715)
from the Swedish Medical Research Council and by a
grant from the Karolinska Institute. J.A. Narvfiez was
supported by the Ministerio de Asuntos Exteriores
(Spain) and M. de Le6n and R. Covefias were sup-
ported by the DGICYT (Spain). We are grateful to Ms.
Anne Edgren for excellent secretarial assistance.
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