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Institute of Physiology, Faculty of Medicine (Charité), Humboldt University, Tucholskystr. 2, 10117 Berlin, Germany
1Correspondence: Institute of Physiology, Faculty of Medicine (Charité), Humboldt University Berlin, Tucholskystr. 2, 10117 Berlin, Germany. E-mail: doris.albrecht{at}charite.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-aminobutyric acid
(GABAA) antagonist. The higher responsiveness of amygdaloid
neurons in transgenic rats as well as the predominance of inhibitory
effects, presumedly mediated by GABAergic interneurons, could change
the output of the amygdala and its influence on thirst, kidney, and
cardiovascular function or on processes of learning and
anxiety.—Albrecht, D., Nitschke, T., and Von Bohlen und Halbach, O.
Various effects of angiotensin II on amygdaloid neuronal activity in
normotensive control and hypertensive transgenic [TGR(mREN-2)27]
rats.
Key Words: amygdala • angiotensin II • AT1 • AT2 • bicuculline • extracellular recording • urethane • iontophoresis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4)
.
Angiotensin II (Ang II) is considered to be one of the most important
hormones in the control of blood pressure. It has many of the
characteristics of a neurotransmitter (1)
. Ang II has
equivalent affinity for both of the major Ang II receptor subtypes,
namely AT1 and AT2
(2
, 4)
. The AT1 receptor mediates
many of the known physiological effects of Ang II, among the most
prominent being those associated with the cardiovascular system and the
kidney. The physiological role of the AT2
receptor has yet to be clearly defined.
Both AT1 (5
, 6)
and
AT2 receptors (7)
were detected
within the amygdala. Moreover, AT1A receptor
messenger RNA (mRNA) is present in the basolateral amygdala
(8)
. In addition, nearly all substructures of the amygdala
display angiotensin-converting enzyme (ACE) activity (9)
.
The amygdala is discussed in terms of its role in receiving afferent
sensory input and in processing information related to hydromineral
balance. Angiotensin acts on and through the amygdala to stimulate
thirst and sodium appetite (10)
. In addition, the
angiotensinergic system seems to play a role in cognition and learning
mechanisms by acting on and through the amygdala (11
12
13)
.
It has also been shown that losartan, an AT1
antagonist, seems to have antianxiety properties in rats
(14)
. The aim of the present study was to investigate the
central effects of Ang II in hypertensive transgenic [TGR(mREN-2)27]
rats (15
, 16)
, which show higher brain Ang II
concentration than Sprague-Dawley (SD) rats (17
, 18)
. An enhanced renal vascular responsiveness to Ang II in
hypertensive [TGR(mREN-2)27] rats has been shown (19)
.
The question arose whether in hypertensive transgenic rats an enhanced
responsiveness to Ang II might occur in a brain structure involved in
the regulation of blood pressure as the amygdala. Therefore, we have
compared the effects of iontophoretically ejected Ang II on single unit
activity in functionally different subnuclei of the amygdala in
normotensive SD and hypertensive transgenic rats.
Furthermore, considering the existence of -aminobutyric acid
(GABAergic) interneurons in the amygdala, we wanted to determine their
possible involvement in the effects of Ang II using the
GABAA receptor antagonist bicuculline.
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MATERIALS AND METHODS |
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Before the experiment, the systolic pressure was determined by
tail-cuff plethysmography (Stoelting, Wheat Lane Wood, Ill.).
The rats were anesthetized with urethane (1.2 g/kg i.p.) and placed in
a stereotaxic instrument. Subsequent injections of urethane were
administered as needed (for details see ref 20
). Rectal
temperature was maintained at 37–38°C with a heating pad. The
electrocardiogram and the EEG from the visual cortex were monitored to
control the level of anesthesia.
Small holes were drilled into the skull at a site 4.5 mm lateral to the midline suture and 6.0 mm anterior to the lambdoid suture. An electrode was lowered 7.5–9.0 mm with a microdrive through the hole to the level of the amygdala.
Recording
Glass microelectrodes for extracellular recording were filled
with saturated trypan blue solution (tip resistance 10–30 M
). The
recorded action potentials were amplified and displayed on an
oscilloscope and, after passing a window discriminator, (World
Precision Instruments, Sarasota, Fla.) were analyzed with the software
spike2 (Cambridge Electronic Design, Cambridge, U.K.) running on a
personal computer. Standardized pulses corresponding to individual
action potentials were used for computing frequency-time histograms
that were displayed on-line during sampling. Data were stored on disc
for subsequent analysis.
Drugs and iontophoresis
Electrodes for iontophoresis were prepared from 5-barrel
micropipettes (World Precision Instruments) with a horizontal puller.
The tips were broken under microscopic visualization (tip diameter 5–7
µm). The recording electrode was affixed to the micropipette assembly
with a tip separation of 20–40 µm.
The following drugs were used: Ang II (100 µM, pH 4.5; RBI, Natick, Mass.), losartan (LOS; angiotensin AT1 receptor antagonist; 100 µM, pH 8.0; Dupont Merck, Wilmington, Del.), PD 123,319 ditrifluoroacetate (AT2 receptor antagonist; 100 µM, pH 4.5; RBI), (-)-bicuculline methiodide (BIC; GABAA receptor antagonist; 5 mM, pH 3; RBI). Retaining currents (4–10 nA) were applied to the pipettes between drug ejections. In a number of experiments, a barrel filled with sodium chloride (165 mM, pH 4.5 or 8) was used for current balance. No significant contribution from current or pH was detected in control experiments.
Experimental program
Iontophoretic ejection of Ang II was delivered repeatedly during
continuous recording of baseline activity. Currents of 20–60 nA were
used, in most cases 40 nA. As tachyphylaxis to Ang II is known to occur
(21)
, and recovery of the surface receptor after removal
of the angiotensin agonist occurs with a half-life of 15 min
(22)
, we ejected angiotensin antagonists concomitant with
Ang II at intervals of at least 15 min (longer intervals in most cases)
to make sure that the disappearance of Ang II effects in the presence
of antagonists was really a blocking effect and not a result of
desensitization of receptors. Moreover, we used angiotensin
concentrations that were 10x lower than those usually administered in
in vivo experiments. The release rate of the barrel should
be <17.9 fmol/min/nA in our experiments. This release rate was
determined for 1 mM solutions of Ang II (pH 4.5) ejected from 5 barrel
glass micropipettes with tip diameters of 4 µm (23)
.
After determination of the Ang II-induced effect, either the blocking potency of the AT1 receptor antagonist or of the AT2 receptor antagonist was tested. When inhibitory effects mediated by Ang II were observed, we tried to test whether bicuculline can abolish the Ang II-induced decrease in the discharge rate. Because not all neurons could be studied with a full experimental program, the number of neurons involved differ in the Results section.
Analysis of neuronal responses
Drug responses were compared with control firing frequency
recorded immediately before drug application. Based on the continuously
recorded rate-meter counts, the average discharge rate of each neuron
was evaluated for 120 s before the iontophoresis. This value
(referred to as ‘control’) was subtracted from all subsequent changes
in firing rate and the results were expressed as ‘% change of
control’. If the average change in the discharge rate during the
entire response time was >40%, the neuron was considered to be
sensitive to the substance applied. This criterion was used to divide
the ‘responders’ from the ‘nonresponders’, taking into account the
results of statistical trend analysis especially for slowly discharging
neurons.
The paired Wilcoxon’s rank-sum test (two-tailed) was used to find out
the predominating effect in a neuronal population, comparing the
spontaneous firing rate with that during administration of the drugs.
The Mann-Whitney U test and the 
2
test were used for statistical analysis of differences between both
groups of rats (SPSS software). The data are presented in the text as
the mean ± SD.
Localization of recording sites
At the end of recording, a small amount of trypan blue was
iontophoretically deposited in the brain by passing a 10 µA negative
current through the recording electrode for ~10 min. On each side of
the brain, recording sites were only stained in one electrode track.
The rat was killed with an overdose of urethane and decapitated, and
the brain was fixed with 10% formaldehyde. Frontal frozen sections
were stained with nuclear red. The location of blue spots within the
amygdala was determined.
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RESULTS |
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).
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Differences between normotensive controls and hypertensive
transgenic rats concerning neuronal firing and the action of Ang II
In general, Ang II could induce excitatory effects (Fig. 1
) or inhibitory effects (Fig. 2A
). Considering a change in the firing rate of >40% as
response, we observed a significant difference in the neuronal
responsiveness between the two strains of rats. The proportion of
responsive amygdaloid neurons was significantly higher in transgenic
rats (41/69 neurons) than in normal animals (40/95 neurons)
(P=0.029, 2 test, cross-table).
This increase in responsiveness was mainly brought about by an increase
in inhibitory effects. The proportion of Ang II-inhibited neurons was
higher in transgenic animals in comparison with controls (25/69 vs.
14/95), whereas the percentage of excited neurons in transgenic animals
was comparable with that obtained in controls (Table 1)
. Thus, the
distribution of nonresponsive, excited, and inhibited amygdaloid
neurons differed significantly between the two strains of rats
(P=0.05, 2 test, cross-table). The
difference in the effects could also be demonstrated by statistical
analysis of the firing rate of all neurons irrespective of the amount
of change. In the amygdala of controls, the predominance of activating
effects was significant (P=0.01, Wilcoxon’s rank-sum test,
n=95). This was not the case for TGR rats. Despite a greater
number of neurons inhibited by Ang II, the Wilcoxon’s rank-sum test
did not reach significance level.
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Analyzing the subdivisions of the amygdala, we also found some
differences. A predominant increase in the firing rate caused by Ang II
could be observed both in the central as well as in the cortical
amygdala of control rats (P=0.019, Wilcoxon’s rank-sum
test, n=43). Inhibitory and excitatory effects occurred to
nearly the same extent in the basolateral complex (n=52). To
the contrary, in transgenic rats the inhibition of the neurons in the
basolateral complex significantly predominated (P=0.05,
Wilcoxon’s rank-sum test, n=45), whereas for the
neuronal group of the cortical and central amygdala (n=24),
the result of the Wilcoxon’s rank-sum test was not significant. The
data for individual nuclei are given in Table 1
.
Comparing the spontaneous activity of all examined neurons we observed
a significant difference between controls and TGRs [0.96±1.83 imp./s
(n=95) vs. 2.03±5.86 imp./s (n=69),
respectively; P=0.008, Mann-Whitney U test]. It
is noteworthy that excitatory effects of Ang II were mainly induced in
slowly firing units, whereas inhibitory effects were more frequently
obtained in neurons with higher spontaneous discharge rates. Thus, the
spontaneous activity of neurons inhibited by Ang II differed
significantly from that of neurons excited by Ang II in
SD and in transgenic rats (P<0.01,
Mann-Whitney U test, see also Fig. 3
).
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There is also a difference between the two groups of rats concerning the duration of the neuronal responses to Ang II. In general, the effects induced by Ang II lasted for several minutes after the end of the iontophoretic ejection. Long-lasting effects were more frequently found in control than in transgenic animals. Thus, in neurons excited by Ang II, significant differences in the duration of the effect could be determined (633±525 s (n=26) vs. 346 ± 329 s (n=16) for controls versus TGRs, respectively; P=0.034, Mann-Whitney U test). The mean iontophoretic current used did not differ in the two strains.
AT1- and AT2-mediated effects of Ang II
within the amygdala
As shown in Figs. 2
and 3
, specific AT1 and
AT2 angiotensin receptor antagonists blocked the
Ang II-induced effects on the firing rate. Losartan antagonized Ang
II-induced increases in the firing rate in six neurons, but it also
blocked Ang II-induced inhibitions in three neurons. The
AT2 antagonist also blocked excitatory
(n=2) as well inhibitory effects (n=6) mediated
by Ang II. In five neurons recorded in different animals it was
possible to test both antagonists. In each case, only one of the
angiotensin antagonists blocked the Ang II-induced effect (see Figs. 1
, 2
). When ejected alone, the AT1 and the
AT2 angiotensin receptor antagonists caused
changes in the discharge rate in three neurons (two increases, one
decrease) and two neurons (increases), respectively. To ensure that
the Ang II receptors were not desensitized by a preceding Ang II
ejection, angiotensin antagonists were tested no earlier than 15 min
after the first administration of Ang II. Therefore, because of the
time needed, the blocking potency of the antagonists could be tested
only in a small sample of neurons.
In nine neurons located within the basolateral amygdala, it was
possible to test the ability of bicuculline to prevent the Ang
II-induced decrease in the discharge rate. For the iontophoresis of
bicuculline, currents were chosen that did not cause a significant
change in the firing rate itself. The GABAA
antagonist blocked the inhibition induced by Ang II in seven of these
neurons recorded in SD as well as in transgenic rats
(Fig. 4
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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as well as with a
recent study on field potentials in the lateral amygdala
(25)
. Because AT1 and
AT2 receptors are present within the amygdala,
various effects of Ang II could be mediated dependent on the receptor
type activated. As shown in the figures, the Ang II-induced effect in
single amygdaloid neurons was mediated either by
AT1 or by AT2 receptors.
Excitatory effects should be mediated by the stimulation of
AT1 receptors. From other brain structures it is
known that Ang II induces via AT1 receptors a
depolarization of the cell membrane by the involvement of different
K+ channels (26
, 27)
. In addition, a
possible modulation of Ca2+ entry via
voltage-dependent Ca2+ channels by Ang II has
been examined (26
, 27)
. An
AT2-mediated opening of potassium channels has
been observed in cocultures of brainstem and hypothalamic neurons
(28)
. Therefore, we would expect an inhibitory action of
Ang II by the activation of AT2 receptors.
However, the application of Ang II to inferior olivar neurons, which
possess only AT2 angiotensin receptors, induced
an increase in neuronal discharges (29)
. Our results
suggest the same excitatory action via AT2
receptors within the amygdala, because PD 123,319 also effectively
blocked Ang II-induced increases in the discharge rate. These data as
well as our recent study (25)
point to an excitatory
action of Ang II mediated by AT1 as well as by
AT2 receptors in the amygdala. Thus, the
direction of the Ang II-induced effects on the firing rate (excitation
or inhibition) does not seem to be angiotensin-receptor specific in our
study. Further studies are needed to clarify the electrophysiological
effect of AT2 receptor stimulation within the
amygdala.
Involvement of GABAergic neurons
We suggest that interneurons are involved in mediating suppressive
effects of Ang II in both strains of rats. Thus, inhibition of relay
cell activity could be explained by Ang II-induced excitation of
interneurons. Moreover, as angiotensins are released from the
multibarrel electrode like other transmitters (23)
, it can
be assumed that effects that were presumed to be direct and that arose
from local drug diffusion were recorded within a zone with 50 µm
radius around the microiontophoretic electrode. There was a distance of
20–40 µm between our recording and the multibarrel electrode.
Alterations in activity recorded at the most distal sites were found
between 100 and 500 µm, whereas synaptic activity induced by remote
drug ejection was seen most frequently within a zone 100–200 µm from
the iontophoretic site (30)
. Because local circuit
interneurons of the basolateral amygdala use GABA as transmitter, we
applied bicuculline to investigate whether Ang II-induced inhibitory
effects can be blocked by this antagonist. Bicuculline effectively
blocked decreases in the discharge rate caused by Ang II. From these
experiments it can be concluded that GABAergic interneurons also
receive an angiotensinergic innervation. An involvement of GABAergic
interneurons in the mediation of inhibitory effects of Ang II was also
found in the thalamus (31)
. For the cortex it has been
also supposed that Ang II might regulate local circuits
(8)
.
Differences between strains of rats
In transgenic rats we found a higher responsiveness of amygdaloid
neurons to Ang II in comparison with SD rats. Considering
the function of the amygdala in fear conditioning, it seems to be of
interest that TGR(mRen2)27 rats showed a greater anxiogenic profile
than control rats (32)
. Administration of captopril to
TGR(mRen2)27 rats reversed the anxiety-like behavior. Okuyama et al.
(33)
demonstrated that AT2-deficient
mice displayed anxiety-like behavior compared with wild-type mice.
Interestingly, in AT2-knockout mice the number of
[3H] prazosin sites was significantly reduced
in the amygdala. Therefore, it cannot be excluded that the interactions
with other neuromodulatory systems, for instance the noradrenergic
system, is altered in TGR(mRen2)27 rats.
Although excitatory as well as inhibitory effects mediated by Ang II
were found both in normotensive SD and in hypertensive TGR
rats, the number of neurons decreasing their firing rate significantly
predominated in transgenic animals. The higher percentage of inhibitory
effects in transgenic animals might be a result of a stronger
innervation of interneurons by angiotensinergic fibers or an
up-regulation of receptors to depress the output to brain stem
structures. On the other hand, the high concentration of Ang II in
transgenic animals (17
, 18)
and thus a possible persistent
activation of GABAergic neurons might lead to a down-regulation of GABA
receptors, explaining the higher baseline activity in hypertensive TGR
rats.
As mentioned above, we found significant differences between
SD and TGR rats concerning their neuronal responsiveness in
the amygdala. A higher responsiveness to Ang II in TGR(mRen2)27 rats
has been found in the thalamus in ACE inhibitor-treated transgenic rats
in comparison with ACE inhibitor-treated SD controls
(34)
. An enhanced sensitivity and responsiveness of
cardiovascular neurons to Ang II has been also observed in
spontaneously hypertensive rats (35)
. In addition, it has
been shown that water deprivation up-regulates angiotensin
AT1 binding and mRNA in rat subfornical organ and
anterior pituitary (36)
.
Responsiveness of neurons in varioius subnuclei of the amygdala
In a recent immunohistochemical study we have determined the
distribution of AT1 receptors within the various
subnuclei of the amygdala of female rats (6)
. This
approach demonstrated high amounts of immunostained cells in the
central amygdala. Within the basolateral amygdaloid group, the lateral
and basolateral nucleus showed moderate staining, while within the
basomedial nucleus of the amygdala only few cells were immunostained.
The low responsiveness to Ang II of basomedial neurons in males
(compare Table 1
) corresponds well with the immunocytochemical results
we got for females. As Ang II stimulates both AT1
and AT2 receptors, the results of our study
correspond well with our fluorescein-coupled Ang II binding study, in
which the lateral, the basolateral, and the central nuclei showed an
Ang II binding to nearly the same extent (37)
.
In summary, the results of this study support the hypothesis that Ang
II plays a role as a neuromodulator in the amygdala. Thus, Ang II
influences not only the striatal parts (38)
of the
amygdala (central and cortical nuclei) that are involved in the
regulation of vegetative functions, but also the cortical part of the
amygdala (basolateral complex), which seems to be involved in cognitive
functions (39)
. The results also suggest that alterations
in the renin-angiotensin system might change the network properties
within the amygdala.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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This article has been cited by other articles:
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  D. Albrecht Angiotensin-(1-7)-induced plasticity changes in the lateral amygdala are mediated by COX-2 and NO Learn. Mem., March 8, 2007; 14(3): 177 - 184. [Abstract] [Full Text] [PDF]
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 D. ALBRECHT, K. HELLNER, T. WALTHER, and O. VON BOHLEN UND HALBACH Angiotensin II and the Amygdala Ann. N.Y. Acad. Sci., April 1, 2003; 985(1): 498 - 500. [Full Text] [PDF]
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 C. Sun, C. Sumners, and M. K. Raizada Chronotropic Action of Angiotensin II in Neurons via Protein Kinase C and CaMKII Hypertension, February 1, 2002; 39(2): 562 - 566. [Abstract] [Full Text] [PDF]
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