6 M
Fig. 4. Bar diagrams showing the effects of injections of Kainic acid Fig. 5. Bar diagrams showing the effects of injections of Piperidine
upper panel and NBQX lower panel into the dPAG on conditioned upper panel and Picrotoxin lower panel into the dPAG on conditioned
inhibition of fear-potentiated startle. The bars represent the mean ASR inhibition of fear-potentiated startle. The bars represent the mean ASR
difference scores after light and noise-light trials, as well as their difference scores after light and noise-light trials, as well as their
differences 6S.E.M.. differences 6S.E.M..
GABA receptor antagonist [10,15,41] were not observed in of fear-potentiated startle; likewise, blockade of the
the present study. AMPA Kainate receptors enhanced fear-potentiated startle.
It is important to note that this inhibition of fear-poten- tiated startle by stimulation of the AMPA Kainate re-
4. Discussion ceptors did not reflect the inhibition by an conditioned
inhibitor. Earlier results from our laboratory indicating a The present study tested the hypothesis that AMPA
role of the GABA receptors within the dPAG in the
A
Kainate receptors and GABA receptors in the dPAG are
expression of conditioned inhibition could not be con-
A
involved in the expression of fear-potentiated startle and firmed in the present study. Both, injections of a GABA
A
or the expression of conditioned inhibition of fear-poten- receptor agonist into the dPAG as well as of a GABA
A
tiated startle. This hypothesis was based on the observa- receptors antagonist neither affected expression nor con-
tions that injections of Kainic acid into the dPAG totally ditioned inhibition of fear-potentiated startle.
block the expression of fear-potentiated startle [48,49] and Since excitation of the PAG using stimulation electrodes
that injections of the GABA receptor antagonist Picrotox- or injections of excitatory amino acids or GABA receptor
A A
in into the dPAG slightly attenuated the expression of antagonists can induce explosive jumps and defensive
conditioned inhibition of fear-potentiated startle [22]. The reactions [3,7,10,14,15] and since these motor behaviours
present study confirmed an important role of AMPA might interfere with the ASR and its modulations [40,51],
Kainate receptor within the dPAG for the expression of the different concentrations of the drugs used in the present
fear-potentiated startle, since stimulation of the AMPA study were chosen using the criteria that no such be-
Kainate receptors within the dPAG blocked the expression havioural effects were elicited.
M . Fendt Brain Research 880 2000 1 –10
7
4.1. The injection sites within the dPAG much more caudal and ventral parts of the PAG compared
with the injection sites of the present study. All injection sites of the present study were located into
the dPAG, i.e. into the dorsomedial and the dorsolateral 4.3. Effects of NBQX injections into the dPAG
region of the PAG. Due to the small number of misplaced injections, it was not possible to statistically analyse their
Injections of NBQX into the dPAG strongly increased effects, but none of the individual animals except those
the expression of fear-potentiated startle. Conditioned with injections into the lateral PAG showed overt be-
inhibition was not affected. Since NBQX had the contrary havioural changes after injections of the different drugs. As
effects of Kainic acid, the important role of the AMPA we previously demonstrated in our laboratory [21,22],
Kainate receptors within the dPAG in the expression of injections of Picrotoxin and also Kainic acid into the
fear-potentiated startle was fully confirmed. lateral PAG 2 animals resulted in an increase in fear-
To the best of our knowledge, this was the first time that potentiated startle, whereas injections of Picrotoxin into
behavioural effects of NBQX injections into the dPAG the deep layers of the superior colliculus 4 animals has
were tested. Furthermore, this is one of the few studies no effects on fear-potentiated startle or conditioned inhibi-
showing an increase in fear-potentiated startle. For exam- tion. Furthermore, injections of Kainic acid into the deep
ple, a previous study of our laboratory reported an increase layers of the superior colliculus did not affect fear-poten-
of fear-potentiated startle after Picrotoxin injection into the tiated startle. Hence, we are confident that the effects
lateral region of the PAG [22]. described in the present experiments are mediated by the
dPAG. 4.4. The role of glutamate within the dPAG
4.2. Effects of Kainic acid injections into the dPAG All types of glutamatergic receptors are abundant in the
dPAG [1,47] and a lot of glutamate-immunopositive termi- Injections of Kainic acid into the dPAG decreased
nals are present in the dPAG [4]. The rostral hypothalamus expression of fear-potentiated startle. This was earlier
seems to be the main source of glutamatergic input into the described by Walker and colleagues [48,49] and confirmed
dPAG [38,46] and it has been suggested that this projection by the present experiments. Since there was a hint that the
is important for the mediation of defensive behaviours. dPAG is involved in conditioned inhibition of fear-poten-
Beitz [6] counted a high number of different brain areas tiated startle [22], the idea came up that the reduction of
projecting with glutamate to the dorsal and lateral parts of fear-potentiated startle by stimulation of the dPAG might
the PAG, but because of the sizes of the injections sites of reflect conditioned inhibition. However, the present study
the retrograde tracer in this study, it was impossible to showed that after injections of Kainic acid into the dPAG,
differentiate between projections to the dPAG and to the conditioned inhibition of fear-potentiated startle was not
other regions of the PAG. The most neurones projecting affected. This was found after infusion of a concentration
with glutamate to the PAG were found in the zona incerta, of Kainic acid which attenuated expression of fear-poten-
hypothalamus, cingulate, perirhinal and frontal cortex, tiated startle, as well as after infusion of a concentration of
cuneiform nucleus,
spinal trigeminal
nucleus, deep
Kainic acid which did not affect expression of fear-poten- mesencephalic nucleus, amygdala, cerebellar nuclei and in
tiated startle. the PAG itself see also [7].
Depaulis and colleagues [13,14] injected Kainic acid The exact role of the glutamatergic transmission within
into the dPAG and observed different defensive reactions the dPAG in the mediation of fear and anxiety is not clear.
e.g. freezing. Kainic acid injection into more caudal and There are some studies e.g. [33] showing that glutamate
more ventral regions of the PAG elicited forward avoid- release within the ventral regions of the PAG is decreased
ance behaviour e.g. forward locomotion, jumps as well as during pain, but it is not clear whether pain also modulates
ultrasonic vocalisation and an increase in arterial pressure. the glutamatergic transmission within the dPAG. Further-
In the present study, none of these active defensive more, the different glutamate receptors seem to be in-
reactions and no changes in automatically recorded sponta- volved differently in the modulation of fear. Whereas the
neous motor activity could be observed. Possible reasons present study and the work of Walker and colleagues
for that might be that during the startle tests the animals [48,49] showed that injections of AMPA Kainate agonists
were put into a little test cage so that the possibility to within the dPAG have an anxiolytic action and AMPA
move is quite limited. Furthermore, the different test Kainate antagonists increase conditioned fear, the NMDA
stimuli startle tone, light CS, noise CI elicited motor receptor seems to mediate the opposite effects. Injections
behaviours startle response, freezing which might inter- of NMDA receptor antagonists [27,45] induced anxiolysis
fere with such defensive reactions. It is important to note in the elevated plus-maze test, whereas agonists of the
that the most prominent defensive reactions in the studies glycine-site of the NMDA receptor had anxiogenic-like
quoted above are elicited by Kainic acid injections into effects [44]. These findings suggest that different gluta-
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mate receptor subtypes mediate different aspects of fear 4.6. Effects of Piperidine injections into the dPAG
which are measured in the different animal models of fear and anxiety. Since NMDA receptors have a low affinity
Piperidine injections into the dPAG neither affected for glutamate if the neurone is not depolarised, this would
expression nor conditioned inhibition of fear-potentiated suggest that the first result of glutamate release in the
startle. Since previous work of our laboratory [22] showed dPAG would be an inhibition of fear via Kainate AMPA
a reduction of conditioned inhibition after blockade of receptors. Different glutamate concentrations within the
GABA receptors within the dPAG, we expected that
A
dPAG could have different effects, e.g. low concentrations Piperidine injections into the dPAG elicits an enhancement
mainly activate the AMPA Kainate receptor, whereas high in conditioned inhibition. To the best of our knowledge,
concentrations mainly activate the NMDA receptor. The there are no previous reports on the behavioural effects of
different glutamate receptors could be located on different Piperidine injections into the dPAG.
neurones of the dPAG and theses neurones could modulate different fear-related behaviours. Consistent with this
considerations, Walker and colleagues [48,49] hypothesised 4.7. The role of GABA within the dPAG
that only high levels of fear activate the dPAG. The present study showed that NMDA receptors within
The present results suggest that the GABAergic trans- the dPAG mediate a reduction of fear-potentiated startle
mission within the dPAG does not affect the expression but are not involved in conditioned inhibition of fear-
and modulation of fear measured by the fear-potentiated potentiated startle. It is important to note that after
startle paradigm. Results of previous studies showing that injection of the highest dose of NBQX the absolute but not
GABA within the dPAG is involved in conditioned inhibi- the percent conditioned inhibition is increased, i.e. that the
tion [22] were not confirmed. Since GABA release within ‘increase’ in the absolute value of conditioned inhibition is
the PAG is decreased during pain [33], it can not be due to the enhancement of fear-potentiated startle. These
excluded that GABA within the dPAG plays a role in the results let suggest that there are different neuronal systems
modulation of fear-related behaviours. So, further work mediating the dPAG inhibition of fera-potentiated startle
using higher concentrations of GABA drugs into the and conditioned inhibition of fear-potentiated startle.
different regions of the PAG and using other animal models of fear and anxiety is necessary.
4.5. Effects of Picrotoxin injections into the dPAG Picrotoxin injections into the dPAG neither affected
5. Conclusion
