Psychobiology Laboratory, Division of Psychology, The Australian National University, Canberra, ACT 0200, Australia
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ABSTRACT |
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Disney, Anita and
Mike B. Calford.
Neurosteroids Mediate Habituation and Tonic Inhibition in the
Auditory Midbrain.
J. Neurophysiol. 86: 1052-1056, 2001.
Habituation of the behavioral response to a
repetitive stimulus is a well-established observation in perceptual
studies and is considered a basic form of nonassociative learning.
There is also a long history of physiological studies suggesting that
central nervous system habituation is mediated by inhibition. At higher levels of the sensory pathways, such inhibition is mainly contributed by GABAa receptor mechanisms. Concepts of modification of synaptic efficacy that apply to excitatory amino acid synaptic transmission do
not have direct parallels with these inhibitory synapses: quantal release of GABA rapidly saturates available receptors at a synapse, placing an upper limit on responsiveness to increased transmitter release. However, pharmacological modulation of GABAa-receptor efficacy
with exogenous agents (e.g., benzodiazepines and -carbolines) is
known to occur through allosteric mechanisms that modulate the
effectiveness (positive and negative) of GABA at this receptor. The
most potent endogenous modulators are 5
-reduced steroids. Production
of these steroids was attenuated in adult rats with systemic injection
of Finasteride, a competitive substrate for 5
-reductase. This
treatment was sufficient to block habituation of the evoked midbrain
response to repetitive presentation of an acoustic click. This result
confirms that simple habituation is due to an increase in active
inhibition, the increase being mediated by steroid modulation of the
GABAa-receptor. Finasteride treatment also brought about a 23%
increase in the evoked response to a click stimulus, suggesting that
5
-reduced steroids normally contribute to tonic inhibition in the
rat inferior colliculus.
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INTRODUCTION |
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For some time it has been known
that the enzymatic machinery for the production of 5-reduced
progesterone-derived steroids, which modulate transmission at
GABAa-synapses, is found throughout most of the mammalian brain.
3
-hydroxy-5
-pregnan-20-one (3
,5
-THP; allopregnanolone) and
3
-tetrahydro-deoxycorticosterone (3
-THDOC) are both known to be
potent positive modulators of inhibition mediated by the binding of
GABA to its a-type receptor (Majewska et al. 1986
). Both
of these steroids can be synthesized de novo, or from intermediate
substrates, in the brain (Hu et al. 1987
; Jung-Testas et al. 1989
). Brain levels of
allopregnanolone have been shown to increase rapidly (<3 min) in
response to anxiety producing stimuli and situations (e.g.,
Barbaccia et al. 1996
; Purdy et al. 1991
)
and in response to ethanol administration (VanDoren et al.
2000
). However, it has not been clear whether these steroids have a role in modulating GABAergic inhibition in neural processing in
the absence of such priming.
The requirement for an endogenous modulator of GABAa-receptor efficacy
is clear. GABA is the major inhibitory neurotransmitter at higher
levels of the CNS. Acting at the ionotropic GABAa receptor, it is
involved in many essential brain processes. Indeed the balance between
inhibition and excitation defines the functioning of many neural
systems, particularly those involved in sensory processing (Calford et al. 1998; Dykes et al. 1984
;
Park and Pollak 1993
). However, the functional need for
modulation of GABAa-receptor-mediated inhibition raises a paradox as
minimal GABA release is considered sufficient for saturation. Quantal
release of GABA rapidly saturates the GABA binding sites of
GABAa-receptors at a synapse and opens a maximal number of
Cl
channels (Mody et al. 1994
),
limiting the effectiveness of increased transmitter release. One
mechanism to control efficacy at such inhibitory synapses is to vary
the number of receptors. With long-term changes in excitability, it is
well established that GABAa-receptor numbers can increase or decrease
(Jones 1993
). However, even under optimal conditions,
this mechanism is too slow (Nusser et al. 1998
) to play
a role in many of the situations in which inhibitory efficacy varies.
Control by phosphorylation of receptors may provide one solution
(Poisbeau et al. 1999
). An alternative possibility is
alteration of the kinetics of the ion channel. Pharmacologically applied GABAa-receptor modulators (benzodiazepines;
-carbolines) that prolong, or shorten, the time during which ions can pass across
the cell membrane are well known. Allopregnanolone and THDOC are the
most potent endogenous positive modulators of the GABAa-receptor
(Majewska et al. 1986
). The present study was designed to establish whether these steroids play a role in modulation of
GABAergic inhibition in physiological circumstances that do not involve
anxiolytic priming or potentially excitotoxic levels of activation.
Habituation to repetitive stimulation is a near-ubiquitous
nervous-system phenomenon. In contrast to adaptation, which occurs in
peripheral structures such as photoreceptors, CNS habituation occurs at
rates of stimulation that do not approach the biophysical limits of
synapses or put metabolic demands on neurons. It is thus considered to
involve an active inhibitory component and is the classical form of
nonassociative learning (Groves and Thompson 1970).
Electrophysiological recording has shown that the amplitudes of
averaged evoked potentials (EPs) recorded in the central auditory pathway decrease over the course of repeated presentations of a
response-eliciting stimulus (Webster 1971
). It has also
been shown that the degree and time course of these decreases in
responsiveness can be manipulated through administering GABAa-receptor
modulators (Jongsma et al. 1998
; Webster and
Aitkin 1971
). In the present study, we determined whether there
is any involvement of neurosteroids in the observed time course of such
response decrements in the rat auditory midbrain nucleus
the inferior
colliculus (IC).
The decreases in EP amplitude in the IC observed in previous
experiments involving repetitive stimulation (Chakravarty and Faingold 1996; Webster 1971
; Webster and
Aitkin 1971
) were maintained during continuous, rapid
presentation of stimuli for between 20 and 30 min. This prolonged
inhibition, already known to be GABAa-receptor mediated, may well be of
the kind that requires positive modulation to be maintained over time.
The synthesis of the 5
-reduced steroids can be blocked using
Finasteride, a competitive inhibitor of 5
-reductase (Faller et al. 1993
). We therefore investigated whether
such blockade alters the course of the response amplitude decrements
recorded during repetitive presentation of an acoustic click stimulus.
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METHODS |
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Single-session, nonrecovery experiments were performed on 32 adult male rats (pigmentedDark Agouti) aged between 8 and 15 wk and
weighing 250-350 g. Anesthesia was induced by intramuscular injection
of ketamine (100 mg/kg; Troy Laboratories) and acepromazine (10 mg/kg;
Troy Laboratories); 10 min prior to anesthetic injections, an analgesic
was administered subcutaneously (carprofen, 3 mg/kg; Zenocarp, Heriot
Agvet). Throughout the experiment, pedal and eye-blink reflexes were
monitored, and supplementary doses of ketamine were given as needed.
Locally derived EPs in response to acoustic click stimuli applied to
the contralateral ear were recorded from the IC through a
microelectrode. During the recording session, animals were held in a
stereotaxic frame inside a sound-attenuating, electrically shielded
room. Stimuli were generated using a 12-bit D/A with controlled output
attenuators (Kaiser Instruments) driven by the MALab
application running on an Apple Macintosh G3. Click stimuli were
produced with 80 µs positive polarity square pulses, passed through a
headphone amplifier (Tucker Davis), driving a piezoelectric transducer
(Motorola) fitted to a hollow ear-bar. Fast-Fourier analysis revealed
that the bulk of the energy of the click was <15 kHz. Locally
manufactured tungsten-in-glass recording electrodes of impedance 1-1.5
M, at 300 Hz, with 5-30 µm of exposed tungsten, were used in all
experiments. Electrodes entered the pia at 1.7 mm rostral and 1.9 mm
lateral of lambda and were advanced at an angle of 43° dorsorostral
to ventrocaudal from vertical to a depth of 3,400 µm. Potentials were
amplified differentially (A-M Systems 1800) with band-pass filtering
100 Hz to 10 kHz. The signal was digitized at 19.5 kHz (MALab event
processor, Kaiser Instruments), viewed on-line (continuous averages),
and stored for off-line analysis.
Preexperimental acoustic stimulation was kept to a minimum. Initially, using low presentation rates, the threshold sound level for a discernible EP was determined. Later recordings were obtained with the stimulus level 35 dB above threshold. After determining thresholds, no stimulus presentations were made for 30 min. No quantitative recordings were made within 1.5 h of induction of anesthesia to allow a return to normal neurosteroid levels following any handling-induced increase.
The basis of the experimental design was to compare the degree of
habituation of the acoustic click EP in the IC of rats with Finasteride
blockade of 5-reductase with that of controls. Figure 2, which
presents a data summary, also presents a summary of the experimental
design. Four groups of animals were used. Two experimental groups
received 60 mg/kg Finasteride
[1,(5
)-andrsotene-4-aza-3-one-N-tert-butyl-17
-carboxaminde, Sigma, 6 mg/ml in 30% wt/vol 2-hydroxypropyl-
-cyclodextrin, Sigma, in 0.9% saline] by intraperitoneal injection; two control groups received only the vehicle solution (2.5-3 ml; 30% wt/vol
2-hydroxypropyl-
-cyclodextrin, Sigma, in 0.9% saline). The
concentration of Finasteride was based on previous reports.
Lephart et al. (1996)
have reported a 60-80% reduction
in enzyme activity and behavioral effects in pregnant rats with 50 mg/kg Finasteride given subcutaneously. Ongoing experiments in this
laboratory with a different paradigm (Saalmann and Calford 1999
) have shown the delivery method and concentration to be
effective in reducing induced inhibition in the IC. A 90-min period
with no acoustic stimulation followed Finasteride or vehicle
injections; this should be sufficient to allow any stress-induced
elevation in neurosteroids to return to normal (Barbaccia et al.
1996
). In one group of experimental animals and one control
group, a baseline measure of habituation was obtained prior to
Finasteride or vehicle injection. All comparisons of EP amplitude were
based on averages of 20 presentations at specified time periods. For graphical presentation, these were referenced to the amplitude at
either the start of the stimulus presentation period under consideration or to the initial value. For statistical comparisons, raw
EP amplitudes were used in a mixed-model analysis-of-covariance design
with the initial EP amplitude or that at the start of a run as the
covariant using the SPSS package.
On completion of recording, the characteristic frequency (CF) and the threshold to a pure tone stimulus were noted before a lethal dose of pentobarbital sodium was administered. The fact that the EP to pure-tone pulses showed frequency tuning consistent (CF near 8 kHz) with the location of the electrode tip (and which varied with depth) confirmed that the EP under study was locally generated. After perfusion/fixation brains were sectioned and stained. Electrode tracks were located by track damage and reconstructed to confirm the recording site as the central nucleus of the inferior colliculus.
Experimental procedures were approved by the Animal Experimentation and Ethics Committee of the Australian National University and conform to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.
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RESULTS |
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The form of the EP obtained in the IC in response to contralateral
click stimuli (Fig. 1A)
closely resembles that obtained by Semple and Aitkin
(1980) to electrical stimulation of the cochlear nucleus. In
that study, manipulation of the rate of electrical stimulation allowed
interpretation of the early wave as presynaptic and the later, and
larger, wave as postsynaptic. Consideration of previous reports
(Webster and Bock 1971
) and preliminary experiments led
to adoption of a 5-Hz presentation rate as sufficient to induce mild
habituation of the postsynaptic EP-component and an insignificant decrement of the presynaptic component. Thus within 2.5 min, the postsynaptic EP showed a rapid and sustained reduction to ~90% of
the initial value that was maintained throughout 25 min of stimulation
(Fig. 1B). Apart from a small initial decrement within the
first minute of presentation, this habituation was completely blocked
in a repeat period of presentation of the click stimulus started 90 min
after Finasteride injection (Fig. 1C). Control animals given
only the vehicle solution showed the normal pattern of habituation as
established in the baseline determination.
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Another striking result of the Finasteride treatment was an increase of
~25% in the EP-amplitude in response to the click stimulus (Fig.
2A). Thus the determination of
a lack of habituation (Fig. 1C) was made with respect to
this increased value. At a later period (145 min after Finasteride
injection; Fig. 2A), presumably when Finasteride inhibition
of 5-reductase became less effective due to synthesis of new enzyme,
a third period of repetitive stimulation revealed a significant
decrement in the EP-amplitude. Interpretation of this later period is
confounded by the dual effects of diminution of the Finasteride induced
effects on habituation and induced enhancement of the EP. Irrespective,
the final level reached was still well above (+23%) the equivalent
level reached at the same time point with the vehicle-injected animals.
Overall, the Finasteride-induced increase in the click EP suggests that
5
-reduced steroids have an ongoing role as positive modulators of
GABAergic inhibition in the IC. A three-way (treatment × run × time) mixed-model ANOVA was performed on the amplitude of the
presynaptic component, and none of the main effects was significant;
specifically the initial 25-min period of repetitive stimulation
resulted in a nonsignificant change to 96% of baseline and 90 min
after Finasteride injection the presynaptic EP was at 101% of
baseline. A difficult to interpret two-way interaction [run × time: F(1,10) = 6.349, P = 0.023] was significant. However, the differences in the presynaptic component change profiles cannot explain the effects observed in the animals given Finasteride: the presynpatic component was at only 101% of
baseline 90 min after Finasteride was administered, while the postsynaptic component had increased to 120% of baseline by this time. This suggests that there was neither clear repetitive stimulation nor Finasteride-induced alterations in the amplitude of the input to
the IC and that the observed changes in the postsynaptic EP probably
originated within the IC.
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The Finasteride-induced increase in the click EP was an unexpected
outcome and provided a potential complication to the interpretation of
the original design, which essentially planned for a within animal
comparison of habituation before and after attenuation of 5-reduced
steroid synthesis. First, it was necessary to establish whether the
90-min period after Finasteride injection was sufficient for the
induced increase to reach a steady state. This was tested in three
animals by sampling the click EP with 4-s presentations of the
repetitive acoustic-click stimulus at 15-min intervals after
Finasteride injection. In each case, a plateau was reached prior to 90 min (Fig. 2C). Hence it is considered unlikely that the lack
of change in the EP with repetitive stimulation in the Finasteride-injected animals resulted from a continual increase in the
base click EP acting against an habituation. Rather the most
parsimonious explanation is that the attenuated levels of 5
-reduced
steroids were responsible for blocking habituation. Second, the
presence of an initial baseline habituation run in the original
experiment complicates the interpretation; despite the fact that EPs
had returned to normal levels after the 90 min break in the
vehicle-injected animals, it may be that in an environment of raised
steroid synthesis (after the initial 25-min presentation) the
subsequent injection of Finasteride had a complex effect. Consequently,
a second set of animals was studied in which both experimental and
control groups received only a 4-s presentation of the repetitive
acoustic-click stimulus prior to Finasteride or vehicle injection (Fig.
2B). This was sufficient to obtain an EP averaged over 20 presentations to establish the base level in each animal. Even with
minimal preinjection acoustic stimulation, the effect of attenuating
the potential synthesis of 5
-reduced steroids resulted in a marked
increase in the click EP. This allows the conclusion that tonic
synthesis of these steroids normally enhances inhibitory components of
the response to acoustic clicks in the IC. In all respects, for both
Finasteride and vehicle treatments, the time course of the changes in
EPs were very similar for the two presentation paradigms (compare Fig.
2, A and B from the time of injections).
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DISCUSSION |
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The effect of interference of 5-reduced neurosteroid synthesis
in the present study adds a new perspective to the understanding of the
roles attributed to neurosteroids (Rupprecht and Holsboer 1999
). It is clear that, in addition to the previously reported roles in the short-term response to anxiety-related stimulation and in
the response to alcohol, 5
-reduced neurosteroids are involved in
modulating inhibition relevant to the processing of sensory stimuli.
Two aspects of this modulation were uncovered in the present study in
which neurosteroid synthesis was attenuated with Finasteride.
Firstly, 5-reduced neurosteroid synthesis appears necessary for
habituation of the response to repetitive stimulation. In animals
treated with Finasteride, there was an initial decrease in EP amplitude
over the first 2 min of repetitive stimulation. Thereafter, the EP
amplitude during the 25 min of stimulation matched that obtained to a
brief presentation. The reported time to increased neurosteroid
synthesis is ~3 min in anxiety-priming paradigms (Purdy et al.
1991
), and thus the initial decrease in EP amplitude is
expected to be controlled by other mechanisms. Elucidation of the
details of the amplitude and timing of this initial effect would
require different methods than those used in this study, where the
summing across animals may be problematic due to small differences in
the timing of the effect. Second, it was found that the amplitude of
the EP to click stimulation is, in the circumstances of the present
paradigm, normally mildly suppressed by the tonic synthesis of
neurosteroids. Both findings suggest that 5
-reduced neurosteroids
have a more subtle role in neuronal functioning than has previously
been thought. The finding of the tonic effect in particular raises the
probability of a role in short- to medium-term gain control of neural
circuits. This is a widespread phenomenon in motor and sensory systems
(e.g., Katz et al. 1999
; Lisberger et al.
1983
). It has previously been reasoned that in some
circumstances changes in response strength and receptive field size are
explicable only with a mechanism for short-term inhibitory synaptic
plasticity that responds to increased excitability (Clarey et
al. 1996
). Such a mechanism appears to require (see
INTRODUCTION) modulation of GABA-synaptic efficacy of the
form provided by the 5
-reduced neurosteroids.
The concept of habituation as a simple nonassociative learning process
(Groves and Thompson 1970) is consistent with mediation by neurosteroid modulation of GABAa-receptors. It is well established that, at most levels, the response of the central auditory pathway to
monaural stimulation involves excitatory and inhibitory components (Clarey et al. 1992
). The present work suggests that
synthesis of neurosteroids by glial cells is induced, or increased, by
repetitive stimulation. The increased neurosteroid concentration
produces a local positive modulation of the effect of GABA on a-type
receptors. In contrast to associative-learning effects, as with
long-term potentiation and depression, this effect would be a
nonselective, spatially limited increase in inhibitory-synapse
efficacy. There is insufficient knowledge at this time to understand
the trigger mechanism for increased synthesis. Where increased
synthesis has resulted from potentially damaging levels of excitation
(Barbaccia et al. 1996
; Purdy et al.
1991
), it appears that increased free-radical concentration can
induce activation (Brown et al. 2000
). However, this is
unlikely to be a factor with the 5-Hz stimulation employed here. It is
known that steroidogenesis is under the control of peripheral-type
benzodiazepine receptor (PBR)-induced transport of cholesterol,
and intermediate steroids, to the inner mitochondrial membrane
(Do-Rego et al. 1998
; Papadopoulos et al.
1997
). Endogenous ligands of PBR are known
(diazepam-binding-inhibitor derived peptides), but their mode of
control and release is not (Do-Rego et al. 1998
; Lacor et al. 1999
). It is possible that excess GABA may
be a stimulating factor because in the retina, GABAa-receptor agonists
have been shown to stimulate steroidogenesis (Guarneri et al.
1995
).
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ACKNOWLEDGMENTS |
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The authors thank B. Gynther and Y. Saalmann for advice in dealing with steroid delivery and R. Tweedale for comments on the manuscript.
This work was supported by National Health and Medical Research Council of Australia Grant 961226.
Present address of A. Disney: Center for Neural Science, New York University, 4 Washington Pl., New York, NY 10003.
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FOOTNOTES |
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Present address and address for reprint requests: M. B. Calford, School of Biomedical Science, Hunter Medical Research Institute, The University of Newcastle, Newcastle, NSW 2308, Australia (E-mail: mike.calford{at}newcastle.edu.au).
Received 26 December 2000; accepted in final form 4 May 2001.
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REFERENCES |
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