Glycine receptors in the caudal pontine reticular formation: are they important for the inhibition of the acoustic startle response?
Michael Koch *, Eckhard Friauf Tierphysiologie, Universitiit Tiibingen, Auf der Morgenstelle 28, D-72076 Tiibingen, Germany
Accepted 25 October 1994
Abstract
The present paper sought to test the hypothesis that inhibitory glycine receptors (GlyRs) on giant neurons of the caudal pontine reticular formation (PnC) are involved in the inhibition of the acoustic startle response (ASR) in rats. First we provided evidence for the presence of the strychnine-sensitive inhibitory GlyR on PnC neurons by immunocytochemical labeling using an antibody against the a I subunit of the GIyR. We then measured the ASR as well as two ASR inhibiting phenomena, short-term habituation and prepulse inhibition, after microinjections of the glycine antagonist strychnine (0, 5 or 10 nmol) or the glycine agonist/3-alanine (0, 50 or 100 nmol) into the PnC. Neither strychnine nor fl-alanine had a measurable influence on any of the parameters of the ASR investigated (amplitude, short-term habituation, prepulse inhibition). In contrast, systemic injection of strychnine (1 mg/kg) markedly increased the ASR amplitude. The systemic administration of strychnine did not impair prepulse inhibition. The human 'startle disease' (hyperekplexia), an exaggerated startle response, is caused by a defect of the a I subunit of the inhibitory GlyR, but it is unclear at which site in the central nervous system this defect ultimately leads to the symptoms of hyperekplexia. Our data indicate that a blockade of the inhibitory GlyRs in the PnC does not affect the ASR of rats, suggesting that deficient GlyRs in rhe PnC might not be involved in the etiology of the human 'startle disease'. We conclude that the inhibitory GlyRs on PnC neurons are not necessary for the inhibition of the ASR and believe that they are involved in another behavioral context.
The acoustic startle response (ASR) is characterized by rapid contractions of facial and skeletal muscles following an unexpected, intense acoustic stimulus. The ASR can be observed in all mammalian species, includ- ing humans, and very likely it subserves a protective function, possibly by facilitating a flight response. The ASR underlies modulatory influences that increase or decrease the response amplitude, a fact which reflects the ability of the organism to adjust its response strength to specific external a n d / o r internal condi- tions. The neural circuitry underlying the ASR, i.e. the primary startle pathway, is already fairly well under- stood. An indispensable sensorimotor interface of this
cochleospinal pathway is the caudal pontine reticular nucleus (PnC), as was shown by electrical stimulation and lesion experiments [6,7]. More specifically, the giant reticulospinal neurons of the PnC appear to mediate and modulate the ASR [16,21,22].
The ASR amplitude is reduced if a non-startling stimulus is presented 30-500 ms before the startle pulse occurs. This phenomenon is termed prepulse inhibition (PPI) and has received considerable atten- tion in recent years as an example of sensorimotor gating [31]. The ASR amplitude can also be reduced by the repetitive presentat ion of startle stimuli, a phe- nomenon termed short-term (or within-session) habitu- ation [8,20]. The neural mechanisms of both of these important phenomena of response suppression are still not understood. Recent experiments suggested that PPI is mediated by a complex neuronal circuitry, in- volving the medial prefrontal cortex [3], the hippocam-
64 M. Koch, E. Friauf / Brain Research 671 (1995) 63-72
pus [4], the nucleus accumbens [31], the ventral pal- lidum [31], the pedunculopontine tegmental nucleus [18,32], and the superior colliculus [10]. The startle reducing effect of acoustic prepulses very likely affects the primary pathway of the ASR at the level of the PnC [22]. One possible mechanism is the activation of an inhibitory cholinergic projection from the peduncu- lopontine tegmental nucleus to the PnC [18]. Neverthe- less, there is the possibility that different parallel PPI circuits may exist, and it is therefore conceivable that additional transmitter systems are involved. The mech- anism underlying short-term habituation is unclear. The decline of the ASR amplitude after repeated presentation of startle stimuli might be due to a reduc- tion of transmitter release or due to blunting of post- synaptic receptor sensitivity within the stimulus-re- sponse pathway, which would represent two forms of intrinsic modifications of the response strength. Alter- natively, a recruitment of inhibitory projections from nuclei outside the stimulus-response pathway would also reduce the gain of the sensorimotor information transfer in the primary response circuit, which would represent a form of extrinsic modulation. Synaptic inhi- bition in the vertebrate central nervous system is mainly mediated by y-aminobutyric acid (GABA) and by glycine [33]. Glycine exerts its inhibitory action after binding to a pentameric receptor protein which subse- quently increases the permeability of the membrane for chloride ions, thereby leading to an inhibition (by hyperpolarization or shunt-inhibition) of the cell [1].
Our interest in the present investigation of the role of lower brainstem GlyRs in the mediation of inhibi- tion of the ASR has been kindled by a recent report [30] that a mutation in the gene encoding for the a 1 subunit of the inhibitory GIyR in humans leads to a disinhibition of sensorimotor transmission, thereby causing a pathologically exaggerated startle response that has been termed hyperekplexia or 'startle disease'. Hyperekplexia is an autosomal hereditary disease char- acterized by sudden jumps and exaggerated flexions of neck and trunk in response to unexpected acoustic, visual, or tactile stimuli. Moreover, the exaggerated startle response fails to habituate [2,12]. It is unclear, however, where in the central nervous system deficient GlyRs ultimately cause a disinhibition of the startle response. The extensive clinical study by Brown and co-workers [2] indicated that the normal startle re- sponse and the exaggerated response found in hyper- ekplectics represent physiological and pathological states, respectively, of the same brainstem efferent system. The authors report that the main clinical fea- tures, as well as imaging studies of hyperekplexia, indicate that the major pathological processes occur in the lower brainstem. In fact, it was concluded that "within the lower brainstem, the pathological startle response in hyperekplexia is most likely to originate in
the medial bulbopontine reticular formation" [2]. Pre- vious animal experiments have already shown that GIyR in the brainstem and/or spinal cord are important for the tonic inhibition of the ASR in rats [13,14]. How- ever, in a later study, a potentiation by the GIyR antagonist strychnine of startle-like responses elicited by electrical stimulation of the primary startle pathway could only be observed after stimulation of the cochlear nucleus or the PnC, suggesting that the inhibitory action of glycine on the ASR appears to be more important 'downstream' from the PnC, i.e. in the spinal cord [9]. Since, however, anatomical data indicate a strong prevalence of inhibitory GlyRs in the PnC of rats ([15,34], and present paper), one could still assume that a tonic inhibitory influence on the ASR, and/or inhibitory input relevant for habituation and PPI, might be exerted on PnC neurons, in such a way that local injections of GIyR ligands into the PnC would affect the ASR. In an attempt to investigate the neural mech- anisms of suppression of the ASR, we made microin- jections of the glycine antagonist strychnine and the glycine agonist fl-alanine into the PnC and tested their effects on the ASR of awake rats. Since in humans a loss of inhibition due to deficient GlyRs leads to hyper- ekplexia, the present animal study could have some bearings on the understanding of the human 'startle disease'.
2. Materials and methods
2.1. Immunolabeling of GlyRs in the brain
Two adult Sprague-Dawley rats were decapitated, their brains were quickly dissected, frozen in liquid nitrogen, and stored at -70°C until further processing. Coronal sections of 12 /~m were cut in a cryostat, thaw-mounted on glass slides, and fixed for 5 min in 5% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4). In order to facilitate the antibody pene- tration into the tissue and to prevent non-specific bind- ing of antibody, the sections were treated for 5 min with 0.5% Triton in PBS and preincubated for 30 min in a carrier solution containing 5% bovine serum albu- min in PBS. The sections were then incubated for 1 h at room temperature in the carrier solution containing the monoclonal primary antibody (GIyR mAb 2b, cour- tesy of Dr. H. Betz, Frankfurt). mAb 2b has been described in detail elsewhere [25,29], and it is selective for the 48 kDa ligand binding subunit (a 1) of the 'adult' isoform of the GIyR. The antibody was obtained as hybridoma supernatant and employed at a dilution of 1:100. After incubation in the primary antibody solution, sections were rinsed several times in PBS and transferred into carrier solution containing the fluores- cent secondary antibody, carboxymethylindocyanine 3
M. Koch, E. Friauf / Brain Research 671 (1995) 63-72 65
(Cy 3)-conjugated goat anti-mouse IgG (Dianova, Hamburg), at a dilution of 1:500. Finally, sections were washed in PBS and mounted without dehydration in glycerine-Mowiol jelly. All histochemical steps were performed at room temperature. Sections were ana- lyzed with a microscope equipped with Plan-Neofluar lenses and with fluorescence illumination, employing a rhodamine filter system, and photomicrographs were made on Kodak TX400 film.
2.2. Behavioral experiments
A total of 25 male; albino Wistar rats (weighing 190-230 g at the time of surgery) were used. Both before and after surgery, the animals were housed in groups of 5 under a 12:12 h light/dark schedule (lights on at 06.00 h) and received rat chow and tap water ad libitum.
The animals were anesthetized with chloral hydrate (420 mg/kg body weight, i.p.), placed in a stereotaxic frame, and implanted with two 23-gauge stainless steel guide cannulae aiming bilaterally at the PnC (stereo- taxic coordinates: 9.7 mm caudal, + 0.8 mm lateral, 9.3 mm ventral from Bregma) according to Paxinos and Watson [23]. The guide cannulae were fixed to the skull with dental cement and two anchoring screws. After surgery, the cannulae were fitted with stylets in order to maintain patency, and the animals were al- lowed to recover for one week.
Drugs Strychnine hydrochloride and /3-alanine (both from
Sigma) were dissolved i(n sterile distilled water. The pH of a 33 mM stock solution of strychnine was 5.8 and the pH of a 0.3 M stock solution of/3-alanine was 7.7.
The animals were divided into two groups, one group receiving the glycine antagonist strychnine and the other group receiving the glycine agonist/3-alanine [24]. Tests for short-term (i.e. within-session) habitua- tion and for PPI were performed under three different doses of the respective drugs, so that each animal was tested on six trials. The ASR was measured after placing the rat in a wire mesh cage (20 x 10 x 12 cm 3) mounted on a piezoelectric accelerometer inside a sound-attenuated charaber. The voltage output of the accelerometer caused by the rat's movements was am- plified, digitized, and fed into a computer for further analysis. Acoustic stimuli were generated by a com- puter using a function synthesizer (Hortmann) and were delivered through a loudspeaker mounted at a distance of 40 cm from the test cage. All intensity measurements were done with a 1/2 inch condenser microphone and a measuring amplifier (Briiel and Kjaer). The noise intensity was determined after band- pass filtering outside the hearing range of the rat (lower cutoff, 250 Hz; upper cutoff, 80 kHz). The
whole-body startle amplitude was calculated from the difference between the maximum voltage output of the accelerometer during 80 ms after and during 80 ms before the onset of the acoustic startle stimulus. The spontaneous motor activity of a rat during testing was calculated as the average root mean square (RMS) value of the voltage output of the accelerometer within a time window of 28 s before each startle stimulus.
Test for drug effects on short-term habituation The animals received injections of strychnine (0
nmol, 5 nmol, 10 nmol) or/3-alanine (0 nmol, 50 nmol, 100 nmol) into the PnC through two 30-gauge injection cannulae. The drugs were administered in a volume of 0.3 /zl using two microliter syringes (1.0 /zl; Scientific Glass Engineering) connected to the injection cannulae by flexible PVC tubing, and the rate of infusion was 0.1 /zl/10 s. The injection cannulae remained in the brain during the experiment. The rats were then placed into the test chamber and, following a 10 min acclimation period during which time the rats received no stimuli except for a continuous white background noise of 55 dB SPL (RMS), they received 40 startle stimuli (10 kHz tone pulses, 100 dB SPL, 20 ms duration including 0.4 ms rise/fall times) at an interstimulus interval of 30 s.
Test for drug effects on prepulse inhibition After drug injection the rats were adapted to the
test chamber as described above. The test session in- cluded an initial startle stimulus followed by four dif- ferent trial types given in a pseudo random order: (1) pulse alone (100 dB SPL broad band noise bursts, 20 ms duration), (2) prepulse (75 dB SPL 10 kHz tone pulse, 20 ms duration, including 0.4 ms rise/fall times) followed by a pulse 100 ms after prepulse onset, (3) prepulse alone, and (4) no stimulus. The intensity of the prepulse was 10 dB below the acoustic startle threshold at 10 kHz [26]. Background noise intensity was 55 dB SPL. A total of 15 presentations of each trial type was given with an interstimulus interval of 30 s. The mean ASR amplitude from the 15 values ob- tained for each of the four trial blocks was calculated, and percent prepulse inhibition (%PPI) was computed as [100 × (startle amplitude on pulse alone t r ia l s - startle amplitude on prepulse plus pulse trials/startle amplitude on pulse alone trials)]. The single pulse at the beginning of the test session normally elicits the largest ASR amplitude. The ASR amplitudes to the subsequent stimuli are more homogeneous and, there- fore, the response to the first pulse was discarded. Finally, five animals were randomly chosen from each of the two experimental groups and these ten rats were tested for their ASR and PPI after systemic injection of 1 mg/kg (i.p.) strychnine or saline.
Upon completion of the tests the animals were sacrificed by an overdose of Nembutal. Their brains
66 M. Koch, E. Friauf /Brain Research 671 (1995) 63-72
were removed, immersion-fixed with 8% paraformal- dehyde solution and cut on a freezing microtome. Coronal sections (50 /xm) were Nissl-stained with thio- nine, the injection sites were localized under a light microscope and traced onto drawings of the rat lower brainstem. The effects of microinjections of drugs into the PnC on the A S R amplitude, on habi tuat ion and on PPI were analyzed using repea ted measures analysis of variance (ANOVA) . Short- term habi tuat ion of the A S R ampli tude was analyzed also with correlat ion analysis, whereby the correlat ion of the data points with a hyperbolic curve was tested with A N O V A of regres-
sion (using the G B - S T A T statistical program). Effects of systemic application of strychnine were analyzed with Student 's t-test and were considered significant if P < 0.05 (two-tailed).
3. Results
3.1. A n a t o m y
Within the pont ine brainstem, strong immuno- reactivity in a puncta te fashion, indicative of a dense
Fig. 1. Fluorescence photomicrographs of a coronal section through the pontine brainstem, illustrating the distribution of GIyR immunoreactivity (A) as well as the staining pattern in the PnC (B,C). Notice densely decorated somata and dendrites of giant neurons in the PnC (arrows). The framed areas in A are enlarged in B and C. Dorsal is to the top, medial to the left. Bars = 500/zm in A, 100 ~m in B,C. DTg, dorsal tegmental nucleus; Mo5, motor trigeminal nucleus; SOC, superior olivary complex.
M. Koch, E. Friau[ / Brain Research 671 (1995) 63-72 67
distribution of GlyRs, was present throughout the mo- tor trigeminal nucleus, the dorsal tegmental nucleus, several nuclei of the superior olivary complex, and the
PnC (Fig. 1A). All of these regions are known to contain glycinergic synapses. In the PnC immuno- reactive puncta were seen in the neuropil, but the
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Fig. 2. Serial drawings of coronal sections through the lower brainstem (rostral to caudal from top to bottom), depicting (A) the injection sites of strychnine (circles) and/3-alanine (triangles) and (B) the location of giant neurons immunolabeled with antibodies against the a 1 subunit of the inhibitory GIyR (asterisks). DMTg, dorsomedial tegmental area; Gi, gigantocellular reticular nucleus; IRt, intermediate reticular nucleus; Mo5, motor trigeminal nucleus; PCRtA, parvoceUular reticular nucleus (pars a); PnC, caudal pontine reticular nucleus; PnO, oral pontine reticular nucleus; Pr5, principal sensory trigeminal nucleus; py, pyramidal tract; s5, sensory root of the trigeminal nerve; SOC, superior olivary complex; SubCV, subceruleus nucleus (ventral); 7, facial nucleus; 7n, facial nerve.
68 M. Koch, E. Friauf / Brain Research 671 (1995) 63-72
highest density of fluorescent puncta was seen around unlabeled cell bodies and proximal dendrites of large PnC neurons (Fig. 1B,C). Many of the large PnC neurons had densely decorated somata, resembling the staining pattern previously reported in the pontine reticular nucleus [15]. The large GlyR immunoreactive PnC neurons most likely represent giant PnC neurons, because the location, size, and soma-dendritic mor- phology of these cells are consistent with that previ- ously described for this cell type [21]. The distribution of giant neurons bearing GlyRs, as well as the sites of drug injections in the pontine reticular formation, are schematically depicted in Fig. 2. We estimated that about 60 giant neurons within the confines of the PnC were strongly labeled by the mAb 2b GIyR antibody.
3.2. Behavior
From the 25 rats which received microinjections into the brainstem, three animals were discarded from the further analysis due to misplaced injection cannulae. In the remaining 22 rats, all injections were made in close vicinity of neurons bearing GlyRs. However, neither strychnine nor/3-alanine had a significant influence on the ASR amplitude (Fig. 3A) after injection into the PnC (ANOVA for strychnine: F2,18 = 1.29, P = 0.3; ANOVA for /3-alanine: F2,22 = 1.6, P = 0.22). More- over, as shown in Fig. 3B, none of the drugs affected PPI (ANOVA for strychnine: F2,18 = 0.45, P = 0.65; ANOVA for fl-alanine: F2,22 = 0.04, P = 0.96). Fig. 4 shows the course of decline of the ASR amplitude observed after repeated testing under different drug conditions. A hyperbolic regression curve was fitted to the data points. The correlation coefficients computed by correlation analysis were r > 0.8, and the ANOVA of regression gave a highly significant correlation of the data points with the hyperbolic model curve in all cases (P < 0.02-P < 0.001). A closer inspection of the time course of the ASR habituation shown in Fig. 4 indi- cated that the decline of the response amplitude was most pronounced between the first and the second block of trials. These data appeared to suggest that both strychnine and /3-alanine facilitated habituation at the lower dose, yet retarded habituation at the higher dose. However, comparing the mean differences between the first and the second block of trials be- tween the different doses of drugs revealed no statisti- cally significant effect (ANOVA for strychnine: F2,18 = 0.71, P = 0.51; ANOVA for /3-alanine: Fe,22 = 1.68, P = 0.21). These results show that neither strychnine nor/3-alanine significantly changed the time course of the ASR amplitude observed after saline injections, indicating that short-term habituation of the ASR was not affected. The spontaneous motor activity measured between the startle stimuli was neither significantly affected by strychnine (F2,18 = 1.14, P = 0.34) nor by
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Fig. 3. Bar diagram showing (A) the mean ASR amplitude ( + S.E.M.) and (B) the mean percent prepulse inhibition ( + S.E.M.) of the ASR after injections of different doses of strychnine (n = 10) and fl-alanine (n = 12) into the PnC. The different treatments did not induce statistically significant effects.
/3-alanine (F2 ,22 = 2.78, P = 0.08) (data not shown). Fig. 5 shows that, in contrast to the local application experi- ments, systemic administration of strychnine (1 mg/kg body weight) significantly increased the ASR (Student's t-test: T = -3 .7 , P < 0.01, pulse strychnine compared to pulse saline). However, this treatment did not affect PPI, as indicated by pairwise comparisons between the ASR amplitudes in the absence and presence of pre- pulses. Presentation of a prepulse reduced the ASR amplitude both in saline-treated rats (%PPI = 65%; T = 2.1, P = 0.05) and in animals systemically treated with strychnine (%PPI = 68%; T = 2.8, P = 0.01). The spontaneous motor activity was not increased in this group compared to saline (T = -0 .02, P = 0.98. Data not shown).
4. Discuss ion
The present study demonstrates that GlyRs occur in abundance on giant neurons in the PnC, a brain region of great importance for the ASR. However, microinjec- tions of the glycine antagonist strychnine or the glycine agonist/3-alanine into the PnC did not affect the ASR. Moreover, neither short-term habituation nor PPI, two
M. Koch, E. Friauf / Brain Research 671 (1995) 63-72 69
prominent phenomena of ASR suppression, were af- fected.
Inhibitory GlyRs in the PnC have already been demonstrated earlier by means of autoradiographical labeling with [3H]strychnine [34]. In the present study, we have focused on the subpopulation of the magno- cellular ('giant') neurons which have mean soma diam- eters of more than 40/~,m. They comprise about 1% of all neurons in the PnC, and have already been charac- terized in detail, both morphologically and physio- logically, as acoustically responsive reticulospinal neu- rons [21,22]. We estimated that about 60 giant neurons within the PnC of an individual rat express the in- hibitory GIyR. As previous cell counts revealed a total of approximately 200-350 giant neurons in the PnC of
a rat [16], we estimate that roughly 1//3 to 1//6 of these cells bear GlyRs. This indicates that a considerable part of these giant neurons should be sensitive to glycinergic drugs. In fact, an inhibitory action of glycine on pontine reticulospinal neurons has already been shown in the cat [11]. However, our behavioral data, fotlowing local drug injections, indicate that inhibitory GlyRs in the PnC are not important for the ASR. These data support the findings of Davis and col- leagues [9], who suggested that spinal GlyRs rather than those located in the brain exert a tonic inhibition of the ASR. The rationale of their experiments was that electrical stimulation of parts of the serially orga- nized ASR pathway elicits startle-like responses and that the amplitude of these responses could only be
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Fig. 4. Diagrams showing short-term habituation of the ASR after injection of strychnine (0, 5, 10 nmol) or/3-alanine (0, 50, 100 nmol) into the PnC. The mean ASR amplitude (n = 10 for strychnine, n = 12 for/3-alanine) is plotted against the stimulus number (in blocks of 5 stimuli) and a hyperbolic regression curve is fitted to the data points. The correlation coefficient r is given in each panel.
70 M. Koch, E. Friauf /Brain Research 671 (1995) 63-72
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Fig. 5. Bar diagram showing the effect of systemic administration of saline or strychnine (1 mg/kg, i.p.) on the ASR amplitude (mean + S.E.M. of n = 10) in the absence (Pulse) or presence (PP-Pulse) of a prepulse given 100 ms prior to the startle pulse. Asterisks indicate a significant difference between the Pulse and PP-Pulse condition (Student's t-test: *P = 0.05, * *P = 0.01). The difference between the ASR amplitudes (Pulse) under saline and under strychnine is also significant (Student's t-test, P < 0.01).
I
modulated by systemic administration of drugs if the action of the drug is 'downstream' from the electrical stimulation site. Their suggestion that supraspinal GlyRs are of minor importance for the ASR was based on the finding that systemically administered strych- nine increased startle-like responses which were elec- trically elicited from the PnC. Our present experiments also failed to show an effect of local injection of GlyR ligands on short-term habituation of the ASR, consis- tent with findings of Kehne and Davis [14] who have shown that systemic administration of strychnine does not alter habituation of the ASR. The neural mecha- nism of short-term habituation of the ASR in rats is still unclear. It can be assumed that the repeated activation of a synapse leads to a process of synaptic depression, by attenuating presynaptic transmitter re- lease or by lowering the sensitivity of postsynaptic receptors, or by both. If these processes of modifica- tion of the response strength act within the stimulus- response pathway itself they are referred to as 'intrin- sic' mechanisms. It has been suggested that short-term habituation of the ASR occurs within the stimulus-re- sponse pathway itself, and not through the action of more rostral brain structures, since chronically decere- brated rats bearing knife cuts at the level of the mid- brain (colliculus inferior), still showed short-term habituation [20]. However, based on decerebration ex- periments, it can only be concluded that brain struc- tures situated rostral to the stimulus-response pathway do not influence short-term habituation, but one can- not yet completely rule out the possibility that an active inhibitory influence accounts for short-term habitua-
tion. Inhibitory neurotransmitters released by segmen- tal interneurons could still influence the stimulus-re- sponse pathway in the case of a transection of the brain at the level of the colliculus inferior, because this segment contains the rostral-most elements of the star- tle pathway, the cochlear nuclei and the PnC. As glycine in the PnC appears to be of no importance for the inhibition of the ASR, GABA would be another possible inhibitory transmitter candidate to be tested in future experiments, since it potently inhibits acousti- cally responsive PnC neurons [19]. Finally, the present study shows that neither local injection of strychnine into the PnC nor systemic application of strychnine impairs PPI. These data are consistent with our previ- ous finding that acetylcholine might be the transmitter of the inhibitory influence of an acoustic prepulse on PnC neurons [18].
Our present finding that GIyR ligands in the PnC do not affect the ASR is somewhat surprising given the presence of glycine release and uptake mechanisms in the pontine reticular formation [5], given the fact that glycine inhibits glutamate-evoked activity of reticu- lospinal neurons in the pontine reticular formation of cats [11], and given the strong prevalence of GlyRs on giant PnC neurons. A role of these giant neurons in the mediation and modulation of the ASR has already been shown by selective lesions [16] and by an intra- cellular recording study, where both PPI-like and habituation-like phenomena have been described to occur in acoustically responsive reticulospinal giant neurons in the PnC [22]. It is very unlikely that proce- dural factors may account for the failure to find an effect of strychnine or /3-alanine here. First, there is the possibility of a ceiling effect masking any increase of the ASR amplitude due to a very intense startle stimulus. This possibility, however, can be excluded, because the systemic administration of strychnine led to a large increase of the ASR and because our previ- ous studies have shown that the ASR amplitude in response to startle stimuli of 100 dB SPL can be increased by different treatments, such as stimulation of the amygdala, by as much as 170% [17]. The second source of error might be an insufficient concentration of the drug solutions used. The highest dose of strych- nine used in our study (3.7/~g/0.3/~1) has been shown to increase the ASR amplitude if administered onto the spinal cord' [13]. In addition, it represents a local concentration of maximally 33 mM, which is far higher than the IC50 of strychnine at the a 1 subunit of the GlyR (about 20 nM [27]) and also higher than the K D for [3H]strychnine binding at the GlyR (approximately 10 nM [24]). fl-Alanine has been reported to exert a lower affinity (K D = 5 /zM) for the GlyR than strych- nine [24] and therefore was used here in a 10-fold higher dose than strychnine. Third, the strychnine solu- tion injected into the PnC was not buffered and the pH
. . M. Koch, E. Friauf/Brain Research 671 (1995) 63-72 71
of 5.8 might be conside, red too low for sufficient bind- ing to the GlyR, since it has been shown that the GlyR function drops to about 50% of its maximal value at a p H of 5.0 [28]. However , Schmieden et al. [28] have also shown that the IC50 of strychnine at a p H of about 6.0 is still lower than 40 nM, indicating that the p H of 5.8 of the solution used~ in the present study would not prevent strychnine f rom blocking the action of glycine. Moreover , the different strychnine solutions used by Kehne and colleagues [13] to effectively increase the A S R after application onto the spinal cord were also unbuffered and the pH[ ranged f rom 5 to 7. The large increase of the A S R ampli tude seen after systemic injection of strychnine confirms the data of Kehne and Davis [14] and extends their findings by showing that PPI is not affected by strychnine. It should be noted that an increase o f the A S R ampli tude has been ob- served after injection of strychnine into the lateral ventricle [13], but it is unclear where in the brain this possibly disinhibitory effect occurs . /3-Alanine has been shown to act as a potent and selective glycine agonist [24], and therefore, the present observat ion that /3- alanine did not affect the A S R ampli tude fur ther sup- ports the conclusion that GlyRs in the PnC do not play an essential role for the inhibition of the ASR.
Shiang et al. [30] have identified point mutat ions in the gene encoding for the a 1 subunit of the GIyR in patients suffering f rom hyperekplexia. Brown and co- workers [2] have sugge,;ted that the exaggerated startle response ampli tude and the poor habi tuat ion of the startle response found in hyperekplexia reflects patho- logical states of disinhibition in the same bulbopont ine reticular format ion pathway that also mediates the normal startle response. Their not ion is based on the overall clinical features (e.g. the pa t te rn of muscle recrui tment in the startle response to acoustic or somesthet ic stimuli), magnet ic resonance imaging, elec- trophysiological data, and on pos tmor tem neuropa tho- logical findings. Our data f rom experiments with rats have shown that b lockade of GlyRs in the PnC does nei ther increase the A S R nor interfere with the inhibi- t ion of the ASR, and, hence, would not suppor t the assumption that hypetekplexia is due to a defect in caudal pont ine bra ins tem GlyRs. The present findings, toge ther with the work: of Kehne et al. [13] and Davis et al. [9], ra ther point ~owards a spinal locus of hyper- ekplexia.
Acknowledgements
We thank Professor H. Betz (Frankfurt) for his generous gift of GIyR m A b 2b antibody, Mr. M. Kun- gel for his helpful corrmaents on the manuscript , Mrs. H. Zillus for expert technical assistance, Mrs. Y. Brockmann for running some of the tests and Mr. M.
Fend t for his help with the Suppor ted by the Deutsche SFB 307 and Fr 772/1-3 .
revision of the paper . Forschungsgemeinschaf t
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