- Email: [email protected]
Lesions of the nucleus basalis magnocellularis do not impair prepulse inhibition and latent inhibition of fear-potentiated startle in the rat
Brain Research 815 Ž1999. 98–105
Research report
Lesions of the nucleus basalis magnocellularis do not impair prepulse inhibition and latent inhibition of fear-potentiated startle in the rat Cornelia Schauz ) , Michael Koch Tierphysiologie, UniÕersitat Auf der Morgenstelle 28, D-72076 Tubingen, Germany ¨ Tubingen, ¨ ¨ Accepted 20 October 1998
Abstract The present study tested if lesions of the nucleus basalis magnocellularis ŽNBM. affect prepulse inhibition ŽPPI. of the acoustic startle response and latent inhibition ŽLI. of fear-potentiated startle. The NBM is known to play an important role in learning and memory. Recently, the interest of research focused on its role in attentional and response selection processes. We here tested the effect of excitotoxic NBM-lesions on PPI, a phenomenon of sensorimotor gating that occurs at early stages of information processing. We also assessed the lesion effects on LI, a phenomenon of reduced conditioning after stimulus preexposure that can be used to measure selective attention. Bilateral infusions into the NBM of 80 nmol of quinolinic acid markedly reduced the number of choline acetyltransferase immunopositive neurons in the NBM and lead to a pronounced reduction of acetylcholine esterase in the cortex and the amygdala. However, no effects on PPI, fear-conditioning, or LI of fear-potentiated startle were found. Therefore, we conclude that there is no NBM-driven attentional or response selection process involved in PPI. Furthermore, the simple association learning in the classical conditioning paradigm used for fear-potentiated startle or LI is unaffected by NBM-lesions. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Acoustic startle response; Fear-potentiated startle; Latent inhibition; Nucleus basalis magnocellularis; Prepulse inhibition; Schizophrenia
1. Introduction The suppression of inappropriate responses is an important principle of behavioral organisation. Two well known phenomena of response suppression are prepulse inhibition ŽPPI. of the acoustic startle response ŽASR. and latent inhibition ŽLI.. PPI is the reduction of the ASR magnitude when a weak stimulus, which by itself does not elicit an ASR, is presented prior to the startling stimulus w17x. PPI is viewed as a phenomenon of sensorimotor gating that occurs at early stages of information processing and probably serves to protect an ongoing stimulus processing routine from disruption by an interfering sensory or motor event. LI refers to the phenomenon of retarded conditioning after repeated non-reinforced presentation of a prospective conditioned stimulus during a preexposure stage w25x. LI is a form of learned inattention or cognitive suppression of a response w23,28x.
) Corresponding author. Fax: [email protected]
q 49-7071-292618;
E-mail:
There is considerable interest in the investigation of the neuronal basis of PPI and LI in rats, because both phenomena of response suppression are impaired in schizophrenics w26,40x. Previous work in rats has shown that PPI and LI are regulated by somewhat overlapping, yet clearly distinguishable cortical, limbic, striatal and tegmental circuitries w22,41,47x. The suppression of inadvertent motor or cognitive events requires the evaluation of the respective stimuli either at the perceptual level or at the cognitive level. Therefore it can be assumed that deficient PPI and LI in schizophrenics reflects dysfunctions in perceptual andror cognitive filter mechanisms of the brain. While most researchers acknowledge a role of attention in fear-conditioning and in LI, the attribution of an attentional deficit to impaired PPI is less clear w7,11,41x. Since treatments that reduce PPI Že.g., apomorphine. do not impair the ASR peak latency reduction by the prepulse it was suggested that PPI deficits are not simply due to a reduced prepulse detection, e.g., through inattention w41x. On the other hand, PPI-disrupting treatments do impair the detection of a prepulse in a combined fear-potentiated ASRrPPI-paradigm w42x indi-
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 1 3 4 - 2
C. Schauz, M. Kochr Brain Research 815 (1999) 98–105
cating that reduced attention might affect PPI performance. In humans PPI is enhanced if the subjects are explicitly instructed to attend to the prepulse w11x. It is noteworthy, however, that the latter paradigms are not the usual procedures of assessing PPI and were constructed to reveal the possible role of attentional mechanisms in PPI. Since the nucleus basalis magnocellularis ŽNBM. plays an important role in the processing of behaviorally relevant stimuli and in selective attention w19,46,49x the present study attempted to assess the role of the NBM in fear-conditioning and LI. Notwithstanding the objections against a prominent role of attention in PPI, we also tested a potential contribution of the NBM to the regulation of PPI. The NBM in rodents consists of large cholinergic neurons, GABAergic neurons and a considerable proportion of not yet identified non-cholinergic neurons w16x. These neurons are dispersed in the basal forebrain along the internal capsule, extending from the medial septum and the nucleus of the diagonal band of Broca Žhorizontal and vertical limbs., invading the substantia innominata and the globus pallidus w1,37x. The NBM has strong projections to the cerebral cortex and also provides the cholinergic input to the lateral and basolateral part of the amygdala w49x. Excitotoxic lesions lead to a significant depletion of cortical acetylcholine esterase ŽAChE.-positive fiber density w27x, but the excitotoxic damage to the cortex or the amygdala differs according to the neurotoxin used, as shown by Boegman et al. w4x. Because in that study quinolinic acid also produced considerable damage to the cholinergic innervation of the amygdala, we have used this toxin for the NBM lesions. In the amygdala acetylcholine ŽACh. is involved in the storage of aversive memories w9x. The involvement of the basal forebrain cholinergic system in cognitive functions and especially its important role in mnemonic processes is known since a long time because of its possible role in cognitive impairments associated with senile dementia and Alzheimer’s disease w15,48x. Recently, the role in attentional processes of the cholinergic neurons of the NBM has been emphasized w29,30, 33,38,44,48x. The cortical and limbic cholinergic projections of the NBM have been suggested to act as specific amplifiers that enhance the salience and the representation in the cortex of behaviorally important stimuli and reduce the behavioral impact of irrelevant stimuli w19,46,49x. The investigation of the role of the basal forebrain cholinergic system in LI yielded inconsistent results. Rochford et al. found a facilitating effect on LI of systemically applied nicotine and nicotinic ACh receptor agonists and this enhancement was reversed by application of nicotinic ACh receptor antagonists w34x. Another experiment by the same group demonstrated an impairment of LI after lesioning of the NBM, however, the effect reported by this group was somewhat difficult to interpret, since lesioned preexposed and non-preexposed rats showed similar behavioral performance as control preexposed rats and not, as would be expected for deficient LI, similarity with
99
control non-preexposed rats w35x. LI of appetitive conditioning was reduced by ACh-specific lesions of the septohippocampal system w2x, but was spared after lesions of the NBM w8x. In the latter study, however, the lesions were produced by 192 IgG-saporin, which does not destroy the cholinergic input to the amygdala. We here tested if NBM lesions with quinolinic acid affects PPI, fear-potentiated startle or LI of fear-potentiated startle.
2. Materials and methods 2.1. BehaÕioral experiments 2.1.1. Animals A total of 42 experimentally naive male Wistar rats Ž200–300 g, Charles River, Sulzfeld, Germany. were used. The animals were kept in groups of 6 per cage in a colony room under a 12 h light–dark cycle Žlights on at 0700 h. with food and water available ad lib. Sixteen rats received PBS injections as controls, 25 rats were bilaterally injected into the NBM with 80 nmol quinolinic acid. The rats were allowed to recover from surgery for 6–7 days. All experiments were done in accordance with international guidelines for the care and use of animals for experiments and were approved by the local council of animal care ŽRegierungsprasidium Tubingen, ZP 4r96.. ¨ ¨ 2.1.2. Surgery Rats were anaesthetized with chloral hydrate Ž420 mgrkg i.p.. and placed in a Kopf stereotaxic frame. After exposition and trepanation of the skull a 30-gauge injection cannula, attached to a microliter syringe, was placed in the substantia innominatarglobus pallidus region, since the magnocellular cholinergic neurons of the NBM are mainly distributed throughout this area. The coordinates were 1.3 mm caudal, "2.3 mm lateral and 7.0 mm ventral to Bregma w32x. In lesion rats 80 nmol of quinolinic acid in 0.3 ml PBS Žadjusted to pH 7.4 with 1 N NaOH. was bilaterally infused, with an infusion rate of 0.1 mlrmin. The injection cannula remained in the brain for another 3 min after infusion was completed to allow for the toxin to diffuse into the parenchyma. In control animals the same volume of PBS was infused. Seven rats died after the surgery because of the lesion effect, one rat was excluded from the study because it did not show PPI in the pretest. PPI was measured before and after surgery. After the PPI measurements were completed the LI procedure began. 2.1.3. Test for prepulse inhibition PPI was measured after 5 min adaptation time to the test chamber and one initial startle stimulus. The test
100
C. Schauz, M. Kochr Brain Research 815 (1999) 98–105
session consisted of five different trial types presented in a randomized order: Ž1. pulse alone Ž100 dB SPL broad band noise bursts, 20 ms duration., Ž2. prepulse and pulse Ž70 dB SPL or Ž3. 60 dB SPL 10 kHz tone prepulse, 20 ms duration, including 0.4 ms riserfall times. followed by a pulse 100 ms after prepulse onset, Ž4. prepulse alone Ž70 dB SPL. and Ž5. no stimulus. Background noise intensity was 55 dB SPL. Twelve presentations of each trial type were given with an interstimulus interval of 30 s. PPI was measured as the difference between the pulse alone trials and the prepulse–pulse trials and the percent PPI was calculated as w100 = Žmean ASR amplitude on pulse alone trials y mean ASR amplitude on prepulse– pulse trials.rmean ASR amplitude on pulse alone trialsx. 2.1.4. Test for latent inhibition of fear-potentiated startle Preexposure and conditioning were performed on two consecutive days, followed by a test session 2 h after the last conditioning trial. 2.1.4.1. Preexposure. Each rat was placed in a dark conditioning box Ž38 = 60 = 28 cm3 . provided with a floor made of steel bars spaced 15 mm apart. Preexposed rats received 40 presentations of the prospective CS Žwhite light, 15 W, duration 3.7 s, interstimulus interval 30 s.. Rats of the non-preexposed group were left in the conditioning box for an identical period of time without receiving the prospective CS. 2.1.4.2. Conditioning. Each rat was trained in the conditioning box to associate the CS with the UCS, a 0.6 mA electric footshock produced by a shock generator Žcustom . presented in the last made at the University of Tubingen ¨ 0.5 s of a 3.7 s white light Ž15 W.. 2.1.4.3. Test. The ASR was measured in a wire mesh cage Ž20 = 10 = 12 cm3 . with a steel floor that was mounted on a piezoelectric accelerometer in a sound-attenuated chamber. The test consisted of 5 min of adaptation to the startle chamber and ten initial acoustic startle stimuli Ž100 dB 10 kHz pure tone of 20 ms duration including 0.4 ms rise and fall times. that were presented to produce a stable baseline of ASR, but were not statistically evaluated. Then 40 acoustic startle stimuli, half of them in darkness Žtone-alone trials., the other half 3.2 s after the onset of the 3.7 s light CS Žlight-tone trials. were presented in a randomized order. Background noise intensity was 55 dB SPL. To assess fear-conditioning the whole-body ASR amplitude in darkness or in the presence of the light was calculated, respectively. LI was inferred from the difference in fearpotentiation between preexposed and non-preexposed rats. 2.2. Histology After completion of the behavioral studies the rats were deeply anaesthetized with nembutal and perfused transcar-
dially with 100 ml PBS followed by 500 ml cold 4% paraformaldehyde with 0.01% glutaraldehyde and then with 0.1 M phosphate buffer ŽPB. with 10% sucrose. The brains were placed in 20% sucrose in PB at 48C over night. Coronal sections Ž50 mm. were cut on a freezing microtome, the sections were divided in three series for Nissl staining, for AChE staining according to a modified version of the protocol of Karnovsky and Roots w18x and for the immunohistochemical detection of choline acetyltransferase ŽChAT.. 2.2.1. AChE staining Sections were left in 0.1 M PB for 4–6 days at 48C before they were incubated for 1 h in an acetylthiocholine solution Ž0.2 mM ethopropazine, 10 mM glycine, 2 mM CuSO4 , 4 mM acetylthiocholine and 50 mM sodium acetate, adjusted to pH 5.0 with drops of glacial acetic acid.. After washing the sections 6 times in dH 2 O they were developed for 1 min in a sodium sulfide solution Ž300 mM, adjusted with 10 M HCl to pH 7.8.. Sections were rinsed again 6 times in dH 2 O and were then incubated in a silver nitrate solution Ž59 mM. for 30 s. After washing the sections in dH 2 O they were mounted on gelatine-coated slides, dehydrated in 100% ethanol Ž1 min., cleared in two changes of xylene and coverslipped with Entellan. 2.2.2. ChAT-immunostaining After washing the slices in PBS they were incubated in a blocking solution Ž3% goat serum in PBS with 0.2% Triton X-100. at room temperature and then incubated in a polyclonal rabbit anti-human ChAT serum Žkindly donated by Dr. Louis B. Hersh. diluted 1:1000 in carrier Ž3% goat serum in PBS. for 48 h at q48C. The sections were washed in PBS several times and then incubated for 1 h in a biotin-labeled goat anti-rabbit IgG antiserum ŽVector., diluted 1:1000 in carrier at room temperature. Sections were rinsed in PBS and incubated for 45 min with an avidin–biotin peroxidase-complex ŽVectastain ABC-Elite kit. diluted 1:100 in 0.01 M PBS containing 0.5 M NaCl. The sections were again rinsed in PBS and were processed in a solution of 0.05% 3,3-diaminobenzidine tetrahydrochloride and 0.01% H 2 O 2 in TBS, to visualize the immunocomplex. Finally, the sections were mounted on gelatine-coated slides, air dried, dehydrated in alcohol, cleared in xylene, and coverslipped with Entellan. 2.3. Statistics The data are presented as mean " S.E.M. All data were analyzed by a multi-variable analysis of variance ŽANOVA.. In PPI experiments treatment Žlesionrcontrol. was the between-subject factor and prepulse intensity Ž60 dBr70 dB. and session Žbefore surgeryrafter surgery. the within-subject factors. Effects on ASR were also computed with treatment and session as factors. In LI experiments treatment Žlesionrcontrol. and stimulus exposure Žpreex-
C. Schauz, M. Kochr Brain Research 815 (1999) 98–105
101
posurerno preexposure. were between-subject factors and nature of trial Žtone-alonerlight-tone. was within-subject factor. ANOVA was followed by Tukey’s protected t-tests for post hoc pairwise comparisons. An alpha value of p - 0.05 was considered to represent a significant difference, a value of p - 0.01 as highly significant. 3. Results 3.1. Anatomy The histological analysis revealed that quinolinic acid infusions into the NBM lead to a destruction of cholinergic neurons in the NBM ŽFig. 1A and B. and to a reduction of AChE mainly in the neocortex and lateral amygdala ŽFig. 1C and D.. Additional to this documented primary lesions we also found extension of the lesion in parts of the globus pallidus, the substantia innominata, the bed nucleus of the stria terminalis and nonspecific destruction, probably secondarily, in different thalamic nuclei Žnot shown..
Fig. 2. Percent PPI in control Ž N s16. and NBM-lesioned Ž N s16. rats. Percent PPI scores are shown before Žblack bars. and after surgery Žwhite bars. following 70 dB or 60 dB acoustic prepulses.
3.2. BehaÕior 3.2.1. Effect of NBM lesions on PPI Fig. 2 shows the percent PPI scores in control and NBM-lesioned rats. ANOVA revealed a significant effect
of prepulse intensity on PPI with lower prepulse intensities yielding lower PPI scores w F Ž1,123. s 46.7; p - 0.01x. No significant effect of treatment w F Ž1,123. s 1.5; p ) 0.1x or
Fig. 1. Photomicrographs of frontal sections through the basal forebrain stained immunocytochemically for ChAT of a NBM-lesioned rat ŽA. and a control rat ŽB.. A loss of ChAT-immunoreactive cells documents the extent of the NBM-lesion. Histochemical AChE-staining of brain sections is shown from a NBM-lesioned rat ŽC. and a control rat ŽD.. The loss of AChE-staining indicates a reduction of the cholinergic innervation of the cortex and the lateral amygdala. Bar s 500 mm.
102
C. Schauz, M. Kochr Brain Research 815 (1999) 98–105
session w F Ž1,123. s 1.1; p ) 0.1x was observed and there was no significant interaction between treatment and session w F Ž1,123. s 0.7; p - 0.5x, indicating that NBM lesions do not reduce PPI at different prepulse intensities. 3.2.2. Effect of NBM lesions on LI The effects of NBM lesions on LI of fear-potentiated startle are shown in Fig. 3A. An overall ANOVA was performed revealing a significant interaction between stimulus exposure and nature of trial w F Ž1,62. s 4.3; p - 0.05x, non-preexposed rats showed a significant increase in light-tone trials compared to tone-alone trials ŽTukey’s protected t-test, p - 0.01. indicative of a state of fear in these rats. In preexposed rats there was no significant difference between light-tone trials and tone-alone trials ŽTukey’s protected t-test; p ) 0.5. indicating LI of fear-
Fig. 4. ASR amplitude in control Ž N s16. and NBM-lesioned Ž N s18. rats before or after lesion or sham-treatment.
potentiated startle. There was no significant interaction between exposure to a stimulus, nature of trial and lesion w F Ž1,62. s 0.03; p ) 0.5x. Fig. 3B shows the absolute differences between tone-alone and light-tone trials in control and lesion groups. ANOVA revealed a significant difference between preexposed and non-preexposed rats in lesioned and control rats w F Ž1,31. s 17.25, p - 0.01x. These results show that NBM lesions do not impair fearpotentiated startle or LI of fear-potentiated startle. 3.2.3. Effect of NBM lesions on ASR We also tested the effect of NBM lesion on the ASR, produced by 100 dB broad band noise pulses. Mean ASR " S.E.M. in control and lesioned rats are shown in Fig. 4. A two-way ANOVA with treatment and session indicated whether an effect of treatment w F Ž1,60. s 0.2; p ) 0.5x or an effect of session w F Ž1,60. s 0.9; p ) 0.1x, nor an interaction between treatment and session w F Ž1,60. s 0.1; p ) 0.8x. We therefore conclude that NBM lesions have no effect on the ASR.
4. Discussion
Fig. 3. ŽA. ASR amplitude Žarbitrary units. of tone-alone and light-tone trials in sham operated Žcontrol. and NBM-lesioned rats, which were either non-preexposed ŽNPE, control N s8, lesion N s8. or preexposed ŽPE, control N s8, lesion N s10.. ŽB. Difference between tone-alone and light-tone trials in controls and NBM-lesioned rats, PE or NPE to the light CS. ŽAsterisks indicate a p- 0.01, Tukey’s t-test after significant main effect of ANOVA..
The present study shows that NBM-lesions that significantly reduce the number of cholinergic cells in the NBM and reduce the presence of AChE in limbic and cortical brain regions do not affect PPI, fear-conditioning, LI of fear-potentiated startle or the ASR. PPI is a measure of sensorimotor gating that is regulated by a complex forebrain circuit including the septohippocampal cholinergic system w6,20x, the basolateral amygdala w45x, the nucleus accumbens w22,41x, and the prefrontal cortex w21x. While PPI is generally related to a sensorimotor gating mechanism that facilitates attention w41x, some authors discuss PPI as a behavioral phe-
C. Schauz, M. Kochr Brain Research 815 (1999) 98–105
nomenon that requires attentional processes w7,11x. NBM lesions disrupt attention but not learning and memory processes in monkeys w44x and an attentional dysfunction was also demonstrated in rats following NBM lesions w29,33x or stimulation of the GABAergic NBM neurons w30x. Based on the assumption that the NBM plays an important role in selective attention w38,48x, we hypothesized that NBM lesions might influence PPI. Our data show that the NBM, which provides a strong cholinergic input to the cortex and the amygdala, is not necessary for this kind of sensorimotor gating. Lesions of the medial septal nucleus, which provides the cholinergic input to the hippocampus, also had no effect on PPI w20x. Taken together with the present data it can be concluded that the forebrain cholinergic system is not necessary for the regulation of PPI, and PPI is not dependent on NBM-driven attention or response selection mechanisms. LI was measured as the reduction of the fear-potentiated ASR by preexposure to the visual CS w39x. In this fear-conditioning paradigm the visual CS is probably conveyed via the perirhinal cortex to the lateralrbasolateral nuclei of the amygdala where the association between neutral and aversive stimulus is presumably formed w36x. These nuclei project to the central amygdaloid nucleus which directly impinges on the primary acoustic startle circuit at the level of the caudal pontine reticular nucleus to facilitate the ASR w10,22x. We hypothesized that in LI of fear-potentiated ASR the disturbance of the expression of fear takes place at the level of the amygdala w39x. Beninger and his group demonstrated that the type of neurotoxin used for the lesions differentially affects the extent of the cholinergic damage to the cortex and the amygdala, and that damage to the amygdala more likely contributes to learning deficits w3x. Our data support the finding that quinolinic acid lesions of the NBM lead to a considerable reduction of AChE in the lateral amygdala. It is already known that ACh plays an important role in the storage of emotional memory in the amygdala w9x. However, our data show that fear-conditioning Žand LI of fear-conditioning. is still possible after impairment of the NBM-amygdaloid cholinergic system. Because a reorganisation of sensory cortical areas has been found after pairing NBM-stimulation with sensory stimuli w19x it seemed reasonable to assume that during preexposure or during conditioning the NBM affects the processing of the visual CS probably at the cortical level. Since NBM lesions did neither disrupt LI nor fear-conditioning, our present findings support the view that the NBM is involved in complex association or spatial learning tasks but not if a simple association between two stimuli is sufficient for learning, as it is in classical conditioning paradigms w5x. A role of the cholinergic forebrain system in LI was discussed recently w47x based on the finding that systemically applied nicotine and nicotinic ACh receptor agonists augment LI and that this enhancement was reversed after
103
application of nicotinic ACh receptor antagonists w34x which is consistent with the assumption that ACh has cognitive enhancing properties w38x. Therefore, the lack of effect of NBM-lesions on LI reported in this paper is surprising, especially in the light of the fact that the NBM has been implicated in attentional processes related to learning w13x and because attention has been considered to be important for LI w23,24,28x. In fact, a disruption by NBM-lesions of LI in a different paradigm has been reported by Rochford et al. w35x. However, the results reported by this group reveal some difficulties of interpretation since the impairment of LI is concluded from the similarity of the behavioral performance of lesioned preexposed and non-preexposed rats with control preexposed rats. Because destruction of the cholinergic cells in the medial septum, which provide the cholinergic input to the hippocampus, was shown to disrupt LI w2x Žbut see Ref. w12x., one can conclude that ACh affects LI at the level of the hippocampus rather than at the cortical level. This conclusion is supported by the finding that immunolesioning of the NBM with 192 IgG-saporin, which lead to a considerable reduction of AChE staining in the neocortex, did not affect LI w8x. It is unlikely that LI is so robust in our paradigm that it cannot be disrupted. This kind of ceiling-effect of LI can be excluded on the basis of the observation that LI of fear-potentiated startle was reduced by a systemic treatment with 1 mgrkg of amphetamine w39x. Because of the scattered distribution of the NBM throughout the basal forebrain, lesions restricted to the cholinergic neurons of the NBM are difficult to perform, as reported previously by other groups w14x. Therefore, we are aware of the fact that the destruction of non-cholinergic cells scattered in or around the NBM remains a problem for the interpretation of our results. The conflicting results following NBM lesions described in the literature are ascribed by some groups to this damage of non-cholinergic cells or other basal forebrain structures. Negative results demand cautious interpretation because of the possibility of methodical fallacies, but our data clearly show that the cholinergic innervation of the cortex and the amygdala is damaged after NBM lesions with quinolinic acid. The concentration of quinolinic acid used here is in accordance with those reported by other groups producing lesions of considerable size w43x and well-documented decreases in cortical and amygdaloid ChAT activity w4,31x. Our lesions extended into parts of the globus pallidus, the substantia innominata, the bed nucleus of the stria terminalis and, probably secondarily Ži.e., transsynaptically and not by diffusion., into parts of the thalamus. This cerebral tissue damage does not produce a sensorimotor gating deficit nor does it affect the simple associative learning in a classical conditioning paradigm. Nevertheless, we conclude that extensive excitotoxic lesions of the NBM did not lead to information processing deficits at the perceptual or cognitive level.
104
C. Schauz, M. Kochr Brain Research 815 (1999) 98–105
Acknowledgements w17x
This study was supported by the DFG ŽSPP 1001 and, Heisenberg Programm for MK.. We are grateful to Dr. H. Herbert and Mr. A. Guthmann for valuable advice with the histology, to Dr. P. Pilz for help with the statistics and to H. Zillus for excellent technical assistance.
w18x w19x w20x
References w1x D.M. Armstrong, C.B. Saper, A.I. Levey, B.H. Wainer, R.D. Terry, Distribution of cholinergic neurons in rat brain: demonstrated by the immunocytochemical localization of choline acetyltransferase, J. Comp. Neurol. 216 Ž1983. 53–68. w2x M.G. Baxter, P.C. Holland, M. Gallagher, Disruption of decrements in conditioned stimulus processing by selective removal of hippocampal cholinergic input, J. Neurosci. 17 Ž1997. 5230–5236. w3x R.J. Beninger, S. Kuhnemann, J.L. Ingles, K. Jhamandas, R.J. ¨ Boegman, Mnemonic deficits in the double Y-maze are related to the effects of nucleus basalis injections of ibotenic and quisqualic acid on choline acetyltransferase in the rat amygdala, Brain Res. Bull. 35 Ž1994. 147–152. w4x R.J. Boegman, J. Cockhill, K. Jhamandas, R.J. Beninger, Excitotoxic lesions of rat basal forebrain: differential effects on choline acetyltransferase in the cortex and amygdala, Neuroscience 51 Ž1992. 129–135. w5x A.E. Butt, G.K. Hodge, Simple and configural association learning in rats with bilateral quisqualic acid lesions of the nucleus basalis magnocellularis, Behav. Brain Res. 89 Ž1997. 71–85. w6x S.B. Caine, M.A. Geyer, N.R. Swerdlow, Hippocampal modulation of acoustic startle and prepulse inhibition in the rat, Pharmacol. Biochem. Behav. 43 Ž1992. 1201–1208. w7x S. Campeau, M. Davis, Prepulse inhibition of the acoustic startle reflex using visual and auditory prepulses: disruption by apomorphine, Psychopharmacology 117 Ž1995. 267–274. w8x A.A. Chiba, D.J. Bucci, P.C. Holland, M. Gallagher, Basal forebrain cholinergic lesions disrupt increments but not decrements in conditioned stimulus processing, J. Neurosci. 15 Ž1995. 7315–7322. w9x C. Dalmaz, I.B. Introini-Collison, J.L. McGaugh, Noradrenergic and cholinergic interactions in the amygdala and the modulation of memory storage, Behav. Brain Res. 58 Ž1993. 167–174. w10x M. Davis, The role of the amygdala in conditioned fear, in: J.P. Aggleton ŽEd.., The Amygdala: Neurobiological Aspects of Emotion, Memory, and Mental Dysfunction, Wiley–Liss Ž1992., pp. 255–305. w11x M.E. Dawson, E.A. Hazlett, D.L. Filion, K.H. Nuechterlein, A.M. Schell, Attention and schizophrenia: impaired modulation of the startle reflex, J. Abnorm. Psychol. 102 Ž1993. 633–641. w12x K.D. Dougherty, D. Salat, T.J. Walsh, Intraseptal injection of the cholinergic immunotoxin 192-IgG saporin fails to disrupt latent inhibition in a conditioned taste aversion paradigm, Brain Res. 736 Ž1996. 260–269. w13x S.B. Dunnett, B.J. Everitt, T.W. Robbins, The basal forebrain–cortical cholinergic system: interpreting the functional consequences of excitotoxic lesions, Trends Neurosci. 14 Ž1991. 494–501. w14x B.J. Everitt, T.W. Robbins, Central cholinergic systems and cognition, Annu. Rev. Psychol. 48 Ž1997. 649–684. w15x H.C. Fibiger, Cholinergic mechanisms in learning, memory and dementia: a review of recent evidence, Trends Neurosci. 14 Ž1991. 220–223. w16x I. Gritti, L. Mainville, M. Mancia, B.E. Jones, GABAergic and other noncholinergic basal forebrain neurons, together with cholinergic
w21x
w22x
w23x w24x w25x
w26x w27x
w28x w29x
w30x
w31x
w32x w33x
w34x
w35x
w36x
w37x
neurons, project to the mesocortex and isocortex in the rat, J. Comp. Neurol. 383 Ž1997. 163–177. H.S. Hoffman, J.R. Ison, Reflex modification in the domain of startle: I. Some empirical findings and their implications for how the nervous system processes sensory input, Psychol. Rev. 87 Ž1980. 175–189. M.J. Karnovsky, L.A. Roots, A ‘direct-coloring’ thiocholine method for cholineesterase, J. Histochem. Cytochem. 12 Ž1964. 219–221. M.P. Kilgard, M.M. Merzenich, Cortical map reorganization enabled by nucleus basalis activity, Science 279 Ž1998. 1714–1718. M. Koch, The septohippocampal system is involved in prepulse inhibition of the acoustic startle response in rats, Behav. Neurosci. 110 Ž1996. 468–477. M. Koch, M. Bubser, Deficient sensorimotor gating after 6-hydroxydopamine lesion of the rat medial prefrontal cortex is reversed by haloperidol, Eur. J. Neurosci. 6 Ž1994. 1837–1845. M. Koch, H.-U. Schnitzler, The acoustic startle response in rats-circuits mediating evocation, inhibition and potentiation, Behav. Brain Res. 89 Ž1997. 35–49. R.E. Lubow, Latent inhibition as a measure of learned inattention: some problems and solutions, Behav. Brain Res. 88 Ž1997. 75–83. R.E. Lubow, Latent Inhibition and Conditioned Attention Theory, Cambridge Univ. Press, Cambridge, 1989. R.E. Lubow, A.U. Moore, Latent inhibition: the effect of nonreinforced pre-exposure to the conditional stimulus, J. Comp. Physiol. Psychol. 52 Ž1959. 415–419. R.E. Lubow, I. Weiner, A. Schlossberg, I. Baruch, Latent inhibition and schizophrenia, Bull. Psychon. Soc. 25 Ž1987. 464–467. J. McGaughy, T. Kaiser, M. Sarter, Behavioral vigilance following infusions of 192 IgG-saporin into the basal forebrain: selectivity of the behavioral impairment and relation to cortical AChE-positive fiber density, Behav. Neurosci. 110 Ž1996. 247–265. J.L. Muir, Attention and stimulus processing in the rat, Cogn. Brain Res. 3 Ž1996. 215–225. J.L. Muir, T.W. Robbins, B.J. Everitt, Disruptive effects of muscimol infused into the basal forebrain on conditional discrimination and visual attention: differential interactions with cholinergic mechanisms, Psychopharmacology 107 Ž1992. 541–550. K. Pang, M.J. Williams, H. Egeth, D.S. Olton, Nucleus basalis magnocellularis and attention: effects of muscimol infusions, Behav. Neurosci. 107 Ž1993. 1031–1038. A.C. Pawley, S. Flesher, R.J. Boegman, R.J. Beninger, K.H. Jhamandas, Differential action of NMDA antagonists on cholinergic neurotoxicity produced by N-methyl-D-aspartate and quinolinic acid, Br. J. Pharmacol. 117 Ž1996. 1059–1064. G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney Ž1986.. T.W. Robbins, B.J. Everitt, H.M. Marston, J. Wilkinson, G.H. Jones, K.J. Page, Comparative effects of ibotenic acid- and quisqualic acid-induced lesions of the substantia innominata on attentional function in the rat: further implications for the role of the cholinergic neurons of the nucleus basalis in cognitive processes, Behav. Brain Res. 35 Ž1989. 221–240. J. Rochford, A.P. Sen, R. Quirion, Effect of nicotine and nicotinic receptor agonists on latent inhibition in the rat, J. Pharmacol. Exp. Ther. 277 Ž1996. 1267–1275. J. Rochford, A.P. Sen, I. Rousse, S.A. Welner, The effect of quisqualic acid-induced lesions of the nucleus basalis magnocellularis on latent inhibition, Brain Res. Bull. 41 Ž1996. 313–317. J.B. Rosen, J.M. Hitchcock, M.J.D. Miserendino, W.A. Falls, S. Campeau, M. Davis, Lesions of the perirhinal cortex but not of the frontal, medial prefrontal, visual, or insular cortex block fear-potentiated startle using a visual conditioned stimulus, J. Neurosci. 12 Ž1992. 4624–4633. C.B. Saper, Organization of cerebral cortical afferent systems in the rat: II. Magnocellular basal nucleus, J. Comp. Neurol. 222 Ž1984. 313–342.
C. Schauz, M. Kochr Brain Research 815 (1999) 98–105 w38x M. Sarter, J.P. Bruno, Cognitive functions of cortical acetylcholine: toward a unifying hypothesis, Brain Res. Rev. 23 Ž1997. 28–46. w39x C. Schauz, M. Koch, Latent inhibition of fear potentiated startle in rats, Behav. Pharmacol. 9 Ž1998. 175–178. w40x N.R. Swerdlow, D.L. Braff, N. Taaid, M.A. Geyer, Assessing the validity of an animal model of deficient sensorimotor gating in schizophrenic patients, Arch. Gen. Psychiatry 51 Ž1994. 139–154. w41x N.R. Swerdlow, S.B. Caine, D.L. Braff, M.A. Geyer, The neural substrates of sensorimotor gating of the startle reflex: a review of recent findings and their implications, J. Psychopharmacol. 6 Ž1992. 176–190. w42x G.B. Varty, R.J. Ralph, M.A. Geyer, Apomorphine and phencyclidine disrupt prepulse inhibition and fear-potentiated startle using a combined paradigm, Soc. Neurosci. Abstr. 23 Ž1997. 1363. w43x P. Winn, T.W. Stone, M. Latimer, M.H. Hastings, A.J.M. Clark, A comparison of excitotoxic lesions of the basal forebrain by kainate, quinolinate, ibotenate, N-methyl-D-aspartate or quisqualate, and the
w44x
w45x
w46x w47x w48x
w49x
105
effects on toxicity of 2-amino-5-phosphonovaleric acid and kynurenic acid in the rat, Br. J. Pharmacol. 102 Ž1991. 904–908. M.L. Voytko, D.S. Olton, R.T. Richardson, L.K. Gorman, J.R. Tobin, D.L. Price, Basal forebrain lesions in monkeys disrupt attention but not learning and memory, J. Neurosci. 14 Ž1994. 167–186. F.-J. Wan, N.R. Swerdlow, The basolateral amygdala regulates sensorimotor gating of acoustic startle in the rat, Neuroscience 76 Ž1997. 715–724. N.M. Weinberger, Learning-induced changes of auditory receptive fields, Curr. Opin. Neurobiol. 3 Ž1993. 570–577. I. Weiner, J. Feldon, The switching model of latent inhibition: an update of neural substrates, Behav. Brain Res. 88 Ž1997. 11–25. G.L. Wenk, The nucleus basalis magnocellularis cholinergic system: one hundred years of progress, Neurobiol. Learn. Memory 67 Ž1997. 85–95. N.J. Woolf, A structural basis for memory storage in mammals, Prog. Neurobiol. 55 Ž1998. 59–77.
Research report
Lesions of the nucleus basalis magnocellularis do not impair prepulse inhibition and latent inhibition of fear-potentiated startle in the rat Cornelia Schauz ) , Michael Koch Tierphysiologie, UniÕersitat Auf der Morgenstelle 28, D-72076 Tubingen, Germany ¨ Tubingen, ¨ ¨ Accepted 20 October 1998
Abstract The present study tested if lesions of the nucleus basalis magnocellularis ŽNBM. affect prepulse inhibition ŽPPI. of the acoustic startle response and latent inhibition ŽLI. of fear-potentiated startle. The NBM is known to play an important role in learning and memory. Recently, the interest of research focused on its role in attentional and response selection processes. We here tested the effect of excitotoxic NBM-lesions on PPI, a phenomenon of sensorimotor gating that occurs at early stages of information processing. We also assessed the lesion effects on LI, a phenomenon of reduced conditioning after stimulus preexposure that can be used to measure selective attention. Bilateral infusions into the NBM of 80 nmol of quinolinic acid markedly reduced the number of choline acetyltransferase immunopositive neurons in the NBM and lead to a pronounced reduction of acetylcholine esterase in the cortex and the amygdala. However, no effects on PPI, fear-conditioning, or LI of fear-potentiated startle were found. Therefore, we conclude that there is no NBM-driven attentional or response selection process involved in PPI. Furthermore, the simple association learning in the classical conditioning paradigm used for fear-potentiated startle or LI is unaffected by NBM-lesions. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Acoustic startle response; Fear-potentiated startle; Latent inhibition; Nucleus basalis magnocellularis; Prepulse inhibition; Schizophrenia
1. Introduction The suppression of inappropriate responses is an important principle of behavioral organisation. Two well known phenomena of response suppression are prepulse inhibition ŽPPI. of the acoustic startle response ŽASR. and latent inhibition ŽLI.. PPI is the reduction of the ASR magnitude when a weak stimulus, which by itself does not elicit an ASR, is presented prior to the startling stimulus w17x. PPI is viewed as a phenomenon of sensorimotor gating that occurs at early stages of information processing and probably serves to protect an ongoing stimulus processing routine from disruption by an interfering sensory or motor event. LI refers to the phenomenon of retarded conditioning after repeated non-reinforced presentation of a prospective conditioned stimulus during a preexposure stage w25x. LI is a form of learned inattention or cognitive suppression of a response w23,28x.
) Corresponding author. Fax: [email protected]
q 49-7071-292618;
E-mail:
There is considerable interest in the investigation of the neuronal basis of PPI and LI in rats, because both phenomena of response suppression are impaired in schizophrenics w26,40x. Previous work in rats has shown that PPI and LI are regulated by somewhat overlapping, yet clearly distinguishable cortical, limbic, striatal and tegmental circuitries w22,41,47x. The suppression of inadvertent motor or cognitive events requires the evaluation of the respective stimuli either at the perceptual level or at the cognitive level. Therefore it can be assumed that deficient PPI and LI in schizophrenics reflects dysfunctions in perceptual andror cognitive filter mechanisms of the brain. While most researchers acknowledge a role of attention in fear-conditioning and in LI, the attribution of an attentional deficit to impaired PPI is less clear w7,11,41x. Since treatments that reduce PPI Že.g., apomorphine. do not impair the ASR peak latency reduction by the prepulse it was suggested that PPI deficits are not simply due to a reduced prepulse detection, e.g., through inattention w41x. On the other hand, PPI-disrupting treatments do impair the detection of a prepulse in a combined fear-potentiated ASRrPPI-paradigm w42x indi-
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 1 3 4 - 2
C. Schauz, M. Kochr Brain Research 815 (1999) 98–105
cating that reduced attention might affect PPI performance. In humans PPI is enhanced if the subjects are explicitly instructed to attend to the prepulse w11x. It is noteworthy, however, that the latter paradigms are not the usual procedures of assessing PPI and were constructed to reveal the possible role of attentional mechanisms in PPI. Since the nucleus basalis magnocellularis ŽNBM. plays an important role in the processing of behaviorally relevant stimuli and in selective attention w19,46,49x the present study attempted to assess the role of the NBM in fear-conditioning and LI. Notwithstanding the objections against a prominent role of attention in PPI, we also tested a potential contribution of the NBM to the regulation of PPI. The NBM in rodents consists of large cholinergic neurons, GABAergic neurons and a considerable proportion of not yet identified non-cholinergic neurons w16x. These neurons are dispersed in the basal forebrain along the internal capsule, extending from the medial septum and the nucleus of the diagonal band of Broca Žhorizontal and vertical limbs., invading the substantia innominata and the globus pallidus w1,37x. The NBM has strong projections to the cerebral cortex and also provides the cholinergic input to the lateral and basolateral part of the amygdala w49x. Excitotoxic lesions lead to a significant depletion of cortical acetylcholine esterase ŽAChE.-positive fiber density w27x, but the excitotoxic damage to the cortex or the amygdala differs according to the neurotoxin used, as shown by Boegman et al. w4x. Because in that study quinolinic acid also produced considerable damage to the cholinergic innervation of the amygdala, we have used this toxin for the NBM lesions. In the amygdala acetylcholine ŽACh. is involved in the storage of aversive memories w9x. The involvement of the basal forebrain cholinergic system in cognitive functions and especially its important role in mnemonic processes is known since a long time because of its possible role in cognitive impairments associated with senile dementia and Alzheimer’s disease w15,48x. Recently, the role in attentional processes of the cholinergic neurons of the NBM has been emphasized w29,30, 33,38,44,48x. The cortical and limbic cholinergic projections of the NBM have been suggested to act as specific amplifiers that enhance the salience and the representation in the cortex of behaviorally important stimuli and reduce the behavioral impact of irrelevant stimuli w19,46,49x. The investigation of the role of the basal forebrain cholinergic system in LI yielded inconsistent results. Rochford et al. found a facilitating effect on LI of systemically applied nicotine and nicotinic ACh receptor agonists and this enhancement was reversed by application of nicotinic ACh receptor antagonists w34x. Another experiment by the same group demonstrated an impairment of LI after lesioning of the NBM, however, the effect reported by this group was somewhat difficult to interpret, since lesioned preexposed and non-preexposed rats showed similar behavioral performance as control preexposed rats and not, as would be expected for deficient LI, similarity with
99
control non-preexposed rats w35x. LI of appetitive conditioning was reduced by ACh-specific lesions of the septohippocampal system w2x, but was spared after lesions of the NBM w8x. In the latter study, however, the lesions were produced by 192 IgG-saporin, which does not destroy the cholinergic input to the amygdala. We here tested if NBM lesions with quinolinic acid affects PPI, fear-potentiated startle or LI of fear-potentiated startle.
2. Materials and methods 2.1. BehaÕioral experiments 2.1.1. Animals A total of 42 experimentally naive male Wistar rats Ž200–300 g, Charles River, Sulzfeld, Germany. were used. The animals were kept in groups of 6 per cage in a colony room under a 12 h light–dark cycle Žlights on at 0700 h. with food and water available ad lib. Sixteen rats received PBS injections as controls, 25 rats were bilaterally injected into the NBM with 80 nmol quinolinic acid. The rats were allowed to recover from surgery for 6–7 days. All experiments were done in accordance with international guidelines for the care and use of animals for experiments and were approved by the local council of animal care ŽRegierungsprasidium Tubingen, ZP 4r96.. ¨ ¨ 2.1.2. Surgery Rats were anaesthetized with chloral hydrate Ž420 mgrkg i.p.. and placed in a Kopf stereotaxic frame. After exposition and trepanation of the skull a 30-gauge injection cannula, attached to a microliter syringe, was placed in the substantia innominatarglobus pallidus region, since the magnocellular cholinergic neurons of the NBM are mainly distributed throughout this area. The coordinates were 1.3 mm caudal, "2.3 mm lateral and 7.0 mm ventral to Bregma w32x. In lesion rats 80 nmol of quinolinic acid in 0.3 ml PBS Žadjusted to pH 7.4 with 1 N NaOH. was bilaterally infused, with an infusion rate of 0.1 mlrmin. The injection cannula remained in the brain for another 3 min after infusion was completed to allow for the toxin to diffuse into the parenchyma. In control animals the same volume of PBS was infused. Seven rats died after the surgery because of the lesion effect, one rat was excluded from the study because it did not show PPI in the pretest. PPI was measured before and after surgery. After the PPI measurements were completed the LI procedure began. 2.1.3. Test for prepulse inhibition PPI was measured after 5 min adaptation time to the test chamber and one initial startle stimulus. The test
100
C. Schauz, M. Kochr Brain Research 815 (1999) 98–105
session consisted of five different trial types presented in a randomized order: Ž1. pulse alone Ž100 dB SPL broad band noise bursts, 20 ms duration., Ž2. prepulse and pulse Ž70 dB SPL or Ž3. 60 dB SPL 10 kHz tone prepulse, 20 ms duration, including 0.4 ms riserfall times. followed by a pulse 100 ms after prepulse onset, Ž4. prepulse alone Ž70 dB SPL. and Ž5. no stimulus. Background noise intensity was 55 dB SPL. Twelve presentations of each trial type were given with an interstimulus interval of 30 s. PPI was measured as the difference between the pulse alone trials and the prepulse–pulse trials and the percent PPI was calculated as w100 = Žmean ASR amplitude on pulse alone trials y mean ASR amplitude on prepulse– pulse trials.rmean ASR amplitude on pulse alone trialsx. 2.1.4. Test for latent inhibition of fear-potentiated startle Preexposure and conditioning were performed on two consecutive days, followed by a test session 2 h after the last conditioning trial. 2.1.4.1. Preexposure. Each rat was placed in a dark conditioning box Ž38 = 60 = 28 cm3 . provided with a floor made of steel bars spaced 15 mm apart. Preexposed rats received 40 presentations of the prospective CS Žwhite light, 15 W, duration 3.7 s, interstimulus interval 30 s.. Rats of the non-preexposed group were left in the conditioning box for an identical period of time without receiving the prospective CS. 2.1.4.2. Conditioning. Each rat was trained in the conditioning box to associate the CS with the UCS, a 0.6 mA electric footshock produced by a shock generator Žcustom . presented in the last made at the University of Tubingen ¨ 0.5 s of a 3.7 s white light Ž15 W.. 2.1.4.3. Test. The ASR was measured in a wire mesh cage Ž20 = 10 = 12 cm3 . with a steel floor that was mounted on a piezoelectric accelerometer in a sound-attenuated chamber. The test consisted of 5 min of adaptation to the startle chamber and ten initial acoustic startle stimuli Ž100 dB 10 kHz pure tone of 20 ms duration including 0.4 ms rise and fall times. that were presented to produce a stable baseline of ASR, but were not statistically evaluated. Then 40 acoustic startle stimuli, half of them in darkness Žtone-alone trials., the other half 3.2 s after the onset of the 3.7 s light CS Žlight-tone trials. were presented in a randomized order. Background noise intensity was 55 dB SPL. To assess fear-conditioning the whole-body ASR amplitude in darkness or in the presence of the light was calculated, respectively. LI was inferred from the difference in fearpotentiation between preexposed and non-preexposed rats. 2.2. Histology After completion of the behavioral studies the rats were deeply anaesthetized with nembutal and perfused transcar-
dially with 100 ml PBS followed by 500 ml cold 4% paraformaldehyde with 0.01% glutaraldehyde and then with 0.1 M phosphate buffer ŽPB. with 10% sucrose. The brains were placed in 20% sucrose in PB at 48C over night. Coronal sections Ž50 mm. were cut on a freezing microtome, the sections were divided in three series for Nissl staining, for AChE staining according to a modified version of the protocol of Karnovsky and Roots w18x and for the immunohistochemical detection of choline acetyltransferase ŽChAT.. 2.2.1. AChE staining Sections were left in 0.1 M PB for 4–6 days at 48C before they were incubated for 1 h in an acetylthiocholine solution Ž0.2 mM ethopropazine, 10 mM glycine, 2 mM CuSO4 , 4 mM acetylthiocholine and 50 mM sodium acetate, adjusted to pH 5.0 with drops of glacial acetic acid.. After washing the sections 6 times in dH 2 O they were developed for 1 min in a sodium sulfide solution Ž300 mM, adjusted with 10 M HCl to pH 7.8.. Sections were rinsed again 6 times in dH 2 O and were then incubated in a silver nitrate solution Ž59 mM. for 30 s. After washing the sections in dH 2 O they were mounted on gelatine-coated slides, dehydrated in 100% ethanol Ž1 min., cleared in two changes of xylene and coverslipped with Entellan. 2.2.2. ChAT-immunostaining After washing the slices in PBS they were incubated in a blocking solution Ž3% goat serum in PBS with 0.2% Triton X-100. at room temperature and then incubated in a polyclonal rabbit anti-human ChAT serum Žkindly donated by Dr. Louis B. Hersh. diluted 1:1000 in carrier Ž3% goat serum in PBS. for 48 h at q48C. The sections were washed in PBS several times and then incubated for 1 h in a biotin-labeled goat anti-rabbit IgG antiserum ŽVector., diluted 1:1000 in carrier at room temperature. Sections were rinsed in PBS and incubated for 45 min with an avidin–biotin peroxidase-complex ŽVectastain ABC-Elite kit. diluted 1:100 in 0.01 M PBS containing 0.5 M NaCl. The sections were again rinsed in PBS and were processed in a solution of 0.05% 3,3-diaminobenzidine tetrahydrochloride and 0.01% H 2 O 2 in TBS, to visualize the immunocomplex. Finally, the sections were mounted on gelatine-coated slides, air dried, dehydrated in alcohol, cleared in xylene, and coverslipped with Entellan. 2.3. Statistics The data are presented as mean " S.E.M. All data were analyzed by a multi-variable analysis of variance ŽANOVA.. In PPI experiments treatment Žlesionrcontrol. was the between-subject factor and prepulse intensity Ž60 dBr70 dB. and session Žbefore surgeryrafter surgery. the within-subject factors. Effects on ASR were also computed with treatment and session as factors. In LI experiments treatment Žlesionrcontrol. and stimulus exposure Žpreex-
C. Schauz, M. Kochr Brain Research 815 (1999) 98–105
101
posurerno preexposure. were between-subject factors and nature of trial Žtone-alonerlight-tone. was within-subject factor. ANOVA was followed by Tukey’s protected t-tests for post hoc pairwise comparisons. An alpha value of p - 0.05 was considered to represent a significant difference, a value of p - 0.01 as highly significant. 3. Results 3.1. Anatomy The histological analysis revealed that quinolinic acid infusions into the NBM lead to a destruction of cholinergic neurons in the NBM ŽFig. 1A and B. and to a reduction of AChE mainly in the neocortex and lateral amygdala ŽFig. 1C and D.. Additional to this documented primary lesions we also found extension of the lesion in parts of the globus pallidus, the substantia innominata, the bed nucleus of the stria terminalis and nonspecific destruction, probably secondarily, in different thalamic nuclei Žnot shown..
Fig. 2. Percent PPI in control Ž N s16. and NBM-lesioned Ž N s16. rats. Percent PPI scores are shown before Žblack bars. and after surgery Žwhite bars. following 70 dB or 60 dB acoustic prepulses.
3.2. BehaÕior 3.2.1. Effect of NBM lesions on PPI Fig. 2 shows the percent PPI scores in control and NBM-lesioned rats. ANOVA revealed a significant effect
of prepulse intensity on PPI with lower prepulse intensities yielding lower PPI scores w F Ž1,123. s 46.7; p - 0.01x. No significant effect of treatment w F Ž1,123. s 1.5; p ) 0.1x or
Fig. 1. Photomicrographs of frontal sections through the basal forebrain stained immunocytochemically for ChAT of a NBM-lesioned rat ŽA. and a control rat ŽB.. A loss of ChAT-immunoreactive cells documents the extent of the NBM-lesion. Histochemical AChE-staining of brain sections is shown from a NBM-lesioned rat ŽC. and a control rat ŽD.. The loss of AChE-staining indicates a reduction of the cholinergic innervation of the cortex and the lateral amygdala. Bar s 500 mm.
102
C. Schauz, M. Kochr Brain Research 815 (1999) 98–105
session w F Ž1,123. s 1.1; p ) 0.1x was observed and there was no significant interaction between treatment and session w F Ž1,123. s 0.7; p - 0.5x, indicating that NBM lesions do not reduce PPI at different prepulse intensities. 3.2.2. Effect of NBM lesions on LI The effects of NBM lesions on LI of fear-potentiated startle are shown in Fig. 3A. An overall ANOVA was performed revealing a significant interaction between stimulus exposure and nature of trial w F Ž1,62. s 4.3; p - 0.05x, non-preexposed rats showed a significant increase in light-tone trials compared to tone-alone trials ŽTukey’s protected t-test, p - 0.01. indicative of a state of fear in these rats. In preexposed rats there was no significant difference between light-tone trials and tone-alone trials ŽTukey’s protected t-test; p ) 0.5. indicating LI of fear-
Fig. 4. ASR amplitude in control Ž N s16. and NBM-lesioned Ž N s18. rats before or after lesion or sham-treatment.
potentiated startle. There was no significant interaction between exposure to a stimulus, nature of trial and lesion w F Ž1,62. s 0.03; p ) 0.5x. Fig. 3B shows the absolute differences between tone-alone and light-tone trials in control and lesion groups. ANOVA revealed a significant difference between preexposed and non-preexposed rats in lesioned and control rats w F Ž1,31. s 17.25, p - 0.01x. These results show that NBM lesions do not impair fearpotentiated startle or LI of fear-potentiated startle. 3.2.3. Effect of NBM lesions on ASR We also tested the effect of NBM lesion on the ASR, produced by 100 dB broad band noise pulses. Mean ASR " S.E.M. in control and lesioned rats are shown in Fig. 4. A two-way ANOVA with treatment and session indicated whether an effect of treatment w F Ž1,60. s 0.2; p ) 0.5x or an effect of session w F Ž1,60. s 0.9; p ) 0.1x, nor an interaction between treatment and session w F Ž1,60. s 0.1; p ) 0.8x. We therefore conclude that NBM lesions have no effect on the ASR.
4. Discussion
Fig. 3. ŽA. ASR amplitude Žarbitrary units. of tone-alone and light-tone trials in sham operated Žcontrol. and NBM-lesioned rats, which were either non-preexposed ŽNPE, control N s8, lesion N s8. or preexposed ŽPE, control N s8, lesion N s10.. ŽB. Difference between tone-alone and light-tone trials in controls and NBM-lesioned rats, PE or NPE to the light CS. ŽAsterisks indicate a p- 0.01, Tukey’s t-test after significant main effect of ANOVA..
The present study shows that NBM-lesions that significantly reduce the number of cholinergic cells in the NBM and reduce the presence of AChE in limbic and cortical brain regions do not affect PPI, fear-conditioning, LI of fear-potentiated startle or the ASR. PPI is a measure of sensorimotor gating that is regulated by a complex forebrain circuit including the septohippocampal cholinergic system w6,20x, the basolateral amygdala w45x, the nucleus accumbens w22,41x, and the prefrontal cortex w21x. While PPI is generally related to a sensorimotor gating mechanism that facilitates attention w41x, some authors discuss PPI as a behavioral phe-
C. Schauz, M. Kochr Brain Research 815 (1999) 98–105
nomenon that requires attentional processes w7,11x. NBM lesions disrupt attention but not learning and memory processes in monkeys w44x and an attentional dysfunction was also demonstrated in rats following NBM lesions w29,33x or stimulation of the GABAergic NBM neurons w30x. Based on the assumption that the NBM plays an important role in selective attention w38,48x, we hypothesized that NBM lesions might influence PPI. Our data show that the NBM, which provides a strong cholinergic input to the cortex and the amygdala, is not necessary for this kind of sensorimotor gating. Lesions of the medial septal nucleus, which provides the cholinergic input to the hippocampus, also had no effect on PPI w20x. Taken together with the present data it can be concluded that the forebrain cholinergic system is not necessary for the regulation of PPI, and PPI is not dependent on NBM-driven attention or response selection mechanisms. LI was measured as the reduction of the fear-potentiated ASR by preexposure to the visual CS w39x. In this fear-conditioning paradigm the visual CS is probably conveyed via the perirhinal cortex to the lateralrbasolateral nuclei of the amygdala where the association between neutral and aversive stimulus is presumably formed w36x. These nuclei project to the central amygdaloid nucleus which directly impinges on the primary acoustic startle circuit at the level of the caudal pontine reticular nucleus to facilitate the ASR w10,22x. We hypothesized that in LI of fear-potentiated ASR the disturbance of the expression of fear takes place at the level of the amygdala w39x. Beninger and his group demonstrated that the type of neurotoxin used for the lesions differentially affects the extent of the cholinergic damage to the cortex and the amygdala, and that damage to the amygdala more likely contributes to learning deficits w3x. Our data support the finding that quinolinic acid lesions of the NBM lead to a considerable reduction of AChE in the lateral amygdala. It is already known that ACh plays an important role in the storage of emotional memory in the amygdala w9x. However, our data show that fear-conditioning Žand LI of fear-conditioning. is still possible after impairment of the NBM-amygdaloid cholinergic system. Because a reorganisation of sensory cortical areas has been found after pairing NBM-stimulation with sensory stimuli w19x it seemed reasonable to assume that during preexposure or during conditioning the NBM affects the processing of the visual CS probably at the cortical level. Since NBM lesions did neither disrupt LI nor fear-conditioning, our present findings support the view that the NBM is involved in complex association or spatial learning tasks but not if a simple association between two stimuli is sufficient for learning, as it is in classical conditioning paradigms w5x. A role of the cholinergic forebrain system in LI was discussed recently w47x based on the finding that systemically applied nicotine and nicotinic ACh receptor agonists augment LI and that this enhancement was reversed after
103
application of nicotinic ACh receptor antagonists w34x which is consistent with the assumption that ACh has cognitive enhancing properties w38x. Therefore, the lack of effect of NBM-lesions on LI reported in this paper is surprising, especially in the light of the fact that the NBM has been implicated in attentional processes related to learning w13x and because attention has been considered to be important for LI w23,24,28x. In fact, a disruption by NBM-lesions of LI in a different paradigm has been reported by Rochford et al. w35x. However, the results reported by this group reveal some difficulties of interpretation since the impairment of LI is concluded from the similarity of the behavioral performance of lesioned preexposed and non-preexposed rats with control preexposed rats. Because destruction of the cholinergic cells in the medial septum, which provide the cholinergic input to the hippocampus, was shown to disrupt LI w2x Žbut see Ref. w12x., one can conclude that ACh affects LI at the level of the hippocampus rather than at the cortical level. This conclusion is supported by the finding that immunolesioning of the NBM with 192 IgG-saporin, which lead to a considerable reduction of AChE staining in the neocortex, did not affect LI w8x. It is unlikely that LI is so robust in our paradigm that it cannot be disrupted. This kind of ceiling-effect of LI can be excluded on the basis of the observation that LI of fear-potentiated startle was reduced by a systemic treatment with 1 mgrkg of amphetamine w39x. Because of the scattered distribution of the NBM throughout the basal forebrain, lesions restricted to the cholinergic neurons of the NBM are difficult to perform, as reported previously by other groups w14x. Therefore, we are aware of the fact that the destruction of non-cholinergic cells scattered in or around the NBM remains a problem for the interpretation of our results. The conflicting results following NBM lesions described in the literature are ascribed by some groups to this damage of non-cholinergic cells or other basal forebrain structures. Negative results demand cautious interpretation because of the possibility of methodical fallacies, but our data clearly show that the cholinergic innervation of the cortex and the amygdala is damaged after NBM lesions with quinolinic acid. The concentration of quinolinic acid used here is in accordance with those reported by other groups producing lesions of considerable size w43x and well-documented decreases in cortical and amygdaloid ChAT activity w4,31x. Our lesions extended into parts of the globus pallidus, the substantia innominata, the bed nucleus of the stria terminalis and, probably secondarily Ži.e., transsynaptically and not by diffusion., into parts of the thalamus. This cerebral tissue damage does not produce a sensorimotor gating deficit nor does it affect the simple associative learning in a classical conditioning paradigm. Nevertheless, we conclude that extensive excitotoxic lesions of the NBM did not lead to information processing deficits at the perceptual or cognitive level.
104
C. Schauz, M. Kochr Brain Research 815 (1999) 98–105
Acknowledgements w17x
This study was supported by the DFG ŽSPP 1001 and, Heisenberg Programm for MK.. We are grateful to Dr. H. Herbert and Mr. A. Guthmann for valuable advice with the histology, to Dr. P. Pilz for help with the statistics and to H. Zillus for excellent technical assistance.
w18x w19x w20x
References w1x D.M. Armstrong, C.B. Saper, A.I. Levey, B.H. Wainer, R.D. Terry, Distribution of cholinergic neurons in rat brain: demonstrated by the immunocytochemical localization of choline acetyltransferase, J. Comp. Neurol. 216 Ž1983. 53–68. w2x M.G. Baxter, P.C. Holland, M. Gallagher, Disruption of decrements in conditioned stimulus processing by selective removal of hippocampal cholinergic input, J. Neurosci. 17 Ž1997. 5230–5236. w3x R.J. Beninger, S. Kuhnemann, J.L. Ingles, K. Jhamandas, R.J. ¨ Boegman, Mnemonic deficits in the double Y-maze are related to the effects of nucleus basalis injections of ibotenic and quisqualic acid on choline acetyltransferase in the rat amygdala, Brain Res. Bull. 35 Ž1994. 147–152. w4x R.J. Boegman, J. Cockhill, K. Jhamandas, R.J. Beninger, Excitotoxic lesions of rat basal forebrain: differential effects on choline acetyltransferase in the cortex and amygdala, Neuroscience 51 Ž1992. 129–135. w5x A.E. Butt, G.K. Hodge, Simple and configural association learning in rats with bilateral quisqualic acid lesions of the nucleus basalis magnocellularis, Behav. Brain Res. 89 Ž1997. 71–85. w6x S.B. Caine, M.A. Geyer, N.R. Swerdlow, Hippocampal modulation of acoustic startle and prepulse inhibition in the rat, Pharmacol. Biochem. Behav. 43 Ž1992. 1201–1208. w7x S. Campeau, M. Davis, Prepulse inhibition of the acoustic startle reflex using visual and auditory prepulses: disruption by apomorphine, Psychopharmacology 117 Ž1995. 267–274. w8x A.A. Chiba, D.J. Bucci, P.C. Holland, M. Gallagher, Basal forebrain cholinergic lesions disrupt increments but not decrements in conditioned stimulus processing, J. Neurosci. 15 Ž1995. 7315–7322. w9x C. Dalmaz, I.B. Introini-Collison, J.L. McGaugh, Noradrenergic and cholinergic interactions in the amygdala and the modulation of memory storage, Behav. Brain Res. 58 Ž1993. 167–174. w10x M. Davis, The role of the amygdala in conditioned fear, in: J.P. Aggleton ŽEd.., The Amygdala: Neurobiological Aspects of Emotion, Memory, and Mental Dysfunction, Wiley–Liss Ž1992., pp. 255–305. w11x M.E. Dawson, E.A. Hazlett, D.L. Filion, K.H. Nuechterlein, A.M. Schell, Attention and schizophrenia: impaired modulation of the startle reflex, J. Abnorm. Psychol. 102 Ž1993. 633–641. w12x K.D. Dougherty, D. Salat, T.J. Walsh, Intraseptal injection of the cholinergic immunotoxin 192-IgG saporin fails to disrupt latent inhibition in a conditioned taste aversion paradigm, Brain Res. 736 Ž1996. 260–269. w13x S.B. Dunnett, B.J. Everitt, T.W. Robbins, The basal forebrain–cortical cholinergic system: interpreting the functional consequences of excitotoxic lesions, Trends Neurosci. 14 Ž1991. 494–501. w14x B.J. Everitt, T.W. Robbins, Central cholinergic systems and cognition, Annu. Rev. Psychol. 48 Ž1997. 649–684. w15x H.C. Fibiger, Cholinergic mechanisms in learning, memory and dementia: a review of recent evidence, Trends Neurosci. 14 Ž1991. 220–223. w16x I. Gritti, L. Mainville, M. Mancia, B.E. Jones, GABAergic and other noncholinergic basal forebrain neurons, together with cholinergic
w21x
w22x
w23x w24x w25x
w26x w27x
w28x w29x
w30x
w31x
w32x w33x
w34x
w35x
w36x
w37x
neurons, project to the mesocortex and isocortex in the rat, J. Comp. Neurol. 383 Ž1997. 163–177. H.S. Hoffman, J.R. Ison, Reflex modification in the domain of startle: I. Some empirical findings and their implications for how the nervous system processes sensory input, Psychol. Rev. 87 Ž1980. 175–189. M.J. Karnovsky, L.A. Roots, A ‘direct-coloring’ thiocholine method for cholineesterase, J. Histochem. Cytochem. 12 Ž1964. 219–221. M.P. Kilgard, M.M. Merzenich, Cortical map reorganization enabled by nucleus basalis activity, Science 279 Ž1998. 1714–1718. M. Koch, The septohippocampal system is involved in prepulse inhibition of the acoustic startle response in rats, Behav. Neurosci. 110 Ž1996. 468–477. M. Koch, M. Bubser, Deficient sensorimotor gating after 6-hydroxydopamine lesion of the rat medial prefrontal cortex is reversed by haloperidol, Eur. J. Neurosci. 6 Ž1994. 1837–1845. M. Koch, H.-U. Schnitzler, The acoustic startle response in rats-circuits mediating evocation, inhibition and potentiation, Behav. Brain Res. 89 Ž1997. 35–49. R.E. Lubow, Latent inhibition as a measure of learned inattention: some problems and solutions, Behav. Brain Res. 88 Ž1997. 75–83. R.E. Lubow, Latent Inhibition and Conditioned Attention Theory, Cambridge Univ. Press, Cambridge, 1989. R.E. Lubow, A.U. Moore, Latent inhibition: the effect of nonreinforced pre-exposure to the conditional stimulus, J. Comp. Physiol. Psychol. 52 Ž1959. 415–419. R.E. Lubow, I. Weiner, A. Schlossberg, I. Baruch, Latent inhibition and schizophrenia, Bull. Psychon. Soc. 25 Ž1987. 464–467. J. McGaughy, T. Kaiser, M. Sarter, Behavioral vigilance following infusions of 192 IgG-saporin into the basal forebrain: selectivity of the behavioral impairment and relation to cortical AChE-positive fiber density, Behav. Neurosci. 110 Ž1996. 247–265. J.L. Muir, Attention and stimulus processing in the rat, Cogn. Brain Res. 3 Ž1996. 215–225. J.L. Muir, T.W. Robbins, B.J. Everitt, Disruptive effects of muscimol infused into the basal forebrain on conditional discrimination and visual attention: differential interactions with cholinergic mechanisms, Psychopharmacology 107 Ž1992. 541–550. K. Pang, M.J. Williams, H. Egeth, D.S. Olton, Nucleus basalis magnocellularis and attention: effects of muscimol infusions, Behav. Neurosci. 107 Ž1993. 1031–1038. A.C. Pawley, S. Flesher, R.J. Boegman, R.J. Beninger, K.H. Jhamandas, Differential action of NMDA antagonists on cholinergic neurotoxicity produced by N-methyl-D-aspartate and quinolinic acid, Br. J. Pharmacol. 117 Ž1996. 1059–1064. G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney Ž1986.. T.W. Robbins, B.J. Everitt, H.M. Marston, J. Wilkinson, G.H. Jones, K.J. Page, Comparative effects of ibotenic acid- and quisqualic acid-induced lesions of the substantia innominata on attentional function in the rat: further implications for the role of the cholinergic neurons of the nucleus basalis in cognitive processes, Behav. Brain Res. 35 Ž1989. 221–240. J. Rochford, A.P. Sen, R. Quirion, Effect of nicotine and nicotinic receptor agonists on latent inhibition in the rat, J. Pharmacol. Exp. Ther. 277 Ž1996. 1267–1275. J. Rochford, A.P. Sen, I. Rousse, S.A. Welner, The effect of quisqualic acid-induced lesions of the nucleus basalis magnocellularis on latent inhibition, Brain Res. Bull. 41 Ž1996. 313–317. J.B. Rosen, J.M. Hitchcock, M.J.D. Miserendino, W.A. Falls, S. Campeau, M. Davis, Lesions of the perirhinal cortex but not of the frontal, medial prefrontal, visual, or insular cortex block fear-potentiated startle using a visual conditioned stimulus, J. Neurosci. 12 Ž1992. 4624–4633. C.B. Saper, Organization of cerebral cortical afferent systems in the rat: II. Magnocellular basal nucleus, J. Comp. Neurol. 222 Ž1984. 313–342.
C. Schauz, M. Kochr Brain Research 815 (1999) 98–105 w38x M. Sarter, J.P. Bruno, Cognitive functions of cortical acetylcholine: toward a unifying hypothesis, Brain Res. Rev. 23 Ž1997. 28–46. w39x C. Schauz, M. Koch, Latent inhibition of fear potentiated startle in rats, Behav. Pharmacol. 9 Ž1998. 175–178. w40x N.R. Swerdlow, D.L. Braff, N. Taaid, M.A. Geyer, Assessing the validity of an animal model of deficient sensorimotor gating in schizophrenic patients, Arch. Gen. Psychiatry 51 Ž1994. 139–154. w41x N.R. Swerdlow, S.B. Caine, D.L. Braff, M.A. Geyer, The neural substrates of sensorimotor gating of the startle reflex: a review of recent findings and their implications, J. Psychopharmacol. 6 Ž1992. 176–190. w42x G.B. Varty, R.J. Ralph, M.A. Geyer, Apomorphine and phencyclidine disrupt prepulse inhibition and fear-potentiated startle using a combined paradigm, Soc. Neurosci. Abstr. 23 Ž1997. 1363. w43x P. Winn, T.W. Stone, M. Latimer, M.H. Hastings, A.J.M. Clark, A comparison of excitotoxic lesions of the basal forebrain by kainate, quinolinate, ibotenate, N-methyl-D-aspartate or quisqualate, and the
w44x
w45x
w46x w47x w48x
w49x
105
effects on toxicity of 2-amino-5-phosphonovaleric acid and kynurenic acid in the rat, Br. J. Pharmacol. 102 Ž1991. 904–908. M.L. Voytko, D.S. Olton, R.T. Richardson, L.K. Gorman, J.R. Tobin, D.L. Price, Basal forebrain lesions in monkeys disrupt attention but not learning and memory, J. Neurosci. 14 Ž1994. 167–186. F.-J. Wan, N.R. Swerdlow, The basolateral amygdala regulates sensorimotor gating of acoustic startle in the rat, Neuroscience 76 Ž1997. 715–724. N.M. Weinberger, Learning-induced changes of auditory receptive fields, Curr. Opin. Neurobiol. 3 Ž1993. 570–577. I. Weiner, J. Feldon, The switching model of latent inhibition: an update of neural substrates, Behav. Brain Res. 88 Ž1997. 11–25. G.L. Wenk, The nucleus basalis magnocellularis cholinergic system: one hundred years of progress, Neurobiol. Learn. Memory 67 Ž1997. 85–95. N.J. Woolf, A structural basis for memory storage in mammals, Prog. Neurobiol. 55 Ž1998. 59–77.