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Selective immunolesioning of cholinergic neurons in nucleus basalis magnocellularis impairs prepulse inhibition of acoustic startle
PII: S 0 3 0 6 - 4 5 2 2 ( 0 1 ) 0 0 4 1 3 - 4
Neuroscience Vol. 108, No. 2, pp. 299^305, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 01 $20.00+0.00
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SELECTIVE IMMUNOLESIONING OF CHOLINERGIC NEURONS IN NUCLEUS BASALIS MAGNOCELLULARIS IMPAIRS PREPULSE INHIBITION OF ACOUSTIC STARTLE M. BALLMAIER,a * F. CASAMENTI,b M. ZOLI,c G. PEPEUb;1 and P. SPANOa;1 a
Division of Pharmacology, Department of Biomedical Sciences and Biotechnologies, Brescia University Medical School, Via Valsabbina 19, 25123 Brescia, Italy b c
Department of Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy
Section of Physiology, Department of Biomedical Sciences, University of Modena, Modena, Italy
AbstractöInformation processing and attentional abnormalities are prominent in neuropsychiatric disorders. Since the cholinergic neurons located in the nucleus basalis magnocellularis have been shown to be involved in attentional performance and information processing, recent e¡orts to analyze the signi¢cance of the basal forebrain in the context of schizophrenia have focused on this nucleus and its projections to the cerebral cortex. We report here that bilateral selective immunolesioning of the cholinergic neurons in the nucleus basalis magnocellularis is followed by signi¢cant de¢cits in sensorimotor gating measured by prepulse inhibition of the startle re£ex in adult rats. This behavioral approach is used in both humans and rodents and has been proposed as a valuable model contributing to the understanding of the neurobiological substrates of schizophrenia. The disruption of prepulse inhibition persisted over repeated testing. The selective lesions were induced by bilateral intraparenchymal infusions of 192 IgG saporin at a concentration having minimal di¡usion into adjacent nuclei of the basal forebrain. The infusions were followed by extensive loss of choline acetyltransferase-immunopositive neurons. Our results show that the cholinergic neurons of the nucleus basalis magnocellularis represent a critical station of the startle gating circuitry and suggest that dysfunction of these neurons may result in impaired sensorimotor gating characteristic of schizophrenia. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: immunotoxin, cholinergic basal forebrain, sensorimotor gating, schizophrenia.
(Sarter and Bruno, 1999). As an integral part of the ascending activating system, the NBM in fact is considered to be important for cortical arousal and for its modulating e¡ects on the thalamic ¢ltering of sensory inputs (Carlsson and Carlsson, 1990; Heimer, 2000). Furthermore, the NBM receives projections from several regions which are concerned mostly with the limbic system, including the medial prefrontal^orbitofrontal cortex, medial temporal lobe structures and ventral striatum (Steriade and Buzsaki, 1990; Wainer and Mesulam, 1990). All these areas are likely to be involved in schizophrenia (Robbins, 1990; Pantelis et al., 1997; Roberts et al., 1997). Thus, it is important to consider that the NBM may be in a position to modulate the activity of cortical substrates highly relevant in the pathophysiology and neurobiology of impaired sensorimotor gating in schizophrenia (Alheid and Heimer, 1988; Heimer et al., 1991). Although the loss of NBM cholinergic neurons is commonly considered responsible for the cognitive de¢cits occurring in dementias (Bartus et al., 1982; Collerton, 1986), the fact that dementias are also characterized by delusions and hallucinations, commonly found in schizophrenia, deserves special attention (Arendt et al., 1983; White and Cummings, 1996; Liberini et al., 1996). Additional evidence for a role of NBM in schizophrenia may be the recent observations that drugs enhancing central
The basal forebrain has long been suggested to play a crucial role in generating the hallmarks of schizophrenia, which include attentional and cognitive de¢cits related to dysfunction in processing of information (Stevens, 1973; Heimer, 2000). So far, the ventral striatopallidal system has been a primary area of research, with special interest devoted to disturbances in dopaminergic and glutamatergic transmission in cortical^subcortical reentrant circuits (Alexander et al., 1986; Groenwegen et al., 1990; Joel and Weiner, 1994). In the viewpoint of schizophrenia as a circuit dysfunction disorder, less well understood is the role of the cholinergic basal forebrain system (Jellinger, 1994) of which the nucleus basalis magnocellularis (NBM) is one of the main components (Bigl et al., 1982). Many of the cells in the NBM project to all cortical regions. It has been suggested that alterations in the cholinergic corticopetal projections are involved in the attentional abnormalities of neuropsychiatric disorders
1 These authors have contributed equally to the work. *Corresponding author. Tel.: +39-30-3717512; fax: +39-303701157. E-mail address: [email protected] (M. Ballmaier). Abbreviations : ANOVA, analysis of variance; ChAT, choline acetyltransferase ; NBM, nucleus basalis magnocellularis; PBS, phosphate-bu¡ered saline; PPI, prepulse inhibition.
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cholinergic transmission substantially reduce the core psychiatric symptoms of Lewy body dementia, including schizophrenia-like psychotic features (McKeith et al., 2000). In view of these observations and of the relationships between the NBM and its related circuits, we examined whether the selective loss of NBM corticopetal cholinergic neurons could produce substantial de¢cits in prepulse inhibition (PPI). Based on the reduction in startle produced by a prepulse stimulus, PPI is a widely used model for measuring impaired sensorimotor gating and information processing in schizophrenia (McGhie and Chapman, 1961; Geyer and Bra¡, 1987; Bra¡, 1999). Selective lesioning is currently assigned an important role in assessing the contribution to cognitive processing by individual nuclei in the basal forebrain (McGaughy et al., 2000). There is consistent evidence that infusions of the immunotoxin 192 IgG saporin directly into the NBM allow signi¢cantly more selective lesioning of cortical cholinergic projection neurons than i.c.v. infusions of the immunotoxin and local excitotoxic lesions. The immunotoxin acts by coupling the ribosome inactivating toxin saporin to an antibody that recognizes low-a¤nity nerve growth factor receptors, which are found in most neurons of the cholinergic basal forebrain. Localized infusions into the NBM have been reported to remove a large part of the cholinergic neurons located in the NBM and projecting to the cerebral cortex, while causing only minimal depletion of hippocampal choline acetyltransferase (ChAT) activity (Berger-Sweeney et al., 1994; Pizzo et al., 1999). Furthermore, since the neurons projecting to the amygdala from the NBM do not bear the low-a¤nity nerve growth factor receptors, the amygdalar cholinergic projections are completely spared following infusions of the immunotoxin (McGaughy et al., 2000). Therefore, taking advantage of the neurochemical selectivity of this method, we studied the e¡ect on PPI by using 192 IgG saporin for localized lesioning of the NBM. So far, studies of the regulation of PPI by the basal forebrain have mainly focused on the nucleus accumbens and the anteromedial striatum (Swerdlow and Geyer, 1998). These regions are considered to be part of a startle gating circuitry connecting limbic cortical regions and subcortical dopaminergic systems, and ultimately innervating the pedunculopontine nucleus via subpallidal e¡erent projections (Swerdlow and Koob, 1987; Geyer and Swerdlow, 1999). At present, it is unclear whether the NBM could be a critical station in this circuitry, which has been speci¢cally implicated in schizophrenia.
EXPERIMENTAL PROCEDURES
Animals and surgery A total of 40 male Sprague^Dawley rats (Harlan, Italy) weighing 220^250 g at the start of the experiment were used in this experiment. Rats were housed in groups of two under reversed light 12-h light^dark circle (lights on at 20.00 h and o¡ at 08.00 h). Methods for housing and all behavioral testing were consistent with the substantial literature of startle measures in
rodents (Swerdlow et al., 2000). Animals were handled individually within 3 days of arrival and daily thereafter. All animal experimentation was conducted in accordance with the Directive for care and use of experimental animals of the European Communities Council 24 November 1986 (86/609/EEC). All e¡orts were made to minimize the number of animals used and their su¡ering. Rats were anesthetized with ketamine/xylazine (80 mg/kg, i.p.) (Sigma, Milan, Italy), and placed in a stereotaxic apparatus. The immunotoxin 192 IgG saporin (Chemicon, Temecula, CA, USA) was diluted in sterile phosphate-bu¡ered saline (PBS, pH 7.4) to the ¢nal concentration. The skull was exposed, and a small hole was drilled on both sides. A 10-Wl Hamilton syringe ¢lled with either saline or a 192 IgG saporin solution (0.4 Wg/Wl) was lowered stereotaxically to the NBM (anteroposterior 30.2, lateral 32.8 mm from bregma and height 7 mm below the dura), according to the atlas of Paxinos and Watson (1982). Either 0.3 Wl of 192 IgG saporin (n = 20) or 0.3 Wl of saline (n = 10) was injected into each side of the NBM region. The toxin was injected over 5 min and the needle was left in place for an additional 5 min (Berger-Sweeney et al., 1994). Ten animals served as unlesioned controls. Rats were behaviorally tested before surgery and at 2, 4 and 6 weeks after surgery. Behavioral apparatus Startle experiments used one startle chamber (SR-LAB; San Diego Instruments, San Diego, CA, USA) housed in a soundattenuated room with a 60-dB ambient noise level. The startle chamber consisted of a Plexiglas cylinder 8.2 cm in diameter resting on a 12.5U25.5-cm Plexiglas frame within a ventilated enclosure. The delivery of acoustic stimuli was controlled by the SR-LAB microcomputer and interface assembly, which also digitized, recti¢ed, and recorded stabilimeter readings, with 100 1-ms readings collected beginning at stimulus onset. Startle amplitude was de¢ned as the average of the 100 readings. Acoustic stimuli and background noise were presented via a Radio Shack Supertweeter mounted 24 cm above the Plexiglas cylinder. Startle magnitude was detected and recorded as transduced cylinder movement via a piezoelectric device mounted below the Plexiglas stand. Startle testing procedure On testing days, approximately 1 h after arrival in the laboratory, each rat was placed in the startle chamber with 70-dB background noise and 5 min later was exposed to ¢ve trial types: (1) pulse (120-dB 40-ms broadband bursts); (2) pulse preceded 100 ms earlier by a 3-, 6- or 12-dB over background 20-ms prepulse; or (3) no stimulus. Sessions consisted of initial and ¢nal blocks of ¢ve pulse trials, and 50 subsequent trials presented in pseudorandom order. Intertrial intervals averaged 15 s. Prepulse intensities were chosen to span a range of relatively weak (3 dB) and intense (12 dB) stimuli. Rats were behaviorally tested before surgery and at 2, 4 and 6 weeks after surgery. Immunohistochemistry At the end of the experiments, the rats were deeply anesthetized and transcardially perfused with ice-cold paraformaldehyde solution (4% in phosphate bu¡er, pH 7.4). The brains were post¢xed for 4 h and cryoprotected in 18% sucrose solution for v48 h. Brains were cut in a cryostat throughout the injected area, in 30-Wm-thick coronal sections, and placed in PBS containing 0.1% sodium azide, and stored at 4³C until used for immunohistochemistry. For detection of cholinergic neurons, the polyclonal antibody raised against the enzyme ChAT (1:1000 goat anti-ChAT, Chemicon, Temecula, CA, USA) was used. The sections were washed in PBS^Triton X-100 0.3% and then incubated free £oating overnight at room temperature with the primary antibody in PBS solutions containing albumin 5 mg/ml, Triton X-100 0.3% and sodium
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Fig. 1. Photomicrographs of ChAT-immunopositive neurons in coronal sections of the NBM. (a) Unlesioned control rats. (b) Saline-injected rats. (c) 192 IgG saporin-injected rats. Note the presence of numerous immunolabelled neurons at the border between the internal capsule (IC) and the globus pallidus (GP) in control unlesioned and saline-injected NBM. A massive loss of cholinergic neurons can be seen in the NBM injected with 192 IgG saporin. (d^f) Higher magni¢cation detail of panels a^c, respectively. Note that both the morphology and staining features of the cells in panels d and e are similar. The spared neurons in 192 IgG saporin-injected NBM (f) appear reduced in size and exhibit paler staining than cells in panels d and e. Scale bars = 350 Wm (a^c); 70 Wm (d^f). CP = caudate^putamen. Brightness and contrast were adjusted using Adobe Photoshop 6.0 software (Adobe Systems, Mountain View, CA, USA) on control and treated slices simultaneously and images were then assembled into montages.
azide 0.1%. On the following day, the sections were washed in PBS and incubated for 1 h with the secondary antibody (1:1000 dilution) (Vector laboratories, Burlingame, CA, USA) in PBS solutions containing albumin 1 mg/ml and sodium azide 0.1%. After incubation with the avidin^biotin^peroxidase complex (Vector kit) for 1 h at room temperature, the primary antibody was localized by 3,3P-diaminobenzidine^H2 O2 -peroxidase color reaction, using NiCl2 as intensi¢er. At the end of the reaction, the sections were mounted on gelatin-coated slides, air dried and then washed, dehydrated and mounted (Scali et al., 2000). For microscope examination and photography, a BX40 microscope equipped with DP10 digital photomicrography system (Olympus Italia, Milan, Italy) was used. Cell counting was performed manually on coded slides under a 10U objective using a calibrated eyepiece grid. NBM ChAT-immunoreactive cells were identi¢ed as purple-black cell bodies with neuronal shape located in the ventral pallidum and the adjoining internal capsule. Only the cells with a well-de¢ned nucleus were included in the counts. A series of ¢ve sections per animal (inter-section
distance = 50^100 Wm), with the intermediate section centered on the needle track, was analyzed. Statistical analysis The initial and ¢nal ¢ve pulse trials permitted analysis of PPI over a more stable range of startle responses, and therefore were not included in any statistical analyses. PPI was de¢ned as the percent reduction in startle magnitude in the presence of the prepulse compared to the magnitude in the absence of the prepulse [1003(100Umagnitude on prepulse trial/magnitude on pulse trial)]. PPI data were analyzed with the general linear model univariate analysis of variance (ANOVA) using PPI values as dependent variable and treatment (immunolesion/saline/ control), prepulse amplitude and session (times after surgery) as ¢xed factors (statistical package, SPSS v. 10). Furthermore, oneway ANOVA was used to test di¡erences between treatments at each prepulse amplitude. Startle amplitude data were analyzed with the general linear model univariate ANOVA, using startle
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amplitude values as dependent variable and treatment (immunolesion/saline/control) and session (times after surgery) as ¢xed factors. The Bonferroni test was used as a post-hoc test for multiple comparisons with P 6 0.05 as threshold for signi¢cant di¡erence. For cell counting, one-way ANOVA followed by Fisher's post-hoc analysis was used.
RESULTS
Immunohistochemical characterization The immunohistochemical analysis was carried out, at 6 weeks after injection, on ¢ve immunotoxin- and three saline-injected rats and three controls, selected at random. In the NBM of control rats, ChAT-immunopositive neurons were localized in intensely labelled magnocellular cells located at the border between the internal capsule and the globus pallidus (Fig. 1a, d). A pronounced loss of ChAT-immunopositive neurons was revealed bilaterally throughout the 192 IgG saporininjected NBM (Fig. 1c, f) while these neurons were largely unaltered, in both number and morphology, following saline injections (Fig. 1b, e). The results of neuron pro¢le counting in both NBM areas are reported in Table 1. Behavioral results Behavioral testing of rats at 2, 4 and 6 weeks after surgery revealed a dramatic e¡ect of the immunotoxin lesion on PPI with no signi¢cant e¡ect on startle amplitude. Statistical analysis showed a signi¢cant e¡ect of treatment (F = 64.7; df 2; P 6 0.001) and prepulse amplitude (F = 44.1; df 2; P 6 0.001), whereas session (F = 1.005; df 2; P = 0.367) and interactions between any of the factors were not signi¢cant. Post-hoc analysis
revealed that the immunotoxin-treated group was signi¢cantly di¡erent from both saline and control groups (P 6 0.001 versus saline or control) (Fig. 2). The profound decrease in PPI persisted over repeated testing. The immunotoxin lesion correlated signi¢cantly with the loss of PPI for all prepulse trial types. The e¡ect of immunotoxin lesions was most evident for weaker prepulse intensities (3 and 6 dB above background) for which PPI in immunotoxin-treated rats was decreased by approximately 50% with respect to saline-treated rats. Importantly, immunotoxin treatment had no significant e¡ect on startle amplitude at any time animals were exposed to testing (P = 0.489 and 0.258 versus saline and control, respectively) (Table 2).
DISCUSSION
This study shows that intraparenchymal administration of the immunotoxin 192 IgG saporin into the NBM produced an extensive loss of cholinergic neurons and a signi¢cant disruption of PPI. The PPI disruption remained stable with repeated testing. Our data further show that this e¡ect is independent of the level of startle amplitude and is most pronounced in a threshold range of PPI produced by weak prepulse intensities (3^6 dB above background). Contribution of lesion selectivity and test parameters to the e¡ect on PPI Our ¢nding is in apparent contrast with a previous study (Schauz and Koch, 1999), which failed to detect any reduction of PPI after quinolinic acid lesions of the NBM. The discrepancy may be explained by several factors, of which the ¢rst implicates the extension of the
Fig. 2. E¡ect of bilateral NBM injection of 192 IgG saporin on PPI. Mean þ S.E.M. values are shown (n = 20 for 192 IgG saporin-injected rats, and 10 for unlesioned control or saline-injected rats). Since no signi¢cant e¡ect of session was present (for further details, see text), for each animal the data of the three testing sessions were pooled. Statistical analysis according to one-way ANOVA, followed by Bonferroni test for multiple comparisons. 12 dB above background, F = 18.8, df 2, P 6 0.001; 6 dB above background, F = 28.8, df 2, P 6 0.001 ; 3 dB above background, F = 19.9, df 2, P 6 0.001. At every prepulse amplitude, PPI values of the immunotoxin-treated group were signi¢cantly di¡erent from saline-treated or control groups (**P 6 0.001 versus saline-treated, ## P 6 0.001 versus control).
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Table 1. E¡ect of bilateral NBM injection of 192 IgG saporin on ChAT-immunopositive neuron number Treatment (n)
Total number of neurons
Change with respect to control rats (%)
Control unlesioned rats (3) Saline-injected rats (3) 192 IgG saporin-injected rats (5)
394 þ 42 365 þ 48 26 þ 4*
37 393
Total neuronal counts in the NBM are expressed as the mean þ S.E.M. of the indicated number (n) of rats. Neurons were counted on both sides of ¢ve sections per animal. Statistical analysis was performed using one-way ANOVA followed by Fisher's post-hoc test: *P 6 0.05, versus control unlesioned rats.
lesion. Localized infusions of 192 IgG saporin into the NBM have been found to speci¢cally and discretely lesion the NBM^cortical cholinergic pathway, while minimizing non-speci¢c tissue damage to adjacent forebrain nuclei (McGaughy et al., 2000); it has further been shown that immunotoxin lesions do not lead to changes in other neurotransmitter systems (Pizzo et al., 1999). In this context, the fact is of importance that the NBM represents a collection of cholinergic and non-cholinergic (e.g. GABA, peptides) neurons (Heimer, 2000). Since the cholinergic projection from the NBM to the cortex is excitatory, while the GABAergic projection is inhibitory, results on sensorimotor gating behavior following a selective 192 IgG saporin-induced lesion of the NBM are likely to be di¡erent from those of non-selective excitotoxic lesions. Moreover, whereas immunotoxininduced lesions largely spare the NBM projection to the amygdala, quinolinic acid has been found to be a more potent neurotoxin of cholinergic neurons innervating the amygdala than those projecting to the cortex (Boegman et al., 1992). The concentration of immunotoxin used in our study has been shown to produce only minor di¡usion into the adjacent diagonal band of Broca and a minimal depletion of hippocampal ChAT activity (Pizzo et al., 1999). The authors of the previous study described extension of the primary lesions into parts of the globus pallidus, the substantia innominata and the bed nucleus of the stria terminalis. Therefore, it is also important to consider the possibility that damage of adjacent forebrain nuclei, in addition to the destruction of non-cholinergic neurons in the NBM, produces adaptive changes in multiple neuronal systems, which ultimately might suppress sensorimotor gating de¢cits. This viewpoint is indirectly supported by a study which showed signi¢cant reduction of PPI after acute administration of cocaine while no e¡ects on PPI were observed after sustained cocaine exposure, which is thought to produce neurochemical changes in several brain regions (Martinez et al., 1999). Together, these observations and considerations suggest that the de¢cits in PPI produced by NBM infusions of 192 IgG saporin are linked to selective and extensive reduction of cholinergic cortical activity. In addition to the extension of the lesion, di¡erences in the parameters used for measurements of startle gating might also account for the discrepancy with the previous report. Schauz and Koch (1999) used 5- and 15-dB prepulses over a background intensity of 55 dB, while we used a more sensitive threshold range of PPI produced by prepulse intensities of 3, 6 and 12 dB above a higher background (70 dB). Previous studies have shown that
de¢cits in PPI are most sensitively detected using weak prepulse stimuli rather than intense prepulse stimuli, thereby avoiding £oor and ceiling e¡ects (Swerdlow and Geyer, 1993). In particular, the suggestion that weak prepulses might be most able to detect de¢cits in PPI is supported by the observation that PPI elicited by weak prepulses is most sensitive to disruption by low doses of apomorphine that do not disrupt PPI that is produced by more intense prepulse stimuli (Davis et al., 1990). This observation merits further investigation and is in accordance with our results suggesting that PPI de¢cits in states of gating circuit dysfunctions might be most sensitively detected in conditions in which the system can exhibit the greatest changes. Possible implications for the understanding of schizophrenia Psychotic symptoms represent frequent neuropsychiatric concomitants of the cognitive dysfunction observed in common forms of dementias, where the loss of cortical cholinergic projections from the NBM is known to be one of the most striking neurochemical alterations (Whitehouse et al., 1982). Given the speci¢c damage to the corticopetal cholinergic projections in the immunotoxin lesion model, our ¢ndings indicate that the NBM may modulate sensorimotor gating via e¡erent projections to the cortical substrates of the startle gating circuitry. It has been shown that the selective loss of corticopetal cholinergic projections is su¤cient to produce impairments in tasks designed to assess various aspects of attentional functions (Chiba et al., 1995, 1999; Bucci et al., 1998; Sarter and Bruno, 1999; McGaughy et al., 2000), including object recognition (Bartolini et al., 1986), a form of non-spatial memory which depends on the activity of prefrontal cortical circuitry (Funahashi et al., 1989). Accordingly, selective immunotoxic lesions of NBM cholinergic neurons impair prefrontal neuronal activity associated with sustained
Table 2. E¡ect of bilateral NBM injection of 192 IgG saporin on startle amplitude (arbitrary units) Time after treatment (weeks)
Saline-injected rats
192 IgG saporin-injected rats
Two Four Six
96.8 þ 38.6 87.9 þ 19.3 81.1 þ 17.6
88.7 þ 26.3 106.6 þ 35.6 149.4 þ 49.8
Mean þ S.E.M. values are shown. Statistical analysis according to one-way ANOVA. No signi¢cant e¡ect was present.
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visual attentional performance (Gill et al., 2000). A role of NBM cholinergic neurons in processing of information has been further emphasized by the ¢nding that pairing NBM stimulation with sensory stimuli results in a global reorganization of sensory cortical maps (Kilgard and Merzenich, 1998). Since the limbic cortex represents a well-characterized substrate of the startle gating circuitry, one might speculate that PPI disruption observed after immunotoxic lesion of NBM principally derives from the loss of the cholinergic modulation of the prefrontal cortex by the NBM; this hypothesis is supported by the observation that infusions into the NBM of 192 IgG saporin show the highest ChAT depletion in the frontal cortex (Pizzo et al., 1999). Prefrontal or limbic cortical dysfunction has long been implicated in the pathophysiology of schizophrenia (Weinberger et al., 1992; Grace, 2000). Furthermore, there is evidence that the limbic involvement is also essential for the psychotic behavior in Alzheimer's disease, with neurochemical alterations a¡ecting the dopaminergic/cholinergic axis likely being central to the clinical manifestations of both disorders (White and Cummings, 1996). Much evidence from PPI studies has demonstrated the importance of abnormal dopamine regulation in the circuitry connecting the limbic cortical regions to the basal forebrain (Swerdlow and Geyer, 1998). In addition, direct manipulations of both glutamatergic and serotonergic activity in the forebrain are also known to a¡ect PPI in rats (Swerdlow and Geyer, 1998; Geyer and Swerdlow, 1999). Therefore, a possible explanation for reduced PPI in the present study may involve direct or indirect interactions of the NBM^cortical cholinergic pathway with other neurotransmitter systems known to a¡ect PPI along the startle gating circuitry characterized by neural connections between limbic cortical areas and subcortical dopaminergic systems, and ultimately the pontine system via GABAergic subpallidal e¡erents to
the pedunculopontine nucleus. Since the NBM also receives projections from cortical areas which are mainly concerned with the limbic system (Wainer and Mesulam, 1990), it is conceivable that these reciprocal connections with limbic cortical targets may account for the modulatory e¡ect of the NBM on PPI. Another possible mechanism by which the NBM may regulate PPI is via indirect connections to the pedunculopontine nucleus through the thalamic reticular nucleus. Since the projection from the NBM to the thalamic reticular nucleus has been proposed to provide an alternative pathway for cortical arousal and to play a role in the thalamic ¢ltering of sensory input (Steriade et al., 1987; Steriade and Buzsaki, 1990; Tourtellotte et al., 1990), one might speculate that PPI de¢cits after NBM lesions may also re£ect changes in the activity of thalamic nuclei impinging on the startle gating circuit at the level of the pedunculopontine nucleus.
CONCLUSIONS
The results presented in this work indicate that selective and discrete lesioning of the NBM leads to a substantial impairment of PPI which persists over repeated testing. Overall, our data suggest that the NBM appears to be ideally located within the basal forebrain for modulating sensorimotor gating abilities that may correlate with important determinants of information processing in schizophrenia. Insights drawn from our results may also enrich the understanding of NBM corticopetal projections within the context of psychotic hallmarks shared by schizophrenia and dementias.
AcknowledgementsöThis work has been supported by grants from the University of Brescia and MURST (PRIN to G.P.). We thank Carla Scali and Rodolfo Mazzoncini for technical assistance.
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Heimer, L., de Olmos, J., Alheid, G.F., Zaborszky, L., 1991. `Perestroika' in the basal forebrain: opening the border between neurology and psychiatry. Prog. Brain Res. 87, 109^165. Jellinger, K., 1994. Neuromorphological background of pathochemical studies in major psychoses. In: Beckmann, H., Riederer, P. (Eds.), Pathochemical Markers in Major Psychoses. Springer, Berlin, pp. 1^23. Joel, D., Weiner, I., 1994. The organization of the basal ganglia-thalamocortical circuits : Open interconnected rather than closed segregated. Neuroscience 63, 363^379. Kilgard, M.P., Merzenich, M.M., 1998. Cortical map reorganization enabled by nucleus basalis activity. Science 279, 1714^1718. Liberini, P., Valerio, A., Memo, M., Spano, P.F., 1996. Lewy-body dementia and responsiveness to cholinesterase inhibitors : a paradigm for heterogeneity of Alzheimer's disease ? Trends Pharmacol. Sci. 17, 155^160. Martinez, Z.A., Ellison, G.D., Geyer, M.A., Swerdlow, N.R., 1999. E¡ects of sustained cocaine exposure on sensorimotor gating of startle in rats. Psychopharmacology 142, 253^260. McGaughy, J., Everitt, B.J., Robbins, T.W., Sarter, M., 2000. The role of cholinergic a¡erent projections in cognition : impact of selective immunotoxins. Behav. Brain Res. 115, 251^263. McGhie, A., Chapman, J., 1961. Disorders of attention and perception in schizophrenia. Br. J. Med. Psychol. 34, 102^116. McKeith, I., Del Ser, T., Spano, P.F., Emre, M., Wesnes, K., Anand, R., Cicin-Sain, A., Ferrara, R., Spiegel, R., 2000. E¤cacy of rivastigmine in dementia with Lewy bodies: a randomised, double-blind, placebo-controlled international study. Lancet 356, 2031^2035. Pantelis, C., Barnes, T.R.E., Nelson, H.E., Tanner, S., Weatherley, L., Owen, A.M., Robbins, T.W., 1997. Frontal-striatal cognitive de¢cits in patients with chronic schizophrenia. Brain 120, 1823^1943. Paxinos, G., Watson, C., 1982. The Rat Brain in Stereotaxic Coordinates. Academic, New York. Pizzo, D.P., Waite, J.J., Thal, L.J., Winkler, J., 1999. Intraparenchimal infusions of 192 IgG-saporin : development of a method for selective and discrete lesioning of cholinergic basal forebrain nuclei. J. Neurosci. Methods 91, 9^19. Robbins, T.W., 1990. The case for frontostriatal dysfunction in schizophrenia. Schizophr. Bull. 16, 391^402. Roberts, G.W., Royston, M.C., Weinberger, D.R., 1997. Schizophrenia. In: Graham, D.I., Lantos, P.L. (Eds.), Green¢eld's Neuropathology, 6th edn. Oxford University, New York, pp. 897^929. Sarter, M., Bruno, J.P., 1999. Abnormal regulation of corticopetal cholinergic neurons and impaired information processing in neuropsychiatric disorders. Trends Neurosci. 22, 67^74. Scali, C., Prosperi, C., Vannucchi, M.G., Pepeu, G., Casamenti, F., 2000. Brain in£ammatory reaction in an animal model of neuronal degeneration and its modulation by an anti-in£ammatory drug: implication in Alzheimer's disease. Eur. J. Neurosci. 12, 1900^1912. Schauz, C., Koch, M., 1999. Lesions of the nucleus basalis magnocellularis do not impair prepulse inhibition and latent inhibition of fearpotentiated startle in the rat. Brain Res. 815, 98^115. Steriade, M., Buzsaki, G., 1990. Parallel activation of thalamic and cortical neurons by brainstem and basal forebrain cholinergic systems. In: Steriade, M., Biesold, D. (Eds.), Brain Cholinergic Systems. Oxford University, New York, pp. 1^52. Steriade, M., Parent, D., Pare, D., Smith, Y., 1987. Cholinergic and non-cholinergic neurons of cat basal forebrain project to reticular and mediodorsal thalamic nuclei. Brain Res. 408, 372^376. Stevens, J.R., 1973. An anatomy of schizophrenia? Arch. Gen. Psychiatry 29, 177^189. Swerdlow, N.R., Geyer, M., 1993. Prepulse inhibition of acoustic startle in rats after lesions of the peduncolopontine tegmental nucleus. Behav. Neurosci. 107, 104^117. Swerdlow, N.R., Geyer, M., 1998. Using an animal model of de¢cient sensorimotor gating to study the pathophysiology and new treatments in schizophrenia. Schizophr. Bull. 24, 285^301. Swerdlow, N.R., Koob, G.F., 1987. Dopamine, schizophrenia, mania and depression : toward a uni¢ed hypothesis of cortico-striato-pallidothalamic function. Behav. Brain Sci. 10, 197^245. Swerdlow, N.R., Martinez, Z.A., Hanlon, F.M., Platten, A., Farid, M., Auerbach, P., Bra¡, D., Geyer, M., 2000. Toward understanding the biology of a complex phenotype: rat strain and substrain di¡erences in the sensorimotor gating-disruptive e¡ects of dopamine agonists. J. Neurosci. 20, 4325^4336. Tourtellotte, W.G., Van Hoesen, G.W., Hyman, B.T., Tikoo, R.K., Damasio, A.R., 1990. Alz-50 immunoreactivity in the thalamic reticular nucleus in Alzheimer's disease. Brain Res. 515, 227^234. Wainer, B.H., Mesulam, M.M., 1990. Ascending cholinergic pathways in the rat brain. In: Steriade, M., Biesold, D. (Eds.), Brain Cholinergic Systems. Oxford University, New York, pp. 65^119. Weinberger, D.R., Berman, K.F., Suddath, R., Torrey, E.F., 1992. Evidence for dysfunction of a prefrontal-limbic network in schizophrenia : an MRI and rCBF study of discordant monozygotic twins. Am. J. Psychiatry 149, 890^897. White, K.E., Cummings, J.L., 1996. Schizophrenia and Alzheimer's disease : clinical and pathophysiologic analogies. Compr. Psychiatry 37, 188^ 195. Whitehouse, P.J., Price, D.L., Struble, R.G., Clark, A.W., Coyle, J.T., DeLong, M.R., 1982. Alzheimer's disease and senile dementia: loss of neurons in the basal forebrain. Science 215, 1237^1239. (Accepted 25 June 2001)
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Neuroscience Vol. 108, No. 2, pp. 299^305, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 01 $20.00+0.00
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SELECTIVE IMMUNOLESIONING OF CHOLINERGIC NEURONS IN NUCLEUS BASALIS MAGNOCELLULARIS IMPAIRS PREPULSE INHIBITION OF ACOUSTIC STARTLE M. BALLMAIER,a * F. CASAMENTI,b M. ZOLI,c G. PEPEUb;1 and P. SPANOa;1 a
Division of Pharmacology, Department of Biomedical Sciences and Biotechnologies, Brescia University Medical School, Via Valsabbina 19, 25123 Brescia, Italy b c
Department of Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy
Section of Physiology, Department of Biomedical Sciences, University of Modena, Modena, Italy
AbstractöInformation processing and attentional abnormalities are prominent in neuropsychiatric disorders. Since the cholinergic neurons located in the nucleus basalis magnocellularis have been shown to be involved in attentional performance and information processing, recent e¡orts to analyze the signi¢cance of the basal forebrain in the context of schizophrenia have focused on this nucleus and its projections to the cerebral cortex. We report here that bilateral selective immunolesioning of the cholinergic neurons in the nucleus basalis magnocellularis is followed by signi¢cant de¢cits in sensorimotor gating measured by prepulse inhibition of the startle re£ex in adult rats. This behavioral approach is used in both humans and rodents and has been proposed as a valuable model contributing to the understanding of the neurobiological substrates of schizophrenia. The disruption of prepulse inhibition persisted over repeated testing. The selective lesions were induced by bilateral intraparenchymal infusions of 192 IgG saporin at a concentration having minimal di¡usion into adjacent nuclei of the basal forebrain. The infusions were followed by extensive loss of choline acetyltransferase-immunopositive neurons. Our results show that the cholinergic neurons of the nucleus basalis magnocellularis represent a critical station of the startle gating circuitry and suggest that dysfunction of these neurons may result in impaired sensorimotor gating characteristic of schizophrenia. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: immunotoxin, cholinergic basal forebrain, sensorimotor gating, schizophrenia.
(Sarter and Bruno, 1999). As an integral part of the ascending activating system, the NBM in fact is considered to be important for cortical arousal and for its modulating e¡ects on the thalamic ¢ltering of sensory inputs (Carlsson and Carlsson, 1990; Heimer, 2000). Furthermore, the NBM receives projections from several regions which are concerned mostly with the limbic system, including the medial prefrontal^orbitofrontal cortex, medial temporal lobe structures and ventral striatum (Steriade and Buzsaki, 1990; Wainer and Mesulam, 1990). All these areas are likely to be involved in schizophrenia (Robbins, 1990; Pantelis et al., 1997; Roberts et al., 1997). Thus, it is important to consider that the NBM may be in a position to modulate the activity of cortical substrates highly relevant in the pathophysiology and neurobiology of impaired sensorimotor gating in schizophrenia (Alheid and Heimer, 1988; Heimer et al., 1991). Although the loss of NBM cholinergic neurons is commonly considered responsible for the cognitive de¢cits occurring in dementias (Bartus et al., 1982; Collerton, 1986), the fact that dementias are also characterized by delusions and hallucinations, commonly found in schizophrenia, deserves special attention (Arendt et al., 1983; White and Cummings, 1996; Liberini et al., 1996). Additional evidence for a role of NBM in schizophrenia may be the recent observations that drugs enhancing central
The basal forebrain has long been suggested to play a crucial role in generating the hallmarks of schizophrenia, which include attentional and cognitive de¢cits related to dysfunction in processing of information (Stevens, 1973; Heimer, 2000). So far, the ventral striatopallidal system has been a primary area of research, with special interest devoted to disturbances in dopaminergic and glutamatergic transmission in cortical^subcortical reentrant circuits (Alexander et al., 1986; Groenwegen et al., 1990; Joel and Weiner, 1994). In the viewpoint of schizophrenia as a circuit dysfunction disorder, less well understood is the role of the cholinergic basal forebrain system (Jellinger, 1994) of which the nucleus basalis magnocellularis (NBM) is one of the main components (Bigl et al., 1982). Many of the cells in the NBM project to all cortical regions. It has been suggested that alterations in the cholinergic corticopetal projections are involved in the attentional abnormalities of neuropsychiatric disorders
1 These authors have contributed equally to the work. *Corresponding author. Tel.: +39-30-3717512; fax: +39-303701157. E-mail address: [email protected] (M. Ballmaier). Abbreviations : ANOVA, analysis of variance; ChAT, choline acetyltransferase ; NBM, nucleus basalis magnocellularis; PBS, phosphate-bu¡ered saline; PPI, prepulse inhibition.
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cholinergic transmission substantially reduce the core psychiatric symptoms of Lewy body dementia, including schizophrenia-like psychotic features (McKeith et al., 2000). In view of these observations and of the relationships between the NBM and its related circuits, we examined whether the selective loss of NBM corticopetal cholinergic neurons could produce substantial de¢cits in prepulse inhibition (PPI). Based on the reduction in startle produced by a prepulse stimulus, PPI is a widely used model for measuring impaired sensorimotor gating and information processing in schizophrenia (McGhie and Chapman, 1961; Geyer and Bra¡, 1987; Bra¡, 1999). Selective lesioning is currently assigned an important role in assessing the contribution to cognitive processing by individual nuclei in the basal forebrain (McGaughy et al., 2000). There is consistent evidence that infusions of the immunotoxin 192 IgG saporin directly into the NBM allow signi¢cantly more selective lesioning of cortical cholinergic projection neurons than i.c.v. infusions of the immunotoxin and local excitotoxic lesions. The immunotoxin acts by coupling the ribosome inactivating toxin saporin to an antibody that recognizes low-a¤nity nerve growth factor receptors, which are found in most neurons of the cholinergic basal forebrain. Localized infusions into the NBM have been reported to remove a large part of the cholinergic neurons located in the NBM and projecting to the cerebral cortex, while causing only minimal depletion of hippocampal choline acetyltransferase (ChAT) activity (Berger-Sweeney et al., 1994; Pizzo et al., 1999). Furthermore, since the neurons projecting to the amygdala from the NBM do not bear the low-a¤nity nerve growth factor receptors, the amygdalar cholinergic projections are completely spared following infusions of the immunotoxin (McGaughy et al., 2000). Therefore, taking advantage of the neurochemical selectivity of this method, we studied the e¡ect on PPI by using 192 IgG saporin for localized lesioning of the NBM. So far, studies of the regulation of PPI by the basal forebrain have mainly focused on the nucleus accumbens and the anteromedial striatum (Swerdlow and Geyer, 1998). These regions are considered to be part of a startle gating circuitry connecting limbic cortical regions and subcortical dopaminergic systems, and ultimately innervating the pedunculopontine nucleus via subpallidal e¡erent projections (Swerdlow and Koob, 1987; Geyer and Swerdlow, 1999). At present, it is unclear whether the NBM could be a critical station in this circuitry, which has been speci¢cally implicated in schizophrenia.
EXPERIMENTAL PROCEDURES
Animals and surgery A total of 40 male Sprague^Dawley rats (Harlan, Italy) weighing 220^250 g at the start of the experiment were used in this experiment. Rats were housed in groups of two under reversed light 12-h light^dark circle (lights on at 20.00 h and o¡ at 08.00 h). Methods for housing and all behavioral testing were consistent with the substantial literature of startle measures in
rodents (Swerdlow et al., 2000). Animals were handled individually within 3 days of arrival and daily thereafter. All animal experimentation was conducted in accordance with the Directive for care and use of experimental animals of the European Communities Council 24 November 1986 (86/609/EEC). All e¡orts were made to minimize the number of animals used and their su¡ering. Rats were anesthetized with ketamine/xylazine (80 mg/kg, i.p.) (Sigma, Milan, Italy), and placed in a stereotaxic apparatus. The immunotoxin 192 IgG saporin (Chemicon, Temecula, CA, USA) was diluted in sterile phosphate-bu¡ered saline (PBS, pH 7.4) to the ¢nal concentration. The skull was exposed, and a small hole was drilled on both sides. A 10-Wl Hamilton syringe ¢lled with either saline or a 192 IgG saporin solution (0.4 Wg/Wl) was lowered stereotaxically to the NBM (anteroposterior 30.2, lateral 32.8 mm from bregma and height 7 mm below the dura), according to the atlas of Paxinos and Watson (1982). Either 0.3 Wl of 192 IgG saporin (n = 20) or 0.3 Wl of saline (n = 10) was injected into each side of the NBM region. The toxin was injected over 5 min and the needle was left in place for an additional 5 min (Berger-Sweeney et al., 1994). Ten animals served as unlesioned controls. Rats were behaviorally tested before surgery and at 2, 4 and 6 weeks after surgery. Behavioral apparatus Startle experiments used one startle chamber (SR-LAB; San Diego Instruments, San Diego, CA, USA) housed in a soundattenuated room with a 60-dB ambient noise level. The startle chamber consisted of a Plexiglas cylinder 8.2 cm in diameter resting on a 12.5U25.5-cm Plexiglas frame within a ventilated enclosure. The delivery of acoustic stimuli was controlled by the SR-LAB microcomputer and interface assembly, which also digitized, recti¢ed, and recorded stabilimeter readings, with 100 1-ms readings collected beginning at stimulus onset. Startle amplitude was de¢ned as the average of the 100 readings. Acoustic stimuli and background noise were presented via a Radio Shack Supertweeter mounted 24 cm above the Plexiglas cylinder. Startle magnitude was detected and recorded as transduced cylinder movement via a piezoelectric device mounted below the Plexiglas stand. Startle testing procedure On testing days, approximately 1 h after arrival in the laboratory, each rat was placed in the startle chamber with 70-dB background noise and 5 min later was exposed to ¢ve trial types: (1) pulse (120-dB 40-ms broadband bursts); (2) pulse preceded 100 ms earlier by a 3-, 6- or 12-dB over background 20-ms prepulse; or (3) no stimulus. Sessions consisted of initial and ¢nal blocks of ¢ve pulse trials, and 50 subsequent trials presented in pseudorandom order. Intertrial intervals averaged 15 s. Prepulse intensities were chosen to span a range of relatively weak (3 dB) and intense (12 dB) stimuli. Rats were behaviorally tested before surgery and at 2, 4 and 6 weeks after surgery. Immunohistochemistry At the end of the experiments, the rats were deeply anesthetized and transcardially perfused with ice-cold paraformaldehyde solution (4% in phosphate bu¡er, pH 7.4). The brains were post¢xed for 4 h and cryoprotected in 18% sucrose solution for v48 h. Brains were cut in a cryostat throughout the injected area, in 30-Wm-thick coronal sections, and placed in PBS containing 0.1% sodium azide, and stored at 4³C until used for immunohistochemistry. For detection of cholinergic neurons, the polyclonal antibody raised against the enzyme ChAT (1:1000 goat anti-ChAT, Chemicon, Temecula, CA, USA) was used. The sections were washed in PBS^Triton X-100 0.3% and then incubated free £oating overnight at room temperature with the primary antibody in PBS solutions containing albumin 5 mg/ml, Triton X-100 0.3% and sodium
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Fig. 1. Photomicrographs of ChAT-immunopositive neurons in coronal sections of the NBM. (a) Unlesioned control rats. (b) Saline-injected rats. (c) 192 IgG saporin-injected rats. Note the presence of numerous immunolabelled neurons at the border between the internal capsule (IC) and the globus pallidus (GP) in control unlesioned and saline-injected NBM. A massive loss of cholinergic neurons can be seen in the NBM injected with 192 IgG saporin. (d^f) Higher magni¢cation detail of panels a^c, respectively. Note that both the morphology and staining features of the cells in panels d and e are similar. The spared neurons in 192 IgG saporin-injected NBM (f) appear reduced in size and exhibit paler staining than cells in panels d and e. Scale bars = 350 Wm (a^c); 70 Wm (d^f). CP = caudate^putamen. Brightness and contrast were adjusted using Adobe Photoshop 6.0 software (Adobe Systems, Mountain View, CA, USA) on control and treated slices simultaneously and images were then assembled into montages.
azide 0.1%. On the following day, the sections were washed in PBS and incubated for 1 h with the secondary antibody (1:1000 dilution) (Vector laboratories, Burlingame, CA, USA) in PBS solutions containing albumin 1 mg/ml and sodium azide 0.1%. After incubation with the avidin^biotin^peroxidase complex (Vector kit) for 1 h at room temperature, the primary antibody was localized by 3,3P-diaminobenzidine^H2 O2 -peroxidase color reaction, using NiCl2 as intensi¢er. At the end of the reaction, the sections were mounted on gelatin-coated slides, air dried and then washed, dehydrated and mounted (Scali et al., 2000). For microscope examination and photography, a BX40 microscope equipped with DP10 digital photomicrography system (Olympus Italia, Milan, Italy) was used. Cell counting was performed manually on coded slides under a 10U objective using a calibrated eyepiece grid. NBM ChAT-immunoreactive cells were identi¢ed as purple-black cell bodies with neuronal shape located in the ventral pallidum and the adjoining internal capsule. Only the cells with a well-de¢ned nucleus were included in the counts. A series of ¢ve sections per animal (inter-section
distance = 50^100 Wm), with the intermediate section centered on the needle track, was analyzed. Statistical analysis The initial and ¢nal ¢ve pulse trials permitted analysis of PPI over a more stable range of startle responses, and therefore were not included in any statistical analyses. PPI was de¢ned as the percent reduction in startle magnitude in the presence of the prepulse compared to the magnitude in the absence of the prepulse [1003(100Umagnitude on prepulse trial/magnitude on pulse trial)]. PPI data were analyzed with the general linear model univariate analysis of variance (ANOVA) using PPI values as dependent variable and treatment (immunolesion/saline/ control), prepulse amplitude and session (times after surgery) as ¢xed factors (statistical package, SPSS v. 10). Furthermore, oneway ANOVA was used to test di¡erences between treatments at each prepulse amplitude. Startle amplitude data were analyzed with the general linear model univariate ANOVA, using startle
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amplitude values as dependent variable and treatment (immunolesion/saline/control) and session (times after surgery) as ¢xed factors. The Bonferroni test was used as a post-hoc test for multiple comparisons with P 6 0.05 as threshold for signi¢cant di¡erence. For cell counting, one-way ANOVA followed by Fisher's post-hoc analysis was used.
RESULTS
Immunohistochemical characterization The immunohistochemical analysis was carried out, at 6 weeks after injection, on ¢ve immunotoxin- and three saline-injected rats and three controls, selected at random. In the NBM of control rats, ChAT-immunopositive neurons were localized in intensely labelled magnocellular cells located at the border between the internal capsule and the globus pallidus (Fig. 1a, d). A pronounced loss of ChAT-immunopositive neurons was revealed bilaterally throughout the 192 IgG saporininjected NBM (Fig. 1c, f) while these neurons were largely unaltered, in both number and morphology, following saline injections (Fig. 1b, e). The results of neuron pro¢le counting in both NBM areas are reported in Table 1. Behavioral results Behavioral testing of rats at 2, 4 and 6 weeks after surgery revealed a dramatic e¡ect of the immunotoxin lesion on PPI with no signi¢cant e¡ect on startle amplitude. Statistical analysis showed a signi¢cant e¡ect of treatment (F = 64.7; df 2; P 6 0.001) and prepulse amplitude (F = 44.1; df 2; P 6 0.001), whereas session (F = 1.005; df 2; P = 0.367) and interactions between any of the factors were not signi¢cant. Post-hoc analysis
revealed that the immunotoxin-treated group was signi¢cantly di¡erent from both saline and control groups (P 6 0.001 versus saline or control) (Fig. 2). The profound decrease in PPI persisted over repeated testing. The immunotoxin lesion correlated signi¢cantly with the loss of PPI for all prepulse trial types. The e¡ect of immunotoxin lesions was most evident for weaker prepulse intensities (3 and 6 dB above background) for which PPI in immunotoxin-treated rats was decreased by approximately 50% with respect to saline-treated rats. Importantly, immunotoxin treatment had no significant e¡ect on startle amplitude at any time animals were exposed to testing (P = 0.489 and 0.258 versus saline and control, respectively) (Table 2).
DISCUSSION
This study shows that intraparenchymal administration of the immunotoxin 192 IgG saporin into the NBM produced an extensive loss of cholinergic neurons and a signi¢cant disruption of PPI. The PPI disruption remained stable with repeated testing. Our data further show that this e¡ect is independent of the level of startle amplitude and is most pronounced in a threshold range of PPI produced by weak prepulse intensities (3^6 dB above background). Contribution of lesion selectivity and test parameters to the e¡ect on PPI Our ¢nding is in apparent contrast with a previous study (Schauz and Koch, 1999), which failed to detect any reduction of PPI after quinolinic acid lesions of the NBM. The discrepancy may be explained by several factors, of which the ¢rst implicates the extension of the
Fig. 2. E¡ect of bilateral NBM injection of 192 IgG saporin on PPI. Mean þ S.E.M. values are shown (n = 20 for 192 IgG saporin-injected rats, and 10 for unlesioned control or saline-injected rats). Since no signi¢cant e¡ect of session was present (for further details, see text), for each animal the data of the three testing sessions were pooled. Statistical analysis according to one-way ANOVA, followed by Bonferroni test for multiple comparisons. 12 dB above background, F = 18.8, df 2, P 6 0.001; 6 dB above background, F = 28.8, df 2, P 6 0.001 ; 3 dB above background, F = 19.9, df 2, P 6 0.001. At every prepulse amplitude, PPI values of the immunotoxin-treated group were signi¢cantly di¡erent from saline-treated or control groups (**P 6 0.001 versus saline-treated, ## P 6 0.001 versus control).
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Table 1. E¡ect of bilateral NBM injection of 192 IgG saporin on ChAT-immunopositive neuron number Treatment (n)
Total number of neurons
Change with respect to control rats (%)
Control unlesioned rats (3) Saline-injected rats (3) 192 IgG saporin-injected rats (5)
394 þ 42 365 þ 48 26 þ 4*
37 393
Total neuronal counts in the NBM are expressed as the mean þ S.E.M. of the indicated number (n) of rats. Neurons were counted on both sides of ¢ve sections per animal. Statistical analysis was performed using one-way ANOVA followed by Fisher's post-hoc test: *P 6 0.05, versus control unlesioned rats.
lesion. Localized infusions of 192 IgG saporin into the NBM have been found to speci¢cally and discretely lesion the NBM^cortical cholinergic pathway, while minimizing non-speci¢c tissue damage to adjacent forebrain nuclei (McGaughy et al., 2000); it has further been shown that immunotoxin lesions do not lead to changes in other neurotransmitter systems (Pizzo et al., 1999). In this context, the fact is of importance that the NBM represents a collection of cholinergic and non-cholinergic (e.g. GABA, peptides) neurons (Heimer, 2000). Since the cholinergic projection from the NBM to the cortex is excitatory, while the GABAergic projection is inhibitory, results on sensorimotor gating behavior following a selective 192 IgG saporin-induced lesion of the NBM are likely to be di¡erent from those of non-selective excitotoxic lesions. Moreover, whereas immunotoxininduced lesions largely spare the NBM projection to the amygdala, quinolinic acid has been found to be a more potent neurotoxin of cholinergic neurons innervating the amygdala than those projecting to the cortex (Boegman et al., 1992). The concentration of immunotoxin used in our study has been shown to produce only minor di¡usion into the adjacent diagonal band of Broca and a minimal depletion of hippocampal ChAT activity (Pizzo et al., 1999). The authors of the previous study described extension of the primary lesions into parts of the globus pallidus, the substantia innominata and the bed nucleus of the stria terminalis. Therefore, it is also important to consider the possibility that damage of adjacent forebrain nuclei, in addition to the destruction of non-cholinergic neurons in the NBM, produces adaptive changes in multiple neuronal systems, which ultimately might suppress sensorimotor gating de¢cits. This viewpoint is indirectly supported by a study which showed signi¢cant reduction of PPI after acute administration of cocaine while no e¡ects on PPI were observed after sustained cocaine exposure, which is thought to produce neurochemical changes in several brain regions (Martinez et al., 1999). Together, these observations and considerations suggest that the de¢cits in PPI produced by NBM infusions of 192 IgG saporin are linked to selective and extensive reduction of cholinergic cortical activity. In addition to the extension of the lesion, di¡erences in the parameters used for measurements of startle gating might also account for the discrepancy with the previous report. Schauz and Koch (1999) used 5- and 15-dB prepulses over a background intensity of 55 dB, while we used a more sensitive threshold range of PPI produced by prepulse intensities of 3, 6 and 12 dB above a higher background (70 dB). Previous studies have shown that
de¢cits in PPI are most sensitively detected using weak prepulse stimuli rather than intense prepulse stimuli, thereby avoiding £oor and ceiling e¡ects (Swerdlow and Geyer, 1993). In particular, the suggestion that weak prepulses might be most able to detect de¢cits in PPI is supported by the observation that PPI elicited by weak prepulses is most sensitive to disruption by low doses of apomorphine that do not disrupt PPI that is produced by more intense prepulse stimuli (Davis et al., 1990). This observation merits further investigation and is in accordance with our results suggesting that PPI de¢cits in states of gating circuit dysfunctions might be most sensitively detected in conditions in which the system can exhibit the greatest changes. Possible implications for the understanding of schizophrenia Psychotic symptoms represent frequent neuropsychiatric concomitants of the cognitive dysfunction observed in common forms of dementias, where the loss of cortical cholinergic projections from the NBM is known to be one of the most striking neurochemical alterations (Whitehouse et al., 1982). Given the speci¢c damage to the corticopetal cholinergic projections in the immunotoxin lesion model, our ¢ndings indicate that the NBM may modulate sensorimotor gating via e¡erent projections to the cortical substrates of the startle gating circuitry. It has been shown that the selective loss of corticopetal cholinergic projections is su¤cient to produce impairments in tasks designed to assess various aspects of attentional functions (Chiba et al., 1995, 1999; Bucci et al., 1998; Sarter and Bruno, 1999; McGaughy et al., 2000), including object recognition (Bartolini et al., 1986), a form of non-spatial memory which depends on the activity of prefrontal cortical circuitry (Funahashi et al., 1989). Accordingly, selective immunotoxic lesions of NBM cholinergic neurons impair prefrontal neuronal activity associated with sustained
Table 2. E¡ect of bilateral NBM injection of 192 IgG saporin on startle amplitude (arbitrary units) Time after treatment (weeks)
Saline-injected rats
192 IgG saporin-injected rats
Two Four Six
96.8 þ 38.6 87.9 þ 19.3 81.1 þ 17.6
88.7 þ 26.3 106.6 þ 35.6 149.4 þ 49.8
Mean þ S.E.M. values are shown. Statistical analysis according to one-way ANOVA. No signi¢cant e¡ect was present.
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M. Ballmaier et al.
visual attentional performance (Gill et al., 2000). A role of NBM cholinergic neurons in processing of information has been further emphasized by the ¢nding that pairing NBM stimulation with sensory stimuli results in a global reorganization of sensory cortical maps (Kilgard and Merzenich, 1998). Since the limbic cortex represents a well-characterized substrate of the startle gating circuitry, one might speculate that PPI disruption observed after immunotoxic lesion of NBM principally derives from the loss of the cholinergic modulation of the prefrontal cortex by the NBM; this hypothesis is supported by the observation that infusions into the NBM of 192 IgG saporin show the highest ChAT depletion in the frontal cortex (Pizzo et al., 1999). Prefrontal or limbic cortical dysfunction has long been implicated in the pathophysiology of schizophrenia (Weinberger et al., 1992; Grace, 2000). Furthermore, there is evidence that the limbic involvement is also essential for the psychotic behavior in Alzheimer's disease, with neurochemical alterations a¡ecting the dopaminergic/cholinergic axis likely being central to the clinical manifestations of both disorders (White and Cummings, 1996). Much evidence from PPI studies has demonstrated the importance of abnormal dopamine regulation in the circuitry connecting the limbic cortical regions to the basal forebrain (Swerdlow and Geyer, 1998). In addition, direct manipulations of both glutamatergic and serotonergic activity in the forebrain are also known to a¡ect PPI in rats (Swerdlow and Geyer, 1998; Geyer and Swerdlow, 1999). Therefore, a possible explanation for reduced PPI in the present study may involve direct or indirect interactions of the NBM^cortical cholinergic pathway with other neurotransmitter systems known to a¡ect PPI along the startle gating circuitry characterized by neural connections between limbic cortical areas and subcortical dopaminergic systems, and ultimately the pontine system via GABAergic subpallidal e¡erents to
the pedunculopontine nucleus. Since the NBM also receives projections from cortical areas which are mainly concerned with the limbic system (Wainer and Mesulam, 1990), it is conceivable that these reciprocal connections with limbic cortical targets may account for the modulatory e¡ect of the NBM on PPI. Another possible mechanism by which the NBM may regulate PPI is via indirect connections to the pedunculopontine nucleus through the thalamic reticular nucleus. Since the projection from the NBM to the thalamic reticular nucleus has been proposed to provide an alternative pathway for cortical arousal and to play a role in the thalamic ¢ltering of sensory input (Steriade et al., 1987; Steriade and Buzsaki, 1990; Tourtellotte et al., 1990), one might speculate that PPI de¢cits after NBM lesions may also re£ect changes in the activity of thalamic nuclei impinging on the startle gating circuit at the level of the pedunculopontine nucleus.
CONCLUSIONS
The results presented in this work indicate that selective and discrete lesioning of the NBM leads to a substantial impairment of PPI which persists over repeated testing. Overall, our data suggest that the NBM appears to be ideally located within the basal forebrain for modulating sensorimotor gating abilities that may correlate with important determinants of information processing in schizophrenia. Insights drawn from our results may also enrich the understanding of NBM corticopetal projections within the context of psychotic hallmarks shared by schizophrenia and dementias.
AcknowledgementsöThis work has been supported by grants from the University of Brescia and MURST (PRIN to G.P.). We thank Carla Scali and Rodolfo Mazzoncini for technical assistance.
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