Effects of genetic background on neonatal Borna disease virus infection-induced neurodevelopmental damage

Brain Research 944 (2002) 97–107 www.elsevier.com / locate / bres

Research report

Effects of genetic background on neonatal Borna disease virus infection-induced neurodevelopmental damage I. Brain pathology and behavioral deficits Mikhail V. Pletnikov a,b , *, Steven A. Rubin b , Michael W. Vogel d , Timothy H. Moran a , Kathryn M. Carbone a,b,c a

Psychiatry and Behavioral Sciences, The Johns Hopkins University School of Medicine, Ross 618, 720 Rutland Avenue, Baltimore, MD 21205, USA Laboratory of Pediatric and Respiratory Viral Diseases, CBER, FDA, Bldg. 29 A, Rm. 1 A-21, HFM-460, 8800 Rockville Pike, Bethesda, MD 20892, USA c Department of Medicine, The Johns Hopkins University School of Medicine, Ross 618, 720 Rutland Avenue, Baltimore, MD 21205, USA d Laboratory of Developmental Neurobiology, Room B-22, Maryland Psychiatric Research Center, University of Maryland, Baltimore, MD 21228, USA

b

Accepted 14 March 2002

Abstract The pathogenic mechanisms of gene–environment interactions determining variability of human neurodevelopmental disorders remain unclear. In the two consecutive papers, we used the neonatal Borna disease virus (BDV) infection rat model of neurodevelopmental damage to evaluate brain pathology, monoamine alterations, behavioral deficits, and responses to pharmacological treatments in two inbred rat strains, Lewis and Fisher344. The first paper reports that despite comparable virus replication and distribution in the brain of both rat strains, neonatal BDV infection produced significantly greater thinning of the neocortex in BDV-infected Fisher344 rats compared to BDV-infected Lewis rats, while no strain-related differences were found in BDV-induced granule cell loss in the dentate gyrus of the hippocampus and cerebellar hypoplasia. Unlike BDV-infected Lewis rats, more severe BDV-induced brain pathology in Fisher344 rats was associated with (1) greater locomotor activity to novelty and (2) impairment of habituation and prepulse inhibition of the acoustic startle response. The present data demonstrate that the same environmental insult can produce differential neuroanatomical and behavioral abnormalities in genetically different inbred rat strains.  2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Developmental disorders Keywords: Borna; Hippocampus; Cerebellum; Startle; Stereotypy; Hyperactivity

1. Introduction A number of neurodevelopmental disorders have been associated with early brain damage following exposure to unfavorable environmental factors [23,39,58]. Since studying the continuum of pathogenic events of developmental disorders in human subjects is difficult, the pathophysiol*Corresponding author. Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Ross 618, Baltimore, MD 21205, USA. Tel.: 11-410-955-2996; fax: 11-410-614-0013. E-mail address: [email protected] (M.V. Pletnikov).

ogy of neurodevelopmental damage remains poorly understood. As a result, treatments to prevent neurodevelopmental damage are lacking or partial [5,59]. Thus, animal models are often used to stimulate studying pathogenic processes and associated behavioral deficits and to identify novel therapeutic regimens [39,58,63]. Current animal models of abnormal brain and behavior development are primarily based on genetic, lesioning and neurotoxic manipulations [4,35]. However, there are relatively few animal models using viruses as teratogens [2,9,17,21,22,51] despite the fact that pre- and postnatal virus infections induce significant developmental CNS injury and behavioral deficits [16,23,32,42,58].

0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )02723-3

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We have developed and characterized a unique model of neurodevelopmental damage using Borna disease virus (BDV) [13], a 8.9-kb nonsegmented, negative-strand, enveloped RNA virus [8,18] as the experimental teratogen. In neonatal Lewis rats, intracranial inoculation with BDV produces a persistent brain infection without global encephalitis [29,44]. Neonatally BDV-infected Lewis rats appear grossly normal, however, they exhibit distinct behavioral deficits similar to some core features of autism spectrum disorders [23,39,50]. For example, locomotor hyperreactivity to novel / aversive stimuli [6,19,30,46], deficient spatial learning and memory [53], and abnormal social (e.g. play) interaction [47] are all manifestations in BDV-infected rats. The observed behavioral alterations may be explained by BDV-induced selective developmental damage to the neocortex, hippocampus and cerebellum [7,20,25,30,61], the regions that have been implicated in autism pathology by a number of neuropathological and MRI studies [23,39,58]. Additionally, BDV-induced neurochemical alterations in the serotonin and norepinephrine brain systems [48] also appear similar to neurochemical alterations observed in neurodevelopmental disorders (e.g. hyperserotoninemia in autism [58,59] and may contribute to the specificity of behavioral deficits exhibited by neonatally BDV-infected rats. In the present study, we used the BDV rat model to address another important aspect of neurodevelopmental damage, the gene–environment interaction that determines disease outcome and differential responses to treatment. Although there is a major genetic contribution to most neurodevelopmental disorders, genetic differences alone do not appear to explain the great variability in clinical conditions, suggesting that an environmental component may also play a role in shaping the pathophysiology of many neurobehavioral diseases. Similarly, this hypothetical model also suggests that exposure to the same environmental factor can result in differential disease outcomes in subjects with different genetic backgrounds [38,45]. In this context, studying abnormal brain and behavior development in genetically different animals (e.g. different rodent strains) can provide more insights into the pathogenic mechanisms of the genetic background–environment interplay. It is well recognized that BDV-induced disease varies by animal species [44,55]. Thus, we sought to characterize neuroanatomical, neurochemical and behavioral BDV-associated abnormalities in two inbred rat strains, Lewis and Fisher344 characterized by differential physiological [24,56], neurochemical [10,36], and behavioral features [15,37] that might play a role in mediating variable disease outcomes in the two rat strains. The present study reports that despite comparable virus replication and brain distribution, weight gain inhibition in two rat strains and similar damage to the dentate gyrus of the hippocampus and cerebellum, neonatal BDV infection produced greater thinning of the neocortex in Fisher344 rats compared to Lewis rats. The differential virus-induced brain pathology

might explain the observed greater novelty-induced hyperactivity and impaired habituation and prepulse inhibition of the acoustic startle response in BDV-infected Fisher344 rats compared to BDV-infected Lewis rats.

2. Materials and methods

2.1. Animals Pregnant Lewis and Fisher344 rats (16–18 days of gestation) were used in the present study (Harlan, Indianapolis, IN, USA). All rat pups were born and reared in the animal vivarium at Johns Hopkins University School of Medicine (Baltimore, MD, USA) and in the animal facility at the Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA) (Bethesda, MD, USA). Both animal facilities have similar conditions for animal housing allowing us to generate consistent experimental data [49]. Following weaning, rats were kept in groups of two to three in 45326323 cm pan-type polypropylene cages with paper-chip bedding and an overhead wire grid supporting food pellets and a water bottle. Cages containing infected animals were kept in a Duo-Flou biosafety cabinet (Bio-Clean Lab. Product, NJ, USA). The sham-inoculated rats were kept in the same room. Rats were maintained on a 10 / 14-h light–dark cycle (lights on at 8 a.m.) and had free access to food and water. Room temperature was maintained at approximately 21 8C. In order to provide direct comparisons with our previous experiments performed on male 4-month-old Lewis rats neonatally infected with BDV [6,46], male Lewis and Fisher344 rats were tested at PND 120, except for the virus titration experiment.

2.2. Inoculation BDV stock was prepared from homogenized BDV-infected rat brain tissue as described earlier [11,12]. Pups were inoculated intracranially via 26-G needle intracranially within 24 h of birth either with 0.02 ml (the titer was 10 4 TICD 50 / g of brain tissue) of CRP3 (He-80) BDV strain (BDV-infected rats) or uninfected inoculum (control rats) [6,12]. The site of the inoculation was the parietal area of the skull, approximately 2 mm from the midline and the midway between the eye and ear. For intracranial inoculation, a pup was taken out of the home cage and placed on warm cloth. An injection of the inoculum was made quickly to minimize pain and stress. Immediately after the injection, a pup was returned to the home cage.

2.3. Infectivity assays BDV-infected Lewis (n54) and Fisher344 (n54) rats were randomly selected at PND 7, 14, 30 and 120 from different litters (one rat per litter), deeply anesthetized with

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Metofane (Pitman-Moore, Mundelein, IL, USA) and sacrificed. Infectivity assays were performed as described elsewhere and viral titer was determined by endpoint dilution [11,12].

2.4. Histopathological examination and anti-BDV immunohistochemistry Control (n56) and BDV-infected (n56) Lewis and control (n56) and BDV-infected (n56) Fisher344 rats were randomly preselected from the pool of rats used in behavioral experiments described in this study. Upon completion of behavioral tests, rats were deeply anesthetized with Metofane (Pitman-Moore, Mundelein, IL, USA) and perfused with phosphate buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde. Brains were removed and postfixed for 24 h, paraffin embedded and cut sagittaly into 8-mm-thick sections. Tissue sections were stained with hematoxylin and eosin for histopathological evaluation. Adjacent sections were stained by avidin– biotin immunohistochemistry (Vector, Burlingame, CA, USA) using a polyclonal rabbit anti-BDV antibody followed by biotinylated anti-rabbit IgG (Vector) as described previously [12].

2.5. Stereology-based count of neurons in the dentate gyrus of the hippocampus Control (n53) and BDV-infected (n53) Lewis and control (n53) and BDV-infected (n53) Fisher344 rats were randomly preselected from the pool of rats used in behavioral experiments described in this study and sacrificed as described above. The cerebrum was dissected, paraffin-embedded and cut sagittally into 25-mm thick sections. The numbers of neurons in the dentate gyrus (DG) of the hippocampus were determined in the right hemispheres. The fractionator sampling scheme and optical dissector were used to quantitatively measure the loss of granule cells in the DG in BDV-infected rats [62]. These measurements were performed using an Olympus microscope with a computer driven X,Y,Z-stage controller (ASI, Eugene, OR, USA) and stereology software (Stereologer, SPA, Alexandria, VA, USA). Fifteen sagittal sections through the hippocampus were selected using a systematic random sampling scheme (e.g. every 15th section with a random start beginning from the most lateral tissue section). Sections were stained with cresyl violet and viewed at a magnification of 463 and the entire DG outlined as the region of interest. Estimates of the numbers of neurons were made by counting neurons within a defined volume in each region using an optical dissector. The dimensions of the optical dissector for DG granule cells was: height 10.00 mm; guard height 5 mm, spacing 200 mm for NL and 100 mm for BDV-infected brains. The spacing of the optical dissectors was decreased for BDV-infected brains in order to increase the sampling frequency to keep the coefficient

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of error (CE) below 10%. To count neurons, an image of counting regions was displayed on the video monitor and a square counting box superimposed on the image using the stereology software STEREOLOGER (SPA Alexandria, VA, USA). Sections were viewed with a 1003 objective (final magnification on the computer screen: 22003). The depth of the box was measured with the z axis encoder attached to the focusing knob. Nomarski optics were used to optically section the tissue within the box. The nuclei of DG granule cells are darkly stained with cresyl violet, so the granule cell nucleus was used as the criterion for determining if a granule cell should be counted. A granule cell was counted if the top of its nuclei came into focus within the box or touched the top and the right edges of the square frame or the lower surface of the box. Nuclei that touched the bottom and the left edges of the box and the upper surface of the box were excluded.

2.6. Neocortex volume measurements The brain hemispheres used for counting neurons in DG were also utilized for measuring cortical volumes with the exception that more brain samples were analyzed for Fisher344 rats. The volume of the neocortex was estimated in control (n53) and BDV infected (n53) Lewis rats and control (n55) and BDV infected (n55) Fisher344 rats with Cavalieri point counting using the STEREOLOGY software Stereologer (SPA). The anterior border of the neocortex was defined by the rhinal fissure, the ventral border by the corpus callosum, and the posterior border by a line drawn between the caudal tip of the corpus callosum and the caudal edge of the cortex. Ten coronal sections from each brain were systematically selected from a random start for the volume estimates and the number of points touching the neocortex counted. Each point represented an area of 1.2 mm 2 . The average coeffecient of variance for volume estimates in both Lewis and Fisher rats was 0.05.

2.7. Behavioral experiments 2.7.1. Novelty-induced hyperactivity Novelty-induced hyperactivity was assessed in control (n58) and BDV-infected (n56) Lewis rats and control (n55) and Fisher344 (n55) rats in an open-field arena that consisted of a Plexiglas square box (60360350 cm) with transparent walls. The floor was divided into 36 sections of equal area by a series of solid lines forming squares. In order to sustain hyperactivity in BDV-infected rats, a 100 W white spotlight brightly illuminated the apparatus and rats were not habituated to the experimental box. After a rat was placed in the open field, behaviors were videotaped for 10 min and scored later for horizontal locomotor activity (number of squares crossed), rearing activity, and stereotypic behaviors. The 0–6 point stereotypy scale was used for measuring stereotypic behaviors in rats [43]: 05asleep or inactive; 15episodes of normal activities;

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25discontinuous activity with bursts of prominent sniffing or rearing; 35continuous stereotyped activity such as sniffing or rearing along a fixed path; 45stereotyped sniffing or rearing fixated in one location; 55stereotyped behavior with bursts of licking or gnawing; 65continuous licking or gnawing.

2.7.2. Acoustic startle response (ASR) Two identical startle chambers (SDI, San Diego, CA, USA) were used for measuring startle reactivity and plasticity. Each rat was placed in a Plexiglas cylinder (9 cm in diameter) within each chamber. A loudspeaker mounted 24 cm above the cylinder provided the broadband background noise and acoustic stimuli. Presentations of the acoustic stimuli were controlled by the SR-LAB software (SDI) and interface system, which also rectified, digitized, and recorded responses from the accelerometer. The maximum voltages within 50-ms reading windows, starting at stimulus onset, were used as the measures of startle amplitudes. Sound levels were measured inside the startle cabinets by means of the digital sound level meter (Realistic, Tandy, Fort Worth, TX, USA). The accelerometer sensitivities within each startle chamber were calibrated regularly and were found to remain constant over the test period. Control (n59) and BDV-infected (n59) Lewis rats and control (n59) and BDV-infected (n59) Fisher344 rats were used in the acoustic startle studies. The experimental session consisted of a 5-min acclimatization period to a 65-dB background noise (continuous throughout the session), followed by the presentation of ten 100-ms 108-dB white noise stimuli at a 20-s interstimulus interval (the habituation session). Since the previous studies have shown significant differences in body weights between BDV-infected and control rats [6,49], amplitudes of the ASR during the habituation session were analyzed and presented as the maximum value of the startle response in relation to the rat’s body weight. These weight-corrected ASRs were determined by dividing the ASR value by the weight in grams of the test subject. Upon the completion of the habituation session, each rat was tested in the prepulse inhibition (PPI) session. During each PPI session, a rat was exposed to ten types of trials: pulse-alone trial (a 108-dB, 100-ms, broadband burst); the omission of stimuli (no-stimulus trial); and four prepulse– pulse combinations (prepulse–pulse trials). A 50-ms broadband burst was used as a prepulse. Prepulse–pulse combinations included the prepulse intensity (10 dB above the background noise) and two prepulse-to-pulse intervals (40 and 80 ms). Each session consisted of eight pulsealone trials, six of each prepulse–pulse trials (combinations), and six no-stimulus trials. All trials were presented in pseudorandom order. In order to reduce a possibility of a ‘floor’ effect in BDV-infected rats, the PPI data were collected in the form of the direct readings from the computer without correc-

tions for weights and with the different sensitivities of the startle platform accelerometer for control rats (5 relative units) and BDV-infected rats (9 relative units). We did not use the highest sensitivity (i.e. 9) of the accelerometer for control rats since it would have limited magnitudes of their ASR. This approach allowed us to demonstrate PPI of the ASR in young and adult Lewis BDV-infected rats [49]. PPI was assessed as the percentage scores of PPI (%PPI): 1003(mean startle amplitude on pulse-alone trials2mean startle amplitude on prepulse-pulse trials / mean startle amplitude on pulse-alone trials) for each animal separately [33,60].The percentage of PPI for each animal was used the dependent variable in the statistical analysis.

2.8. Statistical analyses The data are presented as mean6S.E.M. A three-way ANOVA was used to compare the infectivity levels between two rat strains across the postnatal period, with strain, brain region and postnatal day as independent variables. Effects of BDV infection on weight gain were analyzed with a three-way ANOVA with strain, infection status and postnatal day as independent variables. A twoway ANOVA was used to analyze cell loss in the hippocampus, the volumes of the neocortex and the data from the novelty-induced hyperactivity experiment with strain and infection status as independent variables. A three-way ANOVA was used to analyze the data from the acoustic startle habituation experiment with strain, infection status and startle stimulus as independent variables. Three-way ANOVA was utilized to analyze the data from the prepulse inhibition experiment with strain, infection status and prepulse-to-pulse intervals as independent variables. Tukey’s tests for multiple comparisons were used when applicable. When the data did not pass the normality test and / or equal variance test, the data were subjected to the rank transformation, and ANOVAs were rerun on the transformed data. A P,0.05 was considered as the criterion for statistical significance.

3. Results

3.1. Infectivity assays and body weights Virus replication increased similarly in brain parenchyma in BDV-infected Fisher344 and Lewis rats throughout the postnatal period, with the virus titers in the cerebrum being significantly greater than in the cerebellum for both rat strains, P,0.05 (Table 1). The immunohistochemical distributions of BDV proteins in Lewis and Fisher344 rats were similar with a strong tropism for BDV to neurons in the neocortex, the DG and CA fields of the hippocampus, and PCs of the cerebellum (data not shown). Neonatal BDV infection produced similar inhibition of

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Table 1 Genetic background effects on developmental time course of BDV infectivity in the cerebrum and cerebellum of Lewis and Fisher344 rats Strain

Lewis Fisher344

PND 7

PND 14

PND 30

PND 120

CRB

CRBL

CRB

CRBL

CRB

CRBL

CRB

CRBL

4.860.3 5.260.3

4.360.3 4.560.3

5.760.2 5.860.2

5.060.2 4.760.2

6.060.3 5.760.3

5.060.3 4.860.3

6.460.3 6.260.3

5.760.3 5.660.3

The data are presented as log 10 of tissue culture infectious doses (TCID) 50 / g. Abbreviations: CRB, cerebrum; CRBL, cerebellum.

body weight gain in both rat strains as confirmed by significant effects of infection status, F(1,64)5176.0, P, 0.001 and postnatal day, F(3,64)5815.5, P,0.001. At PND 7, 14, 30 and 120, body weights (g) were: 13.861.1; 22.661.2; 95.863.8; 340627 for control Lewis rats; 12.161.1; 18.461.2; 74.663.4; 212625 for BDV-infected Lewis rats; 13.261.1; 22.661.2; 94.064.6; 324622 for control Fisher344 rats; 12.061.1; 17.361.2; 65.864.6; 205623 for BDV-infected Fisher344 rats.

3.2. BDV-induced neuroanatomical damage As reported previously for neonatally BDV-infected Lewis rats [12,29,44,64], Fisher344 rats showed no clinical or pathological signs of brain inflammatory responses upon examination at PND 120, while neonatal BDV infection produced profound neuroanatomical abnormalities in both Lewis and Fisher344 rats.

3.2.1. Neocortex Neonatal BDV infection differentially affected the cortical development in Lewis and Fisher344 rats. The volumes

of the neocortex in control Lewis and Fisher344 rats were 93.766.2 and 121.164.8 mm 3 , respectively. Neonatal BDV infection produced thinning of the neocortex in both rat strains. The volumes of the neocortex in BDV-infected Lewis and Fisher344 rats were 69.266.2 and 63.264.8 mm 3 , respectively. An analysis of the data for volume of the neocortex revealed no effect of strain, F(1,12)53.68, P50.079, whereas effects of infection status, F(1,12)5 54.7, P,0.001, and the strain by infection status interaction, F(1,12)58.9, P50.011, were significant. Tukey tests showed significantly greater volumes of the neocortex in control Fisher344 rats compared to control Lewis rats, P,0.05, and no significant strain differences in neocortex volumes between BDV-infected rats, indicating that thinning of the neocortex in BDV-infected Fisher344 rats was greater than in BDV-infected Lewis rats.

3.2.2. Hippocampus A quantitative analysis of numbers of neurons in DG of the hippocampus of both strains of BDV-infected rats showed a profound but similar cell loss at PND 120 (Fig. 1). When counted in the hippocampus of right hemisphere,

Fig. 1. BDV-induced loss of neurons in the dentate gyrus of the hippocampus of Lewis and Fisher344 rats at PND 120. Representative images of the hippocampus from control (A) and BDV-infected (B) Fisher344, and control (C) and BDV-infected (D) Lewis rats. Arrows point to the degenerated dentate gyrus in BDV-infected rats. Scale bar, 150 mm.

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the numbers of neurons in DG in control Fisher344 and Lewis rats were 850 000626 000 and 773 000670 000, respectively. The number of neurons in DG in BDVinfected Fisher344 and Lewis rats were significantly decreased by 88.561.4 and 84.165.2%, respectively, P, 0.05, compared to uninfected rats of their respected strains. The average coefficient of error (CE) for granule cell counts in normal and BDV-infected rats was 0.09 and 0.12, respectively.

3.2.3. Cerebellum Comparable profound hypoplasia of the cerebellum and dropout of Purkinje cells (PCs) were found upon qualitative examination of sagittal sections from BDV-infected Lewis and Fisher344 rats at PND 120. Specifically, BDVassociated cerebellar pathology was characterized by thinning of the molecular and internal granule cell layers in two rat strains, and numerous gaps within the Purkinje cell layer, presumably due to missing PCs (Fig. 2). 3.3. BDV-induced behavioral abnormalities 3.3.1. Hyperactivity and stereotypic behaviors BDV-infected Lewis rats showed increased novelty-induced hyperactivity thought to be related to hyper-reactivity to novel and stressful stimulation [6,19]. Thus, we evaluated the effects of genetic background on BDVinduced behavioral deficits using the novelty-induced hyperactivity paradigm. Neonatal BDV infection significantly increased noveltyinduced activity in both rat strains. Compared to their

Table 2 Hyperactivity and stereotypic behaviors in neonatally BDV-infected Lewis and F344 rats Strain

Infection status a

Crossovers (number)

Rearing (number)

Stereotypy (arbitrary units)

Lewis

NL BDV

130.9644.5 566.8651.2*

29.866.9 68.367.9*

0.9560.1 2.6660.1*

Fisher344

NL BDV

80.6656.3 737.6656.3*

27.268.7 86.868.7*

0.9160.1 2.5760.1*

*, P,0.05 vs. NL rats in the same strain group. a NL, control rats; BDV, BDV-infected rats.

respective controls, both BDV-infected Lewis and BDVinfected Fisher344 rats exhibited significantly greater locomotor hyperactivity, both horizontal, F(1,20)5121.1, P,0.001, and vertical, F(1,20)579.0, P,0.001, with BDV-infected Fisher344 rats being somewhat more hyperactive than BDV-infected Lewis rats, P50.094 and P50.072, for horizontal and vertical activities, respectively (Table 2). Similarly, compared to their control groups, BDV-infected rats of both strains demonstrated significantly more stereotypic behaviors, F(1,23)529.5, P, 0.001 (Table 2).

3.3.2. Startle reactivity, habituation, and prepulse inhibition The hippocampus and cerebellum have been implicated in the regulation of habituation and prepulse inhibition (PPI) of the acoustic startle response (ASR) in rats [33]. Moreover, both habituation and PPI of the ASR have been

Fig. 2. BDV-induced loss of the Purkinje cells in the cerebellum of Lewis and Fisher344 rats at PND 120. Representative images of the Purkinje cells layers from control (A) and BDV-infected (B) Fisher344, and control (C) and BDV-infected (D) Lewis rats. Note the layer of the Purkinje cells in control rats (arrowheads, A; C), and rare Purkinje cells in BDV-infected rats (arrows, B; D). Scale bar, 30 mm.

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shown to be affected differentially either by a pharmacological or a developmental manipulation in different rat strains [57,60]. Thus, we sought to analyze effects of genetic background on BDV-associated behavioral deficits using the ASR paradigm. Fig. 3A shows the ASR habituation data as weightadjusted mean startle amplitudes that were registered using the same sensitivity of the accelerometer of the startle chamber. There were no differences in baseline startle responsiveness or habituation of the ASR between control rats of two strains, P.0.05. Neonatal BDV infection was associated with a remarkable decrease in the mean magnitude of the ASR in both rat strains, F(1,280)5475.2, P,0.001. Separate two-way ANOVAs with repeated measures revealed no differences in startle responsiveness and habituation between strains. In contrast, there was a

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significantly greater rate of the within-session habituation of the ASR in BDV-infected Lewis rats compared to BDV-infected Fisher344 rats, P50.032. Neonatal BDV infection differentially impaired PPI of the ASR in Lewis and Fisher344 rats (Fig. 3B). The analysis of the data on PPI of the ASR showed significant effects of strain, F(1,64)58.9, P,0.001, infection status, F(1,64)512.9, P,0.001, prepulse-to-pulse interval, F(1,64)56.8, P50.011, and the strain by infection status interaction, F(1,64)514.6, P,0.001. There were no strainrelated differences in PPI of the ASR between control rats, P.0.05. In contrast, neonatal BDV infection significantly impaired PPI of the ASR in Fisher344 rats and did not affect PPI in Lewis rats. When comparisons were made within each strain, control Fisher344 rats exhibited significantly more PPI of the ASR compared to BDV-infected Fisher344 rats, P,0.05. No difference in PPI of the ASR was found between control and BDV-infected Lewis rats, P.0.05. The mean amplitudes (i.e. the direct readings) of the ASR during pulse-alone trials were 961.06611.6 for control Lewis rats, 178.2628.0 for BDV-infected Lewis rats, 698.54642.1 for control Fisher344 rats and 121.5626.0 for BDV-infected Fisher344 rats.

4. Discussion

Fig. 3. Effects of genetic background on habituation (A) and prepulse inhibition (B) of the acoustic startle response in control (NL) and BDV-infected (BV) Lewis (LEW) and Fisher344 (F344) rats. *, P,0.05 vs. control Fisher344 rats (F344-NL).

We studied the effects of genetic background on neurodevelopmental damage following neonatal BDV infection. Our findings revealed both strain-independent and strainrelated BDV-associated neuroanatomical and behavioral abnormalities in inbred Lewis and Fisher344 rats. There were no effects of genetic background on the virus replication and distribution in the brain as well as no strain effects on BDV-induced inhibition of weight gain, suggesting that the observed strain-related differences in neurobehavioral abnormalities were unlikely to be associated with general physical disability or variation in viral replication. The present results are in line with previous data demonstrating discordance between the levels of virus replication and the severity of neurobehavioral abnormalities. For example, when Lewis and black hooded rats were experimentally inoculated with the virus as adults, disease expression was dramatically different despite comparable virus replication and distribution in the brain. Specifically, while Lewis rats develop T-cell-mediated meningitis and encephalitis leading to severe neurological disease, black hooded rats show minimal, if any, T-cell response to the virus and no neurobehavioral abnormalities [28,29,44]. Thus, the present data demonstrate that host factors can influence the expression of a disease in different species and / or in different strains of the same species despite similar virus replication and distribution in the brain, refuting the notion that the main determinant of clinical picture is high viral load [3,28,32].

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4.1. Effects of genetic background on BDV-induced neuropathology Neonatal BDV infection produces selective developmental damage to the brain regions that continue to develop after birth, i.e. the hippocampus, cerebellum and cortex [7,12,20,25,30,61]. The observation of BDV-associated thinning of the neocortex, degeneration of the DG of the hippocampus and hypoplasia of the cerebellum were noted in both Fisher344 and Lewis rats at PND 120 confirmed and extended the previous findings. Several groups have demonstrated that neonatal BDV infection induces thinning of the neocortex [25,61]. Gonzalez-Dunia et al. have reported cortical shrinkage (by about 30%) and loss of neurons with diameter bigger than 100 mm in 45-day-old neonatally BDV-infected Lewis rats [25]. This observation is consistent with a report of apoptosis of pyramidal neurons in the neocortex of BDVinfected Lewis rats [30]. Here we confirmed those results and, for the first time, show that neonatal BDV infection produced significantly greater damage to the neocortex in Fisher344 rats compared to Lewis rats. It is not clear whether the differential cortical pathology in two rat strains are due to a greater loss of cells or more extensive virus-associated reduction in neuropil density in Fisher344 rats compared to Lewis rats [25]. Continuing degeneration of the DG is a unique neuropathological feature of neonatal BDV infection [12,52,64]. When evaluated by conventional histopathology (e.g. hematoxylin and eosin staining), a reduction in the number of DG neurons was noted as early as the end of the third postnatal week. By the end of the second month of life, the DG is virtually replaced by reactive glial cells [12,30,64]. Among replacing glial cells, microglial cells are thought to prevail over astrocytes [52,64]. In Lewis rats, neurons of the DG have been proposed to die via apoptosis since first TUNEL-positive neurons were found at 3 weeks of life, reaching the peak at 4 weeks, and gradually decreasing in numbers thereafter [61,64]. Our study provided the first quantitative results of neuronal loss in the DG of the hippocampus following BDV infection at PND120. A tremendous loss of granule cells in the DG of both rat strains might be associated with high vulnerability of proliferating neurons to environmental insults [1,26,34,40]. It remains unclear whether this extensive loss of granule cells in the DG is due to direct effects of BDV infection or due to indirect effects of alterations in the level of soluble factors such as cytokines or growth factors [27,41]. Notably, in some situations, such as BDV infection in mice, the infected DG neurons remain viable [54]. Thus, it is possible that the degeneration seen here is independent of direct BDV infection of the DG cells. The possibility of direct BDV-induced damage to hippocampal pyramidal neurons was suggested by prior qualitative data demonstrating that neurons in the CA1-4

regions of the hippocampus showed clear, though less dramatic, loss of cell density [12,52]. However, whether or not neonatal BDV infection causes cell loss in the CA1-4 subfields of the hippocampus remains to be explored using a stereology-based analysis since conventional histopathological analysis is not able to discriminate changes in cell density due to an actual cell loss from a decrease in cell and / or neuropil size. One could hypothesize that compared to Lewis rats, Fisher344 rats might show greater BDVinduced cell loss in the CA subfields of the hippocampus. For example, in an ischemic model, pyramidal neurons of Fisher344 rats have been shown to be more vulnerable than those in other rat strains [31], however, whether strain-related effects of ischemia can be also observed in BDV infection remains to be established. Developmental damage to the cerebellum is another distinctive neuropathological feature of neonatal BDV infection [7,12,20,61]. We described that the cerebella of BDV-infected Lewis rats develop normally until the end of the first postnatal week [7]. By 14 days of life, the cerebella of BDV-infected rats showed evidence of arrested development, stunted size, decreased foliation, and thinned and irregular internal granule cell and molecular layers. By the end of the third postnatal week, the cerebellum in BDV-infected rats is significantly smaller than in control rats, with a profound size reduction in the cerebellar molecular layer and / or internal granule cell layer [7]. BDV infects PCs by day 7 postinfection (p.i.), but does not appear to infect cerebellar granule cells [7]. Although PCs initially survive the BDV infection, many BDV-infected PCs are gradually lost between 27 and 75 days p.i. [7,20,64]. Our results demonstrated that neonatal BDV infection appeared to produce comparable damage to the cerebellum in both rat strains as assessed by qualitative review of sagittal brain sections. Both Lewis and Fisher344 rats showed pronounced hypoplasia of the cerebellum and a significant loss of PCs at PND120. Although it is possible that PCs die by apoptosis, it has not yet been unequivocally demonstrated [30,64].

4.2. Effects of genetic background on BDV-induced behavioral deficits The observed BDV-induced neuropathology in Fisher344 and Lewis rats could underlie observed behavioral deficits, e.g. locomotor hyperactivity and stereotypy, since hippocampal and cerebellar lesions following treatments with more conventional teratogens (e.g. X-irradiation, toxins, malnutrition) also produce an enhanced horizontal and vertical activity in rats [46]. Similar to altered emotionality observed in rats with lesioned hippocampi and / or cerebellum [46], our previous findings suggest that locomotor hyperactivity in BDV-infected Lewis rats may be due to exaggerated locomotor responses to aversive / novel stimulation rather than a general increase in spontaneous activity [46].

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Our data confirmed and extended the previous observations about BDV-induced stereotypic behaviors in Lewis rats. Similar to hyperactivity, stereotypy is a distinct behavioral feature of neonatal and adult BDV infection in Lewis rats [30,44]. Interestingly, compared to neonatally BDV-infected rats, stereotypic movements in adult infected rats appear to be more pronounced and expressed in greater variety [44]. This could be related to the more severe brain pathology produced by adult infection. The present study demonstrates more pronounced behavioral disturbances in BDV-infected Fisher344 rats compared to BDV-infected Lewis rats. For example, greater BDV-induced locomotor hyper-reactivity to novelty was observed in Fisher344 rats compared to Lewis rats. This finding is consistent with the previous report that neonatal lesions to the ventral part of the hippocampus enhanced novelty- and amphetamine-induced locomotion in Fisher344 rats but not in Lewis rats [37], indicating genetic variation in behavioral outcomes of early brain injury. We also found that neonatal BDV infection impaired habituation and PPI of the ASR in Fisher344 rats and did not affect those behaviors in Lewis rats. These data are similar to those for other environmental challenges, e.g. isolation rearing and amphetamine administrations, that produced deficits of PPI in Fisher344 rats but had no effect in Lewis rats [57,60]. Greater BDV-induced thinning of the neocortex in Fisher344 rats might partially contribute to the strainspecific behavioral deficits. A more extensive cell loss in the neocortex of Fisher344 rats might lead to greater disinhibition of the dopamine mesocorticolimbic system [14] activation of which has been shown to enhance locomotor activity and impair PPI and habituation of the ASR in rats [37,60]. The second report that follows the present paper explores putative BDV-induced alterations in the monoamine systems in the two rat strains in an attempt to explain strain-related behavioral deficits. Even if the appropriate methodological adjustments were made to ensure reliability of PPI of the ASR in BDV-infected rats, it still cannot be completely ruled out that a ‘floor’ effect due to low startle amplitude might have confounded the PPI performance in BDV-infected Fisher344 rats. Nonetheless, in spite of the fact that neonatal BDV infection produced low startle responses in both rat strains, BDV-associated impairment of PPI of the ASR was noted in Fisher344 rats only, whereas BDVinfected Lewis rats demonstrated PPI comparable with control animals. In addition, our previous data show that compared to control rats some BDV-infected Lewis rats exhibited a slightly greater PPI when tested at younger age [49]. Thus, the present findings demonstrated strain-specific impairment of PPI of the ASR following neonatal BDV infection, suggesting that the virus infection as an experimental teratogen is capable of producing neurobehavioral alterations similar to more conventional manipulations, e.g. drugs and lesions.

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In conclusion, despite comparable virus replication and distribution and weight gain inhibition, neonatal BDV infection produced differential brain pathology and behavioral deficits in two inbred rat strains, Lewis and Fisher344. Whereas there were no overt strain-related differences in hippocampal and cerebellar pathology, neonatal BDV infection produced greater thinning of the neocortex in Fisher344 rats compared to Lewis rats. This differential neuroanatomical abnormality might be responsible for the observed greater locomotor response to novelty and impaired habituation and prepulse inhibition of the acoustic startle in Fisher344 rats.

Acknowledgements The work was supported by the NIH grant 2R7O1 MH 48948-08A1. The authors wish to thank Michelle Jones (MPRC, University of Maryland) for the technical assistance in neuron counting and behavioral experiments.

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