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SCHRES-06042; No of Pages 8 Schizophrenia Research xxx (2014) xxx–xxx

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Histological correlates of N40 auditory evoked potentials in adult rats after neonatal ventral hippocampal lesion: animal model of schizophrenia A.L. Romero-Pimentel a,b, R.A. Vázquez-Roque b, I. Camacho-Abrego b, K.L. Hoffman a, P. Linares b, G. Flores b,⁎, E. Manjarrez b,⁎ a b

Centro de Investigación en Reproducción Animal (CIRA), Universidad Autónoma de Tlaxcala-CINVESTAV, Tlaxcala, CP 90070, México Instituto de Fisiología, Benemérita Universidad Autónoma de Puebla, 14 Sur 6301, Col. San Manuel, Puebla, Puebla, CP 72570, México

a r t i c l e

i n f o

Article history: Received 9 July 2014 Received in revised form 28 August 2014 Accepted 4 September 2014 Available online xxxx Keywords: Auditory Evoked Potentials N40 wave neonatal ventral hippocampus lesion schizophrenia

a b s t r a c t The neonatal ventral hippocampal lesion (NVHL) is an established neurodevelopmental rat model of schizophrenia. Rats with NVHL exhibit several behavioral, molecular and physiological abnormalities that are similar to those found in schizophrenics. Schizophrenia is a severe psychiatric illness characterized by profound disturbances of mental functions including neurophysiological deficits in brain information processing. These deficits can be assessed by auditory evoked potentials (AEPs), where schizophrenics exhibit abnormalities in amplitude, duration and latency of such AEPs. The aim of the present study was to compare the density of cells in the temporal cerebral cortex and the N40-AEP of adult NVHL rats versus adult sham rats. We found that rats with NVHL exhibit significant lower amplitude of the N40-AEP and a significant lower number of cells in bilateral regions of the temporal cerebral cortex compared to sham rats. Because the AEP recordings were obtained from anesthetized rats, we suggest that NVHL leads to inappropriate innervation in thalamic-cortical pathways in the adult rat, leading to altered function of cortical networks involved in processing of primary auditory information. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The neonatal ventral hippocampal lesion (NVHL) is an established rat neurodevelopmental model of schizophrenia. Rats with NVHL exhibit several behavioral, molecular, and physiological abnormalities which are similar to schizophrenia (for review see Tseng et al., 2009). The NVHL model exhibits reduction in the number of GABAergic neurons in many brain areas (Endo et al., 2007; Straub et al., 2007; Francois et al., 2009), deficits in GABAergic neurotransmission (Endo et al., 2007 and Francois et al., 2009), sensitivity to NMDA antagonists (AlAmin et al., 2000 and Tseng et al., 2007) and a reduction in the number of neurons that integrate some of the part of the limbic system (Vázquez-Roque et al., 2012). Schizophrenia is a devastating mental illness that affects 1% of the world population. It is characterized by brain abnormalities and profound disturbances of mental functions (Harrison and Weinberger, 2005). Patients with schizophrenia show severe neurophysiological deficits in brain information processing, not only at cognitive levels (Goldberg and Gold, 1995) but also at perceptual and

⁎ Corresponding authors at: Instituto de Fisiología, Benemérita Universidad Autónoma de Puebla, 14 sur 6301, Col. San Manuel A.P. 406, C.P. 72570, Puebla, Pue., México. Tel.: +52 22 22 29 5500x7326; fax: +52 22 22 33 4511. E-mail addresses: gonzalo.fl[email protected] (G. Flores), [email protected], [email protected] (E. Manjarrez).

sensory levels (Braff et al., 1991; Branner et al., 2009). Recently, we reported (Valdés-Cruz et al., 2012) that NVHL resulted in a decrease in the absolute power of the parietal and occipital electroencephalographic recordings (at 1–8 Hz, 9–14 Hz, and 15–30 Hz bands). Moreover, the NVHL rats also showed a reduction in exploratory behavior. The N40 waveform of the auditory evoked potential (AEP) in the rat was first described by Knight et al. (1985). The rat N40-AEP is a negative component that is recorded from the cortex 40 to 60 msec following auditory stimulation (Adler et al., 1986; Boutros et al., 1997). The origin of the N40 is unknown but one possible source of this waveform has been localized in the CA3 region of the hippocampus (Bickford-Wimer et al., 1990). This region receives glutamatergic input from the entorhinal cortex and cholinergic input from the medial septum (Insausti et al., 1997; Cenquizca and Swanson, 2007). The rat N40-AEP has mainly been employed for studying mechanisms of sensory gating (De rojas et al., 2013; Okamoto et al., 2012; Chen et al., 2012; Swerdlow et al., 2012; Breier et al., 2010; Vohs et al., 2009; Zhou et al., 2008; Keedy et al., 2007; Hashimoto et al., 2005; Zheng et al., 2005; Siegel et al., 2005; Miyazato et al., 1999; Boutros et al., 1997; Boutros and Kwan1998; Johnson et al., 1998: Stevens et al., 1998; Flach et al., 1996; Shinba et al., 1996; Bickford and Wear, 1995; Campbell et al., 1995; Bickford and Wear, 1995; Luntz-Leybman et al., 1992). Sensory gating can be defined as the ability of the brain to attenuate incoming irrelevant sensory stimuli (Freedman et al., 1987). Schizophrenic patients

http://dx.doi.org/10.1016/j.schres.2014.09.009 0920-9964/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: Romero-Pimentel, A.L., et al., Histological correlates of N40 auditory evoked potentials in adult rats after neonatal ventral hippocampal lesion: animal model of..., Schizophr. Res. (2014), http://dx.doi.org/10.1016/j.schres.2014.09.009

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A.L. Romero-Pimentel et al. / Schizophrenia Research xxx (2014) xxx–xxx

exhibit abnormalities in several characteristics of the AEP sensorial gating (For review see Patterson et al., 2008; Brockhaus-Dumke et al., 2008; Myles-Worsley et al., 2004). These alterations in sensory gating in schizophrenics are similar to those reported in rats with NVHL (Vohs et al., 2009; Swerdlow et al., 2012). However, in such studies the averaged amplitude of the N40 auditory evoked potential has not been employed to examine the integrity of networks underlying the processing of sensory information in NVHL rats. The above-mentioned studies suggest that the NVHL could disrupt the development and function of the circuitry involved in auditory sensorial processing. In this context, the purpose of the present study was to examine auditory processing abnormalities in NVHL rats, by characterizing N40-AEPs and the histological analysis of the temporal cerebral cortex. Specifically, we hypothesized that the N40-AEP in NVHL-rats exhibit lower amplitude compared to the N40-AEP in sham rats, since a consistent characteristic of schizophrenics is the reduced amplitude in the auditory evoked potential (see Fig. 1 from Rosburg et al., 2008). Furthermore, we hypothesized that NVHL rats exhibit a significant reduction in the density of cells located in the temporal cerebral cortex compared with sham rats.

2. Materials and methods 2.1. Animals We performed experiments in rats in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC), the

guidelines contained in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (85–23, revised in 1985) and the “Norma Oficial Mexicana NOM-062-ZOO-1999”. Moreover, the Institute of Physiology from the Benemerita Universidad Autonoma de Puebla, Mexico, approved the present study. Pregnant Sprague–Dawley rats were obtained at gestational Days 14–17 from our animal facilities. The rats were individually housed in a temperature and humidity controlled environment on a 12:12 h light–dark cycle with free access to food and water. We strictly followed the same animal care procedures as in our previous studies (Valdés-Cruz et al., 2012; Vázquez-Roque et al., 2012).

2.2. Neonatal ventral hippocampal lesion (NVHL) At postnatal day (PND) 7, male pups were randomly assigned to either sham or lesioned group. They were hypothermically anesthetized and positioned on a modified platform (Sierra et al., 2009) fixed on a stereotaxic apparatus (Narishige, Scientist Instrument Lab. SN-2 N). For the neonatal ventral hippocampal lesion (NVHL) an incision on the skin overlaying the skull was made, and two 1 mm holes were drilled. A needle connected to an infusion pump through a Hamilton syringe was lowered into each ventral hippocampus coordinate: AP3.0 mm, ML ± 3.5 relative to bregma and VD − 4.9 relative to dura. Ibotenic acid (0.3 μL, 10 μg/μL; Sigma, St Louis, MO) in 0.15 M phosphate buffer saline (PBS) pH 7.4 or vehicle was infused bilaterally at a flow rate of 0.15 μL/min. Pups were monitored and warmed before being returned to their cages. On PD21, the animals were weaned and a

Fig. 1. Schematic drawing of coronal sections illustrating area of the NVHL lesion as determined by Nissl-stained sections of the hippocampus of animals at a postpubertal age. Black, reconstruction of the neuronal loss and gliosis in the hippocampus of the rat with the most widespread lesion. Numbers indicate distance (mm) posterior from bregma according to Paxinos and Watson (1986).



similar number of sham and lesioned rats were placed in cages (four animals per cage). During the NVHL we strictly followed the same care procedures as in our previous studies (Flores et al., 2005; Valdés-Cruz et al., 2012; Vázquez-Roque et al., 2012). 2.3. Recording of Auditory Evoked Potentials 2.3.1. Animal preparation At PD 120–140, sham (n = 4) and lesioned rats (n = 6) that weighed between 350 and 400 g, were anesthetized with a mixture of isoflurane (20 %) and oxygen (80 %). The trachea was cannulated in order to administer the anesthesia directly and to allow artificial ventilation, if needed. The right jugular vein was cannulated in order to administer Atropine (0.05 mg/kg) and Dexamethasone (2 mg/kg). Isoflurane was changed to alpha-cholaroza/borax (60 mg/Kg/1 ml) through the jugular vein after this was cannulated. The rats were placed in a stereotaxic apparatus. The skullcap of the rats was removed between lambda and bregma cranial suture. The meninges were removed and 12 electrodes were placed on the surface of the exposed cerebral cortex. Auditory stimuli were delivered binaurally by means of a STIM2 system from Neuroscan. Two plastic tubes included in the STIM2-earphones were gently introduced in the rat eardrum. The stimuli comprised a click with duration of one millisecond and 1Hz of frequency presentation. It was emitted at magnitudes of 70, 80, 90, 100, 110, 115 and 120 dB. For each magnitude the click was presented 64 times, therefore, in total 448 stimuli were presented per rat. 2.3.2. Recordings and analysis The AEPs were recorded using a Neuroscan Synamps 2 EEG amplifier system from an array of 12 silver-silver chloride Ag/AgCl surface electrodes (200 micrometers of diameter). The reference was an Ag/AgCl electrode inserted into the surrounding muscle tissue. This array of electrodes was designed in our laboratory and adapted to the synamps 2 amplifier according to our previous reports (Manjarrez et al., 2005; Cuellar et al., 2009). All signals were digitalized without filtering in DC mode with a sample rate of 10 kHz, 24-bit A/D conversion and a DC500 Hz band-pass filter. Further, all signals were off-line filtered from 500 Hz to 5 kHz with the same Neuroscan software. Processing included segmentation (0–150 ms before/after the stimuli were presented), baseline correction, artifact rejection and averaging. The N40-AEP, P80-AEP and N120-AEP components in the averaged signals were identified (see magenta arrows in Fig. 3B). We observed a maximal negative peak around 50 ms (i.e., a N40-AEP), which was determined by identifying the most negative deflection between 40 and 60 ms. The amplitude of these AEP components were measured as indicated by the vertical green line in Fig. 3B. Latencies were measured from the time of the stimulus onset to the peak of the maximal AEP negative peak as indicated by the horizontal green line in Fig. 3B. In order to perform a statistical analysis, the electrode located over the temporal hemisphere (which exhibited the maximal amplitude) was selected from the electrode array; subsequently from this unique electrode the averaging of the AEP was obtained. Since the data followed non-normal distribution and to compare latency and N40-AEP component among groups we used the Mann–Whitney U test. 2.3.3. Stereological analysis We performed a stereological analysis of the brains of a subgroup of the same rats employed in the AEP studies. After the AEP experiment, 5 of the 6 NVHL rats and all 4 sham rats were deeply anesthetized with sodium pentobarbital (75 mg/kg ip) and perfused through the heart with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer. Forty-micrometer-thick coronal sections from the primary and secondary auditory temporal cortex were obtained using a vibratome (model 2000; Leica). For stereological analysis of neuronal populations, briefly, sections were mounted on glass slides and stained with cresyl violet and were analyzed with the optical dissector as previously described (Chana et al.,

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2003; Kuczenski et al., 2007; Vázquez-Roque et al., 2012). Cells were sampled within a volume in the primary and secondary auditory temporal cortex by optical sectioning, at a 10 μm distance, within the vibratome section, using an Olympus BH2 microscope with a digital color camera attached to a DataCell computer-assisted image analysis system (Stereo Investigator System; MicroBrightField, Williston, VT) for stereology. From each case, at least five random sections within a given area of about 400 μm were analyzed, and results were averaged and expressed as total number of cells per cubic millimeter. 3. Results 3.1. Verification of the lesion All brains were examined under light microscopy for location of the ibotenic acid lesion. Cresyl violet-stained sections obtained from adult NVHL animals revealed significant bilateral damage of the ventral hippocampus, with neuronal loss, atrophy, and apparent retraction of the hippocampal formation. The brains of the sham animals did not show any morphological alterations (Fig. 1). 3.2. Auditory Evoked Potential The amplitude of the N40-AEP component increased for both groups with the stimulation intensity of 80, 90, 95, 100, 110, 115, 120 dB, as shown in Fig. 2. Fig. 2 illustrates sigmoid curves obtained from averaging of the N40-AEP amplitude (for each auditory intensity we averaged 64 samples) for four sham rats and six NVHL rats. This phenomena shows a Sigmoid distribution (2 parameters sigmoid adjustment test, R N 0.90, p b 0.001 for all the cases). Nevertheless, this intensity dependence was altered by NVHL rats across the stimulation range of 110, 115 and 120 dB. At this range, the N40-AEP component amplitude did not increase, while that of the sham rats showed a clear increase. NVHL rats showed a decrease of the N40-AEP component in most cases, as compared with the sham animal (Fig. 2, 3, Table 1). Fig. 3 shows averaged recordings of AEP for sham (red traces) and NVHL (blue traces) rats. Note that for 100 and 120 dB the NVHL rats exhibited N40-AEPs of lower amplitude than those evoked in sham rats. Mann Whitney U-test indicated that these differences where significant at 90, 95, 100, 110, 115 and 120 dB stimulation (P = b 0.05, for all cases). By contrast, the latency of the N40-AEP component potential did not differ between the two groups (Table 2). We include six tables with quantitative data for positive and negative responses across the AEP recorded (N40, P80 and N120). As

Fig. 2. Grand average of the N40 auditory evoked potential (AEP) amplitude versus the auditory stimulus intensity for sham (red circles) and NVHL (blues circles) rats. The N40 is an AEP component occurring 40 to 60 msec following auditory stimulation. The horizontal line below the asterisk indicates that all the mean amplitude values of the N40-AEP from 90 dB to 120 dB between NVHL and sham rats were statistically significant different (Mann Whitney U-test, *p b 0.05).


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A.L. Romero-Pimentel et al. / Schizophrenia Research xxx (2014) xxx–xxx Table 2 The median, maximums and minimums of the N40-AEP latency for sham and NHVL groups. Mann–Whitney U test statistics values are shown for sham group versus NVHL group latency differences. Stimulus Intensity (dB)

Sham

NVHL

Median (ms)

Max/min (ms)

Median (ms)

Max/min (ms)

80 90 95 100 110 115 120

40.10 40.73 40.17 39.85 39.80 39.77 39.47

40.50/0 40.50/38.90 40.60/38.70 40.70/38.50 40.50/38.00 41.20/37.00 40.50/38.00

38.56 37.77 38.98 38.90 37.30 37.60 38.16

40.90/0 42.50/0 41.40/34.80 41.30/34.50 30.40/39.30 40.50/31.90 40.20/32.40

U

P

8.5 7 7 9.5 4 5.5 8

N0.05 N0.05 N0.05 N0.05 N0.05 N0.05 N0.05

from NVHL rats and red circles from sham rats). We obtained a significant Pearson’s correlation coefficient of r = 0.67 with a p = 0.04, and 7 degrees of freedom. Fig. 5B shows the mean values of the N40-AEP amplitude and the corresponding mean number of cells in both groups of rats (NVHL: blue circle, and sham: red circle). 4. Discussion

We found that rats with NVHL exhibit a significant reduction in the number of cortical cells in bilateral regions of the temporal cerebral cortex compared to sham rats. Fig. 4 illustrates histological pictures of the temporal cerebral cortex in both sham and NVHL rats as indicated. Note the reduced number of cells in the image of the NVHL rats. A statistical analysis of the mean number of cells indicates a significant small number of cells for the NVHL rats compared to the sham rats (p b 0.001, Student’s t-test). In Fig. 5A we illustrate the mean amplitude of the N40-AEP elicited at 120 dB (i.e., the maximal N40-AEP amplitude recorded in 5 NVHL and 4 sham rats) versus the mean number of cells found in the same rats employed in the AEP study (blue circles represent values obtained

We found that adult rats with a NVHL exhibit a significant reduction both in the amplitude of the N40-AEP component as well as in the density of cells in the temporal cerebral cortex. These findings suggest a correlate between the decrease in the amplitude of the N40-AEP and the density of neurons in the temporal lobe of NVHL rats. Our electrophysiological findings parallel those from patients with schizophrenia, in which decreases in N100 amplitude have been reported (Turetsky et al., 2009; Light et al., 2012). These findings are compelling because there are intriguing reports in the literature about an anatomicphysiological correlate in schizophrenic patients in which a reduction in the amplitude of the P300 response is correlated with a reduction in the volume of the left posterior superior temporal gyrus (McCarley et al., 1993; 2002), the anterior medial temporal cortex (Kawasaki et al., 1997) and the anterior cingulate cortex (Preuss et al., 2010). However, there are studies that contradict these findings, showing that correlations between volumes of temporal lobe structures and left P300 amplitudes are low and not significant (Havermans et al., 1999; Meisenzahl et al., 2004). Our findings provide support to the studies by Light et al. (2012), Turetsky et al. (2009), McCarley et al. (1993, 2002)) Kawasaki et al., (1997) and Preuss et al., (2010). Another consistent electrophysiological abnormality in schizophrenic patients is the deficit in sensory gating as measured by the P50 conditioning-testing paradigm, in which two brief auditory stimuli (usually clicks) are presented in rapid succession, typically 500 ms apart. These paired stimuli are presented typically at 10-sec intervals. The amplitude of the P50 response to the second stimulus (S2) in normal subjects is typically attenuated compared to the response to the

Table 1 The median, maximums and minimums of the N40-AEP amplitude for sham and NHVL rats. Mann–Whitney U test statistics values are shown for sham group versus NVHL group amplitude differences. The asterisks indicate statistically significant differences between medians for sham versus NVHL rats.

Table 3 The median, maximums and minimums of the P80-AEP amplitude for sham and NHVL groups. Mann–Whitney U test statistics values are shown for sham group versus NVHL group amplitude differences.

Fig. 3. Grand average of N40-AEP for Sham (red traces) and NVHL (blues traces) rats obtained after 80, 100 and 120 dB stimuli. The arrow indicates the time of application of the auditory stimuli.

illustrated in the Tables 2 to 6 no significant differences were found in P80-AEP and N120-AEP amplitude and latency between the sham and NVHL rats. However, as showed in Table 1, we found significant differences in the N40-AEP amplitude between sham and NVHL rats (* p b 0.05). 3.3. Histological findings on the number of cortical cells in sham versus NVHL rats

Stimulus Intensity (dB)

Sham

NVHL

Median Max/min (μV) (μV)


80 90 95 100 110 115 120

11.74 24.92 28.39 30.96 34.80 40.96 42.44

5.53 9.31 14.27 18.52 23.22 23.17 22.99

16.28/0 29.05/22.80 24.50/39.59 39.59/24.50 40.50/32.48 45.12/38.09 40.14/46.00

U P

13.74/0 18.30/0 24.48/13.52 26.55/13.52 30.81/18.19 33.63/18.13 36.96/15.54

6 0 2 1 0 0 0

N0.05 b0.05* b0.05* b0.05* b0.05* b0.05* b0.05*

Stimulus Intensity (dB)

Sham

NVHL



80 90 95 100 110 115 120

0 3.54 11.99 11.61 12.88 14.72 16.33

2.06 5.34 6.57 8.74 7.39 4.46 8.50

13.08/0 19.92/0 20.45/5.21 22.57/3.39 21.42/9.14 32.20/10.71 35.22/11.00

U

12.16/0 10 12.97/0 11.5 14.18/0.79 6 16.68/7.23 11 11.28/4.07 3 12.30/2.38 2 11.91/7.64 1

P

N0.05 N0.05 N0.05 N0.05 N0.05 N0.05 N0.05


A.L. Romero-Pimentel et al. / Schizophrenia Research xxx (2014) xxx–xxx Table 4 The median, maximums and minimums of the P80-AEP latency for sham and NHVL group. Mann–Whitney U test statistics values are shown for sham group versus NVHL group latency differences. Stimulus Intensity (dB)

Sham

NVHL

Median Max/min (ms) (ms)


80 90 95 100 110 115 120

38.80 75.10 76.65 79.40 76.00 76.10 73.05

0.00 86.95 77.50 76.70 82.30 87.95 87.15

84.30/0 108.90/0 78.60/68.30 86.10/77.30 81.30/67.9 82.20/70.00 76.00/69.20

U

P

92.30/0 10 90.70/0 9.5 90.00/70.00 8 99.70/70.10 10 102.30/70.00 7 99.70/69.00 6 98.45/72.90 4

N0.05 N0.05 N0.05 N0.05 N0.05 N0.05 N0.05

first stimulus (S1), due to inhibitory effects associated with the response to S1. The magnitude of this inhibitory effect is referred to as P50 suppression, or P50 sensory gating, and is reduced in schizophrenic subjects. Independent groups have been able to replicate this basic P50 sensory gating deficit in schizophrenia. Similarly, deficits in sensory gating and reduced paired pulse inhibition (PPI) of startle have been described in NVHL rats (Vohs et al., 2009; Swerdlow et al., 2012). The present results further characterize auditory processing deficits in the context of the N40-AEP of NVHL rats and suggest a possible neuroanatomical correlate of these deficits: the reduced density of cells in the temporal cortex. Moreover, it could be plausible that a reduction in cells in the temporal cortex is associated with a reduction of inhibitory neurons at the cortical level and at the first stages of the sensory processing; which is consistent with recent studies by Macedo et al. (2010) who observed an enhancement of AEPs in the inferior colliculus of NVHL rats, and Chambers et al. (1996) and Swerdlow et al. (2001), who observed an associated gliosis and calcification in the lateral thalamus, thus suggesting that these NVHL rats have alterations in the mechanisms of inhibition in early phases of the acoustic processing. It is important to mention that the group sizes of NVHL (n = 5) and sham (n = 4) were determined according to the following practical, ethical and experimental considerations: 1) the care of these animals with a brain lesion is a demanding task, because the experimenter must take strict care of the animals since the day of the neonatal lesion to the adulthood, 2) the Mexican guidelines for the care and use of laboratory animals recommends to reduce the number of animals for experimentation, 3) all animals that were employed were obtained from different litters of rats; thus indicating that they represent a fair sample of this NVHL animal model. 4) the analysis of our experimental data with this sample size is sufficient to observe statistically significant differences (p b 0.001 and p b 0.05). In humans and rats the acoustic information processing begins in the cochlea and is sent to the cochlear nuclei and to the superior olivary complex, either on the same side or opposite side. Then, the information travels from the superior olivary complex, either on the same side or Table 5 The median, maximums and minimums of the P120-AEP amplitude for sham and NHVL group. Mann–Whitney U test statistics values are shown for sham group versus NVHL group amplitude differences. Stimulus Intensity (dB)

80 90 95 100 110 115 120

Sham

NVHL

Median (μV)

Max/min (μV)

Median (μV)

Max/min (μV)

3.707 1.189 4.150 4.075 2.75 5.97 6.30

7.80/0 2.40/0 10.57/0 5.57/1.98 14.12/2.28 6.27/1.31 10.55/3.42

0 1.869 5.367 6.69 9.00 8.69 9.13

2.33/0 8.45/0 11.43/1.43 15.87/3.27 10.72/5.00 15.82/2.17 16.20/3.31

U

P

8 7 9 7 8 6 9

N0.05 N0.05 N0.05 N0.05 N0.05 N0.05 N0.05

5

Table 6 The median, maximums and minimums of the P120-AEP latency for sham and NHVL group. Mann–Whitney U test statistics values are shown for Sham group versus NVHL group latency differences. Stimulus Intensity (dB)

Sham

NVHL



80 90 95 100 110 115 120

0 0 148.50 140.05 144.00 145.00 148.85

0 145.15 146.85 145.50 132.00 147.80 148.00

137.70/0 149.00/0 160.00/0 147.00/125.40 150.00/104.20 162.50/123.20 160.80/121.50

150.70/0 160.80/0 158.98/124.20 174.20/127.80 163.80/118.00 163.80/127.00 160.00/123.00

U

P

11 4.5 10.5 7 8 10 11

N0.05 N0.05 N0.05 N0.05 N0.05 N0.05 N0.05

opposite side, crossing in the trapezoid body and ascending on the other side. They form a tract called the lateral lemniscus, this carries the auditory information upward through the pons to the inferior colliculus of the midbrain. After this, the information travels to the medial geniculate nucleus of the thalamus. The auditory information finally is projected to the temporal area of the cerebral cortex (Hendelman, 2006). Furthermore, the ventral half of the CA1-hippocampus projects lightly to the primary auditory regions of the cerebral cortex (Cenquizca and Swanson, 2007). This auditory network of communication could be affected by NVHL. In histological studies, NVHL rats show a thin layer of calcium deposits close to the lesioned area, mainly within the auditory thalamus and the zona incerta, in addition to the ventral hippocampus. This abnormality affecting the auditory thalamic region had also been indicated by some other authors, described as a “mild gliosis” in lateral thalamic structures adjacent to the hippocampus (Chambers et al., 1996; Swerdlow et al., 2001; Macedo et al., 2010; Sandner et al., 2010). There are at least three possible causes for these histological findings. First, at the time of the lesion (7 days postnatal), and close to the injection site, a layer of migrating neurons very sensitive to ibotenate could be destroyed, and the resulting sclerotic layer could be calcified (Beas-Zárate et al., 2001). Second, the calcification could be the consequence of glial damage, because some microglial cells have high sensitivity to NMDA agonist during the neonatal period (Tahraoui et al., 2001). Finally, it could be consistent with a calcificated hematoma that could occur subsequently to the destruction of the endothelium of the anterior choroidal artery, which passes throughout the lesioned area (Scremin, 1995). Whatever the cause, these histopathological changes might lead to inappropriate innervation within the thalamic-cortical pathways. Destroying the ventral hippocampus could deprive some important hippocampal afferents as well. These areas could be atrophied because of the loss of a neurotrophic influence mediated by hippocampal afferent axons. Reports in the literature of a reduced number of neurons in the cortex of NVHL rats support this possibility (Halim and Swerdlow, 2000; Flores et al., 2005; Tseng et al., 2008 and Francois et al., 2009). A deficit in dendritic complexity after NVHL must also be considered (Chambers et al., 2010). This atrophy could be determined by the loss of the neurotrophic effect of glutamate, since the neuronal outputs of the ventral hippocampus are primarily glutamatergic (Bardgett and Henry, 1999). For example, a trophic influence has been documented for glutamate in the auditory system. Thus, glutamate that is released when the afferent fibers are active may be necessary to sustain the anatomical structure of the target brain areas (Hyson, 1997). In conclusion, the present results indicate that the NVHL rat model for schizophrenia replicates a robust electrophysiological endophenotype for schizophrenia, of decreased amplitude of the P50 and N100 auditory evoked potential (see Fig. 1 from Rosburg et al., 2008 and compare such figure with our Figs. 1 and 2). Moreover, this alteration in auditory evoked potentials in NVHL rats is associated with a significant decrease in cell number in the temporal cortex. The present results represent an important step in identifying


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Fig. 4. A) Histological pictures of sections of the auditory temporal cortex in sham versus NVHL rats as indicated. B) Comparison between the estimated numbers of cells in both hemispheres relative to the thickness in sham versus NVHL rats. C) The same as B, but for the right auditory temporal cortex. D) The same as B, but for the left auditory temporal cortex. Statistical analyses indicate significantly fewer cells for the NVHL rats compared to the sham rats (** p b 0.001, *p b 0.05).

possible neuroanatomical and neurophysiological bases for the subtle but significant deficits in the primary sensory processing of auditory stimuli in schizophrenia.

Linares P, Flores G, Manjarrez E carried out the experiments and analyzed data. RomeroPimentel AL, Hoffman KL, Flores G, Manjarrez E interpreted data and wrote the manuscript. All authors approved the manuscript.

Role of the funding source The electrophysiological devices and other instruments employed in the experiments were acquired with funds from the following grants: CONACYT F1-153583 (EM) and 138663 (GF) and Cátedra Marcos Moshinsky (EM). RPAL acknowledge fellowship support from CONACyT Mexico.

Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.

Author contributions Romero-Pimentel AL, Hoffman KL, Flores G and Manjarrez E, designed the experiment and methods. Romero-Pimentel AL, Vázquez-Roque RA, Camacho-Abrego I, Hoffman KL,

Acknowledgements This work was supported by the following grants: CONACyT under projects: F1153583 (EM) and 138663 (GF), VIEP-BUAP-00070, PIFI-2008-BUAP and Cátedra Marcos Moshinsky (EM), Mexico. RPAL acknowledge fellowship support from CONACyT Mexico.

Fig. 5. A) Mean amplitude of N40-AEP elicited by auditory stimuli at 120 dB versus the mean number of cells found in both hemispheres of the auditory temporal cortex in NVHL rats (blue circles) and sham rats (red circles). We found a statistically significant Pearson’s correlation coefficient of r = 0.67 with a p = 0.04, and 7 degrees of freedom (gray line). B) Mean values of the pooled data illustrated in panel (A); the blue circle is for NVHL rats and the red circle for sham rats, respectively. Statistical analyses indicate significantly fewer cells (* p b 0.001) and a decreased N40-AEP mean amplitude (* p b 0.05) for the NVHL rats compared to the sham rats.



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