Differential Neuronal Changes in Medial Prefrontal Cortex, Basolateral Amygdala and Nucleus Accumbens after Postweaning Social Isolation Ue-Cheung Ho 1*, Yu-Chun Wang 1, Meng-Ching Ko 2, Chun-Chieh Liao 2, Li-Jen Lee 1,2,3# 1 Department of Medicine, National Taiwan University, Taipei, Taiwan, 2 Graduate Institute of Anatomy and Cell Biology, National Taiwan University, Taipei, Taiwan, 3 Neurobiology and Cognitive Science Center, National Taiwan University, Taipei, Taiwan Materials and Methods Subjects Male Wistar rats of postnatal day (P) 21 were reared either individually or as groups (3 to 4) per cage for 8 weeks. Behavioral tests Individual rats were placed in an open field arena (41 X 37 X 26 cm in length X width X height) and videotaped for 15 min to access their explorative activities. Prepulse inhibition (PPI) was used to measure the startle response. A PPI session consisted of a 20-ms burst of acoustic prepulse (77 or 86 dB) prior to the startle pulse of 115 dB. Measurement of dopamine levels Some brains from group- and isolation-reared rats were used for the measurement of basal dopamine level in the brain using high performance liquid chromatography (HPLC). In brief, tissues from the mPFC, Amy, NAc and VTA were collected and homogenized. After sonication in 0.1 N perchloric acid, the homogenates were centrifuged (14000 rpm at 4 o C for 10 min). The supernatants were used for monoamine analyses through HPLC with an electrochemical detector (Waters, Milford, MA, USA). The pellets were neutralized in 0.1 N NaOH and dissolved in RIPA buffer for protein assay. Total protein was quantified by Bradford method using a protein assay kit (Bio-Rad, Hercules, CA, USA). Finally, dopamine levels were expressed as pg/μg protein. Golgi-Cox impregnation Golgi-Cox method was used to visualize the morphology of neurons in mPFC, Amy and NAc. The rat brains were placed in Golgi-Cox solution at room temperature for 14 days. After impregnation, specimens were cut at thickness of 200 μm with a vibratome. Reconstruction and quantitative measurement of neurons were performed using Neurolucida. Immunohistochemistry Brain tissues obtained from rats were fixed with 4% paraformaldehyde in 0.1M phosphate buffer saline (PBS, pH 7.4) for one day. Coronal sections of ventral tegmental area were cut at thickness of 50 um with a vibratome. Briefly, the sections were placed in 3% hydrogen peroxidase (H 2 O 2 ) for 5 min followed by PBS wash. Non specific immune-reactivity was blocked by normal goat serum (NGS) and bovine serum albumin (BSA) solution containing 0.5% Triton-X100 for 30 minutes. The sections were then incubated overnight at 4 o C with the primary antibody (mouse anti-tyrosine hydroxylase), which was diluted to 1:2000 in PBS. After washed in PBS, the sections were incubated with the second antibody (biotinylated goat anti- mouse, diluted to 1:500 in PBS) for 2 hour. The sections were then incubated with avidin-biotin complex for another 1 hour at room temperature. Immunoreactivity of biotin-peroxidase was then visualized by 3,3-diaminobenzedine tetrahydrochloride and 3% H 2 O 2 in PBS. Cell counting We performed the Stereo Investigator software package (MicroBrightField Inc; Williston, USA) and used the optical fractionators sampling to count the tyrosine hydroxylase positive (TH + ) neurons in VTA of whole brain. Six sections from 1:6 series were analyzed for each brain. By using X10 objective, we traced around the area of VTA according to the visible TH + cell body and atlas of rat brain. In order to determinate the suitable counting frame and sampling grid dimensions, coefficient of error (CE) of each section was controlled below 0.2. Counting frame size was 120μm X 120μm, and sampling grid size was 240μm X 240μm. Finally, X20 objective was used to count the TH + neuron which had >10μm diameter and the obvious nucleus. The estimated CE value range of each brain was 0.08 to 0.06, and the CE mean of total brain was There were no significant difference between the social group (n = 4) and isolated group (n = 7) in estimated population (p = 0.36), volume (p = 0.40) and density (p = 0.51) by using student t test. Figure 1. Increased locomotor activity and impaired prepulse inhibition in social isolation-reared rats. Results Figure 5. Layer II/III pyramidal neurons in the medial prefrontal cortex (mPFC). Summary and conclusion 1.Hyperlocomotor activity and impaired prepulse inhibition were observed in isolation-reared rats. 2.Reduced dopamine level in the BLA of the isolates was found but there was no significant difference in the dopamine level of mPFC and NAc between two groups. 3.In isolated rats, the medium spiny NAc neurons had fewer bifurcating nodes, segments and terminals as wells as lesser total dendritic length. However, the length of both internodal and terminal segments was comparable in the two groups. Reduced dendritic arborization was also found in the layer II/III pyramidal neurons of mPFC whereas dendritic complexity of spiny pyramidal neurons in the BLA was increased. 4.Medium spiny neurons in the NAc are GABAergic inhibitory neurons, reduced dendritic arborization in these neurons suggested altered neuronal excitability which might affect the GABAergic neurotransmission in the limbic system of isolated rats. Besides, exuberant dendrites in the BLA neurons of isolates might reflect enhanced amygdalar output while reduced dendritic arborization in mPFC neurons might be linked to decreased cortical output. These changes may account for the behavioral abnormalities in the schizophrenic animal model. Figure 6. Quantitative study of dendritic segments in the medial prefrontal cortex (mPFC). Abstract In patients of schizophrenia, the mesolimbic circuit is affected and it might account for many symptoms such as sensory-motor gating deficit and hyperactivity. Post-weaning isolation rearing of rodents is a promising model of schizophrenia which displays many schizophrenia- related behavioral changes. However, the structural and functional alterations in the mesolimbic system in this model are not fully characterized. We thus checked the dopamine level in the medial prefrontal cortex (mPFC), basolateral amygdala (BLA) and nucleus accumbens (NAc) and the density of dopaminergic neurons in the ventral tegmental area (VTA) in grouped and isolated rats. We found reduced dopamine level in the BLA of the isolates but no change in the density of VTA dopaminergic neurons. Next, we examined the morphology of the layer II/III pyramidal neurons in the mPFC, spiny pyramidal neurons in the BLA and medium spiny neurons in the NAc. For the mPFC neurons, mild decrease in the segment number and increase in segment length were observed in both apical and basilar dendrites in isolated animals. In the BLA neurons, we found increased branching nodes, dendritic segment and terminal tips in the isolates. For the NAc neurons, the soma size was comparable in both socially and individually reared rats; however, the number of bifurcating nodes and terminals as wells as the total dendritic length were reduced in isolated animals. Together, we found altered dopamine level and neuronal morphology in the mesolimbic system of socially isolated rats, these changes may account for the behavioral abnormalities in this schizophrenic animal model. Figure 2. Dopaminergic neuron in the ventral tegmental area. Figure 7. Increased dendritic branches in BLA neurons of socially deprived rats. Figure 8. Morphometric features of neurons in the nucleus accumbens (NAc). Figure 4. Dopamine level in the medial prefrontal cortex (mPFC), basolateral amygdala (BLA) and nucleus accumbens (NAc). Figure 3. Quantitative study of dopaminergic neuron in the ventral tegmental area.