1. Dendritic complexity is reduced in neurons of nucleus accumbens after social isolation Ue-Cheung Ho 1,2, Yu-Chun Wang 1,2, Chun-Chieh Liao 2 and Li-Jen Lee 2 1 Department of Medicine, National Taiwan University, Taiwan 2 Department of Anatomy and Cell Biology, National Taiwan University, Taipei, Taiwan Abstract In patients of schizophrenia, the mesolimbic circuit is affected and is accounted for many symptoms, such as sensory-motor gating deficit. Post- weaning isolation rearing of rodents is a promising animal model of schizophrenia which also displays sensory-motor gating problem expressed as reduced prepulse inhibition. Nucleus accumbens plays important role in integrating and gating information in the mesolimbic system. However, the structural and functional changes of the neurons within in this schizophrenic animal model are not fully understood. Therefore, we aimed to examine the neuronal morphology in nucleus accumbens in socially isolated rats. Male wistar rats were reared in isolation or in group of 3-4 after weaning. After 8 weeks of isolation, these rats displayed longer travel distance and sign of anxiety in an open field and reduced prepulse inhibition in startle response, but no significant change in the body weight was noted. We then examined the morphology of medium spiny neurons in nucleus accumbens by using Golgi-Cox impregnation method. 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. Moreover, the dendritic complexity revealed by the concentric- ring method of Sholl was significantly reduced particularly in the proximal region of the accumbal neurons. The number of dendritic segment was also decreased after social isolation; whereas the length of each segment was unchanged. The reduced dendritic arborization in medium spiny neurons of nucleus accumbens suggested altered GABAergic neurotransmission in the limbic system of isolated rats. These changes may account for the behavioral abnormalities in this schizophrenic animal model. 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 mins 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. Golgi-Cox impregnation Golgi-Cox method was used to visualize the morphology of medium spiny neurons in nucleus accumbens. 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 software. Statistical analyses All data were presented as mean ± SEM and were compared among groups by using two- tailed unpaired student ’ s t-test and univariate analysis of variance. Asterisks were used to indicate significant differences (*, p<0.05; **, p<0.01; ***, p<0.001) Figure 1. Increased locomotor activity and impaired prepulse inhibition in social isolation-reared rats. (A) Locomotor activities of rats were examined in an open field for 15 minutes. (B) Prepulse inhibition was examined using a brief (20 ms) acoustic prepulse (77 or 86 dB) 100 ms prior to the startle pulse of 115 dB. Results are mean ± SEM. Asterisks indicate significant differences between group-reared (grouped, n = 8) and isolation-reared (isolated, n = 8) rats (** p < 0.01). Figure 2. Medium spiny neurons in the nucleus accumbens (NAc). (A) Sketches of NAc in the coronal sections of a rat brain. The core region of NAc (NAcc) is marked gray. Numbers show distance from Bregma according to the atlas of Paxinos and Watson (1998). (B) Examples of Golgi-Cox impregnated medium spiny cells obtained from the NAcc of socially reared (grouped) and isolated rats. Spines are omitted in this illustration. Bar is 100 mm in B. Figure 3. Dendritic complexity of NAcc medium spiny neurons as estimated by the Sholl methods. (A) Number of intersections between the dendrites and concentric rings. Similarly, the numbers of bifurcating nodes (B) and terminal endings (C) were counted in relation to the distance from the soma. The neurons from isolated rats (n = 15 neurons) had less complicated dendritic arbors than that in the grouped rats (n = 14 neurons). Results are mean ± SEM. Asterisks indicate significant difference (* p < 0.05). Parameters Grouped (n = 14 neurons) Isolated (n = 18 neurons) Primary dendrites4.69±0.294.94±0.31 Bifurcating nodes24.38±2.2917.02±1.89 * Terminal endings29.08±2.2622.01±1.94 * Highest order6.69±0.436.11±0.33 Segment number53.23±5.3239.03±3.82 Total dendritic length (mm)1839.77±141.541407.58±114.31 * Table 1. Morphometric analyses of dendrites of NAcc medium spiny neurons Results are mean ± SEM. Asterisks indicate significant difference (*p < 0.05). Figure 4. Quantitative study of dendritic segments. (A) The number of segments was quantified by dendritic order. Dendritic segments were further divided in to internodal and terminal segments and the length from the first order to the 10th order was then quantified by dendritic order (B) and expressed as averaged values (C). Results are mean ± SEM. Asterisks indicate significant difference (* p < 0.05). Figure 5. Spine density and length of NAcc neurons. (A) The density of dendritic spine in the medium spiny neurons was estimated in different dendritic orders. No significant difference between grouped and isolated rats was observed in any dendritic order. (B) Dendritic spines were classified as branched (B), mushroom (M), stubby (S) and thin (T) types, and the length of each spine was measured. Shorter M-, S-, and T- type spines in the NAcc neurons of isolated rats were observed. Results are mean ± SEM. Asterisks indicate significant difference (*** p < 0.001). Summary and conclusion 1.Hyperlocomotor activity and impaired prepulse inhibition were observed in isolation-reared rats. 2.In isolated rats, the medium spiny NAcc neurons had fewer bifurcating nodes, segments and terminals as wells as lesser total dendritic length. 3.However, the length of both internodal and terminal segments was comparable in the two groups. 4.No significant difference in the spine density was found between grouped and isolated rats in any dendritic order of NAcc neurons. 5.The length of dendritic spine in the isolated animals was reduced especially in the mushroom-, stubby- and thin- types. 6.Medium spiny neurons in the NAcc are GABAergic inhibitory neurons, reduced dendritic arborization and spine length in these neurons suggested altered neuronal excitability which might affect the GABAergic neurotransmission in the limbic system of isolated rats. These changes may account for the behavioral abnormalities in this schizophrenic animal model.