Dendritic complexity is reduced in neurons of nucleus accumbens after social isolation

Ue-Cheung Ho1,2, Yu-Chun Wang1,2, Chun-Chieh Liao2 and Li-Jen Lee2

1Department of Medicine, National Taiwan University, Taiwan2Department 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 dendrites 4.69 ± 0.29 4.94 ± 0.31Bifurcating nodes 24.38 ± 2.29 17.02 ± 1.89 *Terminal endings 29.08 ± 2.26 22.01 ± 1.94 *Highest order 6.69 ± 0.43 6.11 ± 0.33Segment number 53.23 ± 5.32 39.03 ± 3.82Total dendritic length (mm) 1839.77 ± 141.54 1407.58 ± 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.