JOURNHl OF Miiirnnv PHnnivmco-MeBiciNe N°7-8014
DIVERSITY OF MORPHOLOGY AND DISTRIBUTION OF INHIBITORY INTERNEURONS IN THE RAT
SUPERIOR COLLICULUS
Nguyen Van Ba*; Nguyen Nlinli Nui"
SUMMARY
The superior colllculus (SC) is a part of midbrain network that plays a crucial role in attention Inhibitory interneurons in the SC regulate the information streams that involve in sensory processing. Here we demonstrate that the population of Inhibitory Interneurons in the SC can be immunohistochemlcally classified into subgroups based on their expression of calcium-binding proteins such as parvaltjumln (PV) and calblndin (Calb). While PV-immunoreactrve neurons dlstnbutel all over the layers of the SC, Calb-lmmunoreactive neurons appeared only In superficial layer.
Furthennore, Calb-lmmunoreactive neurons in the superficial layer distnbuted In two separate sub layers, grey layer and optic layer. In which, neurons In grey layer were clearly oriented anil polanzed. The number of Calb-lmmunoreactive neurons In the superficial layer was higher than that of PV-immunoreactlve neurons. Furthermore, the size of Calb-lmmunoreactive neurons in the optic layer was bigger than those of grey layer and PV-immunoreactlve neurons in the superficial layer
'Keywords: Supenor colllculus; inhibitory Intemeurons: Parvalbumin: Calblndin.
INTRODUCTION [3]. PV-immunoreactive neurons are involved in the generation of gamma oscillations.
The superior colllculus is a multilayer which nsgulalB worMng memory and irrfomiati™
structure of midbrain networi< that plays a transmission between cortical areas [4].
crucial role in attention [1]. It also participate Nguyen et al [5] had reported that the the subcortical visual pathway to process number of PV- and Calb-immunoreactive fast and coarse information of salient stimuli neurons is decreased in the SC of rat [2]. Previous studies have suggested that the exposed to dioxin
role of inhibitory GABAergic interneurons, x h - „ , , »„ < .u. . . . . . . . . ' , „ , „ ' The purpose of the present study was including pan,album,n (PV) and calblndin to examine the neuroanatomical network (Calb)-,mmunoreactive neurons, is very in subpopulations of inhibitory GABAergic important ,n sensory processing and cognitive interneurons in the SC of adult rat We funrtions. Calcium-binding proteins, such investigated the morphology and distribution as PV and Calb, are commonly used as of PV- and Calb-immunoreactive neurons markers for classifying inhibitory intemeurons in different layers as well as the size and into subgroups in neuroanatomical studies density of these neurons
* Vietnam Military Medical University
"103 Hospital
Conesponding author Nguyen Van Ba ([email protected])
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JOURNRl OF MIUTflRV PHflRMflCO-MeDICINC N°7-20T4 MATERIALS AND METHODS
1. PV and Calb-immunohistochemistry.
* Animals and sample preparation:
In total, 15 adult rats (7 males and 8 females; age: 15 weeks) were subjects for immunohistochemistry. After deep anesthetization with intraperitoneal injection of sodium pentobarbital (50 mg/kg), rats were perfused transcardially with 0.9%
NaCI solution for 10 min, followed by 4%
paraformaldehyde in 0.1 M phosphate buffer (pH 7.2, 10°C) for 20 min.Then, the brains were removed from the skull and fixed in the 4% paraformaldehyde solution and cryoprotected in 20% sucrose solution.
Coronal sections of the SC (40-Mm thick) were cut on a freezing microtome, collected in 0.01 M phosphate-buffered saline (PBS), transferred into a cryoprotectant solution (25% ethylene glycol, 25% glycerin, and 50% 0.1 M PBS), and then stored at -20°C until immunohistochemical staining.
* Immunohistochemistry:
For all staining procedures, free-floating sections were rinsed 3 times in PBS between each incubation step. These sections were quenched for 10 min in 3%
HaO2/20% methanol in PBS and incubated in blocking solution, 3% normal horse serum in PBS-T (0.25% Triton X-100), for 30 min at room temperature. For the PV and Calb immunohistochemistry, the sections were incubated overnight at 4°C with mouse anti-PV monoclonal antibody (1:10,000, Sigma-Aldrich Co. LLC, St. Louis, MO, USA) or mouse antl-Calb monoclonal antibody (1:8000, Swant, Marly, Switzeriand) in 1% blocking solution. After rinsing, the sections were incubated for 1 h at room temperature with biotinylated secondary
antibodies (1:500, Vector Laboratories, Inc, Buriingame, CA, USA) and then incubated in avidin-biotin-peroxidase complex (ABC- Elite, Vector Laboratories, Inc.). The sections were stained with a detection solution (0.25 mg/mL of 3,3-diaminobenzidine and 0.03% H2O2 in PBS). The sections were then rinsed several times in PBS, dehydrated in graded concentrations of ethanol, cleared in xylene, and cover-slipped with entellan.
2. Analysis of the number and size of interneurons in the SC sections.
Images of the sections were obtained with all-in-one fluorescence microscope system (BZ-9000, Keyence Corporation, Osaka, Japan). All images of each section (PV or Calb) were captured at the same light intensity and magnification (optical lens, 10 x; objective lens, 10x). These digital images were analyzed vinth Image software
We counted the stained cells in the superficial layer of the SC at 3 AP levels (-5.80, -6.60, and -7.40 mm from the bregma) in each rat. The estimates of cell number in two dimensions might be erroneous and biased, which was partially overcome by using the Abercromble correction [6, 7], We also classified neurons into two types: small cells were those smaller than 150 pm', big cells were those bigger than 150 pm^
3. Statistical analysis.
The number and average size (area) of the neurons in each area Were calculated from the results obtained from both the left and right hemisphere in each section.
Comparison of number of the neurons in the immunohistochemical analyses was evaluated using two-way analysis of
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JOURNfll OF MIUTBBV PHBRMBCOMePICINC N°7-2014 variance (ANOVA) tests. Comparisons of the percentage of small and big neurons in each groups was evaluated by Chi-test.
All statistical analyses were perfomied
using the SPSS software package (ver, 19, IBM Corporation, Armonk, NY, USA).
Any differences were considered statistically significant with p < 0.05.
RESULTS
1. Distribution of the PV- and Calb-immunoreactive neurons in the SC.
Figure 1: (A) Distribution of PV-immunoreactive neurons was similar in all layers of the SC and did not show polarization and orientation. (B) The Calb-immunoreactive
neurons appeared mainly in superficial layer of the SC and very rare in intermediate and deep layers. Calb- immunoreactive neurons showed bipolar with axon oriented
to the surface of the SC.
There was diversity in the distribution of two kind of inhibitory interneurons. The distribution of PV-immunoreactive neurons was similar in all layers of the SC, but the Calb-immunoreactive neurons appeared mainly in the superficial layer of the SC and
JOURNfll OF miUTRRV PHflRMRCO-meDICINC N°7-2014 very rare in intermediate and deep layers (figure 1). Furthermore, Calb-immunoreactive neurons in the superficial layer distributed in two clear layers: grey layer with bipolar, oriented small neurons and optic layer with muftipolar, non-oriented big neurons. Therefore, we separated the SC into 2 parts, the superficial layer and the intermediate and deep layers, and analyzed the numbers and sizes of the PV- and Calb-immunoreactive neurons in the superficial layer.
2. Analysis of the PV- and Calb-immunoreactive neurons in the superficial layer.
600 .
*^ 500 JO V c 400 c 300 !
p = 0.000
» 200 E
-5.8 -6.6 -7.4 Distance from bregma
Figure 2: Comparisons of numbers of the PV- and Calb-immunoreactive neurons in the superficial layer of the SC. The number of Calb-immunoreactive neurons was significantly higher than that of PV-lmmunoreactive neurons with p < 0.001 (two ways ANOVA).
Figures 2 show comparisons of numbers of the PV- and Calb-immunoreactive neurons in the superficial layer of the SC. The number of Calb-immunoreactive neurons was significantly higher than that of PV-immunoreactive neurons; there were significant main effects of type: F (1,42) = 16.11, p = 0.000 (two ways ANOVA).
PV-neurons Calb-neurons Grey layer Optic layer
i Small cells (<150Mm=) cells (>150[jm')
Figure 3: Description of size of two type neurons in the superficial layer of the SC.
percentage of big cell (size s 150 pm') In the optic layer higher than those in grey layer and PV-neurons in superficial layer (Chi-test, p < 0.01).
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JOURNRl OF MIUTRRV PHBRMRCO-MeDICINC N°7-2014 The description of size of two type neurons in the superficial layer of the SC.
The percentage of small cells in the PV- neurons population was 81.3% and similar to that of Calb-neurons in the grey layer, but higher than that of Calb-neurons in the optic layer (Chi-test, p < 0.01).
OISCUSSION
The SC is a multiiayered, multimodal structure in the midbrain that integrates and process many resources of sensory input and functions as the first relay station of the subcortical visual pathway, which consists of the SC, pulvinar, and amygdala, to detect salient emotional and social stimuli in the environment [2]. PV- and Calb- immunoreactive neurons are putative inhibitory interneurons [8, 9] that regulate the information stream in the brain. The present study demonstrated that there was diversity in the number, morphology and distribution of two subgroups of inhibitory neurons based on their expression of Calcium-binding proteins, such as PV and Calb in the SC. The PV-immunoreactlve neurons located widespread all layers of the SC, non-oriented and non-polarized, but Calb-immunoreactive neurons appeared mainly in the superficial leyer with two sub layers: grey and optic layers, while they were very rare in intermediate and deep layers. The number of Calb-immunoreactive neurons in the superficial layer was higher than that of PV-immunoreactive neurons.
Furthermore, the size of Calb-immunoneactive neurons in the optic layer was bigger than those of grey layer and PV-lmmunoreactive
neurons in the superficial layer. Since inhibitory intemeurons belong to GABAergic system which are usually small and non-oriented, we think that PV-immunoreactive neurons in the SC might be more specific to be considered as inhibitory intemeurons than Caltj-immunoreactive neurons. In contrast, the characteristics of morphology and distribution of Caltj-immunoreactive neurons in the SC may suggest their contribution to population of projection neurons.
CONCLUSION
The present study has provided a description of inhibitory interneurons with detail infomiation of diversity of subgroups which suggest the different roles of the two types of inhibitory interneurons in the SC. By using markers in the expression of Calcium-binding proteins, such as PV and Calb, we classified two types of inhibitory neurons with diversity of morphology and distribution; in which, PV-immunoreactive neurons were more likely to be inhibitory GABAergic interneurons than Calb- immunoreactive neurons and the later may include projection neurons.
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