Anat Cell Biol.  2016 Dec;49(4):259-272. 10.5115/acb.2016.49.4.259.

Hippocampal serotonin-2A receptor-immunoreactive neurons density increases after testosterone therapy in the gonadectomized male mice

• Nikmahzar
• Jahanshahi M
• Ghaemi A
• Naseri GR
• Moharreri
• Lotfinia A

• KMID: 2365556
• DOI: http://doi.org/10.5115/acb.2016.49.4.259

Abstract

The change of steroid levels may also exert different modulatory effects on the number and class of serotonin receptors present in the plasma membrane. The effects of chronic treatment of testosterone for anxiety were examined and expression of 5-HT(2A) serotonergic receptor, neuron, astrocyte, and dark neuron density in the hippocampus of gonadectomized male mice was determined. Thirty-six adult male NMRI mice were randomly divided into six groups: intact-no testosterone treatment (No T), gonadectomy (GDX)-No T, GDX-Vehicle, GDX-6.25 mg/kg testosterone (T), GDX-12.5 mg/kg T, and GDX-25 mg/kg T. Anxiety-related behavior was evaluated using elevated plus maze apparatus. The animals were anesthetized after 48 hours after behavioral testing, and decapitated and micron slices were prepared for immunohistochemical as well as histopathological assessment. Subcutaneous injection of testosterone (25 mg/kg) may induce anxiogenic-like behavior in male mice. In addition, immunohistochemical data reveal reduced expression of 5-HT(2A) serotonergic receptor after gonadectomy in all areas of the hippocampus. However, treatment with testosterone could increase the mean number of dark neurons as well as immunoreactive neurons in CA1 and CA3 area, dose dependently. The density of 5-HT(2A) receptor-immunoreactive neurons may play a crucial role in the induction of anxiety like behavior. As reduction in such receptor expression have shown to significantly enhance anxiety behaviors. However, replacement of testosterone dose dependently enhances the number of 5-HT(2A) receptor-immunoreactive neurons and interestingly also reduced anxiety like behaviors.

Keyword

Gonadectomy; Testosterone; 5-HT(2A) serotonergic receptor; Anxiety; Hippocampus

MeSH Terms

Adult
Animals
Anxiety
Astrocytes
Behavior Rating Scale
Cell Membrane
Hippocampus
Humans
Injections, Subcutaneous
Male*
Mice*
Neurons*
Receptors, Serotonin
Testosterone*
Receptors, Serotonin
Testosterone

Figure

• 

  Fig. 1 Anxiety-related behaviors in mice at 30 minutes after last treatment in the elevated plus maze test. Parameters for assessing anxiety includes percentage of open (A) and closed (B) arm times, percentage of open (C) and closed (D) arm entries, open arm latency (E), and pure index of locomotor activity (F). GDX, gonadectomy; No T, no testosterone treatment; T, testosterone. Values represent mean. *P<0.05 and **P<0.01, means significant.

• 

  Fig. 2 Coronal sections of mice hippocampus (CA1 area) stained by immunohistochemistry with an antibody against a 5-HT2A serotonin receptor. (A) GDX-No T group. (B) GDX-Vehicle group. (C) GDX-6.25 mg/kg T group. (D) GDX-12.5 mg/kg T group. (E) GDX-25 mg/kg T group. (F) Intact-No T group. GDX, gonadectomy; No T, no testosterone treatment; T, testosterone. Scale bars=20 µm (×1,000). Arrows demarcate 5-HT2A receptor-immunoreactive neuron in the hippocampal CA1 area of all groups.

• 

  Fig. 3 Representative PTAH staining of astrocytes in the CA1 area of hippocampus of different groups. (A) GDX-No T group. (B) GDX-Vehicle group. (C) GDX-6.25 mg/kg T group. (D) GDX-12.5 mg/kg T group. (E) GDX-25 mg/kg T group. (F) Intact-No T group. PTAH, phosphotangestic acid hematoxylin; GDX, gonadectomy; No T, no testosterone treatment; T, testosterone. Scale bars=50 µm (×400). Arrows demarcate astrocyte in the hippocampal CA1 area of all groups.

• 

  Fig. 4 The mean density of astrocytes in the hippocampal CA1, CA3, and DG areas. GDX, gonadectomy; No T, no testosterone treatment; T, testosterone. *P<0.05, **P<0.01, and ***P<0.001 means significant.

• 

  Fig. 5 Representative cresyl violet staining of neurons in the CA1 area of hippocampus of different groups. (A) GDX-No T group. (B) GDX-Vehicle group. (C) GDX-6.25 mg/kg T group. (D) GDX-12.5 mg/kg T group. (E) GDX-25 mg/kg T group. (F) Intact-No T group. GDX, gonadectomy; No T, no testosterone treatment; T, testosterone. Scale bars=50 µm (×400). Arrows demarcate neuron in the hippocampal CA1 area of all groups.

• 

  Fig. 6 The effects of fourteen days treatment with different doses of testosterone on the mean density of dark neurons in the hippocampal CA1 area. Light-microscopic appearance of coronal brain sections in GDX-No T (A), GDX-Vehicle (B), GDX-6.25 mg/kg T (C), GDX-12.5 mg/kg T (D), GDX-25 mg/kg T (E), and Intact-No T (F) mice stained by hematoxylin and eosin staining. GDX, gonadectomy; No T, no testosterone treatment; T, testosterone. Scale bars=50 µm (×400). Arrows demarcate neuron in the hippocampal CA1 area of all groups.

• 

  Fig. 7 The bar graphs represent the mean density of dark neurons in GDX-No T, GDX-Vehicle, GDX-6.25 mg/kg T, GDX-12.5 mg/kg T, GDX-25 mg/kg T, and Intact-No T groups in different regions of the hippocampus. GDX, gonadectomy; No T, no testosterone treatment; T, testosterone. Values represent mean. *P<0.05, **P<0.01, and ***P<0.001 means significant.

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