兒茶酚胺及迦瑪胺基丁酸神經元的表現在經急性或重複安非他命注射後的小鼠韁核和海馬結構內的變化

Abstract

側韁核(Lateral habenula, LHb)可以抑制多巴胺的過量釋放，是控制大腦內多巴胺濃度的核區之ㄧ。海馬結構(Hippocampal formation)可分為背側及腹側，背側海馬結構(Dorsal hippocampal formation, DH)的功能主要負責空間記憶及空間導航、而腹側海馬結構(Ventral hippocampal formation, VH)負責防禦行為、情感控制等認知功能。這些區域受損會造成類似精神分裂的症狀，但詳細機制並不清楚。 動物經安非他命處理後，可作為研究藥物成癮或精神分裂的模式。本研究使用成年雄性小鼠，經腹腔一劑或重複注射(每天兩劑，四天共七劑) 5 mg/kg安非他命或等容積的生理食鹽水(對照組)。最後一劑注射半小時或四小時後，小鼠接受灌流固定，以石蠟包埋，製成冠狀石蠟切片(7μm)，使用免疫化學染色法分析韁核及海馬結構的迦瑪胺基丁酸(GABAergic)系統、兒茶酚胺(catecholaminergic)系統，以及鈣結合蛋白(calcium binding proteins)的表現。 單染結果發現 在本實驗探討的區域內，觀察到免疫標誌的GAD67、DBH及AADC的分布型態為點狀神經末梢(punctate)密集圍在神經細胞周圍，或散布於神經毯上。PV的分布型態為點狀末梢、絲狀的神經纖維(process) 密集圍在神經細胞周圍，或散布於神經毯上，以及整顆細胞體(cell body)的染色。 (1)內韁核與側韁核：GAD67、PV、DBH及AADC在內韁核的表現均不受一劑或重複安非他命影響。在側部側韁核內，PV的表現在一劑安非他命處理半小時及四小時後下降，重複處理半小時及四小時後上升。DBH的表現在一劑半小時後下降，一劑四小時後上升。 (2)海馬結構： (i)背側CA1，DBH的表現在重複安非他命處理半小時後上升。腹側CA1，GAD67的表現在一劑處理半小時後上升，四小時後下降；DBH的表現在重複處理半小時後表現下降。 (ii)背側CA2，DBH的表現在一劑安非他命處理半小時及重複處理四小時後上升。 (iii)背側CA3，GAD67的表現在重複安非他命處理後半小時上升；PV的表現在一劑處理半小時後下降，重複處理半小時後上升：DBH的表現在一劑處理半小時及四小時後下降，重複處理半小時後上升。腹側CA3，PV的表現在一劑處理四小時後下降，重複處理半小時後上升，重複處理四小時後下降。AADC的表現在一劑處理半小時後上升，重複處理半小時後下降，重複處理四小時後上升。 (iv)背側CA4，GAD67的表現在一劑安非他命處理半小時、四小時，以及重複處理半小時後上升；PV的表現在一劑處理半小時及四小時後下降。 (v)背側齒狀迴，GAD67的表現在一劑安非他命處理四小時，重複處理半小時及四小時後上升。PV的表現一劑處理四小時後下降，重複處理半小時及四小時後上升。AADC的表現在一劑處理半小時及四小時後下降。腹側齒狀迴，GAD67的表現在一劑處理四小時後下降，重複處理四小時後上升。 雙重染色結果發現 (1)在內韁核以及側韁核中，沒有觀察到GAD67、PV、DBH及AADC神經末梢之間有特別顯著的緊密分布，彼此之間的調控可能並不明顯，仍需要進一步驗證。 (2)在背側及腹側海馬結構中，AADC或DBH會緊密分布在有表現GAD67或是PV的細胞體周圍。並且會在神經細胞的邊緣與GAD67或是PV的神經末梢非常靠近，甚至重疊，可能彼此之間會互相調控。 一劑或重複安非他命會影響側部側韁核內GAD67、PV、DBH以及AADC的表現，但不影響內韁核。這可能代表成癮性藥物對神經系統產生變化，甚至傷害。一劑或重複安非他命處理後，GAD67及PV的表現代表安非他命對側部側韁核及背、腹側海馬結構中GABA系統的影響。GAD67末梢表現上升及PV末梢表現下降，可能代表了GABA的大量釋放。DBH末梢代表正腎上腺素，AADC代表單胺類的神經傳導物質，可能反映了這些興奮性神經傳導物質也在安非他命處理之後產生變化。 已知安非他命可能會影響到韁核控制腦內多巴胺濃度的功能，造成過動等行為異常；也可能會影響到背側及腹側海馬結構，造成注意力、空間記憶及情緒等認知功能異常等現象。本研究發現一劑或重複安非他命處理後，會改變小鼠側部側韁核及背、腹側海馬結構中兒茶酚胺和GABA系統的表現，可能導致側韁核及海馬結構功能改變，代表精神疾病早期致病機轉。

Lateral habenula (LHb) is one of the nuclei regulating the expression of dopaminergic system in brain. Hippocampal formation may be divided into dorsal and ventral parts by differentials and connections. Dorsal hippocampal formation (DH) is mainly responsible for navigation and long-term memory formation, and ventral part (VH) is related to cognitive functions and emotional control. Damages to habenula and hippocampal formation may result in schizophrenia-like syndromes, such as behavioral hyperactivity and impairment of memory. Amphetamine (Amph) treated animals have been used as models to study pathogenesis of addiction and schizophrenia. In previous studies, Amph could alter the expression of catecholaminergic, serotoninergic and GABAergic systems in neocortex, striatum, and hippocampus of the rat. In addition, parvalbumin (PV), a kind of calcium binding proteins (CaBPs), is used to mark a population of GABAergic neurons in central nervous system. Thus, this study investigated the roles of habenula and hippocampal formation in the mechanisms for the action of acute and repeated Amph treatment, by examining the expression of GAD67, PV, AADC and DBH. In this study, male adult SV129 mice received single or multiple (2 doses/day, 7doses in total) injections of saline or Amph, 5mg/kg. At 0.5 or 4 hours after the last injection, the mice were perfused with Bouin’s fixative, followed by preparation of paraffin sections and immunohistochemistry. Our results reveled that: (1)Medial and lateral habenula: There’s no significant alteration in the expression of GAD67, PV, AADC and DBH in the medial habenula after acute or repeated Amph treatment. In the lateral part of lateral habenula, the densities of PV terminals and processes were decreased at 0.5 and 4 h after acute Amph treatment, but increased at 0.5 and 4 h after repeated Amph treatment. (2)Hippocampal formation: (i) In DH CA1 pyramidal cell layer, the density of DBH was increased at 0.5 h after repeated Amph treatment. In VH CA1, the density of GAD67 was increased at 0.5 h after acute but decreased at 4 h after repeated Amph treatment; the density of DBH decreased at 0.5 h after repeated Amph treatment. (ii) In CA2 pyramidal cell layer, the density of DBH was increased at 0.5 h after acute and 4 h after repeated Amph treatment. (iii) In DH CA3 pyramidal cell layer, the density of GAD67 was increased at 0.5 h after repeated Amph treatment; the density of PV was decreased at 0.5 h after acute but increased at 0.5 after repeated Amph treatment; the density of DBH was decreased at 0.5 and 4 h after repeated but decreased at 0.5 h after repeated Amph treatment. In VH CA3, the density of PV was decreased at 4 h after acute but increased at 0.5 h after repeated Amph treatment; the density of AADC was increased at 0.5 h after acute but decreased at 0.5 h amd increased at 4h after repeated Amph treatment. (iv) In DH CA4 pyramidal cell layer, the density of GAD67 was increased at 0.5 and 4 h after acute and at 0.5 h after repeated Amph treatment; the density of PV was decreased at 0.5 and 4 h after acute Amph treatment. (v) In DH dentate gyrus (DG) granule cell layer, the density of GAD67 was increased at 4 h after acute and at 0.5 and 4 h after repeated Amph treatment; the density of AADC was decreased at 0.5 and 4 h after acute Amph treatment. In VH dentate gyrus, the density of GAD67 was decreased at 4 h after acute and increased at 4 h after repeated Amph treatment. The results of double staining: (1) In medial and lateral habenula, we observed the phenomenon that DBH and AADC terminals weren’t significantly colocalized or near GAD67- of PV-ir somata or punctuates. It needs more experiments to identify the connections between the terminals which expressd different transmitters synthesizing enzymes. (2) In dorsal and ventral hippocampal formation, AADC or DBH terminals were distributed closely apposed or overlapped, to GAD67- or PV-ir somata or punctates, implicating interactions. The GABAergic terminals might receive the norepinephrinergic or serotonergic regulation. The alteration of the expression of GAD67, PV, AADC and DBH in habenula and hippocampal formation after acute or repeated Amph treatment would represent that addictive drug could alter, even impair, the nervous system. The up-regulation of GAD67 terminals and down-regulation of PV terminals might be due to the increased release of GABA. DBH punctates represented norepinephrine terminals and AADC punctates stand for monoamine terminals. The alteration of DBH and AADC also revealed that Amph treatment would affect nervous system. It’s known that Amph treatment could disturb the functions of lateral habenula to regulate dopamine release. Amph treatment also affects hippocampal formation to impair navigation and memory. Our results suggested that acute or repeated Amph treatments could change the expression of catecholaminergic and GABAergic systems in habenula and hippocampal formation, leading to functional and behavioral changes, and may implicate early mechanisms of pathogenesis of psychosis.