Obrietan K, van den Pol A N
Department Biological Science, Stanford University, California 94305, USA.
J Neurosci. 1995 Jul;15(7 Pt 1):5065-77. doi: 10.1523/JNEUROSCI.15-07-05065.1995.
GABA is the primary inhibitory transmitter of the adult hypothalamus, synthesized by many neurons and found in 50% of the presynaptic boutons. GABA causes a decrease in Ca2+ in mature hypothalamic neurons in vitro by depressing cellular activity through opening Cl- channels. Despite the early expression of GABAA receptors in the embryonic hypothalamus (E15), the cellular function of GABA in the developing hypothalamus has received little attention. In the present study the role of GABA in modulating intracellular Ca2+ in developing hypothalamic neurons was studied with fura-2 digital imaging. GABA (0.5-500 microM) applied to embryonic hypothalamic neurons elicited a dramatic and rapid increase in intracellular Ca2+ This Ca2+ rise could be completely blocked by the GABAA antagonist bicuculline (20 microM) and persisted in the presence of tetrodotoxin (1 microM). The Ca2+ elevation induced by GABA was greater than that of equimolar concentrations of the excitatory transmitter glutamate in early development. The number of E15 neurons that responded to GABA with a Ca2+ rise increased during the first few days of culture, reaching 78% after 4 d in vitro. The Ca2+ rise was 87% blocked by cadmium (100 microM) and 85% blocked by nimodipine (1 microM), indicating that the mechanism of Ca2+ increase was primarily via L-type voltage operated Ca2+ channels. Addition of bicuculline to synaptically coupled cultures caused a significant decrease in Ca2+ 4-10 d after culturing, indicating hypothalamic neurons were secreting GABA at an early age of development, and that sufficient GABA was released to elicit an increase in Ca2+. This effect was seen even after blocking all glutamatergic activity with glutamate receptor antagonists. In contrast, GABA elicited no Ca2+ rise in older neurons (> 18 d in vitro), and the action of bicuculline reversed and caused a large increase in Ca2+ in spontaneously active neurons. Similar findings were obtained in cultures enriched in GABAergic neurons from the suprachiasmatic nucleus. To determine if the Ca2+ stimulating role of GABA on developing neurons was restricted to the hypothalamus and a few other regions, or whether it might exist throughout the brain, we examined the Ca2+ responses in cultured olfactory bulb, cortex, medulla, striatum, thalamus, hippocampus, and colliculus. The majority (75%) of developing neurons from each region showed a Ca2+ rise in response to GABA. Together these data suggest that GABA elevates Ca2+ in developing, but not mature, neurons from the hypothalamus and all other brain regions examined.(ABSTRACT TRUNCATED AT 400 WORDS)
γ-氨基丁酸（GABA）是成年下丘脑的主要抑制性神经递质，由许多神经元合成，存在于50%的突触前终扣中。在体外，GABA通过打开氯离子通道抑制细胞活性，从而使成熟下丘脑神经元中的钙离子浓度降低。尽管GABAA受体在胚胎下丘脑（E15）中早期就有表达，但GABA在发育中的下丘脑的细胞功能却很少受到关注。在本研究中，我们用fura-2数字成像技术研究了GABA在调节发育中的下丘脑神经元细胞内钙离子方面的作用。将GABA（0.5 - 500微摩尔）应用于胚胎下丘脑神经元，可引起细胞内钙离子急剧快速增加。这种钙离子升高可被GABAA拮抗剂荷包牡丹碱（20微摩尔）完全阻断，并且在存在河豚毒素（1微摩尔）的情况下依然持续存在。在早期发育阶段，GABA诱导的钙离子升高幅度大于等摩尔浓度的兴奋性神经递质谷氨酸。在培养的最初几天里，对GABA产生钙离子升高反应的E15神经元数量增加，体外培养4天后达到78%。钙离子升高有87%被镉（100微摩尔）阻断，85%被尼莫地平（1微摩尔）阻断，这表明钙离子增加的机制主要是通过L型电压门控钙离子通道。在突触耦合培养物中加入荷包牡丹碱，培养4 - 10天后会导致钙离子显著降低，这表明下丘脑神经元在发育早期就分泌GABA，并且释放的GABA足以引起钙离子增加。即使在用谷氨酸受体拮抗剂阻断所有谷氨酸能活性后，这种效应仍然可见。相反，GABA在较成熟的神经元（体外培养>18天）中不会引起钙离子升高，而荷包牡丹碱的作用则相反，会使自发活动的神经元中的钙离子大幅增加。在富含视交叉上核GABA能神经元的培养物中也得到了类似的结果。为了确定GABA对发育中神经元的钙离子刺激作用是否仅限于下丘脑和其他几个区域，还是可能存在于整个大脑中，我们检测了培养的嗅球、皮层、延髓、纹状体、丘脑、海马体和丘脑中的钙离子反应。来自每个区域的大多数（75%）发育中神经元对GABA都有钙离子升高反应。这些数据共同表明，GABA能使下丘脑和所有其他检测的脑区中发育中的神经元而非成熟神经元的钙离子升高。（摘要截取自400字）
