Lane-Ladd S B, Pineda J, Boundy V A, Pfeuffer T, Krupinski J, Aghajanian G K, Nestler E J
Department of Psychiatry, Yale University School of Medicine and Connecticut Mental Health Center, New Haven, Connecticut 06508, USA.
J Neurosci. 1997 Oct 15;17(20):7890-901. doi: 10.1523/JNEUROSCI.17-20-07890.1997.
Chronic morphine administration increases levels of adenylyl cyclase and cAMP-dependent protein kinase (PKA) activity in the locus coeruleus (LC), which contributes to the severalfold activation of LC neurons that occurs during opiate withdrawal. A role for the transcription factor cAMP response element-binding protein (CREB) in mediating the opiate-induced upregulation of the cAMP pathway has been suggested, but direct evidence is lacking. In the present study, we first demonstrated that the morphine-induced increases in adenylyl cyclase and PKA activity in the LC are associated with selective increases in levels of immunoreactivity of types I and VIII adenylyl cyclase and of the catalytic and type II regulatory subunits of PKA. We next used antisense oligonucleotides directed against CREB to study the role of this transcription factor in mediating these effects. Infusion (5 d) of CREB antisense oligonucleotide directly into the LC significantly reduced levels of CREB immunoreactivity. This effect was sequence-specific and not associated with detectable toxicity. CREB antisense oligonucleotide infusions completely blocked the morphine-induced upregulation of type VIII adenylyl cyclase but not of PKA. The infusions also blocked the morphine-induced upregulation of tyrosine hydroxylase but not of Gialpha, two other proteins induced in the LC by chronic morphine treatment. Electrophysiological studies revealed that intra-LC antisense oligonucleotide infusions completely prevented the morphine-induced increase in spontaneous firing rates of LC neurons in brain slices. This blockade was completely reversed by addition of 8-bromo-cAMP (which activates PKA) but not by addition of forskolin (which activates adenylyl cyclase). Intra-LC infusions of CREB antisense oligonucleotide also reduced the development of physical dependence to opiates, based on attenuation of opiate withdrawal. Together, these findings provide the first direct evidence that CREB mediates the morphine-induced upregulation of specific components of the cAMP pathway in the LC that contribute to physical opiate dependence.
长期给予吗啡会增加蓝斑（LC）中腺苷酸环化酶的水平以及环磷酸腺苷（cAMP）依赖性蛋白激酶（PKA）的活性，这有助于在阿片类药物戒断期间发生的LC神经元的数倍激活。有人提出转录因子cAMP反应元件结合蛋白（CREB）在介导阿片类药物诱导的cAMP途径上调中起作用，但缺乏直接证据。在本研究中，我们首先证明，吗啡诱导的LC中腺苷酸环化酶和PKA活性增加与I型和VIII型腺苷酸环化酶以及PKA的催化亚基和II型调节亚基的免疫反应性水平的选择性增加有关。接下来，我们使用针对CREB的反义寡核苷酸来研究该转录因子在介导这些效应中的作用。将CREB反义寡核苷酸直接注入LC（持续5天）可显著降低CREB免疫反应性水平。这种效应具有序列特异性，且与可检测到的毒性无关。注入CREB反义寡核苷酸完全阻断了吗啡诱导的VIII型腺苷酸环化酶上调，但未阻断PKA的上调。注入还阻断了吗啡诱导的酪氨酸羟化酶上调，但未阻断慢性吗啡处理在LC中诱导的其他两种蛋白质Gialpha的上调。电生理研究表明，向LC内注入反义寡核苷酸完全阻止了吗啡诱导的脑片中LC神经元自发放电率的增加。加入8-溴-cAMP（激活PKA）可完全逆转这种阻断作用，但加入福斯可林（激活腺苷酸环化酶）则不能。基于阿片类药物戒断的减轻，向LC内注入CREB反义寡核苷酸也减少了对阿片类药物身体依赖性的发展。总之，这些发现提供了首个直接证据，即CREB介导了吗啡诱导的LC中cAMP途径特定成分上调，这些成分有助于阿片类药物身体依赖性。
