Wallmichrath I, Szabo B
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität, Albertstrasse 25, D-79104, Freiburg i Br, Germany.
Neuroscience. 2002;113(3):671-82. doi: 10.1016/s0306-4522(02)00109-4.
The substantia nigra pars reticulata (SNR) belongs to the brain regions with the highest density of CB(1) cannabinoid receptors. Anatomical studies indicate that the great majority of CB(1) receptors in the SNR are localized on terminals of GABAergic axons arriving from the caudate-putamen (striatonigral axons). The aim of the present experiments was to clarify the role of CB(1) receptors on terminals of striatonigral axons. Oblique sagittal slices, including the caudate-putamen and the substantia nigra, were prepared from brains of young mice. Electrical stimulation in the caudate-putamen elicited GABAergic inhibitory postsynaptic currents (IPSCs) in the SNR, which were studied by patch-clamp techniques. The long latency of IPSCs (14+/-1 ms) suggests that striatonigral axons were indeed activated within the caudate-putamen. The synthetic CB(1)/CB(2) cannabinoid receptor agonist WIN55212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate; 10(-5) M) decreased the amplitude of IPSCs by 93+/-1%. CP55940 ((-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol; 10(-5) M), another CB(1)/CB(2) receptor agonist, also reduced IPSC amplitude, by 76+/-4%. The CB(1) cannabinoid receptor antagonist SR141716A (N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide; 10(-6) M) prevented the inhibition produced by WIN55212-2 (10(-5) M). Depolarization of SNR neurons led to suppression of IPSCs; this suppression was prevented by SR141716A (10(-6) M). Three observations indicate that the agonists inhibited neurotransmission presynaptically. (1) CP55940 (10(-5) M) enhanced the ratio of amplitudes of two IPSCs which were elicited by two electrical stimuli 100 ms apart (paired pulses). (2) WIN55212-2 (10(-5) M) did not change the amplitude of miniature IPSCs recorded in the presence of tetrodotoxin. (3) WIN55212-2 (10(-5) M) also had no effect on currents elicited in SNR neurons by ejection of the GABA(A) receptor agonist muscimol from a pipet. In summary, we have established a method which allows selective examination of GABAergic neurotransmission between striatonigral axons and SNR neurons. Using this method, the function of CB(1) cannabinoid receptors on terminals of striatonigral axons was unequivocally clarified. Activation of these receptors causes strong presynaptic inhibition of GABAergic neurotransmission between striatonigral axons and SNR neurons. This effect may be one explanation of the catalepsy observed in animals after cannabinoid administration. Endocannabinoids released from SNR neurons can modulate striatonigral neurotransmission by inhibiting GABA release from terminals of striatonigral axons.
黑质网状部（SNR）属于脑内CB(1)大麻素受体密度最高的区域。解剖学研究表明，SNR中绝大多数CB(1)受体定位于来自尾状核-壳核（纹状体黑质轴突）的GABA能轴突终末上。本实验的目的是阐明CB(1)受体在纹状体黑质轴突终末上的作用。从幼年小鼠脑中制备包含尾状核-壳核和黑质的斜矢状切片。通过在尾状核-壳核进行电刺激，在SNR中诱发GABA能抑制性突触后电流（IPSCs），并用膜片钳技术进行研究。IPSCs的潜伏期较长（14±1毫秒），提示纹状体黑质轴突确实在尾状核-壳核内被激活。合成的CB(1)/CB(2)大麻素受体激动剂WIN55212-2（R(+)-[2,3-二氢-5-甲基-3-[(吗啉基)甲基]吡咯并[1,2,3-de]-1,4-苯并恶嗪基]-(1-萘基)甲酮甲磺酸盐；10(-5) M）使IPSCs的幅度降低了93±1%。另一种CB(1)/CB(2)受体激动剂CP55940（(-)-顺式-3-[2-羟基-4-(1,1-二甲基庚基)苯基]-反式-4-(3-羟丙基)环己醇；10(-5) M）也使IPSC幅度降低了76±4%。CB(1)大麻素受体拮抗剂SR141716A（N-哌啶基-5-(4-氯苯基)-1-(2,4-二氯苯基)-4-甲基-3-吡唑甲酰胺；10(-6) M）可阻断WIN55212-2（10(-5) M）产生的抑制作用。SNR神经元的去极化导致IPSCs受到抑制；这种抑制作用可被SR141716A（10(-6) M）阻断。三项观察结果表明，激动剂是在突触前抑制神经传递。（1）CP55940（10(-5) M）增加了由间隔100毫秒的两个电刺激诱发的两个IPSCs的幅度比值（成对脉冲）。（2）WIN55212-2（10(-5) M）在存在河豚毒素的情况下不改变微小IPSCs的幅度。（3）WIN55212-2（10(-5) M）对通过微量滴管喷射GABA(A)受体激动剂蝇蕈醇在SNR神经元中诱发的电流也没有影响。总之，我们建立了一种方法，可选择性地检测纹状体黑质轴突与SNR神经元之间的GABA能神经传递。利用该方法，明确阐明了CB(1)大麻素受体在纹状体黑质轴突终末上的功能。这些受体的激活会导致纹状体黑质轴突与SNR神经元之间的GABA能神经传递受到强烈的突触前抑制。这种效应可能是对给予大麻素后动物出现僵住症的一种解释。从SNR神经元释放的内源性大麻素可通过抑制纹状体黑质轴突终末释放GABA来调节纹状体黑质神经传递。
