| The periaqueductal gray (PAG), which is located in the midbrain, is an important structure in consisting of the descending pain inhibitory system (DPIS) in mammalian animals. The function of the PAG is mainly carried out by exciting the 5-HTergic neurons in the ventrolateral column/part of the PAG (vlPAG). After excitation, the 5-HTergic neurons could inhibit directly or indirectly the nociceptive neurons in the spinal dorsal horn and exert antinociceptive effect.There is a sprcial kind local circuit in regulating the activity of the 5-HTergic neurons in the PAG. That is, the widely distributedγ-aminobutyric acid (GABA) like immunoreactivity (LI) neurons can cause a tonic inhibitory effect on the 5-HTergic neurons. The opioid like substances (OLS) can inhibit the activities of the GABA-LI neurons via theμ-opioid receptor (MOR), and finally, to excite indirectly (disinhibit) the activities of the 5-HT-LI neurons.Endomorphin (EM) is a new found member of the endogenous opioid peptide family and also the endogenous ligand for the MOR. With the similar effect in pain inhibition as morphine, EM has much less side effect, indicating their significant clinical potential. EM is consisted of two subtypes—endomorphin 1 (EM1) and endomorphin 2 (EM2). In the brain, EMergic neuronal cell bodies are located mainly in the hypothalamus and the nucleus tractus solitary (NTS), while the EMergic fibers being widely distributed across many structures. There are moderate to dense density of EM1- and EM2-LI fibers and terminals found in different parts the PAG, suggesting EM should be involved in many functions of the PAG. Since the functional role of EM in the PAG has not been reported yet, we studied the possible regulatory mechanisms of EM on the nociceptive transmission in the PAG by modern neuroscience methods. The main problems we met prior to the experiment were as follows:1. Where are the possible origins of the EMergic fibers and terminals in the PAG? Is there any topographical correspondence of the origin areas and the different parts of the PAG?2. Whether EM is involved in the modulation of local pain regulatory circuit in the vlPAG, which is the originating area of the 5-HTegic DPIS?3. What is the possible modulation mechanism of EM on the nociceptive transmission in the vlPAG?Methods and results:1. The origins of the EM-LI fibers and terminals in the PAGBy combining the injection of the retrograde tracer, Fluoro-Gold (FG), into the different columns of the PAG with the EM1 or EM2 immunofluorescent staining, it's found that the EM1/FG and EM2/FG double-labeled neurons were mainly distributed in different nuclei and areas of the hypothalamus, especially in the dorsomedial hypothalamic nucleus (DMH), areas between the ventromedial hypothalamic nucleus and DMH (centromedial hypothalamic region, CMH) and the arcuate nucleus (Arc). The distribution characteristics are as follows:1) For FG injection into the vlPAG, 18% EM1-LI (15.7±6.0) and 14.0% EM2-LI (8.2±2.6) neurons in the DMH, 15% EM1-LI (30.7±5.9) and 18.6% EM2-LI (17.2±4.7) in the CMH and 10.5% EM1-LI (6.3±1.9) and 12.1% EM2-LI (5.7±2.0) in the Arc were labeled with FG; 2) For FG injection into the vlPAG, 14.8% EM1-LI (12.2±4.3) and 10.8% EM2-LI (6.7±2.7) neurons in the DMH, 11.1%的EM1-LI (23.7±7.9) and 10.3% EM2-LI (9.0±3.2) in the CMH and 9.2% EM1-LI (5.2±2.6) and 5.6% EM2-LI (2.8±1.2) in the Arc were labeled with FG;3) For FG injection into the vlPAG, 7.0% EM1-LI (6.2±3.3) and 6.2% EM2-LI (3.8±2.3) neurons in the DMH, 5.3% EM1-LI (11.6±3.6) and 3.4% EM2-LI (3.0±1.6) neurons in the CMH and 2.0% EM1-LI (1.2±0.8) and 2.4% EM2-LI (1.0±0.7) neurons in the Arc were labeled with FG;4) For FG injection into the vlPAG, 8.5% EM1-LI (7.8±2.9) and 7.0% EM2-LI (4.6±1.8) neurons in the DMH, 9.5% EM1-LI (19.2±8.1) and 5.7% EM2-LI (4.8±2.4) in the CMH and 3.5% EM1-LI (2.0±1.2) and 3.9% EM2-LI (1.8±1.8) in the Arc were labeled with FG;Summery:①The number of EM1/FG double-labeled neurons is much larger than that of the EM2/FG double-labeled neurons in the hypothalamus (802 vs. 392);②The number of EM/FG double-labeled neurons in the CMH (480 EM1/FG; 196 EM2/FG) is larger that those in the DMH (237 EM1/FG; 131 EM2/FG) and Arc (85 EM1/FG; 65 EM2/FG);③The greatest number of EM/FG double-labeled neurons appears for FG injection into the vlPAG, indicating that EMergic neurons in the hypothalamus have the most close connection with the vlPAG, followed with the lPAG, dmPAG and dlPAG.The present results indicate that:①EM-LI fiber and terminals in the different parts of the PAG mainly come from the hypothalamus but not from the NTS;②There exists an obvious topographical correspondence between the different nuclei/areas in hypothalamus and different parts of the PAG;③The hypothalamus-PAG projecting EMergic fibers and terminals may be involved in the modulation of the PAG's function. 2. By using morphological and behavioral research works, we studied the mechanism of EM in the modulation of pain regulatory circuit in the vlPAG.2.1 The regulatory circuit constituted by EM-LI structures which might be involved in the vlPAGUnder the electron microscope, the synaptic connections between the EM1- or EM2-LI terminals and the glutamate decarboxylase (GAD), MOR or 5-HT-LI cell bodies and dendrites were observed in the rat vlPAG. The results were as follows:1) There existed many EM1- and EM2-LI axons/terminals and GAD-, MOR- and 5-HT-LI cell bodies/dendrites in the vlPAG;2) EM1- and EM2-LI terminals could make synaptic connections with the MOR-, GAD- or 5-HT-LI cell bodies and dendrites. That is:EM1- and EM2-LI terminals mainly made symmetric/inhibitory synaptic connections with the MOR-LI cell bodies and dendrites (EM1: asymmetric/symmetric=37.8/62.2×100%; EM2: asymmetric/symmetric=41.4/58.6×100%).EM1- and EM2-LI terminals mainly made symmetric synaptic connections with the GAD-LI cell bodies and dendrites (EM1: asymmetric/symmetric=27.5/72.5×100%; EM2: asymmetric/symmetric =23.6/76.4×100%).EM1- and EM2-LI terminals mainly made asymmetric synaptic connections with the 5-HT-LI cell bodies and dendrites (EM1: asymmetric/symmetric=58.3/41.7×100%; EM2: asymmetric/symmetric=56.7/43.3×100%).3) EM2-LI terminals also made synapses with EM2-LI axons and terminals, most of which (92%) were asymmetric. The present results suggest that EM can inhibit the activities of GABA-LI neurons and excite the 5-HT-LI neurons.2.2 The confocal-microscopic observation of the regulatory circuit involved with EM in the vlPAGBy introducing the glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) gene knock-in mouse, we observed the distribution of EM1- or EM2-LI fibers and terminals, GFP-expressing GAD-LI neurons and MOR- or 5-HT-LI neurons. The results were as follows:1) There were extensive co-existences between GFP- and MOR-LI neuronal cell bodies. Almost all of the GFP-LI cell bodies (98%) were labeled with MOR immunoreactivities. EM1- or EM2-LI fibers and terminals could make close contacts with the GFP/MOR double-labeled neurons.2) Fibers from some GFP-LI neuronal cell bodies could make close contacts with the 5-HT-LI cell bodies, while the EM1- or EM2-LI fibers and terminals connected closely with these GFP-LI cell bodies.The present results suggest that there should exist a special kind of regulatory circuit, in which EM might inhibit the GABA (via the MOR) and excite indirectly the 5-HTergic neurons.2.3 The behavioral study of the regulatory circuit involved with EM in the vlPAGAfter EM, antagonist of MOR and antagonist and agonist of the GABAA receptor were injected into the vlPAG via a guide cannula respectively, the rat's paw withdrawal threshold for mechanical stimuli and latency for heat stimuli were measured. The possible mechanisms of EM in the pain regulatory circuit were discussed. The results were as follows: 1) Different doses of EM1 (4, 8, 16 and 32 nmol/0.5μl) and EM2 (2, 4, 8, 16 nmol/0.5μl) can cause obvious analgesia effect in animals, which can be blocked completely by the MOR antagonist;2) These GABAA receptor antagonist can potentiate the analgesia effect of EM, while the GABAA receptor agonist reversed the EM caused analgesia effect.The results indicate that the EM can cause analgesia effect by inhibiting the activity of the GABAergic neurons via the MOR.In summary, the morphological and behavioral studies have demonstrated that the EM can induce obvious analgesia effect in the vlPAG, which is carried out by inhibiting the activity of the GABAergic neurons (via the MOR) and exciting indirectly the activity of the 5-HTergic neurons. |
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