| The lumbosacral dorsal commissural nucleus (DCN) located in the dorsal gray matter of the central canal of the lower lumbar and sacral spinal cord is the main area which receives pelvic visceral information. The intermedial lateral nucleus (IML) of the lower lumbar and sacral spinal cord lies in the seventh layer of spinal cord and is closely related to the lumbosacral DCN in terms of functions. The parabrachial nucleus (PBN) is mainly involved in the regulation of visceral activities. The periaqueductal gray (PAG) and the dorsal raphe nucleus (DR.) are the important components of the descending inhibitory system. The electrical stimulation to the PAG and DR can produce obvious antinociceptive effect.GABA is a crucial inhibitory neurotransmitter in the CNS. Our previous studies have shown that there are not only a vast amount of the cell bodies and dendrites of the GABAa receptor- like immunoreactive (LI) neurons but also many GAD-LI fibers and terminals and GABAergic inhibitory intemeurons (TNINs) in the lumbosacral DCN and IML of the lower lumbar and sacral spinal cord. However,which action sites and GABA receptor subtypes do the GABAergic terminals take action through have been still not clear now.5-HT is a key neurotransmitter of endogenous antinociceptive system and involved in the modulation of neural activities. There are not only plentiful 5-HT-LI fibers and terminals but also large numbers of neurons expressing 5-HT receptors in the lumbosacral DCN and IML of the lower lumbar and sacral spinal cord. However, which action sites and 5-HT receptor subtypes do the 5-HT-LI fibers and terminals exert their functions through remain to be further investingated.The previous morphological studies have proved that the neurons containing 5-HT in the central nervous system (CNS) are mainly distributed in the brainstem raphe nuclei and some 5-HT-LI neurons are also found in the ventrolateral subdivision at the caudal level of the PAG. Furthermore, 5-HT-LI fibers and terminals are also densely distributed in the PBN. The descending inhibitory system originates from the PAG and DR and exerts analgesic effect under the condition of visceral inflammatory stimulation. Until now, there has been little morphological evidence that pelvic visceral inflammatory stimulation activates the 5-HT-LI neurons in the PAG and DR which regulate pelvic visceral nociceptive information in the PBN.Therefore, based on the above questions the present study was made to examine whether 5-HT and GABA are involved in the regulation of formalin-induced visceral inflammatory pain by using retrograde tract-tracing tecnique, histochemistry and immunohistochemistry methods, immunofluorescent multiple-staining technique and GAD67-GFP knock-in mouse. The results showed that:(1) After injection of FG into the unilateral PBN, many neurons retrogradely labeled by FG were observed in the lumbosacral DCN and bilateral IML of the lower lumbar and sacral spinal cord, with a predominance in the ipsilateral side. Afterinstillation of formalin into the colon of the rat, Fos-LI neurons were mainly distributed in the lumbosacral DCN and bilateral ML of the lower lumbar and sacral spinal cord, with a predominance in the ipsilateral side. Some Fos-LI neurons were also scattered in the Laminae L II and deep layers of the ipsilateral dorsal horn. Many 5-HTia receptor > GABAa receptor al subunit> GABAa receptor a3 subunit and GABAa receptor P chain-LI cell bodies % fibers and terminals were observed in the lumbosacral DCN and ML of the lower lumbar and sacral spinal cord. The number of FG/GABAARal/Fos triple-labeled neurons was 9.9% (29/292) to the total FG-labeled neurons, 5.5% (29/529) to the total GABAA receptor al subunit-LI neurons and 4.2% (29/694) to the total Fos-LI neurons, respectively. The number of FG/GABAARa3/Fos triple-labeled neurons was 24.6% (82/333) to the total FG-labeled neurons, 9.8% (82/830) to the total GABAA receptor a3 subunit-LI neurons and 10.0% (82/824) to the total Fos-LI neurons, respectively. The number of FG/GABAARPchain/Fos triple-labeled neurons is was 21.8% (72/330) to the total FG-labeled neurons, 9.1% (72/787) to the total GABAa receptor p chain-LI neurons and 8.8% (72/820) to the total Fos-LI neurons, respectively. The number of FG/5-HTiaR/Fos triple-labeled neurons was 26.3% (81/308) to the total FG-labeled neurons, 11.0% (81/737) to the total 5-HTiA receptor-LI neurons and 10.3% (81/785) to the total Fos-LI neurons, respectively.(2) In order to demonstrate GABAergic ININs better, GAD67-GFP knock-in mouse was used in the present experiment GABAergic ININs were mainly distributed in the superficial layers of the spinal dorsal honu dorsal gray commissure (DGC) and ML. Many 5-HTiA receptor, GABAa receptor (3 chain and Fos-LI neurons were observed in the lumbosacral DCN and ML of the lower lumbar and sacral spinal cord and some of them were 5-HTjaR/GFP and GABAARPchain/GFP double-labeled neurons. The number of 5-HTiaR/GFP double-labeled neurons was15.1% (210/1390) to the total 5-HTiAreceptor-LI neurons and 33.9% (210/618) to the total GFP-LI neurons. The number of GABAARJ3chain/GFP double-labeled neurons was 13.6% (109/798) to the total GABAa receptor P chain-LI neurons and 16.8% (109/647) to the total GFP-LI neurons.(3) After injection of HRP into the unilateral PBN, many neurons retrogradely labeled by HRP were observed in the ventrolateral subdivision at all rostrocaudal levels of the PAG and DPL, predominantly in the ventrolateral subdivision at the caudal level of the PAG. The HRP-labeled neurons were distributed in both sides of the PAG and DR with a predominance in the ipsilateral side. The majority of them were fusiform and triangular (20 to 35 urn in diamter) and some of them were elliptic (15 to 20 um in diamter). After instillation of formalin into the colon of the rat, Fos-LI neurons were mainly distributed in all the subdivisions at all rostrocaudal levels of the PAG, especially in the ventrolateral subdivision and DR. Some HRP-labeled neurons also showed 5-HT and Fos immunoreactivities in the ventrolateral subdivision at the caudal level of the PAG and DR. Totally, 826 HRP-labeled neurons were observed in the ventrolateral subdivision at the caudal level of the PAG of 5 rats. Among them, 320 (38.7%) neurons were 5-HT-LI, 574(69.5%) neurons were Fos-LI and 151 (18.3%) neurons were both 5-HT and Fos immunoreactivities. The number of HRP/5-HT/Fos triple-labeled neurons was 18.3% (151/826) to the total HRP-labeled neurons, 5.2% (151/2912) to the total 5-HT-LI neurons and 4.4% (151/3456) to the total Fos-LI neurons, respectively. There were also many HRP-labeled neurons in the DR. We counted 385 HRP-labeled neurons in the DR of 5 rats. Among them, 280 (72.7%) neurons were 5-HT-LI, 306 (79.5%) neurons were Fos-LI and 106 (27.5%) neurons were both 5-HT and Fos immunoreactivities. The... |
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