Mechanisms Underlying Serotonin Promote γ-aminobutyric Acid (GABA) Synthesis In The Spinal Dorsal Horn

5-hydroxytryptamine (5-HT), an important neurotransmitter in the central nervous system, plays a major role in spinal antinociception, acting on 14 subtypes of 5-HT receptor. In the spinal dorsal horn, previous studies have demonstrated that: (1) Different from the nociceptive effect of peripheral 5-HT, 5-HT mediates antinociception in the spinal level; (2) 5-HT exerts its antinociceptive role by activating inhibitory interneurons or inhibiting excitatory interneurons in the spinal dorsal horn, indirectly; and (3) Several 5-HT receptor subtypes are distributed in the spinal dorsal horn, including 5-HT₁A 5-HT₁B 5-HT₁D 5-HT2A 5-HT2C 5-HT3 and 5-HT4 subtypes. The exact 5-HT receptor subtypes involved in the regulation of 5-HT and the related intracellular signaling pathway, however, remain unclear. The main questions are: (1) lack of powerful tool to investigate the morphological and functional properties of the spinal dorsal neurons; (2) special modulatory effects of 5-HT-mediated facilitation on different inhibitory neurotransmitters are not clear, such as enkephalin (ENK), preprodynorphin (Dyn) and GABA; and (3) cellular mechanisms and related signaling pathways for potenticationmedicated by different 5-HT receptor subtypes need to be elucidated. In the present study, we addressed the above issues by methods of morphology and electrophysiology. The main results are:1. Expression of 5-HT receptor subtype mRNAs in primarily cultured spinal dorsal horn neurons of the rat.In vitro models are useful since they allow for the simplification of in vivo complexity and permit accurately controlled experimental conditions.In order to investigate the receptor subtypes and intracellular signal transduction mechanism of 5-HT in the spinal dorsal horn, the spinal dorsal horn neurons of the embryonic rat were primary cultured. The results showed that primary cultured spinal dorsal horn neurons grew normally and exhibited satisfied morphological features. The expression of 14 subtypes of 5-HT receptor mRNAs in cultured dorsal horn neurons was also examined by using reverse transcription-polymerase chain reaction (RT-PCR). The results indicate that the experimental methods used in the present study were useful to obtain satisfied primary cultured spinal dorsal horn neurons. These neurons express mRNAs for various 5-HT receptor subtypes and provide valuable evidence for the further research on 5-HT in the spinal dorsal horn.2. The effect of 5-HT on several inhibitory neurotransmitters in the rat primarily cultured spinal dorsal horn neurons.Effects of c-fos antisense oligodeoxynucleotide (ASO) on 5-HT-induced upregulation of ppDyn, ppEnk and glutamic acid decarboxylase (GAD) mRNAs in cultured spinal dorsal horn neurons were investigated in order to extend our understanding of expressions of opioid peptides and GABA in spinal cord regulated by the descending serotonergic efferents. RT-PCR revealed a time-course increase in the expression of mRNAs encoding c-fos, ppDyn, ppEnk and GAD after administration of 5-HT (100 nM). Administration of c-fos ASO (0.02 nM) 30 min prior to 5-HT application markedly blocked the expression of c-fos gene. Moreover, c-fos ASOpretreatment significantly decreased the 5-HT-induced upregulation of ppDyn and ppEnk mRNAs, but failed to affect the expression level of GAD mRNA. These results suggest that the raphe-spinal efferents containing 5-HT plays a role in regulating the synthesis of enkephalin, dynorphin and GABA in the spinal dorsal horn neurons. The immediate early gene c-fos might be involved in the 5-HT-induced increase in ppDyn and ppEnk expression. However, under the present experimental conditions, c-fos seems not to be associated with the upregulation of GAD mRNA induced by 5-HT. 3. The mechanism of 5-HT facilitated the synthesis of GABA.In order to understand the morphology and function of GABAergic neurons in the spinal cord moreprecisely, one strain of glutamate decarboxylase 67- green fluorescence protein (GAD67-GFP) knock-in mice were used, in which all GABAergic neurons in the spinal cord were revealed by GFP fluorescence. The distribution pattern of GFP-expressing neurons and the amount of GFP protein in the spinal cord were inconsistent with that of GABAergic neurons and GABA, respectively. The knock-in mouse would be a useful tool for studying the morphology and physiological properties and developmental pattern of GABAergic neurons in the spinal cord. [1] We firstly set up successfully primary cultured spinal dorsal hornneurons of the GAD67-GFP knock-in mice.[2] Western blot revealed that ? 5-HT increased the expression of GFP protein in a time-course and dose-course manner. But the application of 5-HT failed to affect the number of the GFP-positive neurons. (2) Administration of 5-HT 100 nM activated immediately ERK and CREB (within 5 min), and application of MEK inhibiter or PI3 inhibitor 5 min prior to 5-HT administration markedly blocked the expression of phosphoralated ERK and CREB. (3) Moreover, MEK inhibitor or PI3 inhibitor pretreatment significantly decreased the 5-HT-inducedupregulation of GFP.4. Identification of 5-HT receptor subtypes within the spinal GABAergic neurons in GAD67-GFP knock-in mice.[1] In the primarily cultured spinal dorsal horn neurons of the GAD67-GFP knock-in mice, the colocalization of 5-HT|A and 5-HT2A receptor with GFP were identified by double labeling, without the positive results of 5-HT3 receptor subtype.[2] 5-HTia and GFP-positive neurons was distributed in all layers of the spinal dorsal horn, with a denser distribution in the deep laminae. The co-existence of 5-HT2A and GFP were found in the superficial laminae of the spinal dorsal horn.[3] The tissue RT-PCR analysis proved that the expression of GAD67 mRNA and multiple 5-HT receptor subtypes including 5-HTiA, 5-HTib, 5-HTid, 5-HTiF, 5-HT2A, 5-HT2c and 5-HT3AmRNAs, was detected in the spinal dorsal horn. However, the expression of 5-HT2B and 5-HT3B receptor mRNAs was not detected.[4] We explored single-cell RT-PCR to assess gene expression profiles of the 5-HT receptor subtypes (5-HTi³) in individual GABAergic neurons accurately isolated from the spinal dorsal horn of adult the GAD67-GFP knock-in mouse. Within fifty-seven GFP-positive neurons, 5-HTiA, 5-HTib, 5-HT,D, 5-HT2Aand 5-HT3 subtypes mRNAs could be detected with various expression ratio. 5-HTib and 5-HTiD receptor subtypes were observed most frequently (>50%) in GFP-positive neurons. 5-HT)A receptor subtype was present in 47.4% of GFP-positive neurons. About 10.5% and 21.1% of GFP-positive neurons also expressed 5-HTja and 5-HT3A receptor subtypes. The presence of 5-HTif and 5-HT2C receptor subtypes in GFP-positive neurons was not detected.