The Analgesic Effect Of Neurotensin And Its Mechanism In The Rat Spinal Dorsal Horn

Neurotensin (NT) is a tridecapeptide consisted by thirteen amino acids (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH). In the central nervous system, NT paly roles as a neurotransmitter or neuromodulater. Its effects include hypothermial, antinociception, modulation of dopamine neurotransmission and stimulation of anterior pituitary hormone secretion. Microinjection experiments results showed that NT could modulate nociceptive information transmission in several brain regions and pathways involved in the central integration of pain response, including the amygdala, the hypothalamic preoptic nucleus, thalamic nuclei, the periaqueductal gray (PAG), and the rostroventral medulla (RVM). PAG and RVM are important structures of descending pain inhibitoty system. In the RVM, a low dose of NT facilitated pain while a high dose of NT inhibited pain. This phenomenon might be the result of that several NT receptor (NTR) are involved in the pain modulating effect of NT. Up to now, three kinds of NTR have been found and cloned. The NTR1 and NTR2 are G protein coupled receptors with 7 transmembrane structures and mediate NT signaling. The function of NTR3 is still obscure.Although it is clear that NT has antinociceptive effect in superaspinal brain region, previous studies about whether NT also palys an analgesic role at spinal cord level has not draw a consistent conclusion yet. Thus, in the present study, the effect of NT on nociceptive transmission in spinal cord and its mechanism were studied by combined methods of morphology, electrophysiology and behavioural study. There were densely distributed NT positive neuronal cell bodies and fibers as well as terminals and scarcely distributed NTR2 positive neuronal cell bodies and fibers in the superficial layers of spinal dorsal horn (laminae I, II). By using double-immunofluorescence histochemistry for GABA and NT, the close contacts between TRITC labeled neurotensin terminals and FITC labeled GABA neurons were observed under a laser-scanning confocal microscope. By using double-electron-microscopic imrnunohistochemistry, asymmetrical synaptics formed by neurotensin terminals and GABAergic neurons were observed under an electron microscope. NTR2 positive neurons and terminals were observed by using the methods of either immunohistochemistry or in situ hybridization. The result that some of the GABA positive neurons contained NTR2 mRNA suggested the coexistence of GABA and NTR2 in the superficial dorsal horn neurons. No NTR2 positive neuron was observed in the dorsal root ganglia. The dorsal root rhizotomy and/or thoracic cord hemisection had no effect on the expression of NTR2 in the spinal cord dorsal horn. The results above suggested that the NT released in spinal dorsal horn might directly modulate the activity of GABAergic neurons.The whole-cell voltage clamp on spinal substantia gelatinosa (lamina II) neurons was performed to further study the effect of NT on neurotransmitterrelease. In lamina II, NT (2 μM) induced an inward current in only a small part of the neurons (14%). NT did not influent the activities of most neurons. NT (2 μM) has no effect on spontaneous excitatory postsynaptic currents (sEPSCs). While in some of the neurons (54%) recorded, NT increase the frequentcy of either GABAergic or glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) with no effect on its amplitude. In the presence of tetradotoxin (TTX), the facilitaory effect of NT on sIPSCs was blocked. SR 48692 and SR 142948A, two artificial compounds, are non-peptide antagonists of neurotensin. SR 48692 is an antagonist of NTRl, while SR 142948A is an antagonist of both NTRl and NTR2. The results that SR 142948A but not SR 48692 antagonized the facilitatory effect of NT on sIPSCs suggested that NT enhanced inhibitory neurotransmitter GABA and Gly release by activating NTR2 in lamina II of the spinal cord.The role of NT in spinal dorsal horn were studied by heat radiant response and formalin test on SD rats that were implanted with a catheter extended to lumbar enlargement level, through which NT (10 μg) were injection. A RTY-3 heat radiant instrument was used to measure the sensitivity of rat hindpaw to heat stimuli. 5 min after intrathecal injection NT, the heat responding latency of the rat hindpaw was lengthened. This effect of NT was antagonized by SR 142948A instead of SR 48692. In the formalin test, 5 min after NT injection, the formalin (5%) was injected subcutaneously to the rat hind paw. NT significiantly reduced the number of flinches during both the first phase (0-10 min) and the second phase (15-60 min) of formalin test. In the first phase, both SR 48692 and SR 142948A antagonize the effect of NT, while in the second phase, SR 142948A but not SR 48692 antagonize...