Reaction SMILES: CCCN([CH:8]1[CH2:17][C:16]2[C:15]([OH:18])=[CH:14][CH:13]=[CH:12][C:11]=2[CH2:10]C1)CCC.C1C=NC(N2CCN(CCCCN3C(=O)CC4(CCCC4)CC3=O)CC2)=NC=1.N[C@H](C([O-])=O)CCC([O-])=O.C1C=CC2[NH:68][C:66](=[O:67])[N:65](CCN3CCC(C(C4C=CC(F)=CC=4)=O)CC3)[C:63](=[O:64])C=2C=1.CCCN(C1CC2C(OC)=CC=CC=2CC1)CCCl>>[CH3:14][CH2:13][CH2:12][CH:11]([C:16]1([CH2:17][CH3:8])[C:15](=[O:18])[NH:68][C:66](=[O:67])[NH:65][C:63]1=[O:64])[CH3:10]
Reactant: O=C(c1ccc(F)cc1)C1CCN(CCn2c(=O)[nH]c3ccccc3c2=O)CC1
Reactant: CCCN(CCC)C1CCc2cccc(O)c2C1
Agent: CCCN(CCCl)C1CCc2cccc(OC)c2C1
Agent: N[C@@H](CCC(=O)[O-])C(=O)[O-]
Agent: O=C1CC2(CCCC2)CC(=O)N1CCCCN1CCN(c2ncccn2)CC1
Product: CCCC(C)C1(CC)C(=O)NC(=O)NC1=O
Reaction time: 24 h

Procedure:
The mechanism(s) and site(s) whereby 5HT1A agonistic drugs act to improve respiration following SCI remain to be further investigated. However, the beneficial effects of 8-OH-DPAT and buspirone on p.i. respiration that we observed can not be owned to possible neuronal protection for two reasons. First, by 24 hours and 7 days p.i., major neuronal loss at or near injury epicenter already completed in the spinal cord (Noble and Wrathall, 1985 and 1989; Crowe et al., 1997; Teng et al., 1998b). Correspondingly, effects of 8-OH-DPAT in the current study display as a time-dependent phenomenon (FIG. 3), which is contrasted with that of FGF2 (Teng et al., 1999). Secondly, there are no evidences that 5HT1A agonistic drugs spare neurons or neural tissues after traumatic injury. Hence, another possibility for these drugs to work is to act on surviving spinal motoneurons. Yet published data concerning whether 5HT1A agonists postsynaptically stimulate spinal somatomotor neurons do not support this notion. Jackson and White (1990) reported that iontophoretically delivered 8-OH-DPAT into the ventral horn inhibited the glutamate-evoked firing of motoneurons while similarly applied agonists for 5HT1B, 5HT1C and 5HT2 augmented it. In a SCI-related study, increased serotonergic inervation of phrenic motoneurons (located at C3 to C5 of the cervical spinal cord) is identified to be accountable for long-term facilitation of respiratory motor output triggered by episodic hypoxia at 28 days after cervical dorsal rhizotomy (Kinkead et al., 1998). Again, the effect is mediated by 5HT2 receptors in this case since it is blocked by pretreatment of ketanserin, a 5HT2 antagonist (Bach and Mitchell, 1996; Kinkead et al., 1998). Thus, it is conceivable that in our study 8-OH-DPAT and buspirone are not acting on spinal motoneurons. In fact, sites of action for the respiratory effect of 5HT1A agonists were confined within the brainstem to certain degree in literatures describing their cardiovascular effects and their counteraction mechanisms on apneusis. Work by Fozard et al., (1987) show that in conscious, spontaneous hypertension rats, 8-OH-DPAT causes dose-related falls in blood pressure and heart rate. The same effects are not observed in pithed rats. Moreover, the response to 8-OH-DPAT is blocked by intracisternal injection of 8-MeO-CIEPAT, an irreversible 5HT1A receptor antagonist (Fozard et al., 1987). Another report reveals that application of 5-methoxy-N,N-dimethyltrptamine, a 5HT1A agonist to the dorsal surface of the medulla oblongata reverses apneusis produced by pentobarbital (Lalley, 1994). Under this circumstance, the most feasible target for the drug is neurons in the dorsal respiratory group of the medulla. In our earlier study of 5HT1A agonist reversal of morphine-induced apnea (Ferreira et al., 1998), we believe that 5HT1A agonist drugs may act directly on brainstem respiratory rhythm generating centers: the Pre-Botzinger Complex to re-start respiration which was suppressed by morphine (Smith et al., 1991). Indeed, it has been reported that direct application of 5HT to the medulla area embodying Pre-Botzinger Complex increments the frequency of burst discharge of those neurons (Al-Zubaidy et al., 1996; Onimaru et al., 1998). Further, our present data indicate that 5HT1A agonists improve respiration by directly stimulating respiratory neurons and not by other mechanisms such as augmenting sensitivity to CO2. Table 2 exhibits that 8-OH-DPAT treatment does not affect ventilatory response to 7% CO2 in normal rats. Thus, despite we did not directly investigate the mechanism(s) and location(s) for 5HT1A agonists to improve SCI-triggered respiratory dysfunction, our data from 8-OH-DPAT time-course and p-MPPI antagonism experiments clearly suggest that the effect is mediated through 5HT1A receptors. Furthermore, existing evidences indicate that the most likely site(s) for 8-OH-DPAT buspirone to restore p.i. respiratory function to normal is respiratory neurons in the brainstem. Thereafter, we postulate that by stimulating the brainstem respiratory premotoneurons both pre- and post-synoptically, 5HT1A agnistic drugs can increase the activity of phrenic motoneurons which are essentially undamaged by T8 injury. Subsequently, the enchanced diaphragm contraction can compensate activity loss of intercostal muscles resulting from neuronal death in the thoracic spinal cord. We're currently testing this hypothesis by comparing pre-8-OH-DPAT phrenic nerve outflow to that recorded after drug administration in anesthetized rats at different time points p.i.