Effect of DPAT on VMR to CRD
A systemic injection of DPAT increased the VMR to CRD (). Low dose (5µg/kg, i.p., n=6) of DPAT increased the VMR significantly (p<0.05 vs baseline) at distending pressure ≥ 30mmHg (, n=6). However, at higher doses (50 and 250µg/kg, i.p., n=6 and 7, respectively) the VMR increased significantly (p<0.05 vs baseline) at much lower distension pressure (~10mmHg) (). There were no dose-dependent differences in VMRs between 50 and 250µg/kg. Therefore, in subsequent experiments, we used 50µg/kg of DPAT to test the mechanism of action of the drug.
Figure 1 Summary data of viscero-motor responses (VMRs) to colorectal distension (CRD) before and after intra-peritoneal (i.p.) injections of 5µg/kg (A), 50µg/kg (B) and 250µg/kg (C) of selective 5-HT1A receptor agonist DPAT. At low dose (more ...)
To test the receptor-specific action of DPAT, a 5-HT1A receptor selective antagonist WAY-100135 (5mg/kg s.c.) was injected 10 minutes before DPAT injection. The VMR was not attenuated by WAY-100135 alone (, n=3), but DPAT-induced increase in the VMR was completely blocked in the presence of WAY-100135 (, n=5).
Figure 2 (A) VMRs to colon distension before and after subcutaneous (s.c.) injection of selective 5-HT1A receptor antagonist WAY-100135 (5mg/kg). This dose of WAY-10035 did not attenuate VMR. (B) DPAT (50µg/kg, i.p.) failed to increase the VMR in rats (more ...)
To test whether DPAT produces a pronociceptive effect by acting at the spinal level, we injected DPAT (5µmol, i.t.) into the lumbo-sacral (LS) segment of the spinal cord 10 minutes prior to CRD. The drug significantly (p<0.05 vs baseline) increased VMR at all intensities (10 – 60mmHg) of CRD (, n=7).
Intrathecal (i.t.) injection of DPAT (5µmol) into the lumbo-sacral (LS) segment of the spinal cord significantly (*p<0.05 vs baseline) enhanced VMR at all intensities of distension pressure (10 – 60mmHg).
The DPAT-induced increase in the VMR was also blocked when rats were pretreated with the competitive NMDA receptor antagonist AP5 (50µg/kg, i.v.), suggesting that the increase in VMR by DPAT is linked with the activation of NMDA receptors (, n=5). To further investigate the involvement of NMDA receptors, a set of rats were injected with NR1 anti-sense ON (n=5) or mismatch-ON (n=5) into the LS segment of the spinal cord. A baseline VMR was recorded prior to injection and repeated five days later. In the NR1 anti-sense ON-treated group, there was no change in VMR and DPAT failed to increase the VMR (). In contrast, DPAT significantly (p<0.05 vs baseline) increased the VMR at all intensities of distending pressure in NR1 mismatch ON-treated rats ().
Figure 4 Increase in the VMR by DPAT is associated with NMDA receptor mediated visceral hyperalgesia. An injection of competitive NMDA receptor antagonist AP5 (50µg/kg, i.v.) 10 minutes prior to DPAT injection completely blocked the DPAT-induced increase (more ...)
Figure 5 Illustrates the involvement of NMDA receptor in DPAT-induced increase in VMR. A: DPAT (5µmol, i.t.) failed to increase VMR in NMDA-NR1 knock down rats that received repeated injection of NR1 subunit targeted antisense oligoneuleotide (ON). The (more ...)
The DPAT-induced increase in VMR could be either by (1) inhibition of GABAergic transmission through activation of pre-synaptic 5-HT1A receptors in GABAergic interneurons. The activation of 5-HT1A receptor would reduce the release of GABA and remove the inhibitory influence over the excitatory glutamatergic system or (2) by direct activation of 5-HT1A receptors located at the presynaptic terminals of the excitatory glutamatergic neurons in the spinal cord to release glutamate. To test this hypothesis, we designed experiments to block GABA synthesis by injecting γ-amino decarboxylase (GAD) inhibitor allylglycine (AG, 10µg, 2 times for 3 days) and then tested the effects of DPAT (5µmol, i.t.) (, n=4) or its effect following the inhibition of VMR by injecting GABAA receptor agonist muscimol (1.5µmol, i.t) (, n=5). The AG treatment significantly (p<0.05 vs pre-AG baseline) increased the VMRs in these rats suggesting a lack of inhibitory GABAergic tone in the spinal cord to modulate visceral nociception (). Although DPAT injection in these rats slightly increased the VMR, the enhanced response was not statistically significant (, p>0.05). In second set of experiment, i.t. injection of muscimol (1.5µmol, i.t.) in AG-treated rats significantly (p<0.05 vs post-AG) inhibited the VMR and DPAT (5µmol, i.t.) injection slightly reversed the inhibition (). In order to confirm that the pronociceptive effect of AG is by inhibiting GABA synthesis in the spinal cord and not by modulating GABA release via the activation of pre-synaptic 5-HT1A receptor, we tested the effect of i.t. injection of 5-HT1A receptor antagonist WAY-100135 (10µmol). The drug did not attenuate VMRs of AG-treated rats ().
Figure 6 Effects of DPAT (50µg/kg, i.p.) and WAY-100135 (10µmol, i.t.) in chronically allylglycine (AG)-treated rats. Repeated i.t. injection of AG, a GABA synthesis inhibitor, produced significant (*p<0.05 vs baseline) increase in VMRs (more ...)
Effect of DPAT on responses of CRD-sensitive pelvic nerve afferent (PNA) fibers and lumbosacral (LS) spinal neurons
To test whether the increase in VMR to systemic DPAT injection is due to excitation of CRD-sensitive PNA fibers, we tested responses of PNA fibers (n=7) to colon distension before and after the systemic injection of DPAT (50µg/kg, i.p.). The mean baseline spontaneous firing of these fibers before and after DPAT injection were 0.75 ± 0.3 impulses/s and 0.63 ± 0.2 impulses/s, respectively. illustrates a typical increase in firing of a CRD-sensitive PNA fiber to graded CRD (10–60mmHg, 30s). The DPAT injection did not affect responses of these fibers to CRD, suggesting that the increase in VMR by DPAT is not due to hyperexcitation of CRD-sensitive PNA ().
Figure 7 DPAT did not change the mechanotransduction properties of CRD-sensitive pelvic nerve afferent (PNA) fibers. A: illustrates responses of a non-spontaneous CRD-sensitive PNA fiber to graded intensities (20, 40, and 60mmHg) of colon distension before and (more ...)
We then tested the effect of DPAT on responses of CRD- sensitive LS spinal neurons. These experiments were performed in - (1) spinal intact (n=4) and (2) cervical (C1-C2) spinal cord transected rats (n=6). The rationale for recording responses of CRD-sensitive spinal neurons in spinal transected rats was to remove supraspinal influence that may contribute to pronociceptive effect of DPAT. illustrate typical responses of CRD-sensitive LS neurons before and after systemic DPAT (50µg/kg, i.p.) injection. The mean baseline spontaneous firings of these spinal neurons in intact rats before and after DPAT injection were 3.17 ± 1.80 impulses/s and 7.25 ± 4.2 impulses/s, respectively. In spinal transected rats, the mean baseline spontaneous firing significantly increased following DPAT injection (3.15 ± 0.9 impulses/s pre-DPAT vs 11.58 ± 3.76 impulses/s post-DPAT, p<0.05). In both spinal intact and spinal transected rats the drug significantly (p<0.05 vs baseline) increased responses of these neurons to CRD (). Since DPAT increased responses of these neurons in spinal transected rats, it suggests that the excitatory effect of the drug occurs at the spinal cord.
Figure 8 DPAT (50µg/kg, i.p.) significantly increased the responses of CRD-sensitive lumbo-sacral (LS) spinal neurons to colon distension in both spinal cord intact (A & B) and transected (C & D) conditions. Under both conditions the baseline (more ...)
In subsequent experiment, we tested the effect DPAT in rats pretreated with WAY-100135 (5mg/kg, s.c.). The mean baseline spontaneous firings of these spinal neurons before and after WAY-100135 injection were 3.80 ± 1.60 impulses/s and 3.10 ± 1.5 impulses/s, respectively. Following DPAT the mean baseline spontaneous firing of spinal neurons was 4.07 ± 1.40 impulses/s. WAY-100135 alone did not affect the responses of LS neurons to graded CRD, but blocked the excitatory effect of DPAT (, n=3).
Figure 9 The mean SRFs of lumbo-sacral (LS) spinal neurons (n=3) to graded CRD and effects of DPAT (50µg/kg, i.p.) in presence of selective 5-HT1A receptor antagonist WAY-100135 (5mg/kg, s.c.). The excitatory effect of DPAT on responses of CRD-sensitive (more ...)
In another set of experiments, we injected the competitive NMDA receptor antagonist AP5 (50µg/kg, i.v.) prior to testing the effect of DPAT on responses of CRD-sensitive spinal neurons (n=5) in spinal transected rats. The mean baseline spontaneous firings of these neurons before and after AP5 injection were 1.21 ± 0.7 impulses/s and 1.03 ± 0.6 impulses/s, respectively. Following DPAT administration the mean spontaneous firing was 1.64 ± 0.70 impulses/s. While AP5 did not affect responses of CRD-sensitive neurons, preemptive administration of AP5 completely blocked the excitatory effect of DPAT ().
Figure 10 Pretreatment with the competitive NMDA receptor antagonist AP5 (50µg/kg, i.v.) blocked the DPAT-induced excitation of CRD-sensitive lumbo-sacral (LS) spinal neurons to colon distension. A: illustrates an example of CRD-sensitive LS spinal neuron (more ...)