Mechanism Underlying the Analgesic Effect Exerted by Endomorphin-1 in the rat Ventrolateral Periaqueductal Gray

Abstract

The ventrolateral periaqueductal gray (vlPAG) is an important brain area, in which 5-HTergic neurons play key roles in descending pain modulation. It has been proposed that opioid peptides within the vlPAG can excite the 5-HTergic neurons by alleviating tonic inhibition from GABAergic neurons, the so-called disinhibitory effect. However, no direct morphological evidence has been observed for the micro-circuitry among the opioid peptide-, GABA-, and 5-HT-immunoreactive (ir) profiles nor for the functional involvement of the opioid peptides in the intrinsic properties of GABAergic and 5-HTergic neurons. In the present study, through microscopic observation of triple-immunofluorescence, we firstly identified the circuitry among the endomorphin-1 (EM1, an endogenous ligand for the μ-opioid receptor)-ir terminals and GABA-ir and 5-HT-ir neurons within the rat vlPAG. The synaptic connections of these neurons were further confirmed by electron microscopy. Through the in vitro whole-cell patch-clamp method, we showed that EM1 has strong inhibitory effects on the spiking of GABAergic neurons. However, although the resting membrane potential was hyperpolarized, EM1 actually increased the firing of 5-HTergic neurons. More interestingly, EM1 strongly inhibited the excitatory input to GABAergic neurons, as well as the inhibitory input to 5-HTergic neurons. Finally, behavioral results showed that pretreatment with a GABA(A) receptor antagonist potentiated the analgesic effect of EM1, while treatment with a GABA(A) receptor agonist blocked its analgesic effect. In summary, by utilizing morphological and functional methods, we found that the analgesic effect of EM1 is largely dependent on its potent inhibition on the inhibitory inputs to 5-HTergic neurons, which overwhelms EM1's direct inhibitory effect on 5-HTergic neurons.

Endomorphin-2 Inhibits the Activity of the Spinoparabrachial Projection Neuron through Presynaptic Mechanisms in the Spinal Dorsal Horn in Rats

“…Under electron microscope, the EM2-ir reaction products within the confines of large dense-cored vesicles were different from other neurotransmitters, such as glutamate and gamma aminobutyric acid (GABA), which stain the small, round or oval vesicles [6, 12, 22, 51]. Although EM2 is known to exert inhibitory effects in the SDH [23, 31, 50, 52], EM2-ir axon terminals mainly exhibited asymmetric synaptic connections with PNs, and thus it is likely that the synapses are excitatory.…”

“…The sections containing many WGA-HRP-labeled neurons in the SDH were selected under a light microscope by being placed on glass slides. Then, these selected sections were collected in vials containing a mixture of 25% (w/v) sucrose and 10% (v/v) glycerol in 0.05 M PB, and treated by using the freeze-thaw method in liquid nitrogen for enhancement of the penetration of the antibody in the immunohistochemical reaction [6, 39]. Subsequently, the sections were washed three times in 0.05 M Tris-buffered saline (TBS; pH 7.4), incubated with 20% (v/v) NDS in 0.05 M TBS for 30 min to block the non-specific immunoreactivity.…”

“…The procedures for immunoelectron microscopy histochemical staining of EM2-ir profiles were the same as those previously described [6, 39, 40]. Briefly, the sections were incubated for 24 h at RT and then for 72 h at 4°C with rabbit antiserum against EM2 (1: 100), which was diluted in 0.05 M TBS (pH 7.4) containing 2% (v/v) NDS (TBS-NDS).…”

“…EM1 is widely and densely distributed throughout the brain and upper brainstem, whereas EM2 is more prevalent in the spinal cord and lower brainstem [5]. Therefore, EM1 is considered to play more important roles in supraspinal structures such as the periaqueductal gray [6], while EM2 mainly functions in the spinal cord [7-9]. …”

Endomorphins: Promising Endogenous Opioid Peptides for the Development of Novel Analgesics

“…Inhibiting the release of SP from primary afferent terminals via presynaptic MORs [3,27,78]; 2). binding to postsynaptic MORs in GABAergic inhibitory interneurons to exert inhibitory effect on excitatory interneurons [30,70,71,103] and 3) increasing the release of NA and 5-HT in the SDH from the descending terminals of the central endogenous pain control system [30,104]. EM1 and EM2 have been shown to be involved in various rodent models of pain.…”