Alterations in the rostral ventromedial medulla after the selective ablation of μ-opioid receptor expressing neurons

Abstract

The rostral ventromedial medulla (RVM) exerts both inhibitory and excitatory controls over nociceptive neurons in the spinal cord and medullary dorsal horn. Selective ablation of mu-opioid receptor (MOR)-expressing neurons in the RVM using saporin conjugated to the MOR agonist dermorphin-saporin (derm-sap) attenuates stress and injury-induced behavioral hypersensitivity, yet the effect of RVM derm-sap on the functional integrity of the descending inhibitory system and the properties of RVM neurons remain unknown. Three classes of RVM neurons (on-cells, off-cells, and neutral cells) have been described with distinct responses to noxious stimuli and MOR agonists. Using single unit recording in lightly anesthetized rats, RVM neurons were characterized after microinjections of derm-sap or saporin. Derm-sap treatment resulted in a reduction in on-cells and off-cells when compared to saporin controls (P < 0.05). The number of neutral cells remained unchanged. After derm-sap treatment, RVM microinjections of the glutamate receptor agonist homocysteic acid increased tail-flick latencies, whereas the MOR agonist DAMGO had no effect. Furthermore, electrical stimulation of the periaqueductal gray produced analgesia in both derm-sap and saporin controls with similar thresholds. Microinjection of kynurenic acid, a glutamate receptor antagonist, into the RVM disrupted periaqueductal gray stimulation-produced analgesia in both saporin-treated and derm-sap-treated rats. These results indicate that MOR-expressing neurons in the RVM are not required for analgesia produced by either direct or indirect activation of neurons in the RVM.

Figure 1

Tail-flick latencies after rostral ventromedial medulla (RVM) microinjections of DAMGO and homocysteic acid (HCA) in animals pretreated with RVM microinjections of dermorphin–saporin or saporin. (A) The microinjection of DAMGO had no effect in derm-sap–treated animals, yet increased tail-flick latencies in saporin-treated animals. (B) Microinjection of HCA produced an increase in tail-flick latencies in both treatment groups. a, P <0.05 vs baseline (BSL) in saporin-treated animals; b, P < 0.05 vs BSL in derm-sap–treated animals.

Figure 2

Electrical stimulation in the periaqueductal gray (PAG) produced analgesia in derm-sap–treated and saporin-treated animals. (A) Stimulation sites located in the PAG as verified histologically with electrolytic lesions. Lesions in saporin (closed triangles) and derm-sap (open triangles) treated rats were all located in the left ventrolateral PAG. Number to the right of the figure represents the distance of rostral from the interaural line in mm. (B) The microinjection of kynurenic acid into the rostral ventromedial medulla (RVM) produced comparable increases in PAG electrical stimulation–induced analgesia thresholds. (C) Tail-flick latencies preinjection and postinjection of cobalt chloride into the RVM in saporin and derm-sap–treated animals. Cobalt chloride increased tail-flick latencies only in derm-sap–treated animals. a, P < 0.05; aa, P < 0.01 vs pre–kynurenic acid or pre–cobalt chloride injections into the RVM.

Figure 3

Electrophysiological recordings in the rostral ventromedial medulla after saporin and derm-sap injections. (A) A total of 10 recording tracks were made in each animal. Each electrode penetration site is indicated by an “X” and the site of the microinjection cannulas is represented as circles. The number to the right indicates the distance of caudal from the interaural line. (B) The average number of on, off, and neutral cells recorded in each animal. a, P < 0.05 vs the saporin treatment group. (C) The frequency distribution of individual neutral cell ongoing activity in saporin-treated and derm-sap–treated animals.

Figure 4

The properties of on-cells and off-cells recorded in the rostral ventromedial medulla after saporin and derm-saporin treatment. (A) Average on-cell baseline and peak heat-evoked activity. (B) Average off-cell baseline activity and duration of the off-cell pause produced by heating the tail.

Figure 5

Model of the periaqueductal gray rostral ventromedial medulla (PAGRVM)- dorsal horn circuit after the injection of derm-sap into the RVM. Stimulation of the PAG produces analgesia through an excitatory glutamatergic synapse with off-cells in the RVM, a circuit that remains functional even after derm-sap injections. The ablation of μ-opioid receptor–expressing neurons includes pain-facilitating on-cells and possibly an additional population of inhibitory GABAergic neurons that participate in producing the off-cell pause. As a compensatory response, it is proposed that derm-sap treatment increases tonic GABAergic inhibition of off-cells.