Morphological and physiological evidence of a synaptic connection between the lateral parabrachial nucleus and neurons in the A7 catecholamine cell group in rats

Background

The descending noradrenergic (NAergic) system is one of the important endogenous analgesia systems. It has been suggested that noxious stimuli could activate descending NAergic system; nevertheless, the underlying neuronal circuit remains unclear. As NAergic neurons in the A7 catecholamine cell group (A7) are a part of the descending NAergic system and the lateral parabrachial nucleus (LPB) is an important brainstem structure that relays ascending nociceptive signal, we aimed to test whether LPB neurons have direct synaptic contact with NAergic A7 neurons.

Results

Stereotaxic injections of an anterograde tracer, biotinylated dextran-amine (BDA), were administered to LPB in rats. The BDA-labeled axonal terminals that have physical contacts with tyrosine hydroxylase-positive (presumed noadrenergic) neurons were identified in A7. Consistent with these morphological observations, the excitatory synaptic currents (EPSCs) were readily evoked in NAergic A7 neurons by extracellular stimulation of LPB. The EPSCs evoked by LPB stimulation were blocked by CNQX, a non-NMDA receptor blocker, and AP5, a selective NMDA receptor blocker, showing that LPB-A7 synaptic transmission is glutamatergic. Moreover, the amplitude of LPB-A7 EPSCs was significantly attenuated by DAMGO, a selective μ-opioid receptor agonist, which was associated with an increase in paired-pulse ratio.

Conclusions

Taken together, the above results showed direct synaptic connections between LPB and A7 catecholamine cell group, the function of which is subject to presynaptic modulation by μ-opioid receptors.     

c-fos expression in trigeminal spinal nucleus after electrical stimulation of the hypoglossal nerve in the rat

The expression of the immediate early gene, c-fos, was used to determine the distribution of brainstem neurons activated by stimulation of the distal hypoglossal nerve (XIIn) trunk. The traditional view of the XIIn is one of purely motor function; however, stimulation of XIIn excites neurons in the trigeminal spinal nucleus. The rationale for this study was to use c-fos expression as a marker for postsynaptic activity to define the pattern of brainstem neurons excited by XIIn stimulation. It was further hypothesized that if the afferent fibers that course within XIIn supply deep lingual tissues, then c-fos expression after direct stimulation of XIIn should display a pattern similar to that seen after chemical irritant stimulation of the deep tongue muscle. In barbiturate-anesthetized male rats electrical stimulation of XIIn produced a significant increase in Fospositive neurons in the dorsal paratrigeminal nucleus (dPa5) and laminae I-II of caudal subnucleus caudalis (Vc) and upper cervical dorsal horn. Mustard oil injection into the deep tongue muscle also produced an increase in c-fos expression in dPa5; however, the highest density of expression occurred in laminae I-II at the dorsomedial aspect of rostral Vc. Both electrical stimulation of XIIn and mustard oil stimulation of the deep tongue increased c-fos expression in the caudal ventrolateral medulla, an autonomic relay nucleus. These results suggest that one site of innervation for afferent fibers that travel within the distal trunk of XIIn is to supply the deep tongue muscle and to terminate in the dPa5. A second group of postsynaptic neurons activated only by XIIn stimulation was located in lamina I-II in caudal portions of Vc and upper cervical dorsal horn, a laminar distribution consistent with a role for XIIn afferents in sensory or autonomic aspects of lingual function.     

Analysis of the mechanisms underlying the antinociceptive effect of epicatechin in diabetic rats

Aims

The purpose of this study was to investigate the antinociceptive effect of epicatechin as well as the possible mechanisms of action in diabetic rats.

Main methods

Rats were injected with streptozotocin to produce hyperglycemia. The formalin test was used to assess the nociceptive activity.

Key findings

Acute pre-treatment with epicatechin (0.03–30 mg/kg, i.p.) prevented formalin-induced nociception in diabetic rats. Furthermore, daily or every other day treatment for 2 weeks with epicatechin (0.03–30 mg/kg, i.p.) also prevented formalin-induced nociception in diabetic rats. Acute epicatechin-induced antinociception was prevented by l-NAME (N^ω-nitro-l-arginine methyl ester hydrochloride, 1–10 mg/kg, non-selective nitric oxide synthesis inhibitor), 7-nitroindazole (0.1–1 mg/kg, selective neuronal nitric oxide synthesis inhibitor), ODQ (1H-(1,2,4)-oxadiazolo(4,2-a)quinoxalin-1-one, 0.2–2 mg/kg, guanylyl cyclase inhibitor) or glibenclamide (1–10 mg/kg, ATP-sensitive K⁺ channel blocker). Moreover, epicatechin (3 mg/kg)-induced antinociception was fully prevented by methiothepin (0.1–1 mg/kg, serotonergic receptor antagonist), WAY-100635 (0.03–0.3 mg/kg, selective 5-HT_1A receptor antagonist) or SB-224289 (0.03–0.3 mg/kg, selective 5-HT_1B receptor antagonist). In contrast, BRL-15572 (0.03–0.3 mg/kg, selective 5-HT_1D receptor antagonist) only slightly prevented the antinociceptive effect of epicatechin. Naloxone (0.1–1 mg/kg, opioid antagonist) did not modify epicatechin's effect.

Significance

Data suggest the involvement of the nitric oxide–cyclic GMP–K⁺ channel pathway as well as activation of 5-HT_1A and 5HT_1B, and at a lesser extent, 5-HT_1D, but not opioid, receptors in the antinociceptive effect of epicatechin in diabetic rats. Our data suggest that acute or chronic treatment with epicatechin may prove to be effective to treat nociceptive hypersensitivity in diabetic patients.     

The glutamate transporter GLAST is involved in spinal nociceptive processing

GLAST and GLT-1 are the most abundant glutamate transporters in the CNS and protect neurons from glutamate neurotoxicity. Here, we investigated the role of GLAST in spinal nociceptive processing. GLAST protein expression was not altered after treatment of rats with either formalin or zymosan. Surprisingly, knock-down of GLAST in the spinal cord using antisense-oligonucleotides decreased glutamate concentrations in cerebrospinal fluid (CSF) and reduced the nociceptive behaviour in the rat formalin assay. However, it did not influence thermal hyperalgesia in the zymosan-induced paw inflammation model indicating that GLAST is associated with spontaneous rather than inflammatory nociception. Mechanisms that might explain the decreased response in the formalin assay may include compensatory activation of other glutamate transporters, inhibition of glutamate release or disturbance of glutamate recycling. In conclusion, these data suggest that inhibition of GLAST expression in the spinal cord reduces excitatory synaptic activity and thereby spontaneous responses after nociceptive stimulation of the paw.     

Dynamic Processing of Nociception in Cortical Network in Conscious Rats: A Laser-evoked Field Potential Study

(1) Field potential study in conscious rats provides a convenient and effective animal model for pain mechanism and pharmacological research. However, the spatial-temporal character of nociception processing in cortex revealed by field potential technique in conscious rats remains unclear. (2) In the present study, multi-channel field potentials evoked by noxious laser stimulation applied to the hind paw of conscious rats were recorded through 12 chronically implanted skull electrodes. Independent component analysis (ICA) was used to remove possible artifacts and to extract the specific nociception-related component. (3) Two fast sharp responses and one slow blunt response were evoked by noxious laser stimulation. Systemic morphine (5 mg/kg, i.p.) preferentially attenuated the amplitude of the slow blunt response while had no significant effect on the first two sharp responses. ICA revealed that those responses came from activities of contralateral anterior parietal area, medial frontal area and posterior parietal area. A movement artifact was also detected in this study. Partial directed coherence (PDC) analysis showed that there were changes of information flows from medial frontal and posterior parietal area to anterior parietal area after noxious laser stimulation. (4) Characterization of the spatio-temporal responses to noxious laser stimulation may be a valuable model for the study of pain mechanisms and for the assessment of analgesia.     

Microdialysis of excitatory amino acids in the periaqueductal gray of the rat after unilateral peripheral inflammation

Summary This study measured the release of glutamate (Glu) and aspartate (Asp) amino acid transmitters in the ventrocaudal compartment of the rat periaqueductal gray (PAG) following exposure to unilateral peripheral inflammation. The release of endogenous Glu and Asp from the rat ventrocaudal PAG was monitored with the microdialysis technique in unanesthetized, unrestrained rats. There was significant increase (1,300%) in the basal concentrations of Glu release in the 7 days Complete Freund's Adjuvant (CFA) treated group compared to 24h mineral oil control group. Amino acid release was induced by infusing veratridine (75M, a sodium channel activator) directly through the 1 mm long dialysis probe. Perfusion of veratridine into the ventrocaudal PAG resulted in significant elevation of Glu and Asp amino acids. In the 24h and 7 days CFA treated rats, veratridine-evoked release of Glu was significantly decreased in the lateral ventrocaudal PAG compared to control rats injected with mineral oil (CFA vehicle). The peak minus baseline concentrations of Glu in 24h and 7 days CFA treated groups decreased 55.7% and 43.9%, respectively. In contrast, The basal and the peak minus baseline concentrations of Asp showed no significant change between control group and 24h and 7 days CFA treated animals. The results provide direct evidence that Glu excitatory amino acid may be involved in nociception/nociception modulation pathway in the ventrocaudal PAG.     

Modulation of the human nociceptive reflex by cyclic movements

During static conditions the nociceptive reflex is known to vary as a function of, for example, the stimulus position, stimulus intensity, and muscle contraction. The aim of the present human study was to investigate whether the reflex and the corresponding perception of pain are modulated by cyclic movements of the limb involved. Reflexes, evoked by nociceptive electric stimulation of the sural nerve, were recorded from the biceps femoris and the rectus femoris muscles in eight volunteers. Four different experiments were performed to compare the nociceptive reflex and pain score elicited during active isometric/dynamic flexion/extension of the knee joint. The amplitudes of the reflexes were largest for the dynamic conditions. The reflexes, evoked during dynamic extension and isometric contraction of the rectus femoris muscle, had the shortest latencies but the recordings from the biceps femoris muscle were larger than from the rectus femoris muscle. Knee joint angle recordings showed that the largest angle variations occurred for the dynamic conditions and were only marginally disturbed for the isometric conditions. A given stimulus intensity evoked the highest pain intensity during isometric contractions. This indicates that there would seem to be no causal relationship between the size of the nociceptive reflex and the pain intensity.     

The effect of opiates and opiate antagonists on heat latency response in the parasitic nematode Ascaris suum

The effects of the opiates morphine and morphine-6-glucuronide (M6G), the mu opioid receptor specific antagonist D-Phe-Cys-Tyr-D-Trp-Om-Thr-Pen-Thr-NH(2) (CTOP), and the general opiate antagonist naloxone on the latency of response to thermal stimulation were determined in the parasitic nematode Ascaris suum. Thermal detection and avoidance behaviors of the worms were evaluated with a tail flick analgesia meter using a modification of a technique employed for nociception experiments in rodents. Morphine and M6G were shown to have a dose dependent analgesic effect on A. suum's latency of response to heat with morphine being the most potent. The analgesic effect of morphine was reversed by naloxone but not CTOP. Neither naloxone nor CTOP was able to block the analgesia of M6G. CTOP but not naloxone had significant analgesic effects on its own. These findings are generally consistent with previous results on the effects of opiates and nitric oxide release from A. suum tissue. Apparently these nematodes possess opioid receptors that effect nociception.     

On the enigma of pain and hyperalgesia: A molecular perspective

Pain is a common symptom of injuries and inflammatory-related conditions. The perception of pain, commonly known as nociception, depends on integrated receptors and molecular pathways. Inflammatory mediators are involved in the genesis, persistence, and severity of pain. Noxious stimuli can trigger a cascade of inflammatory loops that feedback onto sensory modalities and domains of the CNS, in an attempt to alert the brain of deregulated homeostasis. Understanding the mechanisms of pain continue to make nociception and hyperalgesia a burgeoning field of research.