The intrathecal administration of excitatory amino acid receptor antagonists selectively attenuated carrageenan-induced behavioral hyperalgesia in rats.

A single unilateral injection of carrageenan (4.5-6.0 mg in 0.15-0.20 ml saline) into the rat hindpaw induced behavioral hyperalgesia as evidenced by a significant reduction in hindpaw withdrawal latency to a noxious thermal stimulus. The involvement of N-methyl-D-aspartate (NMDA) receptors in this model of hyperalgesia was examined by intrathecal administration of the selective excitatory amino acid (EAA) receptor antagonists: (+/-)-2-amino-5-phosphonopentanoic acid (AP-5), (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), ketamine hydrochloride (ketamine), 7-chlorokynurenic acid (7-Cl kynurenic acid), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). The effects of dizocilpine maleate (MK-801) were studied under the same conditions and published previously (Ren et al., 1992) and the data are presented for comparison. While the withdrawal latencies of the non-injected paws and of the paws of naive rats were not significantly affected by application of the EAA receptor antagonists at doses tested, the paw withdrawal latencies of the carrageenan-injected paws were elevated dose dependently. The rank order of potency of these agents to reduce hyperalgesia was: MK-801 greater than or equal to AP-5 greater than or equal to CPP = 7-Cl kynurenic acid = ketamine much greater than CNQX greater than 0. In contrast, intrathecal injection of the opioid receptor agonists, [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAMGO, mu-selective) and [D-Pen2,D-Pen5] enkephalin (DPDPE, delta-selective), produced antinociception in both injected and non-injected paws. DAMGO was much more potent, while DPDPE was less potent, than MK-801.(ABSTRACT TRUNCATED AT 250 WORDS)

Neonatal capsaicin treatment attenuates spinal Fos activation and dynorphin gene expression following peripheral tissue inflammation and hyperalgesia.

An animal model of nociception involving unilateral hindpaw inflammation has been used to examine behavioral, molecular, and biochemical aspects of well-characterized spinal cord neural circuits involved in pain transmission. The neurotoxin capsaicin administered neonatally was used to modify this neuronal system by producing a selective destruction of most small, unmyelinated primary afferent axons. Capsaicin had minimal effects on the behavioral hyperalgesia and edema associated with the hindpaw inflammation and on the constitutive expression of preprodynorphin (PPD) mRNA and preproenkephalin mRNA in the spinal cord. However, the inflammation-induced increases in Fos-like immunoreactivity (Fos-LI) and in PPD mRNA were greatly attenuated by neonatal capsaicin treatment. The data indicate that input from small-diameter unmyelinated primary afferents is important for the stimulus-induced increase in Fos-LI and PPD mRNA. Our finding that neonatal capsaicin reduces the levels of Fos-LI and PPD mRNA in a related fashion in the spinal dorsal horn provides further evidence for a relationship between the protein product of the c-fos protooncogene and regulation of dynorphin gene transcription.

Peripheral inflammation is associated with increased glutamic acid decarboxylase immunoreactivity in the rat spinal cord.

We have examined the frequency and distribution of neuron profiles immunoreactive for glutamic acid decarboxylase, a biosynthetic enzyme for the putative inhibitory neurotransmitter, gamma aminobutyric acid, in the lumbar spinal cord of colchicine-treated rats with unilateral inflammation of a hindpaw. Ipsilateral to the inflamed hindpaw, there was an apparent increase in the levels of glutamic acid decarboxylase, as indicated by significant increases in the number of visible glutamic acid decarboxylase-like immunoreactive profiles within the superficial dorsal horn, neck of the dorsal horn and the deep gray matter at L4. An increase limited to the deep gray matter at L6 was also seen. No alteration was identified at L2. These results are the first to demonstrate that peripheral inflammation is associated with altered levels of glutamic acid decarboxylase-like immunoreactivity.

Evidence for calcitonin gene-related peptide contacts on a population of lamina I projection neurons.

Using double-labeling techniques, we evaluated small diameter primary afferent input, as indicated by calcitonin gene-related peptide-immunoreactive varicosities, to a population of lamina I projection neurons in the rat lumbar spinal cord. About one third of the lamina I neurons labeled after injections of a retrograde tracer into the region surrounding the brachium conjunctivum received contacts from immunoreactive varicosities. Significantly fewer immunoreactive varicosities were in apposition to fusiform neurons than pyramidal or flattened neurons. A positive correlation was found between the size of the retrogradely labeled neuron and the number of contacts received. This study demonstrates that a known population of nociceptive lamina I neurons received direct input from presumed nociceptive primary afferents.

Spinal opioid analgesic effects are enhanced in a model of unilateral inflammation/hyperalgesia: possible involvement of noradrenergic mechanisms.

We have examined the spinal analgesic activity of opioid agonists and antagonists in a model of short term, unilateral, carrageenan-induced inflammation/hyperalgesia. Rats received a single s.c. injection of carrageenan (2-6 mg in saline) 3-24 h prior to testing hindpaw withdrawal latencies to noxious thermal stimuli. Dose-response curves for intrathecally administered agonists with mu- and/or delta-opioid activity were shifted to the left for inflamed hindpaws when compared to contralateral non-inflamed paws. The selective kappa-receptor agonist U-50,488H had no activity in this analgesic assay on either inflamed or non-inflamed paws when administered intrathecally. However, systemic administration of U-50,488H did produce significant elevations of paw withdrawal latencies in inflamed paws. The alpha 2-adrenoceptor agonist clonidine also produced dose-dependent antinociception in the paw withdrawal assay after systemic or intrathecal administration. Inflamed hindpaws were significantly more sensitive to the antinociceptive effect of morphine on inflamed hindpaws was blocked by the opioid antagonist naloxone or the alpha 2-adrenoceptor antagonist idazoxan. The effect of clonidine was only blocked by idazoxan. Antagonists alone had no significant effect on withdrawal latencies. The data indicate that the analgesic action of opioids during conditions of inflammation may depend on an interaction with spinal noradrenergic pathways.

Peripheral inflammation is associated with increased dynorphin immunoreactivity in both projection and local circuit neurons in the superficial dorsal horn of the rat lumbar spinal cord.

The present study combined the retrograde transport of fluorescent tracers with the immunocytochemical identification of dynorphin A(1-8) in superficial dorsal horn neurons to examine whether peripheral inflammation-induced dynorphin increases are found in local circuit neurons only or also in neurons projecting at least to the caudal mesencephalon. Evidence is presented that complete Freund's adjuvant-induced inflammation produces a large increase in the number of lamina I dynorphin-containing projection and non-projection neurons, and in the number of lamina II dynorphin local circuit neurons.

Spinal lamina I projection neurons in the rat: collateral innervation of parabrachial area and thalamus.

A major ascending nociceptive pathway from spinal lamina I to the mesencephalon has previously been reported in the cat, rat and monkey. In the present paper, we have used single and double retrograde labeling techniques to describe this projection system and its collateralization to the thalamus in the rat. Injections of wheat germ agglutinin-horseradish peroxidase into the pontomesencephalic parabrachial area labeled cell bodies bilaterally in lamina I and deeper laminae of the spinal cord. Bilateral lesions of the dorsolateral funiculi at thoracic levels reduced labeling of lamina I neurons caudal to the lesions. Combined injections of fluorescent retrograde tracers into the lateral thalamus and parabrachial area resulted in double labeling of projection neurons in lamina I, lamina IV VIII and the lateral spinal nucleus of the cervical and lumbar enlargements. Double-labeled neurons were especially abundant in lamina I. Thus, we have demonstrated a major lamina I projection through the dorsolateral funiculi to the parabrachial area with significant collateralization to the thalamus. Moreover, since more than 80% of retrogradely labeled lamina I spinothalamic tract cells had collaterals to the parabrachial area we have indirectly demonstrated the presence of a dorsolateral funicular pathway for lamina I spinothalamic neurons in the rat. More lamina I neurons were retrogradely labeled from midbrain injections as compared to thalamic injections. The significance of these findings rest on previous work in this and other laboratories and concerns the understanding of spinal nociceptive mechanisms. Lamina I projection neurons are primarily nociceptive-specific in their response properties and have been shown to project to both the midbrain and thalamus via the dorsolateral funiculus in a number of species. The role of this projection system in nociceptive transmission may lie in its ability to distribute precise information to multiple brain stem sites which in turn activate autonomic or affective responses or descending pain modulatory mechanisms.

Altered responses of nociceptive cat lamina I spinal dorsal horn neurons after chronic sciatic neuroma formation.

The activity of lumbar spinal dorsal horn lamina I neurons with afferent drive from the sciatic nerve was studied in intact cats and in cats with acute sciatic nerve transection or chronic sciatic nerve transection with neuroma formation. The majority (51 of 75) of neurons recorded in lamina I ipsilateral to a neuroma had no receptive field and could only be identified by their responses to electrical stimulation of the sciatic nerve. The remainder could be activated by the sciatic nerve, but their responses to mechanical stimulation were irregular in comparison to the stable responses of cells recorded in control animals and to the responses of cells contralateral to chronic nerve lesions. Animals with acute nerve transections demonstrated a loss of sciatic nerve-innervated cells with receptive fields except for those cells located on the lateral edge of the dorsal horn, which had normal, proximal receptive fields and response characteristics. In addition, the characteristic somatotopy of lamina I cells was not observed in some cats with chronic neuromata. The mediolateral distribution of cell types indicated that some cells had altered receptive fields following chronic nerve transection. The data presented for lamina I neurons agrees with the observation of spinal cord plasticity first presented for cat dorsal horn cells. Since there is no evidence for a redistribution of intact afferent fibers following chronic nerve transection in adult mammals, the mechanism of altered somatotopy may involve alterations in synaptic efficacy at existing synapses.

Antinociceptive effect of morphine on rat spinothalamic tract and other dorsal horn neurons.

The importance of the spinothalamic tract in pain transmission makes it an attractive candidate for study with respect to the effects of antinociceptive compounds. We have been interested in the analgesic actions of opioids and noradrenergic agents at the spinal level and have investigated the effects of these agents on extracellularly recorded nociceptive dorsal horn neurons in the rat. Spinothalamic tract cells were identified by antidromic activation from the somatosensory thalamus. Morphine was administered by bathing the spinal cord in an artificial cerebrospinal fluid solution which contained a known concentration of drug. We observed a dose-related inhibition, naloxone-reversible in some cases, of activity produced by spinally administered morphine in identified rat spinothalamic tract cells and dorsal horn nociceptive neurons. Morphine had no effect on stimulus-evoked responses of low threshold dorsal horn neurons.

Physiology and morphology of the lamina I spinomesencephalic projection.

We have examined the physiological and morphological characteristics of spinal dorsal horn lamina I neurons with projections to the midbrain in the cat by combining physiological recording of neurons with the intracellular injection of HRP. Lamina I spinomesencephalic neurons were antidromically activated from the region that included the cuneiform nucleus and lateral periaqueductal gray at the intercollicular level. The majority of mesencephalic projection neurons (50 of 55) responded exclusively to noxious stimulation (nociceptive-specific) of their peripheral receptive fields. Lamina I spinomesencephalic neurons were activated from both the ipsilateral and contralateral midbrain and had slow antidromic conduction velocities (1 to 18 m/second). We identified eight cells with projections to both the midbrain and the thalamus and eight cells that were antidromically activated only from the thalamus. Intracellular injection of HRP revealed that lamina I spinomesencephalic neurons were of diverse morphological types, but generally had extensive, rostrocaudally oriented, dendritic arbors confined to lamina I and the overlying white matter. Axons were observed on nine of the HRP-filled spinomesencephalic neurons; five of the axons issued collateral branches. The morphological characteristics of these neurons did not appear to correlate with functional categories (i.e., wide-dynamic-range- or nociceptive-specific-type neurons). The large number of nociceptive-specific neurons with projections to the midbrain and the interconnections of these midbrain sites with hypothalamic and limbic structures suggest that the lamina I spinomesencephalic pathway plays an important role in the autonomic and affective responses to pain.

Serotonin innervation of physiologically identified lamina I projection neurons.

Physiologically characterized lamina I projection neurons, including antidromically activated spinomesencephalic and spinothalamic tract cells, were intracellularly stained with HRP and then processed and examined for serotonin-immunoreactive contacts. We observed cells with both high and low densities of contacts from serotonergic axons. Serotonin contacts were found on both nociceptive-specific and wide-dynamic-range projection neurons. The density of contacts did not appear to correlate with any physiological characteristic.

Lamina I spinomesencephalic neurons in the cat ascend via the dorsolateral funiculi.

Spinomesencephalic tract neurons in the cat spinal cord were retrogradely labeled following injections of wheatgerm agglutinin conjugated to horseradish peroxidase into the region of the midbrain parabrachial area. Labeled cell bodies were concentrated in lamina I, bilaterally. A more scattered distribution was observed in lamina V and deeper laminae. Bilateral lesions of the dorsolateral funiculus (DLF) at thoracic levels eliminated labeling of lamina I neurons below the lesions, but had no effect on the labeling of the neurons in deeper laminae. Injections of colchicine into the spinal white matter caused the label to accumulate intra-axonally and revealed labeled axons bilaterally in the DLF and ipsilaterally in the ventrolateral and ventral funiculi.

Descending serotonergic fibers in the dorsolateral and ventral funiculi of cat spinal cord.

Although there is much evidence for the presence of serotonergic fibers in the spinal gray matter, there is little evidence for the location of descending serotonergic fiber tracts in the spinal white matter. Using a highly sensitive immunocytochemical technique, we localized serotonin-immunoreactive axons throughout the white and gray matter of the spinal cord. Prominent concentrations of serotonergic axons were found in the dorsolateral and ventral funiculi. It is likely that these two descending fiber tracts contribute serotonergic input to the dorsal and ventral horns, respectively.

Spinal lamina I neurons projecting to the parabrachial area of the cat midbrain.

We have observed a population of lamina I neurons in the cat that has projections to the parabrachial area (parabrachial and cuneiform nuclei). A subpopulation of these neurons also projects to the contralateral thalamus. The majority of projecting cells responded exclusively to noxious stimuli, a few wide-dynamic-range neurons were also observed. Conduction velocities for antidromic activation from the midbrain ranged from 1 to 18 m/s. We stained 14 cells intracellularly with horseradish peroxidase. These findings suggest that a major nociceptive projection pathway originating in lamina I and terminating in the parabrachial area exists in the cat.

Intrathecal serotonin in mice: analgesia and inhibition of a spinal action of substance P.

Serotonin, administered intrathecally in mice, produced dose-related analgesia in the tail flick test and the subcutaneous hypertonic saline assay. Low doses (2.5-5 ng) of serotonin blocked the biting and scratching response elicited by intrathecal substance P. However, higher doses of serotonin itself elicited a behavioral syndrome characterized by scratching of the torso with the hindlimbs. Both the analgesic response and the scratching response due to serotonin were blocked by specific serotonin antagonists and the analgesia is likely mediated by a postsynaptic action on dorsal horn nociceptive neurons.

Pharmacological characterization of substance P-induced nociception in mice: modulation by opioid and noradrenergic agonists at the spinal level.

Mice were tested for antinociceptive activity after intrathecal injection of opioid or noradrenergic agonists by lumbar puncture. Opioid agonists with mu or delta activity and adrenergic agonists with alpha activity demonstrated dose-related, receptor-mediated analgesia in the tail-flick assay, s.c. hypertonic saline assay and the intrathecal substance P behavioral assay. Inhibition of substance P-induced biting and scratching by intrathecally administered antinociceptive agents is likely mediated by post-synaptic receptors. This action of opioids and norepinephrine was antagonized by their respective pharmacological antagonists. Subanalgesic doses of Leu-enkephalin or norepinephrine potentiated the antinociceptive activity of morphine in the substance P assay. Similarly, opioid agonists potentiated the action of norepinephrine. These results suggest that opioid and alpha adrenergic agonists act on separate receptors to produce a synergistic inhibition of the transmission of nociceptive information at the spinal level.

Antinociceptive action of intrathecal neurotensin in mice.

Neurotensin has been demonstrated to be analgesic in rodents. This study used intrathecal injection of neurotensin in unanesthetized mice to evaluate the effect of the peptide at the spinal level on unconditioned behavior. Intrathecal administration of neurotensin produced dose-related inhibition of locomotor activity and of the response elicited by subcutaneous hypertonic saline. The effects of the peptide in the tail flick assay were variable and it produced no inhibition of the behavioral response to intrathecal substance P. The results indicate that neurotensin antinociception at the spinal level does not result from locomotor impairment, may be somewhat selective for chemically induced pain, and may be mediated by a presynaptic action on primary afferent fibers.

Intrathecal opioids block a spinal action of substance P in mice: functional importance of both mu- and delta-receptors.

Inhibition of intrathecal substance P-elicited behavior by mu-, delta- and kappa-opioids was compared. In both the substance P behavioral test and the tail flick antinociceptive test, intrathecal [D-Ala2, Met5]enkephalinamide and [D-Ala2, D-Leu5]enkephalin (DADL) were equipotent, morphine and ethylketazocine were less potent, but nalorphine was inactive. A long-lasting, highly selective, mu-receptor antagonist, beta-funaltrexamine, blocked the inhibition of substance P behaviors by both morphine and ethylketazocine, but did not block the effect of DADL. These results suggest that spinal postsynaptic modulation of nociception is mediated by both delta-type and mu-type opioid agonists.

Intrathecal substance P and somatostatin in rats: behaviors indicative of sensation.

Biochemical, histochemical and neurophysiological data suggest that substance P and somatostatin are neurotransmitters for primary afferent neurons. This study used intrathecal administration of these peptides and others (neurotensin and vasoactive intestinal polypeptide) in chronically catheterized, environmentally adapted, freely moving rats to evaluate their effects on unconditioned behavior. Substance P and somatostatin each elicited behaviors which were dose related. The behaviors included caudally directed biting and licking along with hindlimb scratching, writhing and retching. The behavioral responses were rapid in onset (1 min) and, in the case of substance P, short in duration (3 min). Vehicle, neurotensin and vasoactive intestinal polypeptide were without effect. These results demonstrate the ability of substance P and somatostatin to induce behavior in rats upon intrathecal administration and extend previous studies in mice.

Intrathecal substance P elicits a caudally-directed biting and scratching behavior in mice.

Intrathecal injection of substance P in mice elicited a dose-related biting and scratching response. Systemic but not intrathecal morphine sulfate antagonized this response. We interpret these observations to define a novel nociceptive response to intrathecal substance P.