Tolerance, opioid-induced allodynia and withdrawal associated allodynia in infant and young rats.

Our laboratory has previously characterized age-dependent changes in nociception upon acute morphine withdrawal. This study characterizes changes in mechanical and thermal nociception following acute, intermittent, or continuous morphine administration in infant (postnatal days 5-8) and young (postnatal days 19-21) rats. Morphine was given as a single acute administration (AM), intermittently twice a day for 3 days (IM), or continuously for 72 h via pump (CM). AM did not produce long-term changes in mechanical or thermal nociception in either infant or young rats. CM produced changes in mechanical nociception that included the development of tolerance, opioid-induced mechanical allodynia and withdrawal-associated mechanical allodynia in young rats, but only tolerance and a prolonged withdrawal-associated mechanical allodynia in infant rats. IM produced withdrawal-associated mechanical allodynia in both infant and young rats. Measuring paw withdrawal responses to thermal stimuli, infant and young rats showed tolerance without opioid-induced thermal hyperalgesia or withdrawal-associated thermal hyperalgesia following CM. In contrast to CM, withdrawal-associated thermal hyperalgesia was seen in both ages following IM. In conclusion, CM versus IM differentially modified mechanical and thermal nociception, suggesting that opioid-dependent thermal hyperalgesia and mechanical allodynia can be dissociated from each other in infant and young rats. Furthermore, tolerance, opioid-induced hypersensitivity, and withdrawal-associated hypersensitivity are age-specific and may be mediated by distinct mechanisms.

Anesthetic actions within the spinal cord: contributions to the state of general anesthesia.

The behavioral state known as general anesthesia is the result of actions of general anesthetic agents at multiple sites within the neuraxis. The most common end point used to measure the presence of anesthesia is absence of movement following the presentation of a noxious stimulus. The actions of general anesthetics within the spinal cord have been shown to contribute significantly to the suppression of pain-evoked movements, an important component of clinical anesthesia. Studies in the spinal cord are likely to increase our understanding of the pharmacology by which general anesthetics alter the transmission of somatomotor information. It now appears that the pharmacology responsible for the production of anesthesia is agent- and site-selective, and not the result of a unitary mechanism of action.

Bupivacaine is an effective potassium channel blocker in heart.

The local anesthetic agent bupivacaine increases action potential duration in isolated frog atrial myocytes, and blocks two potassium conductances, IK and IK1. The effective concentrations, particularly for IK, are similar to those which depress the sodium conductance. Potassium channel block may thus contribute to bupivacaine's reported cardiotoxicity.

The NMDA receptor antagonist MK-801 differentially modulates mu and kappa opioid actions in spinal cord in vitro.

We have examined the interactions between NMDA receptors and opioid effects in isolated neonatal rat spinal cord. Electrical stimulation of a lumbar dorsal root evoked a nociceptive-related slow ventral root potential (sVRP) recorded at the corresponding ipsilateral ventral root. The kappa opiate receptor agonist U69,593 (2.5 nM-1 microM) depressed sVRP area by a maximum of 80%, EC50 was approximately 33 nM. Both the non-specific antagonist naloxone and the kappa-specific antagonist nor-binaltorphimine (nor-BNI) antagonized the effects of U69,593. Morphine, a mu agonist, (1 nM-1 microM) depressed sVRP area with an approximate EC50 of 90 nM. The effects of both mu and kappa opioid agonists were selective for the very slow metabotropically mediated components of the sVRP, compared to the relatively fast NMDA receptor-mediated components. The non-competitive N-methyl-D-aspartate (NMDA) antagonist MK-801 (20 nM) had no effect on sVRP area when applied alone but co-applied with morphine significantly potentiated the depressant effects of morphine. In contrast, MK-801 either had no effect on or slightly antagonized the depressant effects of U69,593. Naloxone following morphine produced a significant increase in sVRP area above pre-morphine control values; the increase lasted 30 min or more. Neither naloxone nor nor-BNI was associated with an increase in sVRP area when given alone or following U69,593. MK-801 co-applied with morphine blocked the rebound increase in sVRP area following naloxone. These results suggest that (1) both mu and kappa receptor agonists exert similar selective depressant effects on spinal nociceptive neurotransmission; (2) mu but not kappa agonists exert prolonged excitatory effects that oppose the depression; and (3) NMDA receptors play a role in determining opioid analgesic potency and naloxone-precipitated hyperresponsiveness. The results may be related to initial steps in the development of acute tolerance to mu opioids, and suggest that tolerance to kappa opioids may have a different mechanism.

Substance P and NMDA receptor-mediated slow potentials in neonatal rat spinal cord: age-related changes.

Slow ventral root potentials (slow VRP's) recorded from 1- to 5-day-old rat spinal cords are implicated in nociception, but there is controversy over their origin and persistence in the adult. The present study investigated changes in the role of substance P and NMDA receptors in slow VRP generation during the postnatal period (1-21 days). Through 9 days, dorsal root stimulation elicits slow VRP's with typical peak amplitudes at 3-4 s, decay time constants of 18-20 s, and durations > 20 s. After 11 days, peak amplitude shortens to < 1 s, decay time constant 4-5 s, and duration < 10 s. At 1-6 days, slow VRP's are sensitive to the NMDA receptor antagonist APV and the substance P antagonists spantide and CP 96,345. After 11 days, APV sensitivity is retained, but spantide and ability of substance P to evoke a response are diminished. Abbreviated slow VRP's in post-11-day spinal cords appear to correspond to the early APV-sensitive component of long-duration slow VRP's in younger animals. Attempts to restore long-duration slow VRP's in 12- to 14-day-old rat cords by blocking various inhibitory mechanisms were not successful. The results suggest that a substance P response, some of which is mediated by NK1 receptors, is lost with maturation of the cord. Either a developmental role played by substance P changes with maturity, or the motor neurons of the isolated post-11-day cord lose the capacity to sustain large long-duration plateau potentials.

Substance P and NMDA receptors mediate a slow nociceptive ventral root potential in neonatal rat spinal cord.

Substance P and glutamate actions have separately been implicated in the generation of nociceptive-related slow ventral root potentials (slow VRPs). We report that slow VRPs are dependent on both substance P and NMDA receptor-mediated neurotransmission. Slow VRPs of 10-40 s duration were evoked by electrically stimulating a lumbar dorsal root and recorded at the corresponding ipsilateral ventral root in spinal cords isolated from 1- to 5-day-old rats; the monosynaptic reflex was also recorded. The NMDA receptor antagonist APV (5-20 microM) and the substance P antagonist spantide (10-20 microM) both reversibly depressed the slow VRP without affecting the monosynaptic reflex; spantide and APV applied together nearly abolished the slow VRP. The quisqualate-kainate receptor antagonist CNQX (1-5 microM) reduced the monosynaptic reflex and an early component of the slow VRP. A slow VRP could be elicited by brief (0.1-1.0 s) focal applications of either substance P (2-20 microM) or NMDA (10 microM), and also by CGRP (2-20 microM). Substance P-evoked and NMDA-evoked responses were blocked by their respective antagonists spantide and APV. Each was also cross-sensitive to the other antagonist. Both excitatory amino acids, acting on an NMDA receptor, and substance P, acting on a tachykinin receptor, thus appear to be involved in generating this slow potential. Both NMDA and tachykinin receptors are necessary to generate a full response.

Cholinergic regulation of impulse frequency in peripheral nerve.

Previous studies have demonstrated the existence of receptors for gamma-aminobutyric acid (GABA), beta-adrenergic catecholamines and acetylcholine in vertebrate peripheral nerve, and provided functional correlates for activation of both GABA and beta-adrenergic receptors. The present studies show that a cholinergic receptor present on the nerve can also modify impulse pattern. In frog sciatic nerve, both carbamylcholine and dibutyryl cyclic GMP increased the amplitude of the response to the second stimulus of a train at very short interstimulus intervals. The effect of carbamylcholine was blocked by 4-aminopyridine. The results are consistent with the hypothesis that cholinergic agonists mediate an increase in endogenous cyclic guanosine monophosphate (cGMP), which increases the ability of the nerve to follow closely spaced stimuli by inhibiting potassium channels.

Cyclic AMP reduction of frequency following ability in peripheral axons.

Radioimmunoassays for cAMP demonstrated that a beta-adrenergic agonist, isoproterenol, increased cAMP levels in isolated frog sciatic nerve. Dibutyryl cAMP (db-cAMP) and isoproterenol reduced the amplitude of the compound action potential and decreased the ability of the Xenopus sciatic nerve to follow high frequency stimulation. Similar effects of db-cAMP and a phosphodiesterase inhibitor were seen on intracellularly recorded action potentials of single lobster peripheral axons. These results suggest that cAMP can modulate the electrophysiological response properties of both myelinated and unmyelinated axons.

The alpha 2-adrenoceptor agonist dexmedetomidine increases the apparent potency of the volatile anesthetic isoflurane in rats in vivo and in hippocampal slice in vitro.

Alpha 2-adrenoceptor agonists such as clonidine are sedatives and enhance the effectiveness of several different kinds of anesthetics. This study was performed to quantitate the effect of dexmedetomidine, a novel alpha 2-adrenoceptor agonist, on the action of the volatile anesthetic agent isoflurane in rats in vivo. A separate set of experiments in rat hippocampal slices was designed to determine whether isoflurane and dexmedetomidine exerted similar effects on synaptic transmission in vitro and to examine the interaction between the two agents. In vivo, dexmedetomidine (100 micrograms/kg i.p.) reduced isoflurane minimum alveolar anesthetic requirement (MAC), determined by loss of response to tail pinch, by approximately 90%. In hippocampal CA1 neurons, on the other hand, there was a relatively small potentiation of the effects of isoflurane at the maximally effective dexmedetomidine concentration (1 nM). The hippocampal CA1 area, at least in the slice preparation, may thus not be representative of the CNS site(s) at which alpha 2 adrenoceptor agonists lessen anesthetic requirement in vivo.

Alpha 2-adrenoceptors inhibit a nociceptive response in neonatal rat spinal cord.

Alpha 2-Adrenoceptors mediate analgesia in vivo. The present study explored the actions of the alpha 2-adrenoceptor agonists dexmedetomidine and clonidine on a nociceptive response in isolated neonatal rat spinal cord. Stimulation of a dorsal root generates a slow ventral root potential (slow VRP) at the corresponding ipsilateral ventral root. The slow VRP meets several criteria for a nociceptive response. Dexmedetomidine (10 nM) and clonidine (200 nM) depressed the slow VRP by approximately 80%. Dexmedetomidine's action was approximately linear over the concentration range 0.5-500 nM, whereas clonidine (20 nM-5 microM) exerted biphasic effects. The profile of agonist and antagonist effectiveness characterized the receptor(s) as alpha 2-adrenoceptors; the subtype could not be identified as either alpha 2A or alpha 2B. Naloxone pretreatment partially blocked dexmedetomidine's effect, suggesting a possible endogenous opiate involvement. Dexmedetomidine (0.5-2.0 nM) also depressed the VRP evoked by application of substance P to the cord, implicating postsynaptic as well as possible presynaptic actions. At high concentrations, dexmedetomidine (50-500 nM) depressed the monosynaptic reflex, probably through non-alpha 2-receptor(s). Results from the neonatal spinal cord correlate well with those from in vivo analgesia studies. They suggest an important direct spinal contribution to alpha 2-adrenoceptor-mediated analgesia.

Ethanol as a general anesthetic: actions in spinal cord.

Ethanol, usually studied in relation to intoxication, is also capable of producing general anesthesia. The most common standard of anesthetic potency is the concentration which produces immobility in response to a noxious stimulus. This concentration will be referred to as the anesthetic concentration. Immobilization is a spinal effect. Ethanol effects were studied in spinal cord from 2-7-day-old rats at concentrations which included the anesthetic concentration in both adult rats (97 mM) and 6-7-day-old rats (235 mM). At neonatal but not adult anesthetic concentrations, ethanol depressed monosynaptic reflex amplitude (mediated by glutamate AMPA receptors + compound action potential). At both neonatal and adult anesthetic concentrations ethanol reversibly depressed the population excitatory postsynaptic potential (pEPSP) (glutamate AMPA and NMDA receptors), the slow ventral root potential (NMDA + metabotropic receptors), and the dorsal root potential (GABA(A) receptors, via glutamate-excited interneurons). Effects were greater on NMDA receptor-mediated components than on AMPA-receptor-mediated components of the pEPSP and greater on NMDA than on metabotropic receptor-mediated components of the slow ventral root potential. The profile of ethanol effects on spinal cord resembles that of inhalation general anesthetics. The results show that both AMPA and NMDA receptor-mediated transmission are sensitive to ethanol and that enhancement of GABAergic neurotransmission is overridden by depression of excitation to the interneurons. They provide no obvious explanation for ethanol's lower general anesthetic potency in the neonate.

Correlates of anesthetic properties in isolated spinal cord: cyclobutanes.

Two halogenated cyclobutanes, one anesthetic and one not, were compared on receptor-specific pathways in isolated neonatal rat spinal cord. The anesthetic 1-chloro-1,2,2-trifluorocyclobutane depressed the monosynaptic reflex (glutamate non-NMDA receptors) and abolished a slow ventral root potential (glutamate NMDA, non-NMDA and tachykinin receptors). This compound slightly enhanced the muscimol-evoked dorsal root potential (GABAA) but reversibly depressed the dorsal root potential elicited by dorsal root stimulation. The non-anesthetic 1,2-dichlorohexafluorocyclobutane increased monosynaptic reflex, depressed slow ventral root potential approximately 50%, had little effect on muscimol-evoked dorsal root potential, and irreversibly depressed dorsal root-evoked dorsal root potential. Hypoxia accounts for slow ventral root potential depression, but not monosynaptic reflex enhancement. In this preparation and for this pair of compounds, anesthetic properties are related to blockade of transmission at glutamate synapses, with a small component of GABAA enhancement. Monosynaptic reflex increase may be related to the non-anesthetic cyclobutane's convulsant and anti-anesthetic properties.

Propofol potentiates the depressant effect of alfentanil in isolated neonatal rat spinal cord and blocks naloxone-precipitated hyperresponsiveness.

Our previous studies have shown that a benzodiazepine potentiates opioid actions on spinal cord by blocking a hyperresponsiveness that may be related to the development of opioid tolerance and withdrawal. The present study was designed to test whether propofol, which like benzodiazepines acts on GABA(A) receptors, displays similar interactions with opioids. Spinal cords isolated from 1-7 day old rats were arranged to record the slow ventral root potential (sVRP) elicited by stimulating a lumbar dorsal root. A concentration of propofol which by itself did not depress sVRP significantly enhanced the apparent potency of alfentanil and blocked the increase in sVRP observed when alfentanil is followed by naloxone. The results suggest that enhancement of GABA inhibition may increase opioid potency by inhibiting the development of acute tolerance.

N-methyl-D-aspartate receptors are implicated in hyperresponsiveness following naloxone reversal of alfentanil in isolated rat spinal cord.

In isolated neonatal rat spinal cord, naloxone administered after an opioid increases a nociceptive-related slow ventral root potential (sVRP) to levels above pre-drug controls. We studied the role of N-methyl-D-aspartate (NMDA) receptors in this phenomenon, which may be related to acute tolerance and to hyperalgesia on antagonist-precipitated withdrawal. Naloxone (200 nM) alone produced no significant effect on sVRP area, while naloxone (560 nM) increased area to 121 +/- 17.7% of control (mean +/- SD). Following 200 nM alfentanil, naloxone (200 nM) was associated with a significant rebound in sVRP area to 138 +/- 18.0% of pre-drug control. Hyperresponsiveness developed within 7 min of initial alfentanil exposure. The non-competitive NMDA antagonist MK-801 (20 nM) had no effect on sVRP area when applied alone; higher concentrations produced irreversible depression. MK-801 (20 nM) co-applied with 200 nM alfentanil blocked the rebound increase in sVRP area following naloxone 200 nM and also the increase following naloxone alone (560 nM). The results suggest that alfentanil induces a rapid NMDA receptor-dependent change in spinal cord neuronal excitability.

Descending inhibition in neonatal rat spinal cord: actions of pentobarbital and morphine.

Descending inhibition plays an important role in modulating spinal nociceptive neurotransmission. Barbiturates have been suggested to be poor analgesics or anti-analgesic because they block descending inhibition from supraspinal centers to the spinal cord. Opiate analgesics, on the other hand, are postulated to increase descending inhibition. We tested this hypothesis in an isolated brain stem-spinal cord preparation from neonatal rats, using as the test response a nociceptive-related slow ventral root potential (sVRP) recorded in the lumbar region. Brain stem and spinal cord were separately perfused. Transecting the spinal cord, applying the local anesthetic lidocaine to the brain stem, or cooling the brain stem increased the area of the sVRP, thus demonstrating that tonic descending inhibition is present in this preparation. Pentobarbital (Pb) (1-10 microM) applied to the spinal cord depressed the sVRP in a dose-dependent fashion. Spinal cord transection did not significantly change Pb potency. Pb (5-10 microM) applied to the brain stem alone did not significantly increase sVRP amplitude. Morphine (15-35 nM) applied to the spinal cord also depressed the sVRP but had no effect when applied to the brain stem. The results show that there are functional synaptic connections mediating tonic descending inhibition in the neonatal rat. They do not support interaction with tonic descending inhibition as an explanation for morphine analgesia or as a reason for lack of analgesic properties in the barbiturates.